A

Text book

of

Immunology

Edited by

Arkabrata banerjee

b.sc biotech(h) from the university of burdwan

&

Trained in magenta pigment production from fungus in lab. condition ,shrm biotech

Kolkata

&

Mba from wbut,aicte

……………………1st edition 2011……………………

IMMUNO BIOLOGYAn immune system is a system of biological structures and processes within an organism that protects against disease by identifying and killing pathogens and tumor cells. It detects a wide variety of agents, from viruses to parasitic worms, and needs to distinguish them from the organism's own

healthy cells and tissues in order to function properly. Detection is complicated as pathogens can evolve rapidly, producing adaptations that avoid the immune system and allow the pathogens to successfully infect their hosts.

Immunity is a biological term that describes a state of having sufficient biological defenses to avoid infection, disease, or other unwanted biological invasion. Immunity involves both specific and non-specific components. The non-specific components act either as barriers or as eliminators of wide range of pathogens irrespective of antigenic specificity. Other components of the immune system adapt themselves to each new disease encountered and are able to generate pathogen-specific immunity.

Adaptive immunity is often sub-divided into two major types depending on how the immunity was introduced. Naturally acquired immunity occurs through contact with a disease causing agent, when the contact was not deliberate, whereas artificially acquired immunity develops only through deliberate actions such as vaccination. Both naturally and artificially acquired immunity can be further subdivided depending on whether immunity is induced in the host or passively transferred from a immune host. Passive immunity is acquired through transfer of antibodies or activated T-cells from an immune host, and is short lived -- usually lasting only a few months -- whereas active immunity is induced in the host itself by antigen, and lasts much longer, sometimes life-long. The diagram below summarizes these divisions of immunity.

A further subdivision of adaptive immunity is characterized by the cells involved; humoral immunity is the aspect of immunity that is mediated by secreted antibodies, whereas the protection provided by cell mediated immunity involves T-lymphocytes alone. Humoral immunity is active when the organism generates its own antibodies, and passive when antibodies are transferred between individuals. Similarly, cell mediated immunity is active when the organisms’ own T-cells are stimulated and passive when T cells come from another organism.

Passive immunityPassive immunity is the transfer of active immunity, in the form of readymade antibodies, from one individual to another. Passive immunity can occur naturally, when maternal antibodies are transferred to the fetus through the placenta, and can also be induced artificially, when high levels of human (or horse) antibodies specific for a pathogen or toxin are transferred to non-immune individuals. Passive immunization is used when there is a high risk of infection and insufficient time for the body to

develop its own immune response, or to reduce the symptoms of ongoing or immunosuppressive diseases. Passive immunity provides immediate protection, but the body does not develop memory, therefore the patient is at risk of being infected by the same pathogen later.

Naturally acquired passive immunityMaternal passive immunity is a type of naturally acquired passive immunity, and refers to antibody-mediated immunity conveyed to a fetus by its mother during pregnancy. Maternal antibodies (MatAb) are passed through the placenta to the fetus by an FcRn receptor on placental cells. This occurs around the third month of gestation. IgG is the only antibodyisotype that can pass through the placenta. Passive immunity is also provided through the transfer of IgA antibodies found in breast milk that are transferred to the gut of the infant, protecting against bacterial infections, until the newborn can synthesize its own antibodies.

Artificially acquired passive immunityArtificially acquired passive immunity is a short-term immunization induced by the transfer of antibodies, which can be administered in several forms; as human or animal blood plasma, as pooled human immunoglobulin for intravenous (IVIG) or intramuscular (IG) use, and in the form of monoclonal antibodies (MAb). Passive transfer is used prophylactically in the case of immunodeficiency diseases, such as hypogammaglobulinemia. It is also used in the treatment of several types of acute infection, and to treat poisoning.Immunity derived from passive immunization lasts for only a short period of time, and there is also a potential risk for hypersensitivity reactions, and serum sickness, especially from gamma globulin of non-human origin.

The artificial induction of passive immunity has been used for over a century to treat infectious disease, and prior to the advent of antibiotics, was often the only specific treatment for certain infections. Immunoglobulin therapy continued to be a first line therapy in the treatment of severe respiratory diseases until the 1930’s, even after sulfonamide antibiotics were introduced.

Passive transfer of cell-mediated immunityPassive or "adoptive transfer" of cell-mediated immunity, is conferred by the transfer of "sensitized" or activated T-cells from one individual into another. It is rarely used in humans because it requires histocompatible (matched) donors, which are often difficult to find. In unmatched donors this type of transfer carries severe risks of graft versus host disease. It has, however, been used to treat certain diseases including some types of cancer and immunodeficiency. This type of transfer differs from a bone

marrow transplant, in which (undifferentiated) hematopoietic stem cells are transferred.

Active immunity

The time course of an immune response. Due to the formation of immunological memory, reinfection at later time points leads to a rapid increase in antibody production and

effector T cell activity. These later infections can be mild or even inapparent.

When B cells and T cells are activated by a pathogen, memory B-cells and T- cells develop. Throughout the lifetime of an animal these memory cells will “remember” each specific pathogen encountered, and are able to mount a strong response if the pathogen is detected again. This type of immunity is both active and adaptive because the body's immune system prepares itself for future challenges. Active immunity often involves both the cell-mediated and humoral aspects of immunity as well as input from the innate immune system. The innate system is present from birth and protects an individual from pathogens regardless of experiences, whereas adaptive immunity arises only after an infection or immunization and hence is "acquired" during life.

Naturally acquired active immunityNaturally acquired active immunity occurs when a person is exposed to a live pathogen, and develops a primary immune response, which leads to immunological memory.This type of immunity is “natural” because it is not induced by deliberate exposure. Many disorders of immune system function can affect the formation of active immunity such asimmunodeficiency (both acquired and congenital forms)

and immunosuppression.

Artificially acquired active immunityArtificially acquired active immunity can be induced by a vaccine, a substance that contains antigen. A vaccine stimulates a primary response against the antigen without causing symptoms of the disease. The term vaccination was coined by Edward Jenner and adapted by Louis Pasteur for his pioneering work in vaccination. The method Pasteur used entailed treating the infectious agents for those diseases so they lost the ability to cause serious disease. Pasteur adopted the name vaccine as a generic term in honor of Jenner's discovery, which Pasteur's work built upon.

Layered defenseThe immune system protects organisms from infection with layered defenses of increasing specificity. In simple terms, physical barriers prevent pathogens such as bacteria and virusesfrom entering the organism. If a pathogen breaches these barriers, the innate immune system provides an immediate, but non-specific response. Innate immune systems are found in all plants and animals. If pathogens successfully evade the innate response, vertebrates possess a third layer of protection, the adaptive immune system, which is activated by the innate response. Here, the immune system adapts its response during an infection to improve its recognition of the pathogen. This improved response is then retained

after the pathogen has been eliminated, in the form of an immunological memory, and allows the

adaptive immune system to mount faster and stronger attacks each time this pathogen is encountered.

Components of the immune system

Innate immune system Adaptive immune system

Response is non-specific Pathogen and antigen specific response

Exposure leads to immediate maximal response Lag time between exposure and maximal response

Cell-mediated and humoral components Cell-mediated and humoral components

No immunological memory Exposure leads to immunological memory

Found in nearly all forms of life Found only in jawed vertebrates

Both innate and adaptive immunity depend on the ability of the immune system to distinguish between self and non-self molecules. In immunology, self molecules are those components of an organism's body that can be distinguished from foreign substances by the immune system. Conversely, non-self molecules are those recognized as foreign molecules. One class of non-self molecules are called antigens (short for antibody generators) and are defined as substances that bind to specific immune receptors and elicit an immune respons.

Surface barriersSeveral barriers protect organisms from infection, including mechanical, chemical, and biological barriers. The waxy cuticle of many leaves, the exoskeleton of insects, the shells and membranes of externally deposited eggs, and skin are examples of the mechanical barriers that are the first line of defense against infection. However, as organisms cannot be completely sealed against their environments, other systems act to protect body openings such as the lungs, intestines, and the genitourinary tract. In the lungs, coughing and sneezingmechanically eject pathogens and other irritants from the respiratory tract.

The flushing action of tears and urine also mechanically expels pathogens, while mucus secreted by the

respiratory and gastrointestinal tract serves to trap and entangle microorganisms.

Chemical barriers also protect against infection. The skin and respiratory tract secrete antimicrobial peptides such as the β-defensins. Enzymes such as lysozyme and phospholipase A2 in saliva, tears, and breast milk are also antibacterials. Vaginal secretions serve as a chemical barrier following menarche, when they become slightly acidic, while semencontains defensins and zinc to kill pathogens. In the stomach, gastric acid and proteases serve as powerful chemical defenses against ingested pathogens.

Within the genitourinary and gastrointestinal tracts, commensal flora serve as biological barriers by competing with pathogenic bacteria for food and space and, in some cases, by changing the conditions in their environment, such as pH or available iron. This reduces the probability that pathogens will be able to reach sufficient numbers to cause illness. However, since most antibiotics non-specifically target bacteria and do not affect fungi, oral antibiotics can lead to an “overgrowth” of fungi and cause conditions such as a vaginalcandidiasis (a yeast infection). There is good evidence that re-introduction of probiotic flora, such as pure cultures of the lactobacilli normally found in unpasteurized yoghurt, helps restore a healthy balance of microbial populations in intestinal infections in children and encouraging preliminary data in studies on bacterial gastroenteritis, inflammatory bowel diseases,urinary tract infection and post-surgical infections.

Innate immune system The innate immune system comprises the cells and mechanisms that defend the host from infection by other organisms, in a non-specific manner. This means that the cells of the innate system recognize and respond to pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host. Innate immune systems provide immediate defense against infection, and are found in all classes of plant and animal life.

The innate system is thought to constitute an evolutionarily older defense strategy, and is the dominant immune system found in plants, fungi, insects, and in primitive multicellular organisms.

The major functions of the vertebrate innate immune system include:

Recruiting immune cells to sites of infection, through the production of chemical factors, including specialized chemical mediators, called cytokines.

Activation of the complement cascade to identify bacteria, activate cells and to promote clearance of dead cells or antibody complexes.

The identification and removal of foreign substances present in organs, tissues, the blood and lymph, by specialized white blood cells.

Activation of the adaptive immune system through a process known as  antigen presentation

Cells of the innate immune response

(a) LeukocytesWhite blood cells (WBCs), or leukocytes (also spelled "leucocytes"), are cells of the immune system involved in defending the body against both infectious disease and foreign materials. Five different and diverse types of leukocytes exist, but they are all produced and derived from a multipotent cell in the bone marrow known as a hematopoietic stem cell. Leukocytes are found throughout the body, including the blood andlymphatic system.

The number of WBCs in the blood is often an indicator of disease. There are normally between 4×109 and 1.1×1010 white blood cells in a litreof blood, making up approximately 1% of blood in a healthy adult. An increase in the number of leukocytes over the upper limits is calledleukocytosis, and a decrease below the lower limit is called leukopenia. The physical properties of leukocytes, such as volume, conductivity, and granularity, may change due to activation, the presence of immature cells, or the presence of malignant leukocytes in leukemia.

 scanning electron microscope image of normal circulating human blood. In addition to the irregularly shaped leukocytes, both red blood cells and many small disc-shapedplatelets are visible.

Types

There are several different types of white blood cells. They all have many things in common, but are all distinct in form and function. A major distinguishing feature of some leukocytes is the presence of granules; white blood cells are often characterized as granulocytes or agranulocytes:

Granulocytes (polymorphonuclear leukocytes): leukocytes characterised by the presence of differently staining granules in their cytoplasm when viewed under light microscopy. These granules are membrane-bound enzymes which primarily act in the digestion of endocytosed particles. There are three types of granulocytes: neutrophils, basophils, and eosinophils, which are named according to their staining properties.

Agranulocytes (mononuclear leucocytes): leukocytes characterized by the apparent absence of granules in their cytoplasm. Although the name implies a lack of granules these cells do contain non-specific azurophilic granules, which are lysosomes. The cells include lymphocytes, monocytes, and macrophages.

1.NeutrophilNeutrophil granulocytes are generally referred to as either neutrophils or polymorphonuclear neutrophils (or PMNs), and are subdivided into segmented neutrophils (or segs) and banded neutrophils (or bands). Neutrophils are the most abundant type of white blood cells in mammals and form an essential part of the innate immune system. They form part of the polymorphonuclear cell family (PMNs) together withbasophils and eosinophils.

Neutrophils are normally found in the blood stream. However, during the beginning (acute) phase of inflammation, particularly as a result ofbacterial infection and some cancers, neutrophils are one of the first-responders of inflammatory cells to migrate toward the site of inflammation, firstly through the blood vessels, then through interstitial tissue, following chemical signals (such as Interleukin-8 (IL-8) and C5a) in a process called chemotaxis. They are the predominant cells in pus, accounting for its whitish/yellowish appearance.

Neutrophils are recruited to the site of injury within minutes following trauma and are the hallmark of acute inflammation.

A neutrophil, stained with Wright's stain.  This cell is approximately 12 µm in diameter

With the eosinophil and the basophil, they form the class of polymorphonuclear cells, named for the nucleus's characteristic multilobulated shape (as compared to lymphocytes and monocytes, the other types of white cells). Neutrophils are the most abundant white blood cells in humans (approximately 10^11 are produced daily) ; they account for approximately 70% of all white blood cells (leukocytes).

A minor difference is found between the neutrophils from a male subject and a female subject. The cell nucleus of a neutrophil from a female subject shows a small additional X chromosome structure, known as a "neutrophil drumstick".

The average half-life of non-activated neutrophils in the circulation is about 12 hours. Upon activation, they marginate (position themselves adjacent to the blood vessel endothelium), and undergo selectin-dependent capture followed by integrin-dependent adhesion in most cases, after which they migrate into tissues, where they survive for 1–2 days.

Neutrophils are much more numerous than the longer-lived monocyte/macrophage phagocytes. A pathogen (disease-causing microorganism or virus) is likely to first encounter a neutrophil. Some experts hypothesize that the short lifetime of neutrophils is an evolutionary adaptation. The short lifetime of neutrophils minimizes propagation of those pathogens that parasitize phagocytes because the more time such parasites spend outside a host cell, the more likely they will be destroyed by some component of the body's defenses. Also, because neutrophil antimicrobial products can also damage host tissues, their short life limits damage to the host during inflammation.

Neutrophils will often be phagocytosed themselves by macrophages after digestion of pathogens. PECAM-1 and phosphatidylserine on the cell surface are involved in this process.

Neutrophil granulocyte migrates from the blood vessel to the matrix, sensing proteolytic enzymes, in order to determine intercellular connections (to the improvement of its mobility) and envelop bacteria through phagocytosis

Neutrophils undergo a process called chemotaxis, which allows them to migrate toward sites of infection or inflammation. Cell surface receptors allow neutrophils to detect chemical gradients of molecules such as interleukin-8 (IL-8), interferon gamma (IFN-gamma), and C5a, which these cells use to direct the path of their migration.

Anti-microbial function

Being highly motile, neutrophils quickly congregate at a focus of infection, attracted by cytokines expressed by activated endothelium, mast cells, andmacrophages. Neutrophils express and release cytokines, which in turn amplify inflammatory reactions by several other cell types.

In addition to recruiting and activating other cells of the immune system, neutrophils play a key role in the front-line defence against invading pathogens. Neutrophils have three strategies for directly attacking micro-organisms: phagocytosis (ingestion), release of soluble anti-microbials (including granule proteins) and generation of neutrophil extracellular traps (NETs).

PhagocytosisNeutrophils are phagocytes, capable of ingesting microorganisms or particles. They can internalize and kill many microbes, each phagocytic event resulting in the formation of a phagosome into which reactive oxygen species and hydrolytic enzymes are secreted. The consumption of oxygen during the generation of reactive oxygen species has been termed the "respiratory burst", although unrelated to respiration or energy production.The respiratory burst involves the activation of the enzyme NADPH oxidase, which produces large quantities of superoxide, a reactive oxygen species. Superoxide dismutates, spontaneously or through catalysis via enzymes known as superoxide dismutases (Cu/ZnSOD and

MnSOD), to hydrogen peroxide, which is then converted to hypochlorous acid HClO, by the green heme

enzyme myeloperoxidase. It is thought that the bactericidal properties of HClO are enough to kill bacteria phagocytosed by the neutrophil, but this may instead be step necessary for the activation of proteases.

Role in disease

Low neutrophil counts are termed neutropenia. This can be congenital (genetic disorder) or it can develop later, as in the case of aplastic anemia or some kinds of leukemia. It can also be a side-effect of medication, most prominently chemotherapy. Neutropenia makes an individual highly susceptible to infections. Neutropenia can be the result of colonization by intracellular neutrophilic parasites.

Functional disorders of neutrophils are often hereditary. They are disorders of phagocytosis or deficiencies in the respiratory burst (as in chronic granulomatous disease, a rare immune deficiency, and myeloperoxidase deficiency).

In alpha 1-antitrypsin deficiency, the important neutrophil enzyme elastase is not adequately inhibited by alpha 1-antitrypsin, leading to excessive tissue damage in the presence of inflammation - most prominently pulmonary emphysema.

In Familial Mediterranean fever (FMF), a mutation in the pyrin (or marenostrin) gene, which is expressed mainly in neutrophil granulocytes, leads to a constitutively active acute phase response and causes attacks of fever, arthralgia, peritonitis, and - eventually - amyloidosis

Neutrophil Extracellular Traps(NETs)Zychlinsky and colleagues recently described a new striking observation that activation of neutrophils causes the release of web-like structures of DNA; this represents a third mechanism for killing bacteria. These neutrophil extracellular traps (NETs) comprise a web of fibers composed

of chromatin and serine proteases that trap and kill microbes extracellularly. It is suggested that NETs provide a high local concentration of antimicrobial components and bind, disarm, and kill microbes independent of phagocytic uptake. In addition to their possible antimicrobial properties, NETs may serve as a physical barrier that prevents further spread of pathogens. Trapping of bacteria may be a particularly important role for NETs in sepsis, where NET are formed within blood vessels. Recently, NETs have been shown to play a role in inflammatory diseases, as NETs could be detected in preeclampsia, a pregnancy related inflammatory disorder in which neutrophils are known to be activated.

2.Eosinophil

Eosinophil granulocytes, usually called eosinophils or eosinophiles (or, less commonly, acidophils), are white blood cells that are one of the immune system components responsible for combating multicellular parasites and certain infections in vertebrates. Along withmast cells, they also control mechanisms associated with allergy and asthma. They are granulocytes that develop during haematopoiesisin the bone marrow before migrating into blood.

These cells are eosinophilic or 'acid-loving' as shown by their affinity to coal and tar dyes: Normally transparent, it is this affinity that causes them to appear brick-red after staining with eosin, a red dye, using the Romanowsky method. The staining is concentrated in small granules within the cellular cytoplasm, which contain many chemical mediators, such as histamine and proteins such as eosinophil peroxidase, ribonuclease (RNase), deoxyribonucleases, lipase, plasminogen, and major basic protein. These mediators are released by a process called degranulation following activation of the eosinophil, and are toxic to both parasite and host tissues.

In normal individuals, eosinophils make up about 1-6% of white blood cells, and are about 12-17 micrometers in size. They are found in the medulla and the junction between the cortex and medulla of the thymus, and, in the lower gastrointestinal tract, ovary, uterus, spleen, and lymph nodes, but not in the lung, skin, esophagus, or some other internal organs[vague] under normal conditions. The presence of eosinophils in these latter organs is associated with disease. Eosinophils persist in the circulation for 8–12 hours, and can survive in tissue for an additional 8–12 days in the absence of stimulation. Pioneering work in the 1980s elucidated that eosinophils were unique granulocytes, having the capacity to survive for extended periods of time after their maturation as demonstrated by ex-vivo culture experiments.

Eosinophil under the microscope (40x) from a peripheral blood smear. Red blood cells surround the eosinophil, two platelets at the top left corner.

An increase in eosinophils, i.e., the presence of more than 500 eosinophils/microlitre of blood is called an eosinophilia, and is typically seen in people with a parasitic infestation of theintestines, a collagen vascular disease (such as rheumatoid arthritis), malignant diseases such as Hodgkin's disease, extensive skin diseases (such as exfoliative dermatitis), Addison's disease, in the squamous epithelium of the esophagus in the case of reflux esophagitis, eosinophilic esophagitis, and with the use of certain drugs such as penicillin. In 1989, contaminated L-tryptophan supplements caused a deadly form of eosinophilia known as eosinophilia-myalgia syndrome, which was reminiscent of the Toxic Oil Syndrome in Spain in 1981.

Eosinophil development, migration and activation

Eosinophils develop and mature in bone marrow. They differentiate from myeloid precursor cells in response to the cytokines interleukin 3 (IL-3), interleukin 5 (IL-5), and granulocyte macrophage colony-stimulating factor (GM-CSF). Eosinophils produce and store many secondary granule proteins prior to their exit from the bone marrow. After maturation, eosinophils circulate in blood and migrate to inflammatory sites in tissues, or to sites of helminth infection in response to chemokines like CCL11 (eotaxin-1), CCL24 (eotaxin-2), CCL5 (RANTES), and certain leukotrienes like leukotriene B4 (LTB4) and MCP1/4. At these infectious sites, eosinophils are activated by Type 2 cytokines released from a specific subset ofhelper T cells (Th2); IL-5, GM-CSF, and IL-3 are important for eosinophil activation as well as maturation. There is evidence to suggest that eosinophil granule protein expression is regulated by the non-coding RNA EGOT (gene).

Eosinophil granule proteins

Following activation by an immune stimulus, eosinophils degranulate to release an array of cytotoxic granule cationic proteins that are capable of inducing tissue damage and dysfunction. These include:

major basic protein (MBP)

eosinophil cationic protein (ECP)

eosinophil peroxidase (EPO)

eosinophil-derived neurotoxin (EDN)

Major basic protein, eosinophil peroxidase, and eosinophil cationic protein are toxic to many tissues. Eosinophil cationic protein and eosinophil-derived neurotoxin

are ribonucleaseswith antiviral activity. Major basic protein induces mast cell and basophil degranulation, and is implicated in peripheral nerve remodelling. Eosinophil cationic protein creates toxic pores in the membranes of target cells allowing potential entry of other cytotoxic molecules to the cell, can inhibit proliferation of T cells, suppress antibody production by B cells, induce degranulation by mast cells, and stimulate fibroblast cells to secrete mucus and glycosaminoglycan. Eosinophil peroxidase forms reactive oxygen species and reactive nitrogen intermediates that promote oxidative stress in the target, causing cell death by apoptosis and necrosis.

Functions of eosinophils

Following activation, eosinophils effector functions include production of:

cationic granule proteins and their release by degranulation. reactive oxygen species such as superoxide, peroxide, and hypobromite (hypobromous acid, which is preferentially produced by eosinophil peroxidase).

lipid mediators like the eicosanoids from the leukotriene (e.g., LTC4, LTD4, LTE4) and prostaglandin (e.g., PGE2) families. enzymes, such as elastase.

growth factors such as TGF beta, VEGF, and PDGF.

cytokines such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-13, and TNF alpha.

In addition, eosinophils play a role in fighting viral infections, which is evident from the abundance

of RNAses they contain within their granules, and in fibrin removal during inflammation. Eosinophils along with basophils and mast cells, are important mediators of allergic responses and asthma pathogenesis and are associated with disease severity. They also fighthelminth (worm) colonization and may be slightly elevated in the presence of certain parasites. Eosinophils are also involved in many other biological processes, including postpubertalmammary gland development, oestrus cycling, allograft rejection and neoplasia. They have also recently been implicated in antigen presentation to T cells.

Treatment

Treatments used to combat autoimmune diseases and conditions caused by eosinophils include:

corticosteroids- promote apoptosis. Numbers of eosinophils in blood are rapidly reduced

monoclonal antibody therapy- e.g., mepoluzimab or reslizumab against IL-5, prevents eosinophilopoiesis

antagonists of leukotriene synthesis or receptors

Gleevec (STI571)- inhibits PDGF-BB in hypereosinophilic leukemia

3.BasophilBasophil granulocytes, sometimes referred to as basophils, are the least common of the granulocytes, representing about 0.01% to 0.3% of circulatingwhite blood cells.

The name comes from the fact that these leukocytes are basophilic, i.e., they are susceptible to staining by basic dyes, as shown in the picture.

Basophils contain large cytoplasmic granules which obscure the cell nucleus under the microscope. However, when unstained, the nucleus is visible and it usually has 2 lobes. The mast cell, a cell in tissues, has many similar characteristics. For example, both cell types store histamine, a chemical that is secreted by the cells when stimulated in certain ways (histamine causes some of the symptoms of an allergic reaction). Like all circulating granulocytes, basophils can be recruited out of the blood into a tissue when needed.

Basophil Basophil granulocyte

Basophils of mouse and human have consistent immunophenotypes as follows: FcεRI+, CD123, CD49b(DX-5)+, CD69+, Thy-1.2+, 2B4+, CD11bdull, CD117(c-kit)–, CD24–, CD19–, CD80–,CD14–, CD23–, Ly49c–, CD122–, CD11c–, Gr-1–, NK1.1–, B220–, CD3–,

γδTCR–, αβTCR–, α4 and β4-integrin negative.

Secretions

When activated, basophils degranulate to release histamine, proteoglycans (e.g. heparin and chondroitin), and proteolytic enzymes (e.g. elastase and lysophospholipase). They also secrete lipid mediators like leukotrienes, and several cytokines. Histamine and proteoglycans are pre-stored in the cell's granules while the other secreted substances are newly generated. Each of these substances contributes to inflammation. Recent evidence suggests that basophils are an important source of the cytokine, interleukin-4, perhaps more important than T cells. Interleukin-4 is considered one of the critical cytokines in the development of allergies and the production of IgE antibody by the immune system. There are other substances that can activate basophils to secrete which suggests that these cells have other roles in inflammation.

Basopenia (a low basophil count) is difficult to demonstrate as the normal basophil count is so low; it has been reported in association with autoimmune urticaria (a chronic itching condition). Basophilia is also uncommon but may be seen in some forms of leukaemia or lymphoma.

Function

Basophils appear in many specific kinds of inflammatory reactions, particularly those that cause allergic symptoms. Basophils contain anticoagulant heparin, which prevents blood from clotting too quickly. They also contain the vasodilator histamine, which promotes blood flow to tissues. They can be found in unusually high numbers at sites of ectoparasite infection, e.g.,ticks. Like eosinophils, basophils play a role in both parasitic infections and allergies. They are found in tissues where allergic reactions are occurring and probably contribute to the severity of these reactions. Basophils have protein receptors on their cell surface that bindIgE, an immunoglobulin involved in macroparasite defense and allergy. It is the bound IgE antibody that confers a selective response of these cells to environmental substances, for

example, pollen proteins or helminth antigens. Recent studies in mice suggest that basophils may also regulate the behavior of T cells and mediate the magnitude of the secondary immune response.

4.LymphocytThis is under the adaptive immune system.

A stained lymphocyte surrounded byred blood cells viewed using a light microscope.

A scanning electron microscope(SEM) image of a single human lymphocyte.

A particular class of leukocytes known as lymphocyte mostly carry out the specific acquired immune response.Lymphocytes are much more common in the lymphatic system. Lymphocytes are distinguished by having a deeply staining nucleus which may be eccentric in location, and a relatively small amount of cytoplasm.Lymphocytes provide both the specificity and memory which are characteristic of the adaptive immune response.

Development

Mammalian stem cells differentiate into several kinds of blood cell within the bone marrow. This process is calledhaematopoiesis. All lymphocytes originate, during this process, from a common lymphoid progenitor before differentiating into their distinct lymphocyte types. The differentiation of lymphocytes follows various pathways in a hierarchical fashion as well as in a more plastic fashion. The formation of lymphocytes is known as lymphopoiesis. B cells mature into B lymphocytes in the bone marrow, while T cells migrate to and mature in a distinct organ, called the thymus. Following maturation, the lymphocytes enter the circulation and peripheral lymphoid organs (e.g. the spleen and lymph nodes) where they survey for invading pathogensand/or tumor cells.

The lymphocytes involved in adaptive immunity (i.e. B and T cells) differentiate further after exposure to an antigen; they form effector and memory lymphocytes. Effector lymphocytes function to eliminate the antigen, either by releasing antibodies (in the case of B cells), cytotoxic granules (cytotoxic T cells) or by signaling to other cells of the immune system (helper T cells).Memory cells remain in the peripheral tissues and circulation for an extended time ready to respond to the same antigen upon future exposure.

Characteristics

Microscopically, in a Wright's stained peripheral blood smear, a normal lymphocyte has a large, dark-staining nucleus with little to no eosinophiliccytoplasm. In normal situations, the coarse, dense nucleus of a lymphocyte is approximately the size of a red blood cell (about 7 micrometres in diameter). Some lymphocytes show a clear perinuclear zone (or halo) around the nucleus or could exhibit a small clear zone to one side of the nucleus. Polyribosomes are a prominent feature in the lymphocytes and can be viewed with an electron microscope. The ribosomes are involved in protein synthesis allowing the generation of large quantities of cytokines and immunoglobulins by these cells.

It is impossible to distinguish between T cells and B cells in a peripheral blood smear. Normally, flow cytometry testing is used for specific lymphocyte population counts. This can be used to specifically determine the percentage of lymphocytes that contain a particular combination of specific cell surface proteins, such as immunoglobulins or cluster of differentiation (CD) markers or that produce particular proteins (for example,cytokines using intracellular cytokine staining (ICCS)). In order to study the function of a lymphocyte by virtue of the proteins it generates, other scientific techniques like the ELISPOT or secretion assay techniques can be used.

Lymphocytes and disease

A lymphocyte count is usually part of a peripheral complete blood cell count and is expressed as percentage of lymphocytes to total white blood cells counted.

A general increase in the number of lymphocytes is known as lymphocytosis whereas a decrease is lymphocytopenia.

HighAn increase in lymphocyte concentration is usually a sign of a viral infection (in some rare case, leukemias are found through an abnormally raised lymphocyte count in an otherwise normal person).

LowA low normal to low absolute lymphocyte concentration is associated with increased rates of infection after surgery or trauma.

One basis for low T cell lymphocytes occurs when the human immunodeficiency virus (HIV) infects and destroys T cells (specifically, the CD4+ subgroup of T lymphocytes). Without the key defense that these T cells provide, the body becomes susceptible to opportunistic infections that otherwise would not affect healthy people. The extent of HIV progression is typically determined by measuring the percentage of CD4+ T cells in the patient's blood. The effects of other viruses or lymphocyte disorders can also often be estimated by counting the numbers of lymphocytes present in the blood.

TypesThe blood has three types of lymphocytes:

B cells: B cells make antibodies that bind to pathogens to enable their destruction. (B cells not only make antibodies that bind to pathogens, but after an attack, some B cells will retain the ability to produce an antibody to serve as a 'memory' system.)

T cells:

CD4+ (helper) T cells co-ordinate the immune response and are important in the defense against intracellular bacteria. In acute HIV infection, these T cells are the main index to identify the individual's immune system activity. Research has shown  that CD8+ cells are also another index to identify human's immune activity.

CD8+ cytotoxic T cells are able to kill virus-infected and tumor cells.

γδ T cells possess an alternative T cell receptor as opposed to CD4+ and CD8+ αβ T cells and share characteristics of helper T cells, cytotoxic T cells and natural killer cells.

Natural killer cells: Natural killer cells are able to kill cells of the body which are displaying a signal to kill them, as they have been infected by a virus or have become cancerous.

T cell

Scanning electron micrograph of T lymphocyte (right), a platelet (center) and ared blood cell (left)

T cells or T lymphocytes belong to a group of white blood cells known as lymphocytes, and play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells (NK cells) by the presence of a special receptor on their cell surface called T cell receptors (TCR). The abbreviation T, in T cell, stands for thymus, since this is the principal organ responsible for the T cell's maturation. Several different subsets of T cells have been discovered, each with a distinct function.

Types

HelperT helper cell (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and activation of cytotoxic T cells and macrophages, among other functions. These cells are also known as CD4+ T cells because they express the CD4 protein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules that are expressed on the surface of Antigen Presenting Cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, or TFH, which secrete different cytokines to facilitate a different type of immune response. The mechanism by which T cells are directed into a particular subtype is poorly understood, though signalling patterns from the APC are thought to play an important role.

CytotoxicCytotoxic T cells (TC cells, or CTLs) destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8+ T cells since they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevent autoimmune diseases such as experimental autoimmune encephalomyelitis.

MemoryMemory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with "memory" against past infections. Memory T cells comprise two subtypes: central memory T cells (TCM cells) and effector memory T cells (TEM cells). Memory cells may be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

RegulatoryRegulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus. Two major classes of CD4+ regulatory T cells have been described, including the naturally occurring Treg cells and the adaptive Treg cells. Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus, whereas the adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originate during a normal immune response. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations of the FOXP3 gene can prevent regulatory T cell development, causing the fatal autoimmune disease IPEX.

Natural killerNatural killer T cells (NKT cells) are a special kind of lymphocyte that bridges the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigen presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigen presented by a molecule called CD1d. Once activated, these cells can perform functions ascribed to both Th and Tc cells (i.e., cytokine production and release of cytolytic/cell killing molecules). They are also able to recognize and eliminate some tumor cells and cells infected with herpes viruses.

γδγδ T cells (gamma delta T cells) represent a small subset of T cells that possess a distinct T cell receptor (TCR) on their surface. A majority of T cells have a TCR composed of twoglycoprotein chains called α- and β- TCR chains. However, in γδ T cells, the TCR is made up of one γ-chain and one δ-chain. This group of T cells is much less common (2% of total T cells) than the αβ T cells, but are found at their highest abundance in the gut mucosa, within a population of lymphocytes known as intraepithelial lymphocytes (IELs). The antigenic molecules that activate γδ T cells are still widely unknown. However, γδ T cells are not MHC restricted and seem to be able to recognize whole proteins rather than requiring peptides to be presented by MHC molecules on antigen presenting cells. Some murine γδ T cells recognize MHC class IB molecules though. Human Vγ9/Vδ2 T cells, which constitute the major γδ T cell population in peripheral blood, are unique in that they specifically and rapidly respond to a set of non-peptidic phosphorylated metabolites precursors of cholesterol, collectively named phosphoantigens. Phosphoantigens are produced by virtually all living cells. The most common phosphoantigens from

animal and human cells (including cancer cells) are isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP), while in microbes the most common phosphoantigens are precursors of eubacterial dimethylallyl pyrophosphate (Hydroxy-DMAPP, also known as HMBPP)and

corresponding mononucleotide conjugates. Plant cells produce both types of phosphoantigens. Drugs activating human Vγ9/Vδ2 T cells comprise synthetic phosphoantigens and aminobisphosphonates, which respectively mimick natural phosphoantigens and by up-regulating endogenous IPP/DMAPP.

Typical recognition markers for lymphocytes[

CLASS FUNCTIONPROPORTION

PHENOTYPIC MARKER(S)

NK cellsLysis of virally infected cells and tumour cells

7% (2-13%) CD16 CD56 but not CD3

Helper T cells

Release cytokines and growth factors that regulate other immune cells

46% (28-59%) TCRαβ, CD3 and CD4

Cytotoxic T cells

Lysis of virally infected cells, tumour cells and allografts

19% (13-32%) TCRαβ, CD3 and CD8

γδ T cells Immunoregulation and cytotoxicity TCRγδ and CD3

B cells Secretion of antibodies 23% (18-47%) MHC class II, CD19 and CD21

Development in the thymus

All T cells originate from haematopoietic stem cells in the bone marrow. Haematopoietic progenitors derived from haematopoietic stem cells populate the thymus and expand by cell division to generate a large population of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8, and are therefore classed as double-negative (CD4-CD8-) cells. As they progress through their development they become double-positive thymocytes (CD4+CD8+), and finally mature to single-positive (CD4+CD8- or CD4-

CD8+) thymocytes that are then released from the thymus to peripheral tissues.

About 98% of thymocytes die during the development processes in the thymus by failing either positive selection or negative selection, whereas the other 2% survive and leave the thymus to become mature immunocompetent T cells.

The thymus contributes more naive T cells at younger ages. As the thymus shrinks by about 3% a year throughout middle age, there is a corresponding fall in the thymic production of naive T cells, leaving peripheral T cell expansion to play a greater role in protecting older subjects.

Positive selectionPositive selection "selects for" T-cells capable of interacting with MHC. Double-positive thymocytes (CD4+/CD8+) move deep into the thymic cortex where they are presented with self-antigens (i.e., antigens that are derived from molecules belonging to the host of the T cell) complexed with MHC molecules on the surface of cortical epithelial cells. Only those thymocytes that bind the MHC/antigen complex with adequate affinity will receive a vital "survival signal." The implication of this

binding is that all T cells must be able to recognize self antigens to a certain degree. Developing thymocytes that do not have adequate affinity cannot serve useful functions in the body (i.e. the cells must be able to interact with MHC and peptide complexes in order to effect immune responses). Also, the thymocyte must be able to recognize antigens that are self from non-self.). Because of this, the thymocytes with no affinity for self antigens die by apoptosis and are engulfed by macrophages.

A thymocyte's fate is also determined during positive selection. Double-positive cells (CD4+/CD8+) that are positively selected on MHC class II molecules will eventually become CD4+cells, while cells positively selected on MHC class I molecules mature into CD8+ cells. A T cell becomes a CD4+ cell by downregulating expression of its CD8 cell surface receptors. If the cell does not lose its signal through the ITAM pathway, it will continue downregulating CD8 and become a CD4+, single positive cell. But if there is signal drop, the cell stops downregulating CD8 and switches over to downregulating CD4 molecules instead, eventually becoming a CD8+, single positive cell.

This process does not remove thymocytes that may cause autoimmunity. The potentially autoimmune cells are removed by the process of negative selection (discussed below).

Negative selectionNegative selection removes thymocytes that are capable of strongly binding with "self" peptides presented by MHC. Thymocytes that survive positive selection migrate towards the boundary of the thymic cortex and thymic medulla. While in the medulla, they are again presented with self-antigen in complex with MHC molecules on antigen-presenting cells (APCs) such as dendritic cells and macrophages. Thymocytes that interact too strongly with the antigen receive an apoptotic signal that leads to cell death. The vast majority of all thymocytes end up dying during this process. The remaining cells exit the thymus as mature naive T cells. This process is an important component of immunological tolerance and serves to prevent the formation of self-reactive T cells that are capable of inducing autoimmune diseases in the host.

In summary, positive selection selects for T cells that are capable of recognizing self antigens through MHC. Negative selection selects for T cells that bind too strongly to self antigens. These two selection processes allow for Tolerance of self by the immune system. They do not necessarily occur in a chronological order and can occur simultaneously in the thymus.

Maturation paradoxPositive and negative selection should theoretically kill all developing T cells. The first stage of selection kills all T cells that do not interact with self-MHC, while the second stage selection kills all cells that do. This poses the question: How do we have immunity at all? Currently, two models attempt to explain this:

1. Differential Avidity Hypothesis - the strength of the signal dictates the fate of the T cell.

The differential avidity hypothesis (or simply avidity hypothesis) is one of two models that attempt to

explain how humans have immunity despite such aggressive selection (positive and negative) to kill

developing T cells during their maturation process. The other model is the Differential Signaling

Hypothesis.

The Avidity hypothesis states that the affinity of the T-cell receptor for the MHC:peptide complex along

with the density of the complex provide different signal strength upon binding which in terms dictate the

outcome:

1. strong signal leads to negative selection and thus apoptosis.

2. weak signal leads to positive selection and thus rescued from apoptosis.

2. Differential Signaling Hypothesis - the signals that are transduced differ at each stage.

The differential avidity hypothesis (or simply avidity hypothesis) is one of two models that attempt to

explain how humans have immunity despite such aggressive selection (positive and negative) to kill

developing T cells during their maturation process. The other model is the Differential Signaling

Hypothesis.

The Avidity hypothesis states that the affinity of the T-cell receptor for the MHC:peptide complex along

with the density of the complex provide different signal strength upon binding which in terms dictate the

outcome:

1. strong signal leads to negative selection and thus apoptosis.

2. weak signal leads to positive selection and thus rescued from apoptosis.

Activation

Although the specific mechanisms of activation vary slightly between different types of T cells, the "two-signal model" in CD4+ T cells holds true for most. Activation of CD4+ T cells occurs through the engagement of both the T cell receptor and CD28 on the T cell by the Major histocompatibility complex peptide and B7 family members on the APC, respectively. Both are required for production of an effective immune response; in the absence of CD28 co-stimulation, T-cell receptor signalling alone results in anergy. The signalling pathways downstream from both CD28 and the T cell receptor involve many proteins.

The first signal is provided by binding of the T cell receptor to a short peptide presented by the major histocompatibility complex (MHC) on another cell. This ensures that only a T cell with a TCR specific to that peptide is activated. The partner cell is usually a professional antigen presenting cell (APC), usually a dendritic cell in the case of naïve responses, although B cells and macrophages can be important APCs. The peptides presented to CD8+ T cells by MHC class I molecules are 8-9 amino acids in length; the peptides presented to CD4+ cells by MHCclass II molecules are longer, as the ends of the binding cleft of the MHC class II molecule are open.

The second signal comes from co-stimulation, in which surface receptors on the APC are induced by a relatively small number of stimuli, usually products of pathogens, but sometimes breakdown products of cells, such as necrotic-bodies or heat-shock proteins. The only co-stimulatory receptor expressed constitutively by naïve T cells is CD28, so co-stimulation for these cells comes from

the CD80 and CD86proteins, which together constitute the B7 protein, (B7.1 and B7.2 respectively) on the APC. Other receptors are expressed upon activation of the T cell, such as OX40 and ICOS, but these largely depend upon CD28 for their expression. The second signal licenses the T cell to respond to an antigen. Without it, the T cell becomes anergic, and it becomes more difficult for it to activate in future. This mechanism prevents inappropriate responses to self, as self-peptides will not usually be presented with suitable co-stimulation.

The T cell receptor exists as a complex of several proteins. The actual T cell receptor is composed of two separate peptide chains, which are produced from the independent T cell receptor alpha and beta (TCRα and TCRβ) genes. The other proteins in the complex are the CD3proteins: CD3εγ and CD3εδ heterodimers and, most important, a CD3ζ homodimer, which has a total of six ITAM motifs. The ITAM motifs on the CD3ζ can be phosphorylated by Lck and in turn recruit ZAP-70. Lck and/or ZAP-70 can also phosphorylate the tyrosines on many other molecules, not least CD28, , LAT and SLP-76, which allows the aggregation of signalling complexes around these proteins.

Phosphorylated LAT recruits SLP-76 to the membrane, where it can then bring in PLCγ, VAV1, Itk and potentially PI3K. Both PLCγ and PI3K act on PI(4,5)P2 on the inner leaflet of the membrane to create the active intermediaries diacylglycerol (DAG), inositol-1,4,5-trisphosphate (IP3), and phosphatidlyinositol-3,4,5-trisphosphate (PIP3). DAG binds and activates some PKCs, most important, in T cells PKCθ, a process important for activating the transcription factors NF-κB and AP-1. IP3 is released from the membrane by PLCγ and diffuses rapidly to activate receptors on the ER, which induce the release of calcium. The released calcium then activates calcineurin, and calcineurin activates NFAT, which then translocates to the nucleus. NFAT is a transcription factor, which activates the transcription of a pleiotropic set of genes, most notable, IL-2, a cytokine

that promotes long term proliferation of activated T cells.

T cell activation

B cellB cells are lymphocytes that play a large role in the humoral immune response (as opposed to the cell-mediated immune response, which is governed by T cells). The principal functions of B cells are to make antibodies against antigens, perform the role of antigen-presenting cells(APCs) and eventually develop into memory B cells after activation by antigen interaction. B cells are an essential component of the adaptive immune system.

The abbreviation "B", in B cell, comes from the bursa of Fabricius in birds, where they mature. In mammals, immature B cells are formed in thebone marrow, which is used as a backronym for the cells' name.

The cells of the immune system that make antibodies to invading pathogens like viruses. They form memory cells that remember the same pathogen for faster antibody production in future infections.

B cells exist as clones. All B cells derive from a particular cell, and thus, the antibodies their differentiated progenies (see below) produce can recognize and/or bind the same components (epitope) of a given antigen. Such clonality has important consequences, as immunogenic memory relies on it. The great diversity in immune response comes about because there are up to 109 clones with specificities for recognizing different antigens. A single B cell or a clone of cells with shared specificity upon encountering its specific antigen divides to produce many B cells. Most of such B cells differentiate into plasma cells that secrete antibodies into blood that bind the same epitope that elicited proliferation in the first place. A small minority survives as memory cells that can recognize only the same epitope. However, with each cycle, the number of surviving memory cells increases. The increase is accompanied by affinity maturation which induces the survival of B cells that bind to the particular antigen with high affinity. This subsequent amplification with improved specificity of immune response is known as secondary immune response. B cells that encounter antigen for the first time are known as naive B cells.

Development of B cellsImmature B cells are produced in the bone marrow of most mammals. Rabbits are an exception; their B cells develop in the appendix-sacculus rotundus. After reaching the IgM+ immature stage in the bone marrow, these immature B cells migrate to the spleen, where they are called transitional B cells, and some of these cells differentiate into mature B lymphocytes.

B cell development occurs through several stages, each stage representing a change in the genome content at the antibody loci. An antibody is composed of two identical light (L) and two identical heavy (H) chains, and the genes specifying them are found in the 'V' (Variable) region and the 'C' (Constant) region. In the heavy-chain 'V' region there are three segments; V, D and J, which recombine randomly, in a process called VDJ recombination, to produce a unique variable domain in the immunoglobulin of each individual B cell. Similar rearrangements occur for light-chain 'V' region except there are only two segments involved; V and J. The list below describes the process of immunoglobulin formation at the different stages of B cell development.

When the B cell fails in any step of the maturation process, it will die by a mechanism called apoptosis, here called clonal deletion. B cells are continuously produced in the bone marrow. When B cell receptors on the surface of the cell matches the detected antigens present in the body, the B cell proliferates and secretes a free form of those receptors (antibodies) with identical binding sites as the ones on the original cell surface. After activation, the cell proliferates and B memory cells would form to recognise the same antigen. This information would then be used as a part of the adaptive immune system for a more efficient and more powerful immune response for future encounters with that antigen.

B cell membrane receptors evolve and change throughout the B cell life span. TACI, BCMA and BAFF-R are present on both immature B cells and mature B cells. All of these 3 receptors may be inhibited by Belimumab. CD20 is expressed on all stages of B cell development except the first and last; it is present from pre-pre B cells through memory cells, but not on either pro-B cells or plasma cells.

Immune Tolerance

Like its fellow lymphocyte, the T cell, immature B cells are tested for auto-reactivity by the immune system before leaving the bone marrow. In the bone marrow (the central lymphoid organ), central tolerance is produced. The immature B cells whose B cell Receptors (BCRs) bind too strongly to self antigens will not be allowed to mature. If B cells are found to be highly reactive to self, three mechanisms can occur.

Clonal deletion: the removal, usually by apoptosis, of B cells of a particular self antigen specificity.

Receptor editing: the BCRs of self reactive B cells are given an opportunity to rearrange their conformation. This process occurs via the continued expression of the Recombination activating gene (RAG). Through the help of RAG, receptor editing involves light chain gene rearrangement of the B cell receptor. If receptor editing fails to produce a BCR that is less autoreactive, apoptosis will occur. Note that defects in the RAG-1 and RAG-2 genes are implicated in Severe Combined Immunodeficiency (SCID). The inability to recombine and generate new receptors lead to failure of maturity for both B cells and T cells.

Anergy: B cells enter a state of permanent unresponsiveness when they bind with weakly cross-linking self antigens that are small and soluble.

Functions

The human body makes millions of different types of B cells each day that circulate in the blood and lymphatic system performing the role of immune surveillance. They do not produceantibodies until they become fully activated. Each B cell has a unique receptor protein (referred to as the B cell receptor (BCR)) on its surface that will bind to one particular antigen. The BCR is a membrane-bound immunoglobulin, and it is this molecule that allows the distinction of B cells from other types of lymphocyte, as well as being the main protein involved in B cell activation. Once a B cell encounters its cognate antigen and receives an additional signal from a T helper cell, it can further differentiate into one of the two types of B cells listed below (plasma B cells and memory B cells). The B cell may either become one of these cell types directly or it may undergo an intermediate differentiation step, the germinal centerreaction, where the B cell will hypermutate the variable region of its immunoglobulin gene ("somatic hypermutation") and possibly undergo class switching.

B cell types

. Plasma B cells (also known as plasma cells) are large B cells that have been exposed to antigen and produce and secrete large amounts ofantibodies, which assist in the destruction of microbes by binding to them and making them easier targets for phagocytes and activation of thecomplement system. They are sometimes referred to as antibody factories. An electron micrograph of these cells reveals large amounts of rough endoplasmic reticulum, responsible for synthesizing the antibody, in the cell's cytoplasm. These are short lived cells and undergo apoptosis when the inciting agent that induced immune response is eliminated. This occurs because of cessation of continuous exposure to various colony stimulating factors required for survival.

Memory B cells are formed from activated B cells that are specific to the antigen encountered during the primary immune response. These cells are able to live for a long time, and can respond quickly following a second exposure to the same antigen.

B-1 cells express IgM in greater quantities than IgG and their receptors show polyspecificity, meaning that they have low affinities for many different antigens, but have a preference for other immunoglobulins, self antigens and common bacterial polysaccharides. B-1 cells are present in low numbers in the lymph nodes and spleen and are instead found predominantly in the peritoneal and pleural cavities.

B-2 cells are the conventional B cells.

Marginal-zone B cells

Marginal zone B cells are noncirculating mature B cells that segregate anatomically into the marginal zone (MZ) of the spleen. This region contains multiple subtypes ofmacrophages, dendritic cells, and the MZ B cells; it is not fully formed until 2 to 3 weeks after birth in rodents and 1 to 2 years in humans. The MZ B cells within this region typically express high levels of sIgM, CD21, CD1, CD9 with low to negligible levels of sIgD, CD23, CD5, and CD11b that help to distinguish them phenotypically from FO B cells and B1 B cells.

Similar to B1 B cells, MZ B cells can be rapidly recruited into the early adaptive immune responses in a T cell independent manner. The MZ B cells are especially well positioned as a first line of defense against systemic blood-borne antigens that enter the circulation and become trapped in the spleen. MZ B cells also display a lower activation threshold than their FO B cell counterparts with heightened propensity for PC differentiation that contributes further to the accelerated primary antibody response

Follicular B Cells

Follicular B cells (FO B cells) are a type of B cell that reside in primary and secondary lymphoid follicles (containing germinal centers) of secondary and tertiary lymphoid organs, including spleen and lymph nodes.

The mature B cells from the spleen can be divided into two main populations: the FO B cells, which constitute the majority, and the marginal zone B-cells, lining outside the marginal sinus and border the red pulp. FO B cells express high levels of IgM, IgD, and CD23; lower C21; and no CD1 or CD5, readily distinguishing this compartment from B1 B cells andmarginal zone B-cells . FO B cells organize into the primary follicles of B cell zones focused around follicular dendritic cells in the white pulp of the spleen and the cortical areas of peripheral lymph nodes. Multiphoton-based live imaging of lymph nodes indicate continuous movement of FO B cells within these follicular areas at velocites of ~6 µm per min. Recent studies indicate movement along the processes of FDC as a guidance system for mature resting B cells in peripheral lymph nodes. Unlike their MZ counterpart, FO B cells freely recirculate, comprising >95% of the B cells in peripheral lymph nodes.

The BCR repertoire of the follicular B cell compartment also appears under positive selection pressures during final maturation in the spleen. However, diversity is substantially broader than B1 B and MZ B cell compartments. More importantly, FO B cells require CD40-CD40L dependent T cell help to promote effective primary immune responses and antibody isotype switch and to establish high-affinity B cell memory.

Recognition of antigen by B cells

A critical difference between B cells and T cells is how each lymphocyte recognizes its antigen. B cells recognize their cognate antigen in its native form. They recognize free (soluble) antigen in the blood or lymph using their BCR or membrane bound-immunoglobulin. In contrast, T cells recognize their cognate antigen in a processed form, as a peptide fragment presented by anantigen presenting cell's MHC molecule to the T cell receptor.

Activation of B cells

B cell recognition of antigen is not the only element necessary for B cell activation (a combination of clonal proliferation and terminal differentiation into plasma cells). B cells that have not been exposed to antigen, also known as naïve B cells, can be activated in a T cell-dependent or -independent manner.

B cell activation

T cell-dependent activationOnce a pathogen is ingested by an antigen-presenting cell such as a macrophage or dendritic cell, the pathogen's proteins are then digested to peptides and attached to a class II MHC protein. This complex is

then moved to the outside of the cell membrane. The macrophage is now activated to deliver multiple signals to a specific T cell that recognizes the peptide presented. The T cell is then stimulated to produce autocrines (Refer to Autocrine signalling), resulting in the proliferation and differentiation to effector and memory T cells. Helper T cells (i.e. CD4+ T cells) then activate specific B cells through a phenomenon known as an Immunological synapse. Activated B cells subsequently produce antibodies which assist in inhibiting pathogens until phagocytes (i.e. macrophages, neutrophils) or the complement system for example clears the host of the pathogen(s).

Most antigens are T-dependent, meaning T cell help is required for maximal antibody production. With a T-dependent antigen, the first signal comes from antigen cross linking the B cell receptor (BCR) and the second signal comes from co-stimulation provided by a T cell. T dependent antigens contain proteins that are presented on B cell Class II MHC to a special subtype of T cell called a Th2 cell. When a B cell processes and presents the same antigen to the primed Th cell, the T cell secretes cytokines that activate the B cell. These cytokines trigger B cell proliferation and differentiation into plasma cells. Isotype switching to IgG, IgA, and IgE and memory cell generation occur in response to T-dependent antigens. This isotype switching is known as Class Switch Recombination (CSR). Once this switch has occurred, that particular B cell will usually no longer make the earlier isotypes, IgM or IgD.

T cell-dependent B cell activation, showing a TH2-cell (left), B cell (right), and several interaction molecules

T cell-independent activationMany antigens are T cell-independent in that they can deliver both of the signals to the B cell. Mice without a thymus (nude orathymic mice that do not produce any T cells) can respond to T independent antigens. Many bacteria have repeating carbohydrate epitopes that stimulate B cells, by

cross-linking the IgM antigen receptors in the B cell, responding with IgM synthesis in the absence of T cell help. There are two types of T cell independent activation; Type 1 T cell-independent(polyclonal) activation, and type 2 T cell-independent activation (in which macrophages present several of the same antigen in a way that causes cross-linking of antibodies on the surface of B cells).

The ancestral roots of B cells

In an October 2006 issue of Nature Immunology, certain B cells of basal vertebrates (like fish and amphibians) were shown to be capable of phagocytosis, a function usually associated with cells of the innate immune system. The authors postulate that these phagocytic B cells represent the ancestral history shared between macrophages and lymphocytes. B cells may have evolved from macrophage-like cells during the formation of the adaptive immune system.

B cells in humans (and other vertebrates) are nevertheless able to endocytose antibody-fixed pathogens, and it is through this route that MHC Class II presentation by B cells is possible, allowing Th2 help and stimulation of B cell proliferation. This is purely for the benefit of MHC Class II presentation, not as a significant method of reducing the pathogen load.

B cell-related pathology

Aberrant antibody production by B cells is implicated in many autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus.

5.MonocyteMonocyte is a type of white blood cell, part of the human body's immune system. Monocytes have several roles in the immune system and this includes: (1) replenish resident macrophages and dendritic cells under normal states, and (2) in response to inflammationsignals, monocytes can move quickly (approx. 8-12 hours) to sites of infection in the tissues and divide/differentiate into macrophages and dendritic cells to elicit an immune response. Half of them are stored in the spleen. Monocytes are usually identified in stained smears by their large bilobate nucleus.

Monocytes can be used to generate dendritic cells in vitro by adding cytokines like Granulocyte Monocyte Colony Stimulating Factor (GMCSF) and IL-4.

Monocyte

Physiology

Monocytes are produced by the bone marrow from haematopoietic stem cell precursors called monoblasts. Monocytes circulate in the bloodstream for about one to three days and then typically move into tissues throughout the body. They constitute between three to eight percent of the leukocytes in the blood. Half of them are stored as a reserve in the spleen in clusters in the red pulp's Cords of Billroth. In the tissues monocytes mature into different types of macrophages at different anatomical locations.

Monocytes which migrate from the bloodstream to other tissues will then differentiate into tissue resident macrophages or dendritic cells. Macrophages are responsible for protecting tissues from foreign substances but are also suspected to be the predominant cells involved in triggering atherosclerosis. They are cells that possess a large smooth nucleus, a large area of cytoplasm and many internal vesicles for processing foreign material.

Monocytes and their macrophage and dendritic cell progeny serve three main functions in the immune system. These are phagocytosis, antigen presentation and cytokine production.Phagocytosis is the process of uptake of microbes and particles followed by digestion and destruction of this material. Monocytes can perform phagocytosis using intermediary (opsonising) proteins such as antibodies or complement that coat the pathogen, as well as by binding to the microbe directly via pattern-recognition receptors that recognize pathogens. Monocytes are also capable of killing infected host cells via antibody, termed antibody-mediated cellular cytotoxicity. Vacuolization may be present in a cell that has recently phagocytized foreign matter.

Microbial fragments that remain after such digestion can serve as antigen. The fragments can be incorporated into MHC molecules and then traffic to the cell surface of monocytes (and macrophages and dendritic cells). This process is called antigen presentation and it leads to activation of T lymphocytes, which then mount a specific immune response against the antigen.

Other microbial products can directly activate monocytes and this leads to production of pro-inflammatory and with some delay of anti-inflammatory cytokines. Typical cytokines produced by monocytes are TNF tumor necrosis factor, IL-1 interleukin-1and IL-12 interleukin-12.

Monocyte subpopulations

There are at least three types of monocytes in human blood :

a) the classical monocyte is characterized by high level expression of the CD14 cell surface receptor (CD14++ CD16- monocyte)

b) the non-classical monocyte shows low level expression of CD14 and with additional co-expression of the CD16 receptor (CD14+CD16++ monocyte).

c) the intermediate monocyte with high level expression of CD14 and low level expression of CD16 (CD14++CD16+ monocytes).

There appears to be a developmental relationship in that the classical monocytes develop into the intermediate monocytes to then become the non-classical monocytes CD14+CD16+ monocytes. Hence the non-classical monocytes may represent a more mature version. After stimulation with microbial products the CD14+CD16++ monocytes produce high amounts of pro-inflammatory cytokines like tumor necrosis factor and interleukin-12.

Diagnostic use

A monocyte count is part of a complete blood count and is expressed either as a ratio of monocytes to the total number of white blood cells counted, or by absolute numbers. Both may be useful in determining or refuting a possible diagnosis.

MonocytosisMonocytosis is the state of excess monocytes in the peripheral blood. It may be indicative of various disease states. Examples of processes that can increase a monocyte count include:

chronic inflammation

stress response

hyperadrenocorticism

immune-mediated disease

infectious mononucleosis

pyogranulomatous disease

necrosis

red cell regeneration

Viral Fever

sarcoidosis

A high count of CD14+CD16+ monocytes is found in severe infection (sepsis) and a very low count of these cells is found after therapy with immuno-suppressive glucocorticoids

MonocytopeniaMonocytopenia is a form of leukopenia associated with a deficiency of monocytes.

(b) Mast cellA mast cell (or mastocyte) is a resident cell of several types of tissues and contains many granules rich in histamine andheparin. Although best known for their role in allergy and anaphylaxis, mast cells play an important protective role as well, being intimately involved in wound healing and defense against pathogens.

Mast cells

LocalizationMast cells are found in connective tissues throughout the body,close to blood vessels and particularly areas of the respiratory ,urogenital and gastrointestinal tracks.It has large characteristic electron-dense granules in their cytoplasm,which are very important for their function.the origin of mast cell is uncertain but they probably also originate in the bone marrow.

Classification

Two types of mast cells are recognized, those from connective tissue and a distinct set of mucosal mast cells. The activities of the latter are dependent on T-cells.

Mast cells are present in most tissues characteristically surrounding blood vessels and nerves, and are especially prominent near the boundaries between the outside world and the internal milieu, such as the skin, mucosa of the lungs and digestive tract, as well as in the mouth, conjunctiva, and nose.

Physiology

Mast cells play a key role in the inflammatory process. When activated, a mast cell rapidly releases its characteristic granules and various hormonal mediators into the interstitium. Mast cells can be stimulated to degranulate by direct injury (e.g. physical or chemical [such as opioids, alcohols, and certain antibiotics such as polymyxins]), cross-linking of Immunoglobulin E (IgE) receptors, or by activated complement proteins.

Mast cells express a high-affinity receptor (FcεRI) for the Fc region of IgE, the least-abundant member of the antibodies. This receptor is of such high affinity that binding of IgE molecules is essentially irreversible. As a result, mast cells are coated with IgE. IgE is produced by Plasma cells (the antibody-producing cells of the immune system). IgE molecules, like all antibodies, are specific to one particular antigen.

The role of mast cells in the development of allergy.

In allergic reactions, mast cells remain inactive until an allergen binds to IgE already in association with the cell (see above). Other membrane activation events can either prime mast cells for subsequent degranulation or can act in synergy with FceRI signal transduction. Allergens are generally proteins or polysaccharides. The allergen binds to the antigen-binding sites, which are situated on the variable regions of the IgE molecules bound to the mast cell surface. It appears that binding of two or more IgE molecules (cross-linking) is required to activate the mast cell. The clustering of the intracellular domains of the cell-bound Fc receptors, which are associated with the cross-linked IgE molecules, causes a complex sequence of reactions inside the mast cell that lead to its activation. Although this reaction is most well understood in terms of allergy, it appears to have evolved as a defense system against intestinal worm infestations (tapeworms, etc.).

The molecules thus released into the extracellular environment include:

preformed mediators (from the granules):

serine proteases, such as tryptase

histamine (2-5 pg/cell)

serotonin

proteoglycans, mainly heparin (active as anticoagulant)

newly formed lipid mediators (eicosanoids):

prostaglandin D2

leukotriene C4

platelet-activating factor

cytokines

Eosinophil chemotactic factor

Histamine dilates post capillary venules, activates the endothelium, and increases blood vessel permeability. This leads to local edema(swelling), warmth, redness, and the attraction of other inflammatory cells to the site of release. It also irritates nerve endings (leading to itchingor pain). Cutaneous signs of histamine release are the "flare and wheal"-reaction. The bump and redness immediately following a mosquito bite are a good example of this reaction, which occurs seconds after challenge of the mast cell by an allergen.

Structure of histamine

The other physiologic activities of mast cells are much less well-understood. Several lines of evidence suggest that mast cells may have a fairly fundamental role in innate immunity – they are capable of elaborating a vast array of important cytokines and other inflammatory mediators such as TNFa, they express multiple "pattern recognition receptors" thought to be involved in recognizing broad classes of pathogens, and mice without mast cells seem to be much more susceptible to a variety of infections.[citation

needed]

Mast cell granules carry a variety of bioactive chemicals. These granules have been found to be transferred to adjacent cells of the immune system andneurons via transgranulation via their pseudopodia

Role in disease

Allergic disease

Many forms of cutaneous and mucosal allergy are mediated for a large part by mast cells; they play a central role in asthma, eczema, itch (from various causes) and allergic rhinitis andallergic conjunctivitis. Antihistamine drugs act by blocking the action of histamine on nerve endings. Cromoglicate-based drugs (sodium cromoglicate, nedocromil) block a calcium channel essential

for mast cell degranulation, stabilizing the cell and preventing release of histamine and related mediators. Leukotriene antagonists (such as montelukast andzafirlukast) block the action of leukotriene mediators, and are being used increasingly in allergic diseases.

Anaphylaxis

In anaphylaxis (a severe systemic reaction to allergens, such as nuts, bee stings or drugs), body-wide degranulation of mast cells leads to vasodilation and, if severe, symptoms of life-threatening shock.[citation

needed]

Autoimmunity

Mast cells are implicated in the pathology associated with the autoimmune disorders rheumatoid arthritis, bullous pemphigoid, and multiple sclerosis. They have been shown to be involved in the recruitment of inflammatory cells to the joints (e.g. rheumatoid arthritis) and skin (e.g. bullous pemphigoid) and this activity is dependent on antibodies and complement components. 

Mast cell disorders

Mastocytosis is a rare condition featuring proliferation of mast cells. It exists in a cutaneous and systemic form, with the former being limited to the skin and the latter involving multiple organs. Mast cell tumors are often seen in dogs and cats.

(c)PhagocytePhagocytes are the white blood cells that protect the body by ingesting (phagocytosing) harmful foreign particles, bacteria, and dead or dyingcells. Their name comes from the Greek phagein, "to eat" or "devour", and "-cyte", the suffix in biology denoting "cell", from the Greek kutos, "hollow vessel". They are essential for fighting infections and for subsequent immunity. Phagocytes are important throughout the animal kingdom and are highly developed within vertebrates. One litre of human blood contains about six billion phagocytes. Phagocytes were first discovered in 1882 by Ilya Ilyich Mechnikov while he was studying starfish larvae. Mechnikov was awarded the 1908 Nobel Prize in Physiology or Medicine for his discovery. Phagocytes occur in many species; some amoebae behave like macrophage phagocytes, which suggests that phagocytes appeared early in the evolution of life.

Phagocytes of humans and other animals are called "professional" or "non-professional" depending on how effective they are atphagocytosis. The professional phagocytes include cells called neutrophils, monocytes, macrophages, dendritic cells, and mast cells.The main difference between professional and non-professional phagocytes is that the professional phagocytes have molecules calledreceptors on their surfaces that can detect harmful objects, such as bacteria, that are not normally found in the body. Phagocytes are crucial in fighting infections, as well as in maintaining healthy tissues by removing dead and dying cells that have reached the end of their lifespan.

During an infection, chemical signals attract phagocytes to places where the pathogen has invaded the body. These chemicals may come from bacteria or from other phagocytes already present. The phagocytes move by a method called chemotaxis. When phagocytes come into contact with bacteria, the receptors on the phagocyte's surface will bind to them. This binding will lead to the engulfing of the bacteria by the phagocyte. Some phagocytes kill the ingested pathogen with oxidants and nitric oxide. After phagocytosis, macrophages and dendritic cells can also participate in antigen presentation, a process in which a phagocyte moves parts of the ingested material back to its surface. This material is then displayed to other cells of the immune system. Some phagocytes then travel to the body's lymph nodes and display the material to white blood cells called lymphocytes. This process is important in building immunity. However, many pathogens have evolved methods to evade attacks by phagocytes.

Methods of killing

The killing of microbes is a critical function of phagocytes that is either performed within the phagocyte (intracellular killing) or outside of the phagocyte (extracellular killing).

Simplified diagram of the phagocytosis and destruction of a bacterial cell

Oxygen-dependent intracellular

When a phagocyte ingests bacteria (or any material), its oxygen consumption increases. The increase in oxygen consumption, called arespiratory burst, produces reactive oxygen-containing molecules that are anti-microbial. The oxygen compounds are toxic to both the invader and the cell itself, so they are kept in compartments inside the cell. This method of killing invading microbes by using the reactive oxygen-containing molecules is referred to as oxygen-dependent intracellular killing, of which there are two types.

The first type is the oxygen-dependent production of a superoxide, which is an oxygen-rich bacteria-killing substance. The superoxide is converted to hydrogen peroxide and singlet oxygen by an enzyme called superoxide dismutase. Superoxides also react with the hydrogen peroxide to produce hydroxyl radicals which assist in killing the invading microbe.

The second type involves the use of the enzyme myeloperoxidase from neutrophil granules. When granules fuse with a phagosome, myeloperoxidase is released into the phagolysosome, and this enzyme uses hydrogen peroxide and chlorine to create hypochlorite, a substance used in domestic bleach. Hypochlorite is extremely toxic to bacteria.Myeloperoxidase contains a heme pigment, which accounts for the green color of secretions rich in neutrophils, such as pus and infected sputum.

Oxygen-independent intracellular

Phagocytes can also kill microbes by oxygen-independent methods, but these are not as effective as the oxygen-dependent ones. There are four main types. The first uses electrically charged proteins which

damage the bacterium's membrane. The second type uses lysozymes; these enzymes break down the bacterial cell wall. The third type uses lactoferrins, which are present in neutrophil granules and remove essential iron from bacteria. The fourth type uses proteases and hydrolytic enzymes; these enzymes are used to digest the proteins of destroyed bacteria.

Extracellular

Interferon-gamma—which was once called macrophage activating factor—stimulates macrophages to produce nitric oxide. The source of interferon-gamma can be CD4+ T cells, CD8+ T cells, natural killer cells, B cells, natural killer T cells, monocytes, macrophages, or dendritic cells. Nitric oxide is then released from the macrophage and, because of its toxicity, kills microbes near the macrophage. Activated macrophages produce and secrete tumor necrosis factor. This cytokine—a class of signaling molecule—kills cancer cells and cells infected by viruses, and helps to activate the other cells of the immune system.

In some diseases, e.g., the rare chronic granulomatous disease, the efficiency of phagocytes is impaired, and recurrent bacterial infections are a problem. In this disease there is an abnormality affecting different elements of oxygen-dependent killing. Other rare congenital abnormalities, such as Chediak-Higashi syndrome, are also associated with defective killing of ingested microbes.

Role in apoptosis

Apoptosis (pronounced /ˌæpəˈtoʊsɪs/ or /ˌæpəpˈtoʊsɪs/) is the process of programmed cell death (PCD) that may occur in multicellular organisms. Biochemical events lead to characteristic cell changes (morphology) and death. These changes include blebbing, loss of cell membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. (See also Apoptosis DNA fragmentation.) Apoptosis differs from necrosis, in which the cellular debris can damage the organism.

In an animal, cells are constantly dying. A balance between cell division and cell death keeps the number of cells relatively constant in adults. There are two different ways a cell can die: by necrosis or by apoptosis. In contrast to necrosis, which often results from disease or trauma, apoptosis—or programmed cell death—is a normal healthy function of cells. The body has to rid itself of millions of dead or dying cells every day, and phagocytes play a crucial role in this process.

Dying cells that undergo the final stages of apoptosis display molecules, such as phosphatidylserine, on their cell surface to attract phagocytes. Phosphatidylserine is normally found on the cytosolic surface of the plasma membrane, but is redistributed during apoptosis to the extracellular surface by a hypothetical protein known as scramblase. These molecules mark the cell for phagocytosis by cells that possess the appropriate receptors, such as macrophages. The removal of dying cells by phagocytes occurs in an orderly manner without eliciting an inflammatory response and is an important function of phagocytes.

Apoptosis—phagocytes clear fragments of dead cells from the body.

Interactions with other cells

Phagocytes are usually not bound to any particular organ but move through the body interacting with the other phagocytic and non-phagocytic cells of the immune system. They can communicate with other cells by producing chemicals called cytokines, which recruit other phagocytes to the site of infections or stimulate dormant lymphocytes. Phagocytes form part of the innate immune system which animals, including humans, are born with. Innate immunity is very effective but non-specific in that it does not discriminate between different sorts of invaders. On the other hand, the adaptive immune system of jawed vertebrates—the basis of acquired immunity—is highly specialized and can protect against almost any type of invader. The adaptive immune system is dependent on lymphocytes, which are not phagocytes but produce protective proteins called antibodies which tag invaders for destruction and prevent viruses from infecting cells. Phagocytes, in particular dendritic cells and macrophages, stimulate lymphocytes to produce antibodies by an important process called antigen presentation.

What is opsonization?This is the process of making a microbes easier to phagocytose.Opsonization is a process in which pathogens are coated with a substance called an opsonin, marking the pathogen out for destruction by the immune system. Once a pathogen has been opsonized, it is killed via one of two mechanisms. The pathogen may be ingested and killed by an immune cell, or killed directly withoutingestion.

The process of killing and ingesting a pathogen is called phagocytosis. Cells called phagocytes ingest the pathogens and then kill them by exposing them to toxic chemicals. The chemicals are stored in small membrane-bound parcels within the phagocytes, and these parcels are triggered to open when a phagocyte ingests a pathogen.

Opsonization also leads to pathogen death in a second mechanism called antibody-dependent cellular cytotoxicity, in which immune cells directly kill pathogens without ingesting them. In this process, antibodiesact as opsonins, and then trigger immune cells called granulocytes. These cells then release toxic chemicals into the environment around the pathogens to kill them. In addition to killing pathogens, this process also causes tissue damage via inflammation.

There are several different substances which may act as opsonins; all of these are proteins which are active in the immune system. Two antibody types called IgG and IgA are both opsonins. IgG is active in blood and tissues, and IgA is active in mucosal surfaces such as the airways, urogenital system, and gut. Several proteins which act in the complement system are also opsonins. The complement system is a cascade of reactions between a number of different proteins. The end result of the cascade is opsonization of pathogens, as well as direct pathogen killing via the formation of a protein complex which punctures holes in bacterial cell walls.

PhagocytosisPhagocytosis (from Greek phago, meaning eating, cyte, meaning vessel, and osis meaning process) is the cellular process of engulfing solid particles by the cell membrane to form an internal phagosome by phagocytes and protists. Phagocytosis is a specific form ofendocytosis involving the vesicular internalization of solid particles, such as bacteria, and is, therefore, distinct from other forms of endocytosis such as the vesicular internalization of various liquids. Phagocytosis is involved in the acquisition of nutrients for some cells, and, in the immune system, it is a major mechanism used to remove pathogens and cell debris. Bacteria, dead tissue cells, and small mineral particles are all examples of objects that may be phagocytosed.

The process is homologous to eating only at the level of single-celled organisms; in multicellular animals, the process has been adapted to eliminate debris and pathogens, as opposed to taking in fuel for cellular processes, except in the case of the Trichoplax.

Phagocytosis in three steps:1. Unbound phagocyte surface receptors do not trigger phagocytosis.2. Binding of receptors causes them to cluster.3. Phagocytosis is triggered and the particle is taken up by the phagocyte.

In immune system

Phagocytosis in mammalian immune cells is activated by attachment to Pathogen-associated molecular patterns (PAMPS), which leads toNF-κB activation. Opsonins such as C3b and antibodies can act as attachment sites and aid phagocytosis of pathogens.

Engulfment of material is facilitated by the actin-myosin contractile system. The phagosome of ingested material is then fused with the lysosome, leading to degradation.

Degradation can be oxygen-dependent or oxygen-independent.

Oxygen-dependent degradation depends on NADPH and the production of reactive oxygen species. Hydrogen peroxide andmyeloperoxidase activate a halogenating system, which leads to the destruction of bacteria.

Oxygen-independent degradation depends on the release of granules, containing proteolytic enzymes such as defensins, lysozyme, and cationic proteins. Other antimicrobial peptides are present in these granules, including lactoferrin, which sequesters iron to provide unfavourable growth conditions for bacteria.

It is possible for cells other than dedicated phagocytes (such as dendritic cells) to engage in phagocytosis.

In apoptosis

Following apoptosis, the dying cells need to be taken up into the surrounding tissues by macrophages in a process called Efferocytosis. One of the features of an apoptotic cell is the presentation of a variety of intracellular molecules on the cell surface, such as Calreticulin, Phosphatidylserine (From the inner layer of the plasma membrane), Annexin A1, and oxidisedLDL. These molecules are recognised by receptors

on the cell surface of the macrophage such as the Phosphatidylserine Receptor, or by soluble (free floating) receptors such asThrombospondin 1, Gas-6, and MFG-E8, which themselves, then, bind to other receptors on the macrophage such as CD36 and Alpha-V Beta-3 Integrin.

In protists

Protists (pronounced /ˈproʊtɨst/) are a diverse group of eukaryotic microorganisms. Historically, protists were treated as the kingdomProtista, which includes mostly unicellular organisms that do not fit into the other kingdoms, but this group is contested in modern taxonomy. Instead, it is "better regarded as a loose grouping of 30 or 40 disparate phyla with diverse combinations of trophic modes, mechanisms of motility, cell coverings and life cycles. The protists do not have much in common besides a relatively simple organization—either they are unicellular, or they are multicellularwithout specialized tissues. This simple cellular organization distinguishes the protists from other eukaryotes, such as fungi, animals andplants.

Protists live in almost any environment that contains liquid water. Many protists, such as the algae, are photosynthetic and are vitalprimary producers in ecosystems, particularly in the ocean as part of the plankton. Other protists, such as the Kinetoplastids andApicomplexa, are responsible for a range of serious human diseases, such as malaria and sleeping sickness.

In many protists, phagocytosis is used as a means of feeding, providing part or all of their nourishment. This is called phagotrophic nutrition, as distinguished from osmotrophic nutrition, which takes place by absorption.

In some, such as amoeba, phagocytosis takes place by surrounding the target object with pseudopods, as in animal phagocytes. In humans, Entamoeba histolytica can phagocytose red blood cells. This process is known as "erythrophagocystosis", and is considered the only reliable way to distinguish Entamoeba histolytica from noninvasive species such as Entamoeba dispar.

Ciliates also engage in phagocytosis. In ciliates there is a specialized groove or chamber in the cell where phagocytosis takes place, called the cytostome or mouth.

The resulting phagosome may be merged with lysosomes containing digestive enzymes, forming a phagolysosome. The food particles will then be digested, and the released nutrients are diffused or transported into the cytosol for use in other metabolic processes.

Mixotrophy can involve phagotrophic nutrition and phototrophic nutrition.

(d)MacrophageMacrophages (Greek: big eaters, from makros "large" + phagein "eat"; abbr. MΦ) are white blood cells within tissues, produced by the differentiation of monocytes. Human macrophages are about 21 micrometres (0.00083 in) in diameter. Monocytes and macrophages arephagocytes, acting in both non-specific defense (innate immunity) as well as to help initiate specific defense mechanisms (adaptive immunity) of vertebrate animals. Their role is to phagocytose (engulf and then digest) cellular debris and pathogens either as stationary or as mobile cells, and to stimulate lymphocytes and other immune cells to respond to the pathogen. They can be identified by specific expression of a number of proteins including CD14, CD11b, F4/80 (mice)/EMR1 (human), Lysozyme M, MAC-1/MAC-3 and CD68 by flow cytometry or immunohistochemical staining. They move by action of Amoeboid movement.

Macrophage

Life cycle

When a leukocyte enters damaged tissue through the endothelium of a blood vessel (a process known as the leukocyte extravasation), it undergoes a series of changes to become a macrophage. Monocytes are attracted to a damaged site by chemical substances through chemotaxis, triggered by a range of stimuli including damaged cells, pathogens and cytokinesreleased by macrophages already at the site. At some sites such as the testis, macrophages have been shown to populate the organ through proliferation. Unlike short-lived neutrophils, macrophages survive longer in the body up to a maximum of several months.

Function

1.PhagocytosisOne important role of the macrophage is the removal of necrotic cellular debris in the lungs. Removing dead cell material is important in chronic inflammation, as the early stages of inflammation are dominated by neutrophil granulocytes, which are ingested by macrophages if they come of age (see CD-31 for a description of this process.)

The removal of necrotic tissue is, to a greater extent, handled by fixed macrophages, which will stay at strategic locations such as the lungs, liver, neural tissue, bone, spleen and connective tissue, ingesting foreign materials such as pathogens, recruiting additional macrophages if needed.

When a macrophage ingests a pathogen, the pathogen becomes trapped in a phagosome, which then fuses with a lysosome. Within the phagolysosome, enzymes and toxic peroxides digest the pathogen. However, some bacteria, such as Mycobacterium tuberculosis, have become resistant to these methods of digestion. Macrophages can digest more than 100 bacteria before they finally die due to their own digestive compounds.

Steps of a macrophage ingesting a pathogen:a. Ingestion through phagocytosis, a phagosome is formedb. The fusion of lysosomes with the phagosome creates a phagolysosome; the pathogen is broken down by enzymesc. Waste material is expelled or assimilated (the latter not pictured)Parts:1. Pathogens2. Phagosome3. Lysosomes4. Waste material5. Cytoplasm6. Cell membrane

2.Role in adaptive immunityMacrophages are versatile cells that play many roles. As scavengers, they rid the body of worn-out cells and other debris. They are foremost among the cells that "present" antigen, a crucial role in initiating an immune response. As secretory cells, monocytes and macrophages are vital to the regulation of immune responses and the development of inflammation; they produce a wide array of powerful chemical substances (monokines) including enzymes, complement proteins, and regulatory factors such as interleukin-1. At the same time, they carry receptors for lymphokines that allow them to be "activated" into single-minded pursuit of microbes and tumour cells.

After digesting a pathogen, a macrophage will present the antigen (a molecule, most often a protein found on the surface of the pathogen, used by the immune system for identification) of the pathogen to the correspondinghelper T cell. The presentation is done by integrating it into the cell membrane and displaying it attached to an MHC class II molecule, indicating to other white blood cells that the macrophage is not a pathogen, despite having antigens on its surface.

Eventually, the antigen presentation results in the production of antibodies that attach to the antigens of pathogens, making them easier for macrophages to adhere to with their cell membrane and phagocytose. In some cases, pathogens are very resistant to adhesion by the macrophages.

The antigen presentation on the surface of infected macrophages (in the context of MHC class II) in a lymph node stimulates TH1 (type 1 helper T cells) to proliferate (mainly due to IL-12secretion from the macrophage). When a B-cell in the lymph node recognizes the same unprocessed surface antigen on the bacterium with its surface bound antibody, the antigen is endocytosed and processed. The processed antigen is then presented in MHCII on the surface of the B-cell. TH1 receptor that has proliferated recognizes the antigen-MHCII complex (with co-stimulatory factors- CD40 and CD40L) and causes the B-

cell to produce antibodies that help opsonisation of the antigen so that the bacteria can be better cleared byphagocytes.

Macrophages provide yet another line of defense against tumor cells and somatic cells infected with fungus or parasites. Once a T cell has recognized its particular antigen on the surface of an aberrant cell, the T cell becomes an activated effector cell, chemical mediators known as lymphokines that stimulate macrophages into a more aggressive form. These activated macrophages can then engulf and digest affected cells much more readily. The macrophage does not generate a response specific for an antigen, but attacks the cells present in the local area in which it was activated.

3.Role in tissue reorganizationMacrophages secrete not only cytotoxic and inflammation controlling mediators but also substances participating in tissue reorganization. They include enzymes, as hyaluronidase, elastase, and collagenase, inhibitors of some of them (antiproteases), regulatory growth factors and others. Hyaluronidase, by destroying hyaluronic acid, an important component of connective tissue, reduces viscosity and thus permits greater spreading of material in tissue spaces. Hyaluronidase is therefore sometimes designated the ''spreading factor''. Elastase and collagenase are enzymes capable to split collagen and elastin, the basic members of connective proteins.

4.Producer of arachidonic acid its metabolitesMacrophages are important producers of arachidonic acid and its metabolites. Upon phagocytosis macrophages release up to 50% of their arachidonic acid from membranous esterified glycerol phospholipid. It is immediately metabolized into different types of prostanoids. From them prostaglandins, especially PGE , and prostacyclin (PGI ) are characterized as pro-inflammatory agents: they induce vasodilatation, act synergeticly with complement component C5a and LTB , mediate fever and myalgia response to IL-1, in the combination with bradykinin and histamine they contribute to erythema, oedema, and pain induction. Tromboxan TXA  is considered as an inflammatory mediator; it facilitates platelet aggregation and triggers vasoconstriction. LTB  is the efficient chemoatractant substance. A mixture of LTC , LTD  and LTE  became known as slow-reacting substance of anaphylaxis (SRS-A). These leukotrienes are important mediators of bronchial asthma, since they provoke long-term contractions of bronchial smooth muscles.

5.As killer cellIn addition, macrophages are important killer cells (K cells); by means of antibody-dependent cell-mediated cytotoxicity (ADCC) they are able to kill or damage extracellular targets. They also take part in the initiation of T cell activation by processing and presenting antigen. Finally they are central effector and regulatory cells of the inflammatory response. To fulfil these functions, macrophages in their activated state are able to produce more than one hundred of different substances.

Involvement in symptoms of diseases

Due to their role in phagocytosis, macrophages are involved in many diseases of the immune system. For example, they participate in the formation of granulomas, inflammatory lesions that may be caused by a large number of diseases.

Some disorders, mostly rare, of ineffective phagocytosis and macrophage function have been described.[citation needed]

Macrophages are the predominant cells involved in creating the progressive plaque lesions of atherosclerosis.[citation needed]

Macrophages also play a role in Human Immunodeficiency Virus (HIV) infection. Like T cells, macrophages can be infected with HIV, and even become a reservoir of ongoing virus replication throughout the body.

Macrophages are believed to help cancer cells proliferate as well. They are attracted to oxygen-starved (hypoxic) tumour cells and promote chronic inflammation. Inflammatory compounds such as Tumor necrosis factor (TNF) released by the macrophage activates the gene switch nuclear factor-kappa B. NF-κB then enters the nucleus of a tumour cell and turns on production of proteins that stop apoptosis and promote cell proliferation and inflammation.

Recent investigations point a link between streptococcal infection and autoimmune behaving microglia which cause OCD

(d)Dendritic cellsDendritic cells (DCs) are immune cells that form part of the mammalian immune system. Their main function is to process antigen material and present it on the surface to other cells of the immune system, thus functioning as antigen-presenting cells. They act as messengers between the innate and adaptive immunity.

Dendritic cells are present in small quantities in tissues that are in contact with the external environment, mainly the skin (where there is a specialized dendritic cell type called Langerhans cells) and the inner lining of the nose, lungs, stomach and intestines. They can also be found in an immature state in the blood. Once activated, they migrate to the lymphoid node where they interact with T cells and B cells to initiate and shape the adaptive immune response. At certain development stages they grow branched projections, the dendrites, that give the cell its name. However, these do not have any special relation with neurons, which also possess similar appendages. Immature dendritic cells are also called veiled cells, in which case they possess large cytoplasmic 'veils' rather than dendrites.

A dendritic cell

Types of dendritic cells

In all dendritic cells, the similar morphology results in a very large contact surface to their surroundings compared to overall cell volume.

In vivo - primate

The most common division of dendritic cells is "myeloid" vs. "plasmacytoid":

Name Description Secretion Toll-like

receptors

Myeloid dendritic cell (mDC)

Most similar to monocytes. mDC are made up of at least two subsets:(1) the more common mDC-1, which is a major stimulator of T cells(2) the extremely rare mDC-2, which may have a function in fighting wound infection

IL-12TLR 2, TLR 4

Plasmacytoid dendritic cell (pDC)

Look like plasma cells, but have certain characteristics similar to myeloid dendritic cells.

Can produce high amounts of interferon-alpha and thus became known as IPC (interferon-producing cells) before their dendritic cell nature was revealed.

TLR 7, TLR 9

The markers BDCA-2, BDCA-3, and BDCA-4 can be used to discriminate among the types.

Lymphoid and myeloid DCs evolve from lymphoid or myeloid precursors respectively and thus are of hematopoietic origin. By contrast, follicular dendritic cells (FDC) are probably ofmesenchymal rather than hematopoietic origin and do not express MHC class II, but are so named because they are located in lymphoid follicles and have long "dendritic" processes.

In vitroIn some respects, dendritic cells cultured in vitro do not show the same behaviour or capability as dendritic cells isolated ex vivo. Nonetheless, they are often used for research as they are still much more readily available than genuine DCs.

Mo-DC or MDDC refers to cells matured from monocytes

HP-DC refers to cells derived from hematopoietic progenitor cells.

NonprimateWhile humans and non-human primates such as Rhesus macaques appear to have DCs divided into these groups, other species (such as the mouse) have different subdivisions of DCs.

Life cycle There are 3 main kinds of dendritic cells which are found in skin and in T cell and B cell areas of lymphoyid tissues.Dendritic cell are so called because of their many surface membrane folds,similar in

appearance to dendrite of the nervous system.these fold allow maximum interaction with other cells of the immune system.

Most denritic cells possess high levels of surface MHC class II molecules and process and present peptide antigens to T cells.Their role is to recognize microbial antigens through innate receptors and process and present them to T cell of the adaptive immune system.follicular dendritic cells hold intact antigens in specialized areas of lymphoid tissues.

Dendritic cells and cytokines

The dendritic cells are constantly in communication with other cells in the body. This communication can take the form of direct cell-to-cell contact based on the interaction of cell-surface proteins. An example of this includes the interaction of the receptor B7 of the dendritic cell with CD28 present on the lymphocyte. However, the cell-cell interaction can also take place at a distance via cytokines.

For example, stimulating dendritic cells in vivo with microbial extracts causes the dendritic cells to rapidly begin producing IL-12. IL-12 is a signal that helps send naive CD4 T cells towards a Th1 phenotype. The ultimate consequence is priming and activation of the immune system for attack against the antigens which the dendritic cell presents on its surface. However, there are differences in the cytokines produced depending on the type of dendritic cell. The lymphoid DC has the ability to produce huge amounts of type-1 IFN's, which recruit more activated macrophage to allow phagocytosis.

Relationship to HIV, allergy, and autoimmune diseases

HIV, which causes AIDS, can bind to dendritic cells via various receptors expressed on the cell. The best studied example is DC-SIGN (usually on MDC subset 1, but also on other subsets under certain conditions; since not all dendritic cell subsets express DC-SIGN, its exact role in sexual HIV-1 transmission is not clear). When the dendritic cell takes up HIV and then travels to the lymph node, the virus is able to move to helper T-cells, and this infection of helper T-cells is the major cause of disease. This knowledge has vastly altered our understanding of the infectious cycle of HIV since the mid-1990s, since in the infected dendritic cells, the virus possesses a reservoir which also would have to be targeted by a therapy. This infection of dendritic cells by HIV explains one mechanism by which the virus could persist after prolonged HAART. Many other viruses, such as the SARS virus seems to use DC-SIGN to 'hitchhike' to its target cells. However, most work with virus binding to DC-SIGN expressing cells has been conducted using in vitro derived cells such as moDCs. The physiological role of DC-SIGN in vivo is more difficult to ascertain.

Altered function of dendritic cells is also known to play a major or even key role in allergy and autoimmune diseases like lupus erythematosus and inflammatory bowel diseases (Crohn's disease and ulcerative colitis).

(e)Natural killer cellNatural killer cells (or NK cells) are a type of cytotoxic lymphocyte that constitute a major component of the innate immune system. NK cells play a major role in the rejection oftumors and cells infected by viruses. They kill cells by releasing small cytoplasmic granules of proteins called perforin and granzyme that cause the target cell to die by apoptosis.

NK cells are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes..They do not express T-cell antigen receptors (TCR) or Pan T marker CD3 or surface immunoglobulins (Ig) B cell

receptors but they usually express the surface markers CD16 (FcγRIII) and CD56 in humans, and NK1.1/NK1.2 in certain strains of mice. Up to 80% of NK cells also express CD8.

They were named "natural killers" because of the initial notion that they do not require activation in order to kill cells that are missing "self" markers of major histocompatibility complex(MHC) class I.

Activation

Given their strong cytolytic activity and the potential for auto-reactivity, NK cell activity is tightly regulated. NK cells must receive an activating signal, which can come in a variety of forms, the most important of which are listed below.

. Cytokines

The cytokines play a crucial role in NK cell activation. As these are stress molecules released by cells upon viral infection, they serve to signal to the NK cell the presence of viral pathogens. Cytokines involved in NK activation include IL-12, IL-15, IL-18, IL-2, and CCL5.

Fc receptor

NK cells, along with macrophages and several other cell types, express the Fc receptor (FcR) molecule (FC-gamma-RIII = CD16), an activating biochemical receptor that binds theFc portion of antibodies. This allows NK cells to target cells against which a humoral response has been mobilized and to lyse cells through Antibody-dependent cellular cytotoxicity (ADCC).

. Activating and inhibitory receptors

Aside from the Fc receptor, NK cells express a variety of receptors that serve either to activate or to suppress their cytolytic activity. These receptors bind to various ligands on target cells, both endogenous and exogenous, and have an important role in regulating the NK cell response.

Mechanism

Schematic diagram indicating the complementary activities of cytotoxic T-cellsand NK cells.

NK cells are cytotoxic; small granules in their cytoplasm contain proteins such as perforin and proteasesknown as granzymes. Upon release in close proximity to a cell slated for killing, perforin forms pores in the cell membrane of the target cell, creating an aqueous channel through which the granzymes and associated molecules can enter, inducing either apoptosis or osmotic cell lysis. The distinction between apoptosis and celllysis is important in immunology: lysing a virus-infected cell would only release the virions, whereas apoptosis leads to destruction of the virus inside.

NK cells are activated in response to interferons or macrophage-derived cytokines. They serve to contain viralinfections while the adaptive immune response is generating antigen-specific cytotoxic T cells that can clear the infection. Patients deficient in NK cells prove to be highly susceptible to early phases of herpes virus infection.

In order for NK cells to defend the body against viruses and other pathogens, they require mechanisms that enable the determination of whether a cell is infected or not. The exact mechanisms remain the subject of current investigation, but recognition of an "altered self" state is thought to be involved. To control their cytotoxic activity, NK cells possess two types of surface receptors: activating receptors and inhibitory receptors. Most of these receptors are not unique to NK cells and can be present in some T cell subsets as well.

These inhibitory receptors recognize MHC class I alleles, which could explain why NK cells kill cells possessing low levels of MHC class I molecules. This inhibition is crucial to the role played by NK cells. MHC class I molecules consist of the main mechanism by which cells display viral or tumor antigens to cytotoxic T-cells. A common evolutionary adaption to this seen in both intracellular microbes and tumours is a chronic down-regulation of these MHC I molecules, rendering the cell impervious to T-cell mediated immunity. It is believed that NK cells, in turn, evolved as an evolutionary response to this adaption, as the loss of the MHC would deprive these cells of the inhibitory effect of MHC and render these cells vulnerable to NK cell mediated apoptosis.

Receptor types

NK cell receptor types (with inhibitory as well as some activating members) are differentiated by structure:

1. CD94 : NKG2 (heterodimers) — a C-type lectin family receptor, conserved in both rodents and primates and identifies non-classical (also non-polymorphic) MHC I molecules likeHLA E. Though indirect, this is a way to survey the levels of classical (polymorphic) HLA molecules, however, because expression of HLA-E at the cell surface is dependent upon the presence of classical MHC class I leader peptides.

2. Ly49 (homodimers) — a relatively ancient, C-type lectin family receptor; are of multigenic presence in mice, while humans have only one pseudogenic Ly49; the receptor for classical (polymorphic) MHC I molecules.

3. KIR (Killer-cell immunoglobulin-like receptors) — belong to a multigene family of more recently-evolved Ig-like extracellular domain receptors; are present in non-human primates; and are the main receptors for both classical MHC I (HLA-A, HLA-B, HLA-C) and also non-classical HLA-G in primates. Some KIRs are specific for certain HLA subtypes.

4. ILT or LIR (leukocyte inhibitory receptors) — are recently-discovered members of the Ig receptor family.

NK cell receptors can also be differentiated based on function. Natural cytotoxicity receptors directly induce apoptosis after binding to ligands that directly indicate infection of a cell. The MHC dependent receptors (described above) use an alternate pathway to induce apoptosis in infected cells.

γδ T cellsγδ T cells (gamma delta T cells) represent a small subset of T cells that possess a distinct T cell receptor (TCR) on their surface. A majority of T cells have a TCR composed of twoglycoprotein chains called α- and β- TCR chains. In contrast, in γδ T cells, the TCR is made up of one γ-chain and one δ-chain. This group of T cells is usually much less common than αβ T cells, but are found at their highest abundance in the gut mucosa, within a population of lymphocytes known as intraepithelial lymphocytes (IELs).

The antigenic molecules that activate γδ T cells are still largely unknown. However, γδ T cells are peculiar in that they do not seem to require antigen processing and MHC presentation of peptide epitopes although some recognize MHC class IB molecules. Furthermore, γδ T cells are believed to have a prominent role in recognition of lipid antigens.

There also exists a γδ T cell sub-population within the epidermal compartment of the skin. Named Dendritic Epidermal γδ T cells (DETC), in mice, these cells arise during fetal development and express an invariant and canonical Vγ3 Vδ1 T cell receptor (TCR)[using Garman nomenclature].

γδ T cells in innate and adaptive immunityThe conditions that lead to responses of γδ T cells are not fully understood, and current concepts of γδ T cells as 'first line of defense', 'regulatory cells', or 'bridge between innate and adaptive responses' only

address facets of their complex behavior. In fact, γδ T cells form an entire lymphocyte system that develops under the influence of other leukocytes, in thethymus and in the periphery. Mature γδ T cells are divided into functionally distinct subsets that obey their own (mostly unknown) rules and that have countless direct and indirect effects on healthy tissues and immune cells, on pathogens and tissues enduring infections and the host responses to them.

Like other 'unconventional' T cell subsets bearing invariant TCRs, such as CD1d-restricted Natural Killer T cells, γδ T cells exhibit several characteristics that place them at the border between the more evolutionarily primitive innate immune system that permits a rapid beneficial response to a variety of foreign agents, and the adaptive immune system, where B and T cells coordinate a slower but highly antigen-specific immune response leading to long-lasting memory against subsequent challenges by the same antigen.

On one hand, γδ T cells may be considered a component of adaptive immunity in that they rearrange TCR genes to produce junctional diversity and will develop a memory phenotype.

However, the various subsets may also be considered part of the innate immunity where a restricted TCR may be used as a pattern recognition receptor. For example, according to this paradigm, large numbers of (human) Vγ9/Vδ2 T cells respond within hours to common molecules produced by microbes, and highly restricted intraepithelial Vδ1 T cells will respond to stressed epithelial cells bearing sentinels of danger.

Recent work has shown that human Vγ9/Vδ2 T cells are also capable of phagocytosis, a function previously exclusive to innate myeloid lineage cells such as neutrophils, monocytes and dendritic cells 

Clearly, the complexity of γδ T cell biology spans definitions of both innate and adaptive immune responses.

Types in humans

Human Vδ2+ T cellsVγ9/Vδ2 T cells are unique to humans and primates and represent a minor and unconventional constituent of the leukocyte population in peripheral blood (0.5-5%); yet they are assumed to play an early and essential role in sensing 'danger' by invading pathogens as they expand dramatically in many acute infections and may exceed all other lymphocytes within a few days,e.g. in tuberculosis, salmonellosis, ehrlichiosis, brucellosis, tularemia, listeriosis, toxoplasmosis, and malaria. Of note, all Vγ9/Vδ2 T cells recognize the same small microbial compound (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), a natural intermediate of the non-mevalonate pathway of isopentenyl pyrophosphate (IPP) biosynthesis. HMB-PP is an essential metabolite in most pathogenic bacteria including Mycobacterium tuberculosis and malaria parasites, but is absent from the human host. Bacterial species that lack the non-mevalonate pathway and synthesize IPP via the classical mevalonate pathway instead, such as Streptococcus, Staphylococcus, and Borrelia, are unable to produce HMB-PP and do not specifically activate Vγ9/Vδ2 T cells.

Human non-Vδ2+ T cellsThe extensive structural diversity of Vδ1 and Vδ3 TCRs and the existence of Vδ1+ clones reactive against MHC, MHC-like, or non-MHC molecules suggest recognition of a highly diverse and heterogeneous set of antigens by non-Vδ2 cells, although cognate interactions between non-Vδ2 TCRs and any of these antigens have not been shown yet. MHC class-I-chain-related gene A (MICA) has also

been proposed as an important tumor antigen recognized by Vδ1+ T cells. However, the very low affinity of MICA–Vδ1 TCR interactions estimated by surface plasmon resonance analyses raises doubts about the functional relevance of MICA or MHC class-I-chain-related gene B (MICB) recognition by Vδ1+ TCRs.

Non-Vδ2 γδ T cells are expanded in various infectious contexts involving intracellular bacteria (Mycobacteria and Listeria) as well as extracellular bacteria, such as Borrelia burgdorferiand viruses (HIV, cytomegalovirus). In most instances, the stimuli that trigger Vd1 expansion are not derived from pathogens but instead correspond to endogenous gene products presumably upregulated on infection. The antigens recognized by non-Vδ2 T cells expanded in the above infectious contexts have not been characterized, but the fact that Vδ1+ T-cell responses are not blocked by monoclonal antibody directed against known classical or non-classical MHC molecules suggests recognition of a new class of conserved stress-induced antigens.

Overview table

Type

Microscopic

Appearance

Diagram

Approx. %

in adultsSee also:

Blood valu

es

Diameter (μm)

Main targetsNucle

usGranules

Lifetime

Neutrophil

54–62%

10–12

bacteria

fungi

multilobed

fine, faintly pink (H&E Stain)

6 hours–few days(days inspleen and other tissue)

Eosinophil

1–6% 10–12

larger parasites

modulate allergic inflammatory responses

bi-lobed

full of pink-orange (H&E Stain)

8–12 days (circulate for 4–5 hours)

Basophil

<1% 12–15 release histamine for infla

mmatory responses

bi-lobed ortri-lobed

large blue

a few hours to a few days

Lymphocyte

25–33%

7–8 B cells: releases antibodies and assists activation of T cells

T cells:

Th (T helper) cells:

deeply staining, eccentric

NK-cells and Cytotoxic (CD8

weeks to years

activate and regulate T and B cells

CD8+ cytotoxic T cells: virus-infected and tumor cells.

γδ T cells:

Regulatory (suppressor) T cells: Returns the functioning of the immune system to normal operation after infection; preventsautoimmunity

Natural killer cells: virus-infected and tumor cells.

+) T-cells 

Monocyte

2–10% 14–17

Monocytes migrate from the bloodstream to other tissues and differentiate into tissue resident macrophages or dendritic cells.

kidney shaped

none

hours to days

Macrophage

21 (human)

Phagocytosis (engulfment and digestion) of cellular debris andpathogens, and stimulation of lymphocytes and other immune cells that respond to the pathogen.

activated: daysimmature: months to years

Dendritic cells

Main function is as an antigen-presenting cell (APC) that activates T lymphocytes.

similar to macrophages

What is interferon?Interferons (IFNs) are proteins made and released by lymphocytes in response to the presence of pathogens—such as viruses,bacteria, or parasites—or tumor cells. They allow communication between cells to trigger the protective defenses of the immune system that eradicate pathogens or tumors.

IFNs belong to the large class of glycoproteins known as cytokines. Interferons are named after their ability to "interfere" with viral replication within host cells. IFNs have other functions: they activate immune cells, such as natural killer cells and macrophages; they increase recognition of infection or tumor cells by up-regulating antigen presentation to T lymphocytes; and they increase the ability of uninfected host cells to resist new infection by virus. Certain host symptoms, such as aching muscles and fever, are related to the production of IFNs during infection.

About ten distinct IFNs have been identified in mammals; seven of these have been described for humans. They are typically divided among three IFN classes: Type I IFN, Type II IFN, and Type III IFN. IFNs belonging to all IFN classes are very important for fighting viral infections.

Types of interferon

Based on the type of receptor through which they signal, human interferons have been classified into three major types.

Interferon type I: All type I IFNs bind to a specific cell surface receptor complex known as the IFN-α receptor (IFNAR) that consists of IFNAR1 and IFNAR2 chains. The type I interferons present in humans are IFN-α, IFN-β and IFN-ω.

Interferon type II: Binds to IFNGR. In humans this is IFN-γ.

Interferon type III: Signal through a receptor complex consisting of IL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12). Acceptance of this classification is less universal than that of type I and type II, and unlike the other two, it is not currently included in Medical Subject Headings.

Function

All interferons share several common effects; they are antiviral agents and can fight tumors.

As an infected cell dies from a cytolytic virus, viral particles are released that can infect nearby cells. However, the infected cell can warn neighboring cells of a viral presence by releasing interferon. The neighboring cells, in response to interferon, produce large amounts of an enzyme known as protein kinase R (PKR). This enzyme phosphorylates a protein known as eIF-2in response to new viral infections; eIF-2 is a eukaryotic translation initiation factor that forms an inactive complex with another protein, called eIF2B, to reduce protein synthesis within the cell. Another cellular enzyme, RNAse L—also induced following PKR activation—destroys RNA within the cells to further reduce protein synthesis of both viral and host genes. Inhibited protein synthesis destroys both the virus and infected host cells. In addition, interferons induce production of hundreds of other proteins — known collectively as interferon-stimulated genes (ISGs)—that have roles in combating viruses. They also limit viral spread by increasing p53 activity, which kills virus-infected cells by promoting apoptosis. The effect of IFN on p53 is also linked to its protective role against certain cancers.

Another function of interferon is to upregulate major histocompatibility complex molecules, MHC I and MHC II, and increase immunoproteasome activity. Higher MHC I expression increases presentation of viral peptides to cytotoxic T cells, while the immunoproteasome processes viral peptides for loading onto the MHC I molecule, thereby increasing the recognition and killing of infected cells by T cells. Higher MHC II expression increases presentation of viral peptides to helper T cells; these cells release cytokines that signal to and co-ordinate the activity of other cells of the immune system. Interferons directly activate some other immune cells, such as macrophages and natural killer cells.

Induction of interferons

Production of interferons predominantly occurs in response to microbes, such as viruses and bacteria, and their products. Binding of molecules uniquely found in microbes—viralglycoproteins, viral RNA, bacterial endotoxin (lipopolysaccharide), bacterial flagella, CpG motifs--by pattern recognition receptors, such as membrane bound Toll like receptors or the cytoplasmic receptors RIG-I or MDA5, can trigger release of IFNs. Toll Like Receptor 3 (TLR3) is important for inducing interferon in response to the presence of double-stranded RNA viruses; the ligand for this receptor is double-stranded RNA (dsRNA). After binding dsRNA, this receptor activates the transcription factors IRF3 and NF-κB, which are important for initiating synthesis of many inflammatory proteins. Release of IFN from cells is also induced by mitogens. Other cytokines, such as interleukin 1, interleukin 2, interleukin-12, tumor necrosis factor and colony-stimulating factor, can also enhance interferon production.

Virus resistance to interferons

Many viruses have evolved mechanisms to resist interferon activity. They circumvent the IFN response by blocking downstream signaling events that occur after the cytokine binds to its receptor, by preventing further IFN production, and by inhibiting the functions of proteins that are induced by IFN. Viruses that inhibit IFN signaling include Japanese EncephalitisVirus (JEV), dengue type 2 virus (DEN-2) and viruses of the herpesvirus family, such as human cytomegalovirus (HCMV) and Kaposi's sarcoma-associated herpesvirus (KSHV or HHV8). Viral proteins proven to affect IFN signaling include EBV nuclear antigen 1 (EBNA1) and EBV nuclear antigen 2 (EBNA-2) from Epstein-Barr virus, the large T antigen ofPolyomavirus, the E7 protein of Human papillomavirus (HPV), and the B18R protein of vaccinia virus. Reducing IFN-α activity may prevent signaling via STAT1, STAT2, or IRF9 (as with JEV infection) or through the JAK-STAT pathway (as with DEN-2 infection). Several poxviruses encode soluble IFN receptor homologs—like the B18R protein of the vaccinia virus—that bind to and prevent IFN interacting with its cellular receptor, impeding communication between this cytokine and its target cells. Some viruses can encode proteins that bind todouble-stranded RNA (dsRNA) to prevent the activity of RNA-dependent protein kinases; this is the mechanism reovirus adopts using its sigma 3 (σ3) protein, and vaccinia virus employs using the gene product of its E3L gene, p25. The ability of interferon to induce protein production from interferon stimulated genes (ISGs) can also be affected. Production ofprotein kinase R, for example, can be disrupted in cells infected with JEV or flaviviruses. Some viruses escape the anti-viral activities of interferons by gene (and thus protein) mutation. The H5N1 influenza virus, also known as bird flu, has resistance to interferon and other anti-viral cytokines that is attributed to a single amino acid change in its Non-Structural Protein 1 (NS1), although the precise mechanism of how this confers immunity is unclear.

Interferon therapy

DiseasesThe immune effects of interferons have been exploited to treat several diseases. Agents that activate the immune system, such as smallimidazoquinoline molecules that activate TLR7, can induce IFN-α. Imidazoquinoline is the main ingredient of Aldara (Imiquimod) cream, a treatment approved in the United States by the Food and Drug Administration (FDA) for actinic keratosis, superficial basal cell carcinoma,papilloma and external genital warts. Synthetic IFNs are also made, and administered as antiviral, antiseptic and anticarcinogenic drugs, and to treat some autoimmune diseases.

Interferon beta-1a and interferon beta-1b are used to treat and control multiple sclerosis, an autoimmune disorder. This treatment is effective for slowing disease progression and activity in relapsing-remitting multiple sclerosis and reducing attacks in secondary progressive multiple sclerosis.

Interferon therapy is used (in combination with chemotherapy and radiation) as a treatment for many cancers. This treatment is most effective for treating hematological malignancy; leukemia and lymphomas including hairy cell leukemia, chronic myeloid leukemia, nodular lymphoma, cutaneous T-cell lymphoma. Patients with recurrent melanomas receive recombinant IFN-α2b.

Both hepatitis B and hepatitis C are treated with IFN-α, often in combination with other antiviral drugs. Some of those treated with interferon have a sustained virological response and can eliminate hepatitis virus. The most harmful strain - hepatitis C genotype I virus - can only be treated around 50% of time by the standard of care treatment of interferon-α/ribavirin. Given the treatment, biopsies show reductions in liver damage and cirrhosis. Some evidence shows giving interferon immediately following infection can prevent chronic hepatitis C, although diagnosis early in infection is difficult since physical symptoms are sparse in early hepatitis C infection. Control of chronic hepatitis C by IFN is associated with reduced hepatocellular carcinoma.

Administered intranasally in very low doses, interferon is extensively used in Eastern Europe and Russia as a method to prevent and treat viral respiratory diseases such as cold and flu. However, mechanisms of such action of interferon are not well understood; it is thought that doses must be larger by several orders of magnitude to have any effect on the virus. Although most Western scientists are skeptical of any claims of good efficacy, recent findings suggest that interferon applied to mucosa may act as an adjuvant against influenza virus, boosting the specific immune system response against the virus.  A flu vaccine that uses interferon as adjuvant is currently under clinical trials in the US.

When used in the systemic therapy, IFNs are mostly administered by an intramuscular injection. The injection of IFNs in the muscle, in the vein, or under skin is generally well tolerated. The most frequent adverse effects are flu-like symptoms: increased body temperature, feeling ill, fatigue, headache, muscle pain, convulsion, dizziness, hair thinning, and depression.Erythema, pain and hardness on the spot of injection are also frequently observed. IFN therapy causes immunosuppression, in particular through neutropenia and can result in some infections manifesting in unusual ways.

Drug formulations

Pharmaceutical forms of interferons

Generic name Trade name

Interferon alpha 2a Roferon A

Interferon alpha 2b Intron A/Reliferon

Human leukocyte Interferon-alpha (HuIFN-alpha-Le) Multiferon

Interferon beta 1a, liquid form Rebif

Interferon beta 1a, lyophilized Avonex

Interferon beta 1a, biogeneric (Iran) Cinnovex

Interferon beta 1b Betaseron / Betaferon

Interferon beta 1b, biosimilar (Iran) ZIFERON

PEGylated interferon alpha 2a Pegasys

PEGylated interferon alpha 2a (Egypt) Reiferon Retard

PEGylated interferon alpha 2b PegIntron

PEGylated interferon alpha 2b plus ribavirin (Canada) Pegetron

Several different types of interferon are now approved for use in humans. By March 10, 2009, MultiferonTM — known generically as human leukocyte interferon-alpha (HuIFN-alpha-Le) — was being used in 14 European countries. This drug was approved for treatment of patients with high risk (stage IIb-III)cutaneous melanoma, after 2 treatment cycles with dacarbazine, following a clinical trial performed in Germany.

What is Innate immune evasion?Cells of the innate immune system effectively prevent free growth of bacteria within the body; however, many pathogens have evolved mechanisms allowing them to evade the innate immune system.

Evasion strategies that circumvent the innate immune system include intracellular replication, such as in Salmonella, or a protective capsule that prevents lysis by complement and by phagocytes, as in Mycobacterium tuberculosis. Bacteroides species are normally mutualistic bacteria, making up a substantial portion of the mammalian gastrointestinal flora.Some species (B. fragilis, for example) are opportunistic pathogens, causing infections of the peritoneal cavity. These species evade the immune

system through inhibition of phagocytosis by affecting the receptors that phagocytes use to engulf bacteria or by mimicking host cells so that the immune system does not recognize them as foreign.Staphylococcus aureus inhibits the ability of the phagocyte to respond to chemokine signals. Other organisms such as M. tuberculosis, Streptococcus pyogenes and Bacillus anthracisutilize mechanisms that directly kill the phagocyte.

Bacteria and fungi may also form complex biofilms, providing protection from the cells and proteins of the immune system; recent studies indicate that such biofilms are present in many successful infections, including the chronic Pseudomonas aeruginosa and Burkholderia cenocepacia infections characteristic of cystic fibrosis.

What is Anatomical barriers?The epithelial surfaces form a physical barrier that is very impermeable to most infectious agents, acting as the first line of defense against invading organisms. Desquamation of skin epithelium also helps remove bacteria and other infectious agents that have adhered to the epithelial surfaces. In the gastrointestinal and respiratory tract, movement due to peristalsis or cilia helps removing infectious agents. Also, mucus traps infectious agents. The gut flora can prevent the colonization of pathogenic bacteria by secreting toxic substances or by competing with pathogenic bacteria for nutrients or attachment to cell surfaces. The flushing action of tears and saliva helps prevent infection of the eyes and mouth.

Anatomical barrier Additional defense mechanims

Skin Sweat, desquamation, flushing, organic acids

Gastrointestinal tractPeristalsis, gastric acid, bile acids, digestive enzyme,flushing, thiocyanate, defensins, gut flora

Respiratory airways and lungs Mucociliary elevator, surfactant, defensins

Nasopharynx Mucus, saliva, lysozyme

Eyes Tears

Adaptive immune systemThe adaptive immune system is composed of highly specialized, systemic cells and processes that eliminate or prevent pathogenic challenges. Thought to have arisen in the first jawed vertebrates, the adaptive or "specific" immune system is activated by the “non-specific” and evolutionarily older innate immune system (which is the major system of host defense against pathogens in nearly all other living things). The adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens (to generate immunity), and to mount stronger attacks each time the pathogen is encountered. It is adaptive immunity because the body's immune system prepares itself for future challenges.

The system is highly adaptable because of somatic hypermutation (a process of accelerated somatic mutations), and V(D)J recombination (an irreversible genetic recombination of antigen receptor gene segments). This mechanism allows a small number of genes to generate a vast number of different antigen receptors, which are then uniquely expressed on each individual lymphocyte. Because the gene rearrangement leads to an irreversible change in the DNA of each cell, all of the progeny (offspring) of that cell will then inherit genes encoding the same receptor specificity, including the Memory B cells and Memory T cells that are the keys to long-lived specific immunity. Immune network theory is a theory of how the adaptive immune system works, that is based on interactions between the variable regions of the receptors of T cells, B cells and of molecules made by T cells and B cells that have variable regions.

Functions

Adaptive immunity is triggered in vertebrates when a pathogen evades the innate immune system and generates a threshold level of antigen.

The major functions of the adaptive immune system include:

the recognition of specific “non-self” antigens in the presence of “self”, during the process of antigen presentation.

the generation of responses that are tailored to maximally eliminate specific pathogens or pathogen infected cells.

the development of immunological memory, in which each pathogen is “remembered” by a signature antibody. These memory cells can be called upon to quickly eliminate a pathogen should subsequent infections occur.

Effector cells

The cells of the adaptive immune system are a type of leukocyte, called a lymphocyte. B cells and T cells are the major types of lymphocytes. The human body has about 2 trillion lymphocytes, constituting 20-40% of white blood cells (WBCs); their total mass is about the same as the brain or liver. The peripheral blood contains 20–50% of circulating lymphocytes; the rest move within the lymphatic system.

B cells and T cells are derived from the same multipotent hematopoietic stem cells, and are indistinguishable from one another until after they are activated. B cells play a large role in the humoral immune response, whereas T-cells are intimately involved in cell-mediated immune responses. However, in nearly all other vertebrates, B cells (and T-cells) are produced by stem cells in the bone marrow. T-cells

travel to and develop in the thymus, from which they derive their name. In humans, approximately 1-2% of the lymphocyte pool recirculates each hour to optimize the opportunities for antigen-specific lymphocytes to find their specific antigen within the secondary lymphoid tissues.

In an adult animal, the peripheral lymphoid organs contain a mixture of B and T cells in at least three stages of differentiation:

naive cells that have matured, left the bone marrow or thymus, have entered the lymphatic system, but that have yet to encounter their cognate antigen,

effector cells that have been activated by their cognate antigen, and are actively involved in eliminating a pathogen and,

memory cells – the long-lived survivors of past infections.

CD8+ T lymphocytes and cytotoxicityCytotoxic T cells (also known as TC, killer T cell, or cytotoxic T-lymphocyte (CTL)) are a sub-group of T cells which induce the death of cells that are infected with viruses (and other pathogens), or are otherwise damaged or dysfunctional.

Killer T cells—also called cytotoxic T lymphocytes or CTL-directly attack other cells carrying certain foreign or abnormal molecules on their surfaces.

Naive cytotoxic T cells are activated when their T-cell receptor (TCR) strongly interacts with a peptide-bound MHC class I molecule. This affinity depends on the type and orientation of the antigen/MHC complex, and is what keeps the CTL and infected cell bound together. Once activated, the CTL undergoes a process called clonal expansion in which it gains functionality, and divides rapidly, to produce an army of “armed”-effector cells. Activated CTL will then travel throughout the body in search of cells bearing that unique MHC Class I + peptide.

When exposed to these infected or dysfunctional somatic cells, effector CTL release perforin and granulysin: cytotoxins which form pores in the target cell's plasma membrane, allowing ions and water to flow into the infected cell, and causing it to burst or lyse. CTL releasegranzyme, a serine protease that enters cells via pores to induce apoptosis (cell death). To limit extensive tissue damage during an infection, CTL activation is tightly controlled and generally requires a very strong MHC/antigen activation signal, or additional activation signals provided by "helper" T-cells (see below).

Upon resolution of the infection, most of the effector cells will die and be cleared away by phagocytes, but a few of these cells will be retained as memory cells. Upon a later encounter with the same antigen, these

memory cells quickly differentiate into effector cells, dramatically shortening the time required to mount an effective response.

Helper T-cellsCD4+ lymphocytes, or helper T cells, are immune response mediators, and play an important role in establishing and maximizing the capabilities of the adaptive immune response. These cells have no cytotoxic or phagocytic activity; and cannot kill infected cells or clear pathogens, but, in essence "manage" the immune response, by directing other cells to perform these tasks.

Helper T cells express T-cell receptors (TCR) that recognize antigen bound to Class II MHC molecules. The activation of a naive helper T-cell causes it to release cytokines, which influences the activity of many cell types, including the APC that activated it. Helper T-cells require a much milder activation stimulus than cytotoxic T-cells. Helper T-cells can provide extra signals that "help" activate cytotoxic cells.

Th1 and Th2: helper T cell responsesTwo types of effector CD4+ T helper cell responses can be induced by a professional APC, designated Th1 and Th2, each designed to eliminate different types of pathogens. The factors that dictate whether an infection will trigger a Th1 or Th2 type response are not fully understood, but the response generated does play an important role in the clearance of different pathogens.

The Th1 response is characterized by the production of Interferon-gamma, which activates the bactericidal activities of macrophages, and induces B-cells to make opsonizing (coating) antibodies, and leads to "cell-mediated immunity" . The Th2 response is characterized by the release of Interleukin 4, which results in the activation of B-cells to make neutralizing (killing) antibodies, leading to "humoral immunity". Generally, Th1 responses are more effective against intracellular pathogens (viruses and bacteria that are inside host cells), while Th2 responses are more effective against extracellular bacteria, parasites and toxins. Like cytotoxic T-cells, most of the CD4+ helper cells will die upon resolution of infection, with a few remaining as CD4+ memory cells.

HIV is able to subvert the immune system by attacking the CD4+ T cells, precisely the cells that could drive the destruction of the virus, but also the cells that drive immunity against all other pathogens encountered during an organisms' lifetime.

A third type of T lymphocyte, the regulatory T cells (Treg), limits and suppresses the immune system, and may control aberrant immune responses to self-antigens; an important mechanism in controlling the development of autoimmune diseases.

T cells never act as antigen-presenting cells.

γδ T cellsγδ T cells (gamma delta cells) possess an alternative T cell receptor (TCR) as opposed to CD4+ and CD8+ αβ T cells and share characteristics of helper T cells, cytotoxic T cells and natural killer cells. Like other 'unconventional' T cell subsets bearing invariant TCRs, such as CD1d-restricted Natural Killer T cells, γδ T cells exhibit characteristics that place them at the border between innate and adaptive immunity. On one hand, γδ T cells may be considered a component of adaptive immunity in that they rearrange TCR genes via V(D)J recombination, which also produces junctional diversity, and develop a memory phenotype. On the other hand however, the various subsets may also be considered part of the innate immune system where a restricted TCR and/or NK receptors may be used as a pattern recognition receptor. For example, according to this paradigm, large numbers of Vγ9/Vδ2 T cells respond within hours to common molecules produced by microbes, and highly restricted intraepithelial Vδ1 T cells will respond to stressed epithelial cells.

B lymphocytes and antibody production

B Cells are the major cells involved in the creation of antibodies that circulate in blood plasma and lymph, known as humoral immunity. Antibodies (or immunoglobulin, Ig), are large Y-shaped proteins used by the immune system to identify and neutralize foreign objects. In mammals there are five types of antibody: IgA, IgD, IgE, IgG, and IgM, differing in biological properties, each has evolved to handle different kinds of antigens. Upon activation, B cells produce antibodies, each of which recognizes a unique antigen, and neutralize specific pathogens.

Like the T cell receptor, B cells express a unique B cell receptor (BCR), in this case, an immobilized antibody molecule. The BCR recognizes and binds to only one particular antigen. A critical difference between B cells and T cells is how each cell "sees" an antigen. T cells recognize their cognate antigen in a processed form - as a peptide in the context of an MHC molecule, while B cells recognize antigens in their native form. Once a B cell encounters its cognate (or specific) antigen (and receives additional signals from a helper T cell(predominately Th2 type)), it further differentiates into an effector cell, known as a plasma cell.

Plasma cells are short lived cells (2-3 days) which secrete antibodies. These antibodies bind to antigens, making them easier targets for phagocytes, and trigger the complement cascade. About 10% of plasma cells will survive to become long-lived antigen specific memory B cells. Already primed to produce specific antibodies, these cells can be called upon to respond quickly if the same pathogen re-infects the host; while the host experiences few, if any, symptoms.

Antigen presentationAdaptive immunity relies on the capacity of immune cells to distinguish between the body's own cells and unwanted invaders.

Association between TCR and MHC class I or MHC class II

The host's cells express "self" antigens. These antigens are different from those on the surface of bacteria ("non-self" antigens) or on the surface of virally infected host cells (“missing-self”). The adaptive response is triggered by recognizing non-self and missing-self antigens.

With the exception of non-nucleated cells (including erythrocytes), all cells are capable of presenting antigen and of activating the adaptive response. Some cells are specially equipped to present antigen, and to prime naive T cells. Dendritic cells and B-cells (and to a lesser extent macrophages) are equipped with special immunostimulatory receptors that allow for enhanced activation of T cells, and are termed professional antigen presenting cells (APC).

Several T cells subgroups can be activated by professional APCs, and each type of T cell is specially equipped to deal with each unique toxin or bacterial and viral pathogen. The type of T cell activated, and the type of response generated depends, in part, on the context in which the APC first encountered the antigen.

Exogenous antigensDendritic cells engulf exogenous pathogens, such as bacteria, parasites or toxins in the tissues and then migrate, via chemotactic signals, to the T cell enriched lymph nodes. During migration, dendritic cells undergo a process of maturation in which they lose most of their ability to engulf other pathogens and develop an ability to communicate with T-cells. The dendritic cell usesenzymes to chop the pathogen into

smaller pieces, called antigens. In the lymph node, the dendritic cell will display these "non-self" antigens on its surface by coupling them to a "self"-receptor called the Major histocompatibility complex, or MHC (also known in humans as Human leukocyte antigen (HLA)). This MHC:antigen complex is recognized by T-cells passing through the lymph node. Exogenous antigens are usually displayed on MHC class II molecules, which activate CD4+ helper T-cells.

Antigen presentation stimulates T cells to become either "cytotoxic" CD8+ cells or "helper" CD4+ cells

Alternative adaptive immune system

Although the classical molecules of the adaptive immune system (e.g. antibodies and T cell receptors) exist only in jawed vertebrates, a distinct lymphocyte-derived molecule has been discovered in primitive jawless vertebrates, such as the lamprey and hagfish. These animals possess a large array of molecules called variable lymphocyte receptors (VLRs for short) that, like the antigen receptors of jawed vertebrates, are produced from only a small number (one or two) of genes. These molecules are believed to bind pathogenic antigens in a similar way to antibodies, and with the same degree of specificity.

Lymphoid organs and tissues(in adaptive immune

system)/Lymphatic system

The lymphatic system in animals is part of the immune system, made up of a network of conduits that

carry a clear fluid called lymph(from Latin lympha "water"). It also includes the lymphoid tissue and

lymphatic vessels through which the lymph travels in a one-way system in which lymph flows only toward

the heart. Lymphoid tissue is found in many organs, particularly the lymph nodes, and in thelymphoid

follicles associated with the digestive system such as the tonsils. The system also includes all the

structures dedicated to the circulation and production of lymphocytes, which includes

the spleen, thymus, bone marrow and the lymphoid tissue associated with thedigestive system. The

lymphatic system as we know it today was first described independently by Olaus Rudbeck and Thomas

Bartholin.

The blood does not directly come in contact with the parenchymal cells and tissues in the body, but

constituents of the blood first exit the microvascular exchange blood vessels to become interstitial fluid,

which comes into contact with the parenchymal cells of the body. Lymph is the fluid that is formed

when interstitial fluid enters the initial lymphatic vessels of the lymphatic system. The lymph is then

moved along the lymphatic vessel network by either intrinsic contractions of the lymphatic vessels or by

extrinsic compression of the lymphatic vessels via external tissue forces (e.g. the contractions of skeletal

muscles).

lymphatic_system

Function

The lymphatic system has multiple interrelated functions:

it is responsible for the removal of interstitial fluid from tissues

it absorbs and transports fatty acids and fats as chyle to the circulatory system

it transports immune cells to and from the lymph nodes in to the bone

The lymph transports antigen-presenting cells (APCs), such as dendritic cells, to the lymph nodes

where an immune response is stimulated.

The lymph also carries lymphocytes from the efferent lymphatics exiting the lymph nodes.

Clinical significance

The study of lymphatic drainage of various organs is important in diagnosis, prognosis, and treatment of

cancer. The lymphatic system, because of its physical proximity to many tissues of the body, is

responsible for carrying cancerous cells between the various parts of the body in a process

called metastasis. The intervening lymph nodes can trap the cancer cells. If they are not successful in

destroying the cancer cells the nodes may become sites of secondary tumors.

Diseases and other problems of the lymphatic system can cause swelling and other symptoms. Problems

with the system can impair the body's ability to fight infections.

Organization

The lymphatic system can be broadly divided into the conducting system and the lymphoid tissue.

The conducting system carries the lymph and consists of tubular vessels that include the lymph

capillaries, the lymph vessels, and the right and left thoracic ducts.

The lymphoid tissue is primarily involved in immune responses and consists of lymphocytes and

other white blood cells enmeshed in connective tissue through which the lymph passes. Regions of

the lymphoid tissue that are densely packed with lymphocytes are known as lymphoid follicles.

Lymphoid tissue can either be structurally well organized as lymph nodes or may consist of loosely

organized lymphoid follicles known as the mucosa-associated lymphoid tissue (MALT)

Lymphoid tissue

Lymphoid tissue associated with the lymphatic system is concerned with immune functions in defending

the body against the infections and spread of tumors. It consists of connective tissue with various types of

white blood cells enmeshed in it, most numerous being the lymphocytes.

The lymphoid tissue may be primary, secondary, or tertiary depending upon the stage of lymphocyte

development and maturation it is involved in. (The tertiary lymphoid tissue typically contains far fewer

lymphocytes, and assumes an immune role only when challenged with antigens that result

in inflammation. It achieves this by importing the lymphocytes from blood and lymph.)

Primary lymphoid organs

The central or primary lymphoid organs generate lymphocytes from immature progenitor cells.

These are bone marrow and thymus.These are called primary lymphoid argans T and B cells must first

undergo maturation in these tissues before migrating to the 2nd-dary lymphoid tissues such as the

spleen,lymph nodes,mucosa associated lymphoid tissues(MALT).

(a)Bone marrowBone marrow is a special, spongy, fatty tissue that houses stem cells, located inside a few large bones. These stem cells transform themselves into white and red blood cells and platelets, essential for immunity and circulation. Anemia, leukemia, and other lymphoma cancers can compromise the resilience of bone marrow.Bone marrow transplants are a growing treatment for these conditions of the lymphatic system that can't be otherwise cured.

Our skull, sternum, ribs, pelvis, and femur bones all contain bone marrow, but other smaller bones do not. Inside this special tissue, immature stems cells reside, along with extra iron. While they are undifferentiated, the stem cells wait until unhealthy, weakened, or damaged cells need to be replaced. A stem cell can turn itself into a platelet, a white blood cell like a T-cell, or a red blood cell. This is the only way such cells get replaced to keep our body healthy.

Bone marrow is the primary source of pluripotent stem ceels that gives rise to all hematopoietic cells includind lymphocydes.The bone marrow is the major organ for B cell maturation and give rice to precursor cells of the thymic lymphocytes.

Bone marrow is the flexible tissue found in the hollow interior of bones. In adults, marrow in large bones produces new blood cells. It constitutes 4% of the total body weight of humans, i.e. approximately 2.6 kg (5.7 lbs.) in adults.

Ceels in bone marrow

Marrow typesThere are two types of bone marrow: red marrow (consisting mainly of hematopoietic tissue) and yellow marrow (consisting mainly of fat cells).Red blood cells, platelets and most white blood cells arise in red marrow. Both types of bone marrow contain numerous blood vessels and capillaries.

At birth, all bone marrow is red. With age, more and more of it is converted to the yellow type. About half

of adult bone marrow is red. Red marrow is found mainly in the flat bones, such as the hip bone, breast

bone, skull, ribs, vertebrae and shoulder blades, and in the cancellous ("spongy") material at

the epiphyseal ends of the long bones such as the femur and humerus. Yellow marrow is found in the

hollow interior of the middle portion of long bones.

In cases of severe blood loss, the body can convert yellow marrow back to red marrow to increase blood

cell production.

Stroma

The stroma of the bone marrow is all tissue not directly involved in the primary function of hematopoiesis.

The yellow bone marrow belongs here, and makes the majority of the bone marrow stroma, in addition to

stromal cells located in the red bone marrow. Yellow bone marrow is found in the Medullary cavity.

Still, the stroma is indirectly involved in hematopoiesis, since it provides the hematopoietic

microenvironment that facilitates hematopoiesis by theparenchymal cells. For instance, they

generate colony stimulating factors, affecting hematopoiesis.

Cells that constitute the bone marrow stroma are:

fibroblasts (reticular connective tissue)

macrophages

adipocytes

osteoblasts

osteoclasts

endothelial cells forming the sinusoids

Macrophages contribute especially to red blood cell production. They deliver iron for hemoglobin-

production.

Bone marrow barrierThe blood vessels constitute a barrier, inhibiting immature blood cells from leaving the bone marrow. Only

mature blood cells contain the membrane proteins required to attach to and pass the blood

vessel endothelium.

Hematopoietic stem cells may also cross the bone marrow barrier, and may thus be harvested from

blood.

Stem cellsThe bone marrow stroma contain mesenchymal stem cells (also called marrow stromal cells). These cells are multipotent stem cells that can differentiate into a variety of cell types. Cell types that MSCs have been shown to differentiate into in vitro or in vivo include osteoblasts, chondrocytes, myocytes, adipocytes, and, as described lately, beta-pancreatic islets cells. They can also transdifferentiate into neuronal cells.

Mesenchyme is embryonic connective tissue that is derived from the mesoderm and that

differentiates into hematopoietic and connective tissue, whereas MSCs do not differentiate into

hematopoietic cells.

Stromal cells are connective tissue cells that form the supportive structure in which the functional

cells of the tissue reside. While this is an accurate description for one function of MSCs, the term fails

to convey the relatively recently-discovered roles of MSCs in the repair of tissue.

Because the cells, called MSCs by many labs today, can encompass multipotent cells derived

from other non-marrow tissues, such as adult muscle or the dental pulp of deciduousbaby teeth, yet

do not have the capacity to reconstitute an entire organ, the term Multipotent Stromal Cell has been

proposed as a better replacement. An extremely rich source for mesenchymal stem cells is the

developing tooth bud of the mandibular third molar. While considered multipotent, they may prove to

be pluripotent. The stem cells eventually form enamel, dentin,blood vessels, dental pulp, nervous

tissues, including a minimum of 29 different unique end organs. Because of extreme ease in

collection at 8–10 years of age before calcification and minimal to no morbidity they will probably

constitute a major source for personal banking, research and multiple therapies. These stem cells

have been shown capable of producing hepatocytes.

Types of stem cells

Bone marrow contains three types of stem cells:

Hematopoietic stem cells give rise to the three classes of blood cells that are found in the

circulation: white blood cells (leukocytes), red blood cells (erythrocytes), and platelets (thrombocytes).

Mesenchymal stem cells are found arrayed around the central sinus in the bone marrow. They

have the capability to differentiate intoosteoblasts, chondrocytes, myocytes, and many other types of

cells. They also function as "gatekeeper" cells of the bone marrow.

Endothelial stem cells

Differentiation capacityMSCs have a large capacity for self-renewal while maintaining their multipotency. Beyond that, there is

little that can be definitively said. The standard test to confirm multipotency is differentiation of the cells

into osteoblasts, adipocytes, and chondrocytes as well as myocytes and possibly neuron-like cells.

However, the degree to which the culture will differentiate varies among individuals and how

differentiation is induced, e.g., chemical vs. mechanical; and it is not clear whether this variation is due to

a different amount of "true" progenitor cells in the culture or variable differentiation capacities of

individuals' progenitors. The capacity of cells to proliferate and differentiate is known to decrease with the

age of the donor, as well as the time in culture. Likewise, whether this is due to a decrease in the number

of MSCs or a change to the existing MSCs is not known.

Immunomodulatory effectsNumerous studies have demonstrated that human MSC avoid allorecognition, interfere with dendritic

cell and T-cell function, and generate a local immunosuppressive microenvironment by

secreting cytokines. It has also been shown that the immunomodulatory function of human MSC is

enhanced when the cells are exposed to an inflammatory environment characterised by the presence of

elevated local interferon-gamma levels. Other studies contradict some of these findings, reflecting both

the highly heterogeneous nature of MSC isolates and the considerable differences between isolates

generated by the many different methods under development.

Clinical use

The mesenchymal stem cells can be activated and mobilized if needed. However, the efficiency is very

low. For instance, damage to muscles heals very slowly. However, if there were a method of activating

the mesenchymal stem cells, then such wounds would heal much faster.

Direct injection or placement of cells into a site in need of repair may be the preferred method of

treatment, as vascular delivery suffers from a "pulmonary first pass effect" where intravenous injected

cells are sequestered in the lungs. Clinical case reports in orthopedic applications have been published,

though the number of patients treated is small and these methods still lack rigorous study demonstrating

effectiveness. Wakitani has published a small case

series of nine defects in five knees involving surgical transplantation of mesenchymal stem cells with

coverage of the treated chondral defects.

Diseases involving the bone marrow

The normal bone marrow architecture can be displaced by malignancies or infections such

as tuberculosis, leading to a decrease in the production of blood cells and blood platelets. In addition,

cancers of the hematologic progenitor cells in the bone marrow can arise; these are the leukemias.

To diagnose diseases involving the bone marrow, a bone marrow aspiration is sometimes performed.

This typically involves using a hollow needle to acquire a sample of red bone marrow from the crest of the

ilium under general or local anesthesia. The average number of cells in a leg bone is about

440,000,000,000 (440x109).

Exposure to radiation or chemotherapy will kill many of the rapidly dividing cells of the bone marrow and

will therefore result in a depressed immune system. Many of the symptoms ofradiation sickness are due

to damage to the bone marrow cells.

Donation and transplantation of bone marrow

It is possible to take hematopoietic stem cells from one person and then infuse them into another person

(Allogenic) or into the same person at a later time (Autologous). If donor and recipient are compatible,

these infused cells will then travel to the bone marrow and initiate blood cell production.

Transplantation from one person to another is performed in severe cases of disease of the bone marrow.

The patient's marrow is first killed off with drugs or radiation, and then the new stem cells are introduced.

Before radiation therapy or chemotherapy in cases of cancer, some of the patient's hematopoietic stem

cells are sometimes harvested and later infused back when the therapy is finished to restore the immune

system.

Bone marrow as a food

Many cultures utilize bone marrow as a food. The Vietnamese prize beef bone as the soup base for their

national staple phở; Alaskan Natives eat the bone marrow of caribou and moose; Indians use slow-

cooked marrow as the core ingredient of the Indian dish Nalli Nihari; Mexicans use beef bone marrow

from leg bones, called tuétano, which is cooked and served as filling for tacos or tostadas; it is also

considered to be the highlight of the Italian dish ossobuco (braised veal shanks); beef marrowbones are

often included in the French pot-au-feu broth, the cooked marrow being traditionally eaten on toasted

bread with sprinkled coarse sea salt, in Iranian cuisine lamb shanks are usually broken before cooking to

allow diners to suck out and eat the marrow when the dish is served. Though once used in various

preparations, including pemmican, bone marrow has fallen out of favor as a food in the United States,

though bone marrow is used in many gourmet restaurants and is popular among foodies. In the

Philippines, the soup "Bulalo" is made primarily of beef stock and marrow bones, seasoned with

vegetables and boiled meat. In Hungary tibia is a main ingredient of beef soup; the bone is chopped into

short (10-15cm) pieces and the ends are covered with salt to prevent the marrow from leaving the bone

while cooking. Upon serving the soup the marrow is usually spread on toast.

Diners in the 18th century used a marrow scoop (or marrow spoon), often of silver and with a long thin

bowl, as a table implement for removing marrow from a bone.

Some anthropologists believe that early humans were scavengers rather than hunters. Marrow would

then have been a major protein source for tool-using hominids, who were able to crack open the bones of

carcasses left by top predators such as lions.

(b)ThymusThe thymus is a specialized organ in the immune system. The functions of the thymus are the production

of T-lymphocytes (T cells), which are critical cells of the adaptive immune system, and the production and

secretion of thymosins, hormones which control T-lymphocyte activities and various other aspects of the

immune system. The thymus is composed of two identical lobes and is located anatomically in the

anterior superior mediastinum, in front of the heart and behind the sternum.

Histologically, the thymus can be divided into a central medulla and a peripheral cortex which is

surrounded by an outer capsule. The cortex and medulla play different roles in the development of T-

cells. Cells in the thymus can be divided into thymic stromal cells and cells of hematopoietic origin

(derived from bone marrow resident hematopoietic stem cells). Developing T-cells are referred to

as thymocytesand are of hematopoietic origin. Stromal cells include thymic cortical epithelial cells, thymic

medullary epithelial cells, and dendritic cells.

The thymus provides an inductive environment for development of T-lymphocytes from hematopoietic

progenitor cells. In addition, thymic stromal cells allow for the selection of a functional and self-tolerant T-

cell repertoire. Therefore, one of the most important roles of the thymus is the induction of central

tolerance.

The thymus is largest and most active during the neonatal and pre-adolescent periods. By the early

teens, the thymus begins to atrophyand thymic stroma is replaced by adipose (fat) tissue. Nevertheless,

residual T lymphopoiesis continues throughout adult life.

The two main components of the thymus, the lymphoid thymocytes and the thymic epithelial cells, have distinct

developmental origins. The thymic epithelium is the first to develop, and appears in the form of two flask-shape

endodermal diverticula, which arise, one on either side, from the third branchial pouch (pharyngeal pouch), and extend

lateralward and backward into the surrounding mesoderm and neural crest-derived mesenchyme in front of the

ventral aorta.

Structure

Each lateral lobe is composed of numerous lobules held together by delicate areolar tissue; the entire

organ being enclosed in an investingcapsule of a similar but denser structure. The primary lobules vary in

size from that of a pin's head to that of a small pea, and are made up of a number of

small nodules or follicles.

The follicles are irregular in shape and are more or less fused together, especially toward the interior of

the organ. Each follicle is from 1 to 2 mm in diameter and consists of a medullary and a cortical portion,

and these differ in many essential particulars from each other.

Cortex

The cortical portion is mainly composed of lymphoid cells, supported by a network of finely-

branched epithelial reticular cells, which is continuous with a similar network in the medullary portion. This

network forms an adventitia to the blood vessels.

The cortex is the location of the earliest events in thymocyte development, where T cell receptor gene

rearrangement and positive selection takes place.

Medulla

In the medullary portion, the reticulum is coarser than in the cortex, the lymphoid cells are relatively fewer

in number, and there are found peculiar nest-like bodies, the concentric corpuscles of Hassall. These

concentric corpuscles are composed of a central mass, consisting of one or more granular cells, and of a

capsule formed of epithelioid cells. They are the remains of the epithelial tubes, which grow out from the

third branchial pouches of the embryo to form the thymus. Each follicle is surrounded by

a vascular plexus, from which vessels pass into the interior, and radiate from the periphery toward the

center, forming a second zone just within the margin of the medullary portion. In the center of the

medullary portion there are very few vessels, and they are of minute size.

The medulla is the location of the latter events in thymocyte development. Thymocytes that reach the

medulla have already successfully undergone T cell receptor gene rearrangement and positive selection,

and have been exposed to a limited degree of negative selection. The medulla is specialised to allow

thymocytes to undergo additional rounds of negative selection to remove auto-reactive T-cells from the

mature repertoire. The gene AIRE is expressed by the thymic medullary epithelium, and drives the

transcription of organ-specific genes such as insulin to allow maturing thymocytes to be exposed to a

more complex set of self-antigens than is present in the cortex.

Vasculature

The arteries supplying the thymus are derived from the internal mammary, and from the superior

thyroid and inferior thyroids.

The veins end in the left brachiocephalic vein (innominate vein) , and in the thyroid veins.

The nerves are exceedingly minute; they are derived from the vagi and sympathetic nervous system.

Branches from the descendens hypoglossi and phrenic reach the investing capsule, but do not penetrate

into the substance of the organ.

Function

In the two thymic lobes, hematopoietic precursors from the bone-marrow, referred to as thymocytes,

mature into T-cells. Once mature, T-cells emigrate from the thymus and constitute the peripheral T-cell

repertoire responsible for directing many facets of the adaptive immune system. Loss of the thymus at an

early age through genetic mutation (as in DiGeorge Syndrome) results in severe immunodeficiency and a

high susceptibility to infection.

The stock of T-lymphocytes is built up in early life, so the function of the thymus is diminished in adults. It

is largely degenerated in elderly adults and is barely identifiable, consisting mostly of fatty tissue, but it

continues to function as an endocrine gland important in stimulating the immune system. Involution of the

thymus has been linked to loss of immune function in the elderly, susceptibility to infection and to cancer.

The ability of T-cells to recognize foreign antigens is mediated by the T cell receptor. The T cell

receptor undergoes genetic rearrangement during thymocyte maturation, resulting in each T-cell bearing

a unique T-cell receptor, specific to a limited set of peptide:MHC combinations. The random nature of the

genetic rearrangement results in a requirement of central tolerance mechanisms to remove or inactivate

those T cells which bear a T cell receptor with the ability to recognise self-peptides.

Disease Associations

Immunodeficiency

As the thymus is the organ of T-cell development, any congenital defect in thymic genesis or a defect

in thymocyte development can lead to a profound T cell primary immunodeficiency. Defects that affect

both the T cell and B cell lymphocyte lineages result in Severe Combined Immunodeficiency Syndrome

(SCID). Acquired T cell deficiencies can also affect thymocytedevelopment in the thymus.

DiGeorge Syndrome

DiGeorge Syndrome is a genetic disorder caused by the deletion of a small section of chromosome 22.

This results in a midline congenital defect including thymic aplasia, or congenital deficiency of a thymus.

Patients may present with a profound immunodeficiency disease, due to the lack of T cells. No other

immune cell lineages are affected by the congenital absence of the thymus. DiGeorge Syndrome is the

most common congenital cause of thymic aplasia in humans. In mice, the nude mouse strain are

congenitally thymic deficent. These mice are an important model of primary T cell deficiency.

SCID

Severe combined immunodeficiency syndromes (SCID) are group of rare congenital genetic diseases that

result in combined T lymphocyte and B lymphocyte deficencies. These syndromes are cause by

defective hematopoietic progenitor cells which are the precursors of both B- and T-cells. This results in a

severe reduction in developing thymocytes in the thymus and consequently thymic atrophy. A number of

genetic defects can cause SCID, including IL-7 receptor deficiency, common gamma chain deficiency,

and Recombination activating gene deficiency.

HIV / AIDS

The HIV virus causes an acquired T-cell immunodeficiency syndrome (AIDS) by specifically killing CD4+

T-cells. Whereas, the major effect of the virus is on mature peripheral T-cells, the HIV virus can also infect

developing thymocytes in the thymus, most of which express CD4.

Autoimmune Disease

Autoimmune diseases are caused by a hyperactive immune system that instead of attacking

foreign pathogens reacts against the host organism (self) causing disease. One of the primary functions

of the thymus is to prevent autoimmunity through the process of central tolerance, immunologic tolerance

to self antigens.

APECED

Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy (APECED) is an extremely rare

genetic autoimmune syndrome. However, this disease highlights the importance of the thymus in

prevention of autoimmunity. This disease is caused by deficiency of the Autoimmune Regulator (AIRE)

gene in the thymus. AIRE allows for the ectopic expression of tissue-specific proteins in the thymus

medulla, such as proteins that would normally only be expressed in the eye or pancreas. This expression

in the thymus, allows for the deletion of autoreactive thymocytes by exposing them to self-antigens during

their development, a mechansism of central tolerance. Patients with APECED develop an autoimmune

disease that affects multiple endocrine tissues.

Myasthenia gravis

Myasthenia gravis is an autoimmune disease caused by antibodies that block acetylcholine receptors.

Myasthenia gravis is often associated with thymic hypertrophy. Thymectomy may be necessary to treat

the disease.

Cancer

Two primary forms of tumours originate in the thymus.

Thymomas

Tumours originating from the thymic epithelial cells are called thymomas, and are found in about 10-15%

of patients with myasthenia gravis. Symptoms are sometimes confused withbronchitis or a strong cough

because the tumour presses on the recurrent laryngeal nerve. All thymomas are potentially cancerous,

but they can vary a great deal. Some grow very slowly. Others grow rapidly and can spread to

surrounding tissues. Treatment of thymomas often requires surgery to remove the entire thymus.

Lymphomas

Tumours originating from the thymocytes are called

thymic lymphomas. Lymphomas or leukemias of thymocyte origin are classified as Precursor T acute

lymphoblastic leukemia/lymphoma (T-ALL).

People with an enlarged thymus, particularly children, were treated with intense radiation in the years

before 1950. There is an elevated incidence of thyroid cancer and leukemia in treated individuals.

Second thymus

The thymus is also present in most vertebrates, with similar structure and function as the human thymus.

Some animals have multiple secondary (smaller) thymi in the neck; this phenomenon has been reported

for mice  and also occurs in 5 out of 6 human fetuses. As in humans, the Guinea pig's thymus naturally

atrophies as the animal reaches adulthood, but in the athymic hairless guinea pig (which arose from a

spontaneous laboratory mutation) possessed no thymic tissue whatsoever, and the organ cavity is

replaced with cysticspaces.

Animal thymic tissue sold in a butcher shop or at a meat counter is known as sweetbread.

In animalsThymus is present in mammals, where it plays the same immunological function as in human beings.

Secondary lymphoid organs

Secondary or peripheral lymphoid organs maintain mature naive lymphocytes and initiate an adaptive

immune response. The peripheral lymphoid organs are the sites of lymphocyte activation by antigen.

Activation leads to clonal expansion and affinity maturation. Mature Lymphocytes recirculate between the

blood and the peripheral lymphoid organs until they encounter their specific antigen.

Secondary lymphoid tissue provides the environment for the foreign or altered native molecules

(antigens) to interact with the lymphocytes. It is exemplified by the lymph nodes, and the lymphoid follicles

in tonsils, Peyer's patches, spleen, adenoids, skin, etc. that are associated with the mucosa-associated

lymphoid tissue (MALT).

Lymph nodeA lymph node (pronounced /ˈlɪmf.noʊd/) is a small ball-shaped organ of the immune system, distributed

widely throughout the body and linked by lymphatic vessels. Lymph nodes are garrisons of B, T, and

other immune cells. Lymph nodes are found all through the body, and act as filters or traps for foreign

particles. They are important in the proper functioning of the immune system.

Lymph nodes also have clinical significance. They become inflamed or enlarged in various conditions,

which may range from trivial, such as a throat infection, to life-threatening such as cancers. In the latter,

the condition of lymph nodes is so significant that it is used forcancer staging, which decides the

treatment to be employed, and for determining the prognosis.

Lymph nodes can also be diagnosed by biopsy whenever they are inflamed. Certain diseases affect

lymph nodes with characteristic consistency and location.

Function

Pathogens, or germs, can set up infections anywhere in the body. However, lymphocytes, a type of white

blood cell, will meet the antigens, or proteins, in the peripheral lymphoid organs, which includes lymph

nodes. The antigens are displayed by specialized cells in the lymph nodes. Naive lymphocytes (meaning

the cells have not encountered an antigen yet) enter the node from the bloodstream, through specialized

capillary venules. After the lymphocytes specialize they will exit the lymph node through the efferent

lymphatic vessel with the rest of thelymph. The lymphocytes continuously recirculate the peripheral

lymphoid organs and the state of the lymph nodes depends on infection. During an infection, the lymph

nodes can expand due to intense B-cell proliferation in the germinal centers, a condition commonly

referred to as "swollen glands".

Structure

The lymph node is surrounded by a fibrous capsule, and inside the lymph node the

fibrous capsule extends to form trabeculae. The substance of the lymph node is divided into the

outer cortex and the inner medulla surrounded by the former all around except for at the hilum, where the

medulla comes in direct contact with the surface.

Thin reticular fibers, elastin and reticular fibers form a supporting meshwork called reticular network (RN)

inside the node, within which the white blood cells (WBCs), most prominently, lymphocytes are tightly

packed as follicles in the cortex. Elsewhere, there are only occasional WBCs. The RN provides not just

the structural support, but also provide surface for adhesion of thedendritic cells, macrophages and

lymphocytes. It allows for exchange of material with blood through the high endothelial venulesand

provides the growth and regulatory factors necessary for activation and maturation of immune cells.

The number and composition of follicles can change especially when challenged by an antigen, when

they develop a germinal center.

A lymph sinus is a channel within the lymph node lined by the endothelial cells along with fibroblastic

reticular cells and allows for smooth flow of lymph through them. Thus, subcapsular sinus is a sinus

immediately deep to the capsule, and its endothelium is continuous with that of the afferent lymph vessel.

It is also continuous with similar sinuses flanking the trabeculae and within the cortex (cortical sinuses).

The cortical sinuses and that flanking the trabeculae drain into the medullary sinuses, from where the

lymph flows into the efferent lymph vessel.

Multiple afferent lymph vessels that branch and network extensively within the capsule bring lymph into

the lymph node. This lymph enters the subcapsular sinus. The innermost lining of the afferent lymph

vessels is continuous with the cells lining the lymph sinuses. The lymph gets slowly filtered through the

substance of the lymph node and ultimately reaches the medulla. In its course it encounters the

lymphocytes and may lead to their activation as a part of adaptive immune response.

The concave side of the lymph node is called the hilum. The efferent attaches to the hilum by a relatively

dense reticulum present there, and carries the lymph out of the lymph node.

Schematic diagram of lymph node showing the flow of lymph through the lymph sinuses. Note: Outflowing lymph has

more lymphocytes

Cortex

In the cortex, the subcapsular sinus drains to trabecular sinuses, and then the lymph flows into the

"medullary sinuses".

The outer cortex consists mainly of the B cells arranged as follicles, which may develop a germinal center

when challenged with an antigen, and the deeper cortex mainly consisting of the T cells. There is a zone

known as the subcortical zone where T-cells (or cells that are mainly red) mainly interact with dendritic

cells, and where the reticular network is dense.

Medulla

There are two named structures in the medulla:

The medullary cords are cords of lymphatic tissue, and include plasma cells and B cells

The medullary sinuses (or sinusoids) are vessel-like spaces separating the medullary cords. The

Lymph flows into the medullary sinuses from cortical sinuses, and into efferent lymphatic vessels.

Medullary sinuses contain histiocytes (immobile macrophages) and reticular cells.

Shape and size

Human lymph nodes are bean-shaped and range in size from a few millimeters to about 1–2 cm in their

normal state. They may become enlarged due to a tumor or infection. Lymphocytes, also known as white

blood cells are located within honeycomb structures of the lymph nodes. Lymph nodes are enlarged when

the body is infected, primarily because there is an elevated rate of trafficking of lymphocytes into the node

from the blood, exceeding the rate of outflow from the node, and secondarily as a result of the activation

and proliferation of antigen-specific T and B cells (clonal expansion). In some cases they may feel

enlarged because of a previous infection; although one may be healthy, one may still feel them residually

enlarged.

Lymphatic circulation

Lymph circulates to the lymph node via afferent lymphatic vessels and drains into the node just beneath

the capsule in a space called the subcapsular sinus. The subcapsular sinus drains into trabecular sinuses

and finally into medullary sinuses. The sinus space is criss-crossed by

the pseudopods of macrophages which act to trap foreign particles and filter the lymph. The medullary

sinuses converge at the hilum and lymph then leaves the lymph node via the efferent lymphatic

vessel towards either a more central lymph node or ultimately for drainage into a central venous

subclavian blood vessel, most via the postcapillary venules, and cross its wall by the process

of diapedesis.

The B cells migrate to the nodular cortex and medulla.

The T cells migrate to the deep cortex ("paracortex").

When a lymphocyte recognizes an antigen, B cells become activated and migrate to germinal centers (by

definition, a "secondary nodule" has a germinal center, while a "primary nodule" does not). When

antibody-producing plasma cells are formed, they migrate to the medullary cords. Stimulation of the

lymphocytes by antigens can accelerate the migration process to about 10 times normal, resulting in

characteristic swelling of the lymph nodes.

The spleen and tonsils are large lymphoid organs that serve similar functions to lymph nodes, though the

spleen filters blood cells rather than lymph.

Distribution

Regional lymph tissue

Humans have approximately 500-600 lymph nodes distributed throughout the body, with clusters found in

the underarms, groin, neck, chest, and abdomen.

Lymph nodes of the head and neck

Cervical lymph nodes

Anterior cervical: These nodes, both superficial and deep, lie above and beneath

the sternocleidomastoid muscles. They drain the internal structures of the throat as well as part of

the posterior pharynx, tonsils, and thyroid gland.

Posterior cervical: These nodes extend in a line posterior to the sternocleidomastoids but

in front of the trapezius, from the level of theMastoid portion of the temporal bone to the clavicle.

They are frequently enlarged during upper respiratory infections.

Tonsillar (sub mandibular): These nodes are located just below the angle of the mandible. They

drain the tonsillar and posterior pharyngeal regions.

Sub-mandibular: These nodes run along the underside of the jaw on either side. They drain the

structures in the floor of the mouth and the maxillary anterior, bicuspid and 1st and 2nd molars. They

also drain all of the mandibular teeth except the central incisors.

Retropharyngeal: Drains lymph from the soft palate and the 3rd molars.

Sub-mental: These nodes are just below the chin. They drain the central incisors and midline of

lower lip and tip of the tongue.

Supraclavicular lymph nodes: These nodes are in the hollow above the clavicle, just lateral to

where it joins the sternum. They drain a part of the thoracic cavity and abdomen. Virchow's node is a

left supraclavicular lymph node which receives the lymph drainage from most of the body (especially

the abdomen) via the thoracic duct and is thus an early site of metastasis for various malignancies.

Lymph nodes of the thorax

Lymph nodes of the lungs: The lymph is drained from the lung tissue

through subsegmental, segmental, lobar and interlobar lymph nodes to the hilar lymph nodes, which are

located around the hilum (the pedicle, which attaches the lung to the mediastinal structures, containing

the pulmonary artery, the pulmonary veins, the main bronchus for each side, some vegetative nerves and the

lymphatics) of each lung. The lymph flows subsequently to the mediastinal lymph nodes.

Mediastinal lymph nodes: They consist of several lymph node groups, especially along the trachea (5

groups), along the esophagus and between the lung and the diaphragm. In the mediastinal lymph nodes arises

lymphatic ducts, which draines the lymph to the left subclavian vein (to the venous angle in the confluence of the

subclavian and deep jugular veins).

The mediastinal lymph nodes along the esophagus are in tight connection with the abdominal lymph nodes along

the esophagus and the stomach. That fact facilitates spreading of tumors cells through these lymphatics in cases of

cancers of the stomach and particularly of the esophagus. Through the mediastinum, the main lymphatic drainage

from the abdominal organs goes via the thoracic duct (ductus thoracicus), which drains majority of the lymph from the

abdomen to the above mentioned left venous angle.

Lymph nodes of the arm

These drain the whole of the arm, and are divided into two groups, superficial and deep. The superficial

nodes are supplied by lymphatics which are present throughout the arm, but are particularly rich on the

palm and flexor aspects of the digits.

Superficial lymph glands of the arm:

Supratrochlear glands: Situated above the medial epicondyle of the humerus, medial to

the basilic vein, they drain the C7 and C8 dermatomes.

Deltoideopectoral glands: Situated between the pectoralis

major and deltoid muscles inferior to the clavicle.

Deep lymph glands of the arm: These comprise the axillary glands, which are 20-30 individual

glands and can be subdivided into:

Lateral glands

Anterior or pectoral glands

Posterior or subscapular glands

Central or intermediate glands

Medial or subclavicular glands

Lower limbs

Superficial inguinal lymph nodes

Deep inguinal lymph nodes

Popliteal lymph nodes

What is immunological memory?When B cells and T cells are activated some will become memory cells. Throughout the lifetime of an animal these memory cells form a database of effective B and T lymphocytes. Upon interaction with a previously encountered antigen, the appropriate memory cells are selected and activated. In this manner, the second and subsequent exposures to an antigen produce a stronger and faster immune response. This is "adaptive" because the body's immune system prepares itself for future challenges. Immunological memory can either be in the form ofpassive short-term memory or active long-term memory.

Passive memory

Passive memory is usually short-term, lasting between a few days and several months. Newborn infants have had no prior exposure to microbes and are particularly vulnerable to infection. Several layers of passive protection are provided by the mother. In utero, maternal IgG is transported directly across the placenta, so that at birth, human babies have high levels of antibodies, with the same range of antigen specificities as their mother. Breast milk contains antibodies that are transferred to the gut of the infant, protecting against bacterial infections, until the newborn can synthesize its own antibodies.

This is passive immunity because the fetus does not actually make any memory cells or antibodies, it only borrows them. Short-term passive immunity can also be transferred artificially from one individual to another via antibody-rich serum.

Active Memory

Active immunity is generally long-term and can be acquired by infection followed by B cells and T cells activation, or artificially acquired by vaccines, in a process called immunization.

What is immunization ?Historically, infectious disease has been the leading cause of death in the human population. Over the last century, two important factors have been developed to combat their spread;sanitation and immunization. Immunization (commonly referred to as vaccination) is the deliberate induction of an immune response, and represents the single most effective manipulation of the immune system that scientists have developed. Immunizations are successful because they utilize the immune system's natural specificity as well as its inducibility.

The principle behind immunization is to introduce an antigen, derived from a disease causing organism, that stimulates the immune system to develop protective immunity against that organism, but which does not itself cause the pathogenic effects of that organism. An antigen (short for antibody generator), is defined as any substance that binds to a specific antibody and elicits an adaptive immune response.

Most viral vaccines are based on live attenuated viruses, while many bacterial vaccines are based on acellular components of micro-organisms, including harmless toxin components.Many antigens derived from acellular vaccines do not strongly induce an adaptive response, and most bacterial vaccines require the addition of adjuvants that activate the antigen presenting cells of the innate immune system to enhance immunogenicity

Adaptive immunity during pregnancyThe cornerstone of the immune system is the recognition of "self" versus "non-self". Therefore, the mechanisms which protect the human fetus (which is considered "non-self") from attack by the immune system, are particularly interesting. Although no comprehensive explanation has emerged to explain this mysterious, and often repeated, lack of rejection, two classical reasons may explain how the fetus is tolerated. The first is that the fetus occupies a portion of the body protected by a non-immunological barrier, the uterus, which the immune system does not routinely patrol. The second is that the fetus itself may promote local immunosuppression in the mother, perhaps by a process of active nutrient depletion. A more modern explanation for this induction of tolerance is that specific glycoproteins expressed in the uterus during pregnancy suppress the uterine immune response (see eu-FEDS).

During pregnancy in viviparous mammals (all mammals except Monotremes), endogenous retroviruses are activated and produced in high quantities during the implantation of the embryo. They are currently known to possess immunosuppressive properties, suggesting a role in protecting the embryo from its mother's immune system. Also viral fusion proteins apparently cause the formation of the placental syncytium in order to limit the exchange of migratory cells between the developing embryo and the body of the mother (something an epitheliumwill not do sufficiently, as certain blood cells are specialized to be able to insert themselves between adjacent epithelial cells). The immunodepressive action was the initial normal behavior of the virus, similar to HIV, the fusion proteins were a way to spread the infection to other cells by simply merging them with the infected one (HIV does this too). It is believed that the ancestors of modern viviparous mammals evolved after an infection by this virus, enabling the fetus to survive the immune system of the mother.

The human genome project found several thousand ERVs classified into 24 families

What’s the meaning of Cell mediated immunity?

Cell-mediated immunity is an immune response that does not involve antibodies or complement but

rather involves the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-

lymphocytes, and the release of various cytokines in response to an antigen. Historically, the immune

system was separated into two branches: humoral immunity, for which the protective function of

immunization could be found in the humor (cell-free bodily fluid or serum) and cellular immunity, for

which the protective function of immunization was associated with cells. CD4 cells or helper T cells

provide protection against different pathogens.

Cellular immunity protects the body by:

1. activating antigen-specific cytotoxic T-lymphocytes that are able to induce apoptosis in body cells

displaying epitopes of foreign antigen on their surface, such as virus-infected cells, cells with

intracellular bacteria, and cancer cells displaying tumor antigens;

2. activating macrophages and natural killer cells, enabling them to destroy intracellular pathogens;

and

3. stimulating cells to secrete a variety of cytokines that influence the function of other cells involved

in adaptive immune responses and innate immune responses.

Cell-mediated immunity is directed primarily at microbes that survive in phagocytes and microbes that

infect non-phagocytic cells. It is most effective in removing virus-infected cells, but also participates in

defending against fungi, protozoans, cancers, and intracellular bacteria. It also plays a major role

in transplant rejection.

What’s the meaning of Humoral immunity?

The Humoral Immune Response (HIR) is the aspect of immunity that is mediated by

secreted antibodies (as opposed to cell-mediated immunity, which involves T lymphocytes) produced in

the cells of the B lymphocyte lineage (B cell). B Cells (with co-stimulation) transform into plasma cells

which secrete antibodies. The co-stimulation of the B cell can come from another antigen presenting cell,

like a dendritic cell. This entire process is aided by CD4+ T-helper 2 cells, which provide co-stimulation.

Secreted antibodies bind to antigens on the surfaces of invading microbes (such as viruses or bacteria),

which flags them for destruction. Humoral immunity is so named because it involves substances found in

the humours, or body fluids.

The study of the molecular and cellular components that comprise the immune system, including their

function and interaction, is the central science of immunology. The immune system is divided into a more

primitive innate immune system, and acquired or adaptive immune system of vertebrates, each of which

contains humoral and cellular components.

Humoral immunity refers to antibody production and the accessory processes that accompany it,

including: Th2 activation and cytokine production, germinal center formation and isotypeswitching, affinity

maturation and memory cell generation. It also refers to the effector functions of antibody, which include

pathogen and toxin neutralization, classical complementactivation, and opsonin promotion

of phagocytosis and pathogen elimination

Major discoveries in the study of humoral immunity

Substance Activity Discovery

Alexin(s)Complement

Soluble components in the serumthat are capable of killing microorganisms

Buchner (1890),Ehrlich (1892)

AntitoxinsSubstances in the serum that can neutralizethe activity of toxins, enabling passive immunization

von Behring and Kitasato (1890)

BacteriolysinsSerum substances that work with thecomplement proteins to induce bacterial lysis

Richard Pfeiffer (1895)

Bacterial agglutinins& precipitins

Serum substances that agglutinate bacteriaand precipitate bacterial toxins

von Gruber and Durham (1896),

Kraus (1897)

HemolysinsSerum substances that work with complementto lyse red blood cells

Belfanti and Carbone (1898)Jules Bordet (1899)

Opsoninsserum substances that coat the outer membraneof foreign substances and enhance the rate ofphagocytosis by macrophages

Wright and Douglas (1903)

Antibodyformation (1900), antigen-antibody bindinghypothesis (1938), produced by B cells (1948),structure (1972), immunoglobulin genes (1976)

Founder: P Ehrlich

2nd Unit

Antibodies

Antibodies are immune system-related proteins called immunoglobulins. Each antibody consists of four polypeptides– two heavy chains and two light chains joined to form a "Y" shaped molecule.

The amino acid sequence in the tips of the "Y" varies greatly among different antibodies. This variable region, composed of 110-130 amino acids, give the antibody its specificity for binding antigen. The variable region includes the ends of the light and heavy chains. Treating the antibody with a protease can cleave this region, producing Fab or fragment antigen binding that include the variable ends of an antibody. Material used for the studies shown below originated from Fab.

Antibodies (also known as immunoglobulins, abbreviated Ig) are gamma globulin proteins that are found in blood or other bodily fluids of vertebrates, and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses. They are typically made of basic structural units—each with two large heavy chains and two small light chains—to form, for example,monomers with one unit, dimers with two units or pentamers with five units. Antibodies are produced by a kind of white blood cell called a plasma cell. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals, which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter.

Though the general structure of all antibodies is very similar, a small region at the tip of the protein is

extremely variable, allowing millions of antibodies with slightly different tip structures, or antigen binding

sites, to exist. This region is known as the hypervariable region. Each of these variants can bind to a

different target, known as an antigen. This huge diversity of antibodies allows the immune system to

recognize an equally wide variety of antigens. The unique part of the antigen recognized by an antibody is

called the epitope. These epitopes bind with their antibody in a highly specific interaction, called induced

fit, that allows antibodies to identify and bind only their unique antigen in the midst of the millions of

different molecules that make up an organism. Recognition of an antigen by an antibodytags it for attack

by other parts of the immune system. Antibodies can also neutralize targets directly by, for example,

binding to a part of a pathogen that it needs to cause an infection.

The large and diverse population of antibodies is generated by random combinations of a set

of gene segments that encode different antigen binding sites (or paratopes), followed by

random mutations in this area of the antibody gene, which create further diversity.Antibody genes also re-

organize in a process called class switching that changes the base of the heavy chain to another, creating

a different isotype of the antibody that retains the antigen specific variable region. This allows a single

antibody to be used by several different parts of the immune system. Production of antibodies is the main

function of the humoral immune system

Each antibody binds to a specific antigen; an interaction similar to a lock and key.

Basic structure of immunoglobulins/antigenDifferent immunoglobulins can differ structurally, they all are built from the same basic units.

A. Heavy and Light Chains All immunoglobulins have a four chain structure as their basic unit. They are composed of two identical light chains (23kD) and two identical heavy chains (50-70kD)

Heavy chain

There are five types of mammalian Ig heavy chain denoted by the Greek letters: α, δ, ε, γ, and μ. The

type of heavy chain present defines theclass of antibody; these chains are found in IgA, IgD, IgE, IgG,

and IgM antibodies, respectively. Distinct heavy chains differ in size and composition; α and γ contain

approximately 450 amino acids, while μ and ε have approximately 550 amino acids.

Each heavy chain has two regions, the constant region and the variable region. The constant region is

identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains

γ, α and δ have a constant region composed of three tandem (in a line) Igdomains, and a hinge region for

added flexibility; heavy chains μ and ε have a constant region composed of four immunoglobulin

domains.The variable region of the heavy chain differs in antibodies produced by different B cells, but is

the same for all antibodies produced by a single B cell or B cell clone. The variable region of each heavy

chain is approximately 110 amino acids long and is composed of a single Ig domain.

Light chain

In mammals there are two types of immunoglobulin light chain, which are called lambda (λ) and kappa

(κ). A light chain has two successive domains: one constant domain and one variable domain. The

approximate length of a light chain is 211 to 217 amino acids. Each antibody contains two light chains that

are always identical; only one type of light chain, κ or λ, is present per antibody in mammals. Other types

of light chains, such as the iota (ι) chain, are found in

lower vertebrates like Chondrichthyes and Teleostei.

B. Disulfide bonds

1. Inter-chain disulfide bonds - The heavy and light chains and the two heavy chains are held together by inter-chain disulfide bonds and by non-covalent interactions The number of inter-chain disulfide bonds varies among different immunoglobulin molecules.

2. Intra-chain disulfide binds - Within each of the polypeptide chains there are also intra-chain disulfide bonds.

C. Variable (V) and Constant (C) Regions When the amino acid sequences of many different heavy chains and light chains were compared, it became clear that both the heavy and light chain could be divided into two regions based on variability in the amino acid sequences. These are the:

1. Light Chain - VL (110 amino acids) and CL (110 amino acids)

2. Heavy Chain - VH (110 amino acids) and CH (330-440 amino acids)

D. Hinge Region This is the region at which the arms of the antibody molecule forms a Y. It is called the hinge region because there is some flexibility in the molecule at this point.

E. Domains Three dimensional  images of the immunoglobulin molecule show that it is not straight. Rather, it is folded into globular regions each of which contains an intra-chain disulfide bond. These regions are called domains.

1. Light Chain Domains - VL and CL

2. Heavy Chain Domains - VH, CH1 - CH3 (or CH4)

F. Oligosaccharides Carbohydrates are attached to the CH2 domain in most immunoglobulins. However, in some cases carbohydrates may also be attached at other locations.

 

1. Fab region2. Fc region3. Heavy chain with one variable (VH) domain followed by a constant domain (CH1), a hinge region, and two more constant (CH2 and CH3) domains.4. Light chain with one variable (VL) and one constant (CL) domain5. Antigen binding site (paratope)6. Hinge regions.

Structure of the variable region

A. Hypervariable (HVR) or complementarity determining regions (CDR)

Comparisons of the amino acid sequences of the variable regions of immunoglobulins show that most of the variability resides in three regions called the hypervariable regions or the complementarity determining regions. Antibodies with different specificities (i.e. different combining sites) have different complementarity determining regions while antibodies of the exact same specificity have identical complementarity determining regions (i.e. CDR is the antibody combining site). Complementarity determining regions are found in both the H and the L chains.

B. Framework regions

The regions between the complementarity determining regions in the variable region are called the framework regions. Based on similarities and differences in the framework regions the immunoglobulin heavy and light chain variable regions can be divided into groups and subgroups. These represent the products of different variable region genes.

IMMUNOGLOBULIN FRAGMENTS STRUCTURE/FUNCTION RELATIONSHIPSImmunoglobulin fragments produced by proteolytic digestion have proven very useful in elucidating structure/function relationships in immunoglobulins.

A.  Fab Digestion with papain breaks the immunoglobulin molecule in the hinge region before the H-H inter-chain disulfide bond. This results in the formation of two identical fragments that contain the light chain and the VH and CH1 domains of the heavy chain.

Antigen binding - These fragments were called the Fab fragments because they contained the antigen binding sites of the antibody. Each Fab fragment is monovalent whereas the original molecule was divalent. The combining site of the antibody is created by both VH and VL. An antibody is able to bind a particular antigenic determinant because it has a particular combination of VH and VL. Different combinations of a VH and VL result in antibodies that can bind a different antigenic determinants.

B. Fc Digestion with papain also produces a fragment that contains the remainder of the two heavy chains each containing a CH2 and CH3 domain. This fragment was called Fc because it was easily crystallized.

 Effector functions - The effector functions of immunoglobulins are mediated by this part of the molecule. Different functions are mediated by the different domains in this fragment. Normally the ability of an antibody to carry out an effector function requires the prior binding of an antigen; however, there are exceptions to this rule.

B. F(ab')2 Treatment of immunoglobulins with pepsin results in cleavage of the heavy chain after the H-H inter-chain disulfide bonds resulting in a fragment that contains both antigen binding sites. This fragment was called F(ab')2because it is divalent. The Fc region of the molecule is digested into small peptides by pepsin. The F(ab')2binds antigen but it does not mediate the effector functions of antibodies.

GENERAL FUNCTIONS OF IMMUNOGLOBULINS

A. Antigen binding Immunoglobulins bind specifically to one or a few closely related antigens. Each immunoglobulin actually binds to a specific antigenic determinant. Antigen binding by antibodies is the primary function of antibodies and can result in protection of the host.  The valency of antibody refers to the number of antigenic determinants that an individual antibody molecule can bind. The valency of all antibodies is at least two and in some instances more.

B. Effector Functions Frequently the binding of an antibody to an antigen has no direct biological effect. Rather, the significant biological effects are a consequence of secondary "effector functions" of antibodies. The immunoglobulins mediate a variety of these effector functions. Usually the ability to carry out a particular effector function requires that the antibody bind to its antigen. Not every immunoglobulin will mediate all effector functions. Such effector functions include:

1. Fixation of complement - This results in lysis of cells and release of biologically active molecules.

2. Binding to various cell types - Phagocytic cells, lymphocytes, platelets, mast cells, and basophils have receptors that bind immunoglobulins. This binding can activate the cells to perform some function. Some immunoglobulins also bind to receptors on placental trophoblasts, which results in transfer of the immunoglobulin across the placenta. As a result, the transferred maternal antibodies provide immunity to the fetus and newborn

C.Activation of complement

Antibodies that bind to surface antigens on, for example, a bacterium attract the first component of

the complement cascade with their Fc region and initiate activation of the "classical" complement

system. This results in the killing of bacteria in two ways. First, the binding of the antibody and

complement molecules marks the microbe for ingestion by phagocytes in a process called opsonization;

these phagocytes are attracted by certain complement molecules generated in the complement cascade.

Secondly, some complement system components form a membrane attack complex to assist antibodies

to kill the bacterium directly.

D.Natural antibodies

Humans and higher primates also produce “natural antibodies” which are present in serum before viral

infection. Natural antibodies have been defined as antibodies that are produced without any previous

infection, vaccination, other foreign antigen exposure or passive immunization. These antibodies can

activate the classical complement pathway leading to lysis of enveloped virus particles long before the

adaptive immune response is activated. Many natural antibodies are directed against the

disaccharide galactose α(1,3)-galactose (α-Gal), which is found as a terminal sugar on glycosylated cell

surface proteins, and generated in response to production of this sugar by bacteria contained in the

human gut. Rejection ofxenotransplantated organs is thought to be, in part, the result of natural antibodies

circulating in the serum of the recipient binding to α-Gal antigens expressed on the donor tissue.

Isotypes/Classes of Antibody

Antibodies can come in different varieties known as isotypes or classes. Inplacental mammals there are five antibody

isotypes known as IgA, IgD, IgE, IgG and IgM. They are each named with an "Ig" prefix that stands for

immunoglobulin, another name for antibody, and differ in their biological properties, functional locations and ability to

deal with different antigens, as depicted in the table.

The antibody isotype of a B cell changes during cell development andactivation. Immature B cells, which have never

been exposed to an antigen, are known as naïve B cells and express only the IgM isotype in a cell surface bound

form. B cells begin to express both IgM and IgD when they reach maturity—the co-expression of both these

immunoglobulin isotypes renders the B cell 'mature' and ready to respond to antigen. B cell activation follows

engagement of the cell bound antibody molecule with an antigen, causing the cell to divide and differentiate into an

antibody producing cell called a plasma cell. In this activated form, the B cell starts to produce antibody in

a secretedform rather than a membrane-bound form. Some daughter cells of the activated B cells undergo isotype

switching, a mechanism that causes the production of antibodies to change from IgM or IgD to the

other antibody isotypes, IgE, IgA or IgG, that have defined roles in the immune system.

Antibody isotypes of mammals

Name Types Description Antibody Complexes

IgA 2

Found in mucosal areas, such as the gut, respiratory tractand urogenital tract, and prevents colonization bypathogens. Also found in saliva, tears, and breast milk.

IgD 1

Functions mainly as an antigen receptor on B cells that have not been exposed to antigens. It has been shown to activate basophils and mast cells to produce antimicrobial factors.

IgE 1Binds to allergens and triggers histamine release from mast cells and basophils, and is involved in allergy. Also protects against parasitic worms.

IgG 4

In its four forms, provides the majority of antibody-based immunity against invading pathogens. The only antibody capable of crossing the placenta to give passive immunity to fetus.

IgM 1

Expressed on the surface of B cells and in a secreted form with very high avidity. Eliminates pathogens in the early stages of B cell mediated (humoral) immunity before there is sufficient IgG.

Immunoglobulin A

Immunoglobulin A (IgA) is an antibody that plays a critical role in mucosal immunity. More IgA is produced in mucosal linings than all other types of antibody combined; between 3 and 5g is secreted into the intestinal lumen each day. IgA has two subclasses (IgA1 and IgA2) and can exist in a dimeric form called secretory IgA (sIgA). In its secretory form, IgA is the main immunoglobulin found in mucous secretions, including tears, saliva, colostrum and secretions from the genito-urinary tract, gastrointestinal tract, prostate and respiratory epithelium. It is also found in small amounts in blood. The secretory component of sIgA protects the immunoglobulin from being degraded by proteolytic enzymes, thus sIgA can survive in the harsh gastrointestinal tract environment and provide protection against microbes that multiply in body secretions. IgA is a poor activator of the complement system, and opsonises only weakly. Its heavy chains are of the type α.

The dimeric IgA molecule.1 H-chain2 L-chain3 J-chain4 secretory component

FormsIgA1 vs. IgA2

It exists in two isotypes, IgA1 (90%) and IgA2 (10%):

IgA1 is found in serum and made by bone marrow B cells.

In IgA2, the heavy and light chains are not linked with disulfide but with noncovalent bonds. IgA2 is made by

B cells located in the mucosae and has been found to secrete into colostrum, maternal milk, tears and saliva.

Serum vs. secretory IgA

It is also possible to distinguish forms of IgA based upon their location - serum IgA vs. secretory IgA.

In secretory IgA, the form of IgA that is found in secretions, polymers of 2-4 IgA monomers are linked by two

additional chains. One of these is the J chain (joining chain), which is a polypeptide of molecular mass 15kD, rich

with cysteine and structurally completely different from other immunoglobulin chains. This chain is formed in the IgA-

secreting cells.

The oligomeric forms of IgA in the external (mucosal) secretions also contain a polypeptide of a much larger

molecular mass (70 kD) called the secretory component that is produced byepithelial cells. This molecule originates

from the poly-Ig receptor (130 kD) that is responsible for the uptake and transcellular transport of oligomeric (but not

monomeric) IgA across the epithelial cells and into secretions such as tears, saliva, sweat, and gut fluid.

IgA activity

he high prevalence of IgA in mucosal areas is a result of a cooperation between plasma cells that produce polymeric

IgA (pIgA), and mucosal epithelial cells that express an immunoglobulin receptor called the polymeric Ig

receptor (pIgR). pIgA is released from the nearby activated plasma cells and binds to pIgR. This results in

transportation of IgA across mucosal epithelial cells and its cleavage from pIgR for release into external secretions.

In the blood, IgA interacts with an Fc receptor called FcαRI (or CD89), which is expressed on immune effector cells,

to initiate inflammatory reactions. Ligation of FcαRI by IgA containing immune complexes causes antibody-dependent

cell-mediated cytotoxicity (ADCC), degranulation

of eosinophils and basophils, phagocytosis by monocytes, macrophages,neutrophils and eosinophils, and triggering

of respiratory burst activity by polymorphonuclear leukocytes.[

IgA’s Properties

a) IgA is the 2nd most common serum Ig.

b) IgA is the major class of Ig in secretions - tears, saliva, colostrum, mucus. Since it is found in secretions secretory IgA is important in local (mucosal) immunity.

c) Normally IgA does not fix complement, unless aggregated.

d) IgA can binding to some cells - PMN's and some lymphocytes.

Immunoglobulin D

Immunoglobulin D (IgD) is an antibody isotype that makes up about 1% of proteins in the plasma membranes of mature B-lymphocytes where it is usually coexpressed with another cell surface antibody called IgM. IgD is also produced in a secreted form that is found in very small amounts in blood serum. Secreted IgD is produced as a monomeric antibody with two heavy chains of the delta (δ) class, and two Ig light chains.

Function

IgD's function has always been a puzzle in immunology since its discovery in 1964. IgD was recently found to be

present in species from cartilaginous fish to human (probably with the exception of birds). This nearly ubiquitous

appearance in species with an adaptive immune system demonstrates that IgD is as ancient as IgM and suggests the

notion that IgD has important immunological functions.

In B cells, IgD's function is to signal the B cells to be activated. By being activated, they are ready to take part in the

defense of the body in the immune system. During B-cell differentiation, IgM is the exclusive isotype expressed by

immature B cells. IgD starts to be expressed when the B-cell exits the bone marrow to populate peripheral lymphoid

tissues. When a B-cell reaches its mature state, it co-expresses both IgM and IgD. It is not well understood whether

IgM and IgD antibodies are functionally different on B cells. Cδ Knockout mice have no major B-cell intrinsic defects.

Recently, IgD was found to bind to basophils and mast cells and activate these cells to produce antimicrobial factors

to participate in respiratory immune defense in human. It also stimulates basophils to release B-cell homeostatic

factors. This is consistent with the reduction in the number of peripheral B cells, reduced serum IgE level and

defective primary IgG1 response in IgD knockout mice.

Properties

a) IgD is found in low levels in serum; its role in serum uncertain.

b) IgD is primarily found on B cell surfaces where it functions as a receptor for antigen. IgD on the surface of B cells has extra amino acids at C-terminal end for anchoring to the membrane. It also associates with the Ig-alpha and Ig-beta chains.

c) IgD does not bind complement.

 Immunoglobulin EIn biology, Immunoglobulin E (IgE) is a class of antibody (or immunoglobulin "isotype") that has only been found

in mammals. IgE is a monomeric antibody with 4 Ig-like domains (CH1->CH4).  It plays an important role in allergy,

and is especially associated with type 1hypersensitivity. IgE has also been implicated in immune system responses to

most parasitic worms like Schistosoma mansoni,Trichinella spiralis, and Fasciola hepatica, and may be important

during immune defense against certain protozoan parasites such asPlasmodium falciparum.

Although IgE is typically the least abundant isotype - blood serum IgE levels in a normal ("non-atopic") individual are

only 0.05% of the IgG concentration, compared to 10 mg/ml for the IgGs (the isotypes responsible for most of the

classical adaptive immune response) - it is capable of triggering the most powerful immune reactions.

Role in disease

Atopic individuals can have up to 10 times the normal level of IgE in their blood (as do sufferers of hyper-IgE

syndrome). However, this may not be a requirement for symptoms to occur as has been seen in asthmatics with

normal IgE levels in their blood - recent research has shown that IgE production can occur locally in the nasal

mucosa.

IgE that can specifically recognise an "allergen" (typically this is a protein, such as dust mite DerP1, cat FelD1, grass

or ragweed pollen, etc.) has a unique long-lived interaction with its high affinity receptor, FcεRI, so

that basophils and mast cells, capable of mediating inflammatory reactions, become "primed", ready to release

chemicals like histamine, leukotrienesand certain interleukins, which cause many of the symptoms we associate with

allergy, such as airway constriction in asthma, local inflammation in eczema, increased mucus secretion in allergic

rhinitis and increased vascular permeability, ostensibly to allow other immune cells to gain access to tissues, but

which can lead to a potentially fatal drop in blood pressure as in anaphylaxis. Although the mechanisms of each

response are fairly well understood, why some allergics develop such drastic sensitivities when others merely get a

runny nose is still one of science's hot topics. Regulation of IgE levels through control of B cell differentiation to

antibody-secreting plasma cells is thought to involve the "low affinity" receptor, FcεRII orCD23[citation needed]. CD23 may

also allow facilitated antigen presentation, an IgE-dependent mechanism whereby B cells expressing CD23 are

able to present allergen to (and stimulate) specific T helper cells, causing the perpetuation of a Th2 response, one of

the hallmarks of which is the production of more antibodies.

Pharmacology

IgE may be an important target in treatments for allergy and asthma.

Currently, severe allergy and asthma is usually treated with drugs (like anti-histamines) that damp down the late

stages of inflammation and relax airway smooth muscle. Unfortunately, these treatments are fairly broad in their

action, and so many have unpleasant side effects; they may also inhibit important protective responses.[citation needed]

In 2002, researchers at The Randall Division of Cell and Molecular Biophysics determined the structure of IgE.

Understanding of this structure (which is atypical of other isotypes in that it is highly bent and asymmetric), and of the

interaction of IgE with receptor FcεRI will enable development of a new generation of allergy drugs that seek to

interfere with the IgE-receptor interaction. A new treatment, omalizumab, a monoclonal antibody, recognises IgE not

bound to its receptor and is used to neutralise or mop-up existing IgE and prevent it from binding to cells. It may be

possible to design treatments cheaper than monoclonal antibodies (for instance, small molecule drugs) that use a

similar approach to inhibit IgE binding to its receptor.

Properties

a) IgE is the least common serum Ig since it binds very tightly to Fc receptors on basophils and mast cells even before interacting with antigen.

b) Involved in allergic reactions - As a consequence of its binding to basophils an mast cells, IgE is involved in allergic reactions. Binding of the allergen to the IgE on the cells results in the release of various pharmacological mediators that result in allergic symptoms.

c) IgE also plays a role in parasitic helminth diseases. Since serum IgE levels rise in parasitic diseases, measuring IgE levels is helpful in diagnosing parasitic infections. Eosinophils have Fc receptors for IgE and binding of eosinophils to IgE-coated helminths results in killing of the parasite.

d) IgE does not fix complement.

 Immunoglobulin GImmunoglobulin G (IgG) are antibody molecules. Each IgG is composed of four peptide chains -- two heavy

chains γ and two light chains. Each IgG has two antigen binding sites. Other Immunoglobulins may be described in

terms of polymers with the IgG structure considered the monomer.

IgG is the most abundant immunoglobulin and is approximately equally distributed in blood and in tissue liquids,

constituting 75% of serumimmunoglobulins in humans. IgG molecules are synthesized and secreted by plasma B

cells.

Functions

IgG antibodies are predominantly involved in the secondary immune response (the main antibody involved in primary

response is IgM). The presence of specific IgG generally corresponds to maturation of the antibody response.

IgG is the only isotype that can pass through the human placenta, thereby providing protection to the fetus in utero.

Along with IgA secreted in the breast milk, residual IgG absorbed through the placenta provides

the neonate with humoral immunity before its own immune system develops. Colostrum contains a high percentage

of IgG, especially in bovine colostrum.

IgG can bind to many kinds of pathogens, for example viruses, bacteria, and fungi, and protects the body against

them by agglutination and immobilization, complement activation(classical

pathway), opsonization for phagocytosis and neutralization of their toxins. It also plays an important role in Antibody-

dependent cell-mediated cytotoxicity(ADCC).

IgG is also associated with Type II and Type III Hypersensitivity.

Subclasses

There are four IgG subclasses (IgG1, 2, 3 and 4) in humans, named in order of their abundance

in serum (IgG1 being the most abundant).

Name PercentCrosses placenta easily

Complement activatorBinds to Fc receptor on phagocytic cells

IgG1 66% Yes second highest high affinity

IgG2 23% No third highest extremely low affinity

IgG3 7% Yes highest high affinity

IgG4 4% Yes no intermediate affinity

Note: IgG affinity to Fc receptors on phagocytic cells is specific to individual species from which the antibody comes

as well as the class. The structure of the hinge regions gives each of the 4 IgG classes its unique biological profile.

Even though there is about 95% similarity between their Fc regions, the structure of the hinge regions is relatively

different.

Properties

IgG is the most versatile immunoglobulin because it is capable of carrying out all of the functions of immunoglobulin molecules.

a) IgG is the major Ig in serum - 75% of serum Ig is IgG

b) IgG is the major Ig in extra vascular spaces

c) Placental transfer - IgG is the only class of Ig that crosses the placenta. Transfer is mediated by a receptor on placental cells for the Fc region of IgG. Not all subclasses cross equally well; IgG2 does not cross well.

d) Fixes complement - Not all subclasses fix equally well; IgG4 does not fix complement

e) Binding to cells - Macrophages, monocytes, PMNs and some lymphocytes have Fc receptors for the Fc region of IgG. Not all subclasses bind equally well; IgG2 and IgG4 do not bind to Fc receptors. A consequence of binding to the Fc receptors on PMNs, monocytes and macrophages is that the cell can now internalize the antigen better. The antibody has prepared the antigen for eating by the phagocytic cells. The term opsonin is used to describe substances that enhance phagocytosis. IgG is a good opsonin. Binding of IgG to Fc receptors on other types of cells results in the activation of other functions.

Immunoglobulin MImmunoglobulin M, or IgM for short, is a basic antibody that is produced by B cells. It is the primary antibody against A and B antigens onred blood cells. IgM is by far the physically largest antibody in the human circulatory system. It is the first antibody to appear in response to initial exposure to antigen.

IgM (Immunoglobulin M) antibody molecule consisting of 5 base units.1: Base unit.2: Heavy chains.3: Light chains.4: J chain.5: Intermolecular disulfide bonds.

Structure and function

IgM forms polymers where multiple immunoglobulins are covalently linked together with disulfide bonds, mostly as a

pentamer but also as a hexamer. IgM has a molecular mass of approximately 900 kD (in its pentamer form). Because

each monomer has two antigen binding sites, a pentameric IgM has 10 binding sites. Typically, however, IgM cannot

bind 10 antigens at the same time because the large size of most antigens hinders binding to nearby sites.

The J chain is found in pentameric IgM but not in the hexameric form, perhaps due to space constraints in the

hexameric complex. Pentameric IgM can also be made in the absence of J chain. At present, it is still uncertain what

fraction of normal pentamer contains J chain, and to this extent it is also uncertain whether a J chain-containing

pentamer contains one or more than one J chain. 

Because IgM is a large molecule, it cannot diffuse well, and is found in the interstitium only in very low quantities. IgM

is primarily found inserum; however, because of the J chain, it is also important as a secretory immunoglobulin.

Due to its polymeric nature, IgM possesses high avidity, and is particularly effective at complement activation.

Propertiesa) IgM is the third most common serum Ig.

b) IgM is the first Ig to be made by the fetus and the first Ig to be made by a virgin B cells when it is stimulated by antigen.

c) As a consequence of its pentameric structure, IgM is a good complement fixing Ig. Thus, IgM antibodies are very efficient in leading to the lysis of microorganisms.

d) As a consequence of its structure, IgM is also a good agglutinating Ig . Thus, IgM antibodies are very good in clumping microorganisms for eventual elimination from the body.

e) IgM binds to some cells via Fc receptors.

f) B cell surface Ig Surface IgM exists as a monomer and lacks J chain but it has an extra 20 amino acids at the C-terminus to anchor it into the membrane. Cell surface IgM functions as a receptor for antigen on B cells. Surface IgM is noncovalently associated with two additional proteins in the membrane of the B cell called Ig-alpha and Ig-beta as indicated in. These additional proteins act as signal transducing molecules since the cytoplasmic tail of the Ig molecule itself is too short to transduce a signal. Contact between surface immunoglobulin and an antigen is required before a signal can be transduced by the Ig-alpha and Ig-beta chains. In the case of T-independent antigens, contact between the antigen and surface immunoglobulin is sufficient to activate B cells to differentiate into antibody secreting plasma cells. However, for T-dependent antigens, a second signal provided by helper T cells is required before B cells are activated.

3rd Unit Complement systemThe complement system is a biochemical cascade that helps, or “complements”, the ability of antibodies

to clear pathogens from an organism. It is part of the immune system called the innate immune

systemthat is not adaptable and does not change over the course of an individual's lifetime. However, it

can be recruited and brought into action by the adaptive immune system.

The complement system consists of a number of small proteins found in the blood, generally synthesized

by the liver, and normally circulating as inactive precursors (pro-proteins). When stimulated by one of

several triggers, proteases in the system cleave specific proteins to release cytokines and initiate an

amplifying cascade of further cleavages. The end-result of this activation cascade is massive amplification

of the response and activation of the cell-killing membrane attack complex. Over 25 proteins and protein

fragments make up the complement system, including serum proteins, serosal proteins, and cell

membrane receptors. They account for about 5% of the globulin fraction of blood serum.

Three biochemical pathways activate the complement system: the classical complement pathway,

thealternative complement pathway, and the mannose-binding lectin pathway

Sometimes the interaction of antibodies with antigen is useful by itself. For example,

coating a virus or bacterium thus preventing it from binding to — and invading — a host cell (e.g., antipolio antibodies);

binding to a toxin molecule (e.g., diphtheria or tetanus toxin) thus keeping the toxin from entering a cell where it does its dirty work.

But most of the time, the binding of antibodies to antigen performs no useful function until and unless it can activate an effector mechanism. The complement system serves several effector roles.

So,

the complement system provides the actual protection from the response while the interaction of antibodies and antigen provides the specificity of the response.

Put another way, antibodies "finger" the target, complement destroys it.

Features of the system

The complement system consists of some 30 proteins circulating in blood plasma. Most of these are inactive until

o they are cleaved by a protease which, in turn,

o converts them into a protease.

Thus many components of the system serve as the substrate of a prior component and then as an enzyme to activate a subsequent component.

This pattern of sequential activation produces an expanding cascade of activity (reminiscent of the operation of the blood clotting system

Functions of the Complement

The following are the basic functions of the complement

1. Opsonization - enhancing phagocytosis of antigens

2. Chemotaxis - attracting macrophages and neutrophils

3. Lysis - rupturing membranes of foreign cells

4. Clumping of antigen-bearing agents

5. Altering the molecular structure of viruses

Overview

The proteins and glycoproteins that constitute the complement system are synthesized by the liver

hepatocytes. But significant amounts are also produced by tissue macrophages, blood monocytes, and

epithelial cells of the genitourinal tract and gastrointestinal tract. The three pathways of activation all

generate homologous variants of the protease C3-convertase. The classical complement pathway

typically requires antigen:antibody complexes for activation (specific immune response), whereas the

alternative and mannose-binding lectin pathways can be activated by C3 hydrolysis or antigens without

the presence of antibodies (non-specific immune response). In all three pathways, a C3-convertase

cleaves and activates component C3, creating C3a and C3b, and causing a cascade of further cleavage

and activation events. C3b binds to the surface of pathogens, leading to greater internalization

byphagocytic cells by opsonization. C5a is an important chemotactic protein, helping recruit inflammatory

cells. Both C3a and C5a have anaphylatoxin activity, directly triggeringdegranulation of mast cells as well

as increasing vascular permeability and smooth muscle contraction. C5b initiates the membrane attack

pathway, which results in the membrane attack complex (MAC), consisting of C5b, C6, C7, C8, and

polymeric C9. MAC is the cytolytic endproduct of the complement cascade; it forms a transmembrane

channel, which causes osmotic lysis of the target cell. Kupffer cells and other macrophage cell types help

clear complement-coated pathogens. As part of the innate immune system, elements of the complement

cascade can be found in species earlier than vertebrates; most recently in the protostome horseshoe crab

species, putting the origins of the system back further than was previously thought.

Table 1. Proteins of the Complement system

Classical PathwayLectin

PathwayAlternative

PathwayLytic

Pathway

Activation Proteins:

C1qrs, C2, C3, C4

 

Control Proteins:

C1-INH, C4-BP

 

Mannan binding protein (MBP), mannan-asociated serine protease (MASP, MASP2)

 

C3, Factors B & D*, Properdin (P)

 

 

Factors I* & H, decay accelerating factor (DAF), Complement receptor 1(CR1),etc.

 

C5, C6, C7, C8, C9

 

 

Protein S

Components underlined acquire enzymatic activity when activated.

Components marked with an asterisk have enzymatic activity in their native form.

PATHWAYS OF COMPLEMENT ACTIVATION

Complement activation can be divided into four pathways: the classical pathway, the lectin pathway, the alternative pathway and the membrane attack (or lytic) pathway. Both classical and alternative pathways lead to the activation of C5 convertase and result in the production of C5b which is essential for the activation of the membrane attack pathway.

 

CLASSICAL PATHWAY

C1 activationC1, a multi-subunit protein containing three different proteins (C1q, C1r and C1s), binds to the Fc region of IgG and IgM antibody molecules that have interacted with antigen.  C1 binding does not occur to antibodies that have not complexed with antigen and binding requires calcium and magnesium ions.  (N.B.  In some cases C1 can bind to aggregated immunoglobulin [e.g. aggregated IgG] or to certain pathogen surfaces in the absence of antibody).  The binding of C1 to antibody is via C1q and C1q must cross link at least two antibody molecules before it is firmly fixed.  The binding of C1q results in the activation of C1r which in turn activates C1s.  The result is the formation of an activated “C1qrs”, which is an enzyme that cleaves C4 into two fragments C4a and C4b. 

C4 and C2 activation (generation of C3 convertase)The C4b fragment binds to the membrane and the C4a fragment is released into the microenvironment.  Activated “C1qrs” also cleaves C2 into C2a and C2b.  C2a binds to the membrane in association with C4b, and C2b is released into the microenvironment. The resulting C4bC2a complex is a C3 convertase, which cleaves C3 into C3a and C3b. 

C3 activation (generation of C5 convertase)C3b binds to the membrane in association with C4b and C2a, and C3a is released into the microenvironment.  The resulting C4bC2aC3b is a C5 convertase.  The generation of C5 convertase is the end of the classical pathway. 

      Several of the products of the classical pathway have potent biological activities that contribute to host defenses.  Some of these products may also have detrimental effects if produced in an unregulated manner.  Table 2 summarizes the biological activities of classical pathway components.

Table 2.  Biological Activity of classical pathway products

Component

Biological Activity

C2bProkinin; cleaved by plasmin to yield kinin, which  results in edema

C3a

Anaphylotoxin; can activate basophils and mast cells to degranulate resulting in increased vascular permeability and contraction of smooth muscle cells, which may lead toanaphylaxis

C3b

Opsonin; promotes phagocytosis by binding to complement receptors

Activation of phagocytic cells

C4a Anaphylotoxin (weaker than C3a)

C4bOpsonin; promotes phagocytosis by binding to complement receptors

If the classical pathway were not regulated there would be continued production of C2b, C3a, and C4a.  Thus, there must be some way to regulate the activity of the classical pathway.

Table 3 summarizes the ways in which the classical pathway is regulated. 

Table 3.   Regulation of the Classical Pathway

Component

Regulation

All C1-INH; dissociates C1r and C1s from C1q

C3aC3a inactivator (C3a-INA;Carboxypeptidase B); inactivates C3a

C3bFactors H and I; Factor H facilitates the degradation of C3b by Factor I

C4a C3-INA

C4b

C4 binding protein(C4-BP) and Factor I; C4-BP facilitates degradation of C4b by Factor I; C4-BP also prevents association of C2a with C4b thus blocking the formation of C3 convertase

The importance of C1-INH in regulating the classical pathway is demonstrated by the result of a deficiency in this inhibitor.  C1-INH deficiencies are associated with the development of hereditary angioedema.

Figur4. Activation of C3 by the classical pathway

ALTERNATIVE PATHWAY The alternative pathway begins with the activation of C3 and requires Factors B and D and Mg++ cation, all present in normal serum.

1. Amplification loop of C3b formationIn serum there is low level spontaneous hydrolysis of C3 to produce C3i. Factor B binds to C3i and becomes susceptible to Factor D, which cleaves Factor B into Bb. The C3iBb complex acts as a C3 convertase and cleaves C3 into C3a and C3b. Once C3b is formed, Factor B will bind to it and becomes susceptible to cleavage by Factor D. The resulting C3bBb complex is a C3 convertase that will continue to generate more C3b, thus amplifying C3b production. If this process continues unchecked, the result would be the consumption of all C3 in the serum. Thus, the spontaneous production of C3b is tightly controlled.

Spontaneous activation of C3

2. Control of the amplification loop

As spontaneously produced C3b binds to autologous host membranes, it interacts with DAF (decay accelerating factor), which blocks the association of Factor B with C3b thereby preventing the formation of additional C3 convertase. In addition, DAF accelerates the dissociation of Bb from C3b in C3 convertase that has already formed, thereby stopping the production of additional C3b. Some cells possess complement receptor 1 (CR1). Binding of C3b to CR1 facilitates the enzymatic degradation of C3b by Factor I. In addition, binding of C3 convertase (C3bBb) to CR1 also dissociates Bb from the complex. Thus, in cells possessing complement receptors, CR1 also plays a role in controlling the amplification loop. Finally, Factor H can bind to C3b bound to a cell or in the in the fluid phase and facilitate the enzymatic degradation of C3b by Factor I. Thus, the amplification loop is controlled by either blocking the formation of C3 convertase, dissociating C3 convertase, or by enzymatically digesting C3b. The importance of controlling this amplification loop is illustrated in patients with genetic deficiencies of Factor H or I. These patients have a C3 deficiency and increased susceptibility to certain infections. 

3. Stabilization of C convertase by activator (protector) surfaces

When bound to an appropriate activator of the alternative pathway, C3b will bind Factor B, which is enzymatically cleaved by Factor D to produce C3 convertase (C3bBb).  However, C3b is resistant to degradation by Factor I and the C3 convertase is not rapidly degraded, since it is stabilized by the activator surface.  The complex is further stabilized by properdin binding to C3bBb.  Activators of the alternate pathway are components on the surface of pathogens and include: LPS of Gram-negative bacteria and the cell walls of some bacteria and yeasts.  Thus, when C3b binds to an activator surface, the C3 convertase formed will be stable and continue to generate additional C3a and C3b by cleavage of C3.

Stabilization of C3 convertase

4. Generation of C5 convertase

Some of the C3b generated by the stabilized C3 convertase on the activator surface associates with the C3bBb complex to form a C3bBbC3b complex.  This is the C5 convertase of the alternative pathway.  The generation of C5 convertase is the end of the alternative pathway.  The alternative pathway can be activated by many Gram-negative (most significantly, Neisseria meningitidis and N. gonorrhoea), some Gram-positive bacteria and certain viruses and parasites, and results in the lysis of these organisms. Thus, the alternative pathway of C activation provides another means of protection against certain pathogens before an antibody response is mounted.  A deficiency of C3 results in an increased susceptibility to these organisms.  The alternate pathway may be the more primitive pathway and the classical and lectin pathways probably developed from it.

Remember that the alternative pathway provides a means of non-specific resistance against infection without the participation of antibodies and hence provides a first line of defense against a number of infectious agents.

Many gram negative and some gram positive bacteria, certain viruses, parasites, heterologous red cells, aggregated immunoglobulins (particularly, IgA) and some other proteins (e.g. proteases, clotting pathway products) can activate the alternative pathway. One protein, cobra venom factor (CVF), has been extensively studied for its ability to activate this pathway.

 

Stabilized C5 convertase of the alternative pathway.

MEMBRANE ATTACK (LYTIC)  PATHWAY

C5 convertase from the classical (C4b2a3b), lectin (C4b2a3b) or alternative (C3bBb3b) pathway cleaves C5 into C5a and C5b. C5a remains in the fluid phase and the C5b rapidly associates with C6 and C7 and inserts into the membrane. Subsequently C8 binds, followed by several molecules of C9. The C9 molecules form a pore in the membrane through which the cellular contents leak and lysis occurs. Lysis is not an enzymatic process; it is thought to be due to physical damage to the membrane. The complex consisting of C5bC6C7C8C9 is referred to as the membrane attack complex (MAC).

C5a generated in the lytic pathway has several potent biological activities. It is the most potent anaphylotoxin. In addition, it is a chemotactic factor for neutrophils and stimulates the respiratory

burst in them and it stimulates inflammatory cytokine production by macrophages. Its activities are controlled by inactivation by carboxypeptidase B (C3-INA).

Some of the C5b67 complex formed can dissociate from the membrane and enter the fluid phase. If this were to occur it could then bind to other nearby cells and lead to their lysis. The damage to bystander cells is prevented by Protein S (vitronectin). Protein S binds to soluble C5b67 and prevents its binding to other cells.

The lytic pathway.

BIOLOGICALLY ACTIVE PRODUCTS OF COMPLEMENT ACTIVATION

Activation of complement results in the production of several biologically active molecules which

contribute to resistance,anaphylaxis and inflammation.

Kinin productionC2b generated during the classical pathway of C activation is a prokinin which becomes biologically active following enzymatic alteration by plasmin. Excess C2b production is prevented by limiting C2 activation by C1 inhibitor (C1-INH) also known as serpin which displaces C1rs from the C1qrs complex. A genetic deficiency of C1-INH results in an overproduction of C2b and is the cause of hereditary angioneurotic edema. This condition can be treated with Danazol which promotes C1-INH production or with ε-amino caproic acid which decreases plasmin activity.

AnaphylotoxinsC4a, C3a and C5a (in increasing order of activity) are all anaphylotoxins which cause basophil/mast cell degranulation and smooth muscle contraction. Undesirable effects of these peptides are controlled by carboxypeptidase B (C3a-INA).

Chemotactic FactorsC5a and MAC (C5b67) are both chemotactic. C5a is also a potent activator of neutrophils, basophils and macrophages and causes induction of adhesion molecules on vascular endothelial cells.

OpsoninsC3b and C4b in the surface of microorganisms attach to C-receptor (CR1) on phagocytic cells and promote phagocytosis.

Other Biologically active products of C activationDegradation products of C3 (iC3b, C3d and C3e) also bind to different cells by distinct receptors and modulate their functions.

 

In summary, the complement system takes part in both  specific and non-specific resistance and generates a number of products of biological and pathophysiological significance (Table 4).

There are known genetic deficiencies of most individual C complement components, but C3 deficiency is most serious and fatal. Complement deficiencies also occur in immune complex diseases (e.g., SLE) and acute and chronic bacterial, viral and parasitic infections.

Regulation of C1rs (C4 convertase ) by C1-INH

Table 4.  Activities of Complement Activation Products and their Control Factors

Fragment Activity EffectControl Factor (s)

C2aProkinin, accumulation of fluids

Edema C1-INH

C3a

Basophil and mast cells degranulation; enhanced vascular permeability, smooth muscle contraction

Anaphylaxis C3a-INA

C3bOpsonin, phagocyte activation

Phagocytosis

Factors H and I

C4a

Basophil and mast cells degranulation; enhanced vascular permeability, smooth muscle contraction

Anaphylaxis

(least potent)

 

C3a-INA

C4b OpsoninPhagocytosis

C4-BP and Factor I

C5a

Basophil and mast cells degranulation; enhanced vascular permeability, smooth muscle contraction

Anaphylaxis

(most potent)

C3a-INAChemotaxis, stimulation of respiratory burst, activation of phagocytes, stimulation of inflammatory cytokines

Inflammation

C5bC6C7

Chemotaxis InflammationProtein S (vitronectin)Attaches to other

membranesTissue damage

 

Table 5. Complement deficiencies and disease

Pathway/ Disease Mechanism

Component

Classical Pathway  

   C1INHHereditary angioedema

Overproduction of C2b (prokinin)

  C1, C2, C4Predisposition to SLE

Opsonization of immune complexes help keep them soluble, deficiency results in increased precipitation in tissues and inflammation

Lectin Pathway  

MBL

Susceptibility to bacterial infections in infants or immunosuppressed

Inability to initiate the lectin pathway

Alternative Pathway  

   Factors B or D

Susceptibility to pyogenic (pus-forming) bacterial infections

Lack of sufficient opsonization of bacteria

  C3Susceptibility to bacterial infections

Lack of opsonization and inability to utilize the membrane attack pathway

  C5, C6, C7 C8, and C9

Susceptibility to Gram-negative infections

Inability to attack the outer membrane of Gram-negative bacteria

 Properdin (X-linked)Susceptibility meningococcal meningitis

Lack of opsonization of bacteria

 Factors H or IC3 deficiency and susceptibility to bacterial infections

Uncontrolled activation of C3 via alternative pathway resulting in depletion of C3

 

Activation of complements by antigen-associated antibody

In the classical pathway, C1 binds with its C1q subunits to Fc fragments (made of CH2 region) of IgG or

IgM, which has formed a complex with antigens. C4b and C3b are also able to bind to antigen-associated

IgG or IgM, to its Fc portion (See below figure)

Such immunoglobulin-mediated binding of the complement may be interpreted as that the complement

uses the ability of the immunoglobulin to detect and bind to non-self antigens as its guiding stick. The

complement itself is able to bind non-self pathogens after detecting their pathogen-associated molecular

patterns (PAMPs), however, utilizing specificity of antibody, complements are able to detect non-self

enemies much more specifically. There must be mechanisms that complements bind to Ig but would not

focus its function to Ig but to the antigen.

Below figure shows the classical and the alternative pathways with the late steps of complement

activation schematically. Some components have a variety of binding sites. In the classical pathway C4

binds to Ig-associated C1q and C1r2s2 enzyme cleave C4 to C4b and 4a. C4b binds to C1q, antigen-

associated Ig (specifically to its Fc portion), and even to the microbe surface. C3b binds to antigen-

associated Ig and to the microbe surface. Ability of C3b to bind to antigen-associated Ig would work

effectively against antigen-antibody immune complexes to make them soluble. In the figure, C2b refers to

the larger of the C2 fragments.

Regulation of the complement system

The complement system has the potential to be extremely damaging to host tissues, meaning its

activation must be tightly regulated. The complement system is regulated bycomplement control proteins,

which are present at a higher concentration in the blood plasma than the complement proteins

themselves. Some complement control proteins are present on the membranes of self-cells preventing

them from being targeted by complement. One example is CD59, also known as protectin, which inhibits

C9 polymerisation during the formation of the membrane attack complex.

Role in disease

It is thought that the complement system might play a role in many diseases with an immune component,

such as Barraquer-Simons Syndrome, asthma, lupus erythematosus,glomerulonephritis, various forms

of arthritis, autoimmune heart disease, multiple sclerosis, inflammatory bowel disease, and ischemia-

reperfusion injuries.  and rejection of transplanted organs.

The complement system is also becoming increasingly implicated in diseases of the central nervous

system such as Alzheimer's disease and other neurodegenerative conditions such as spinal cord injuries.

Deficiencies of the terminal pathway predispose to both autoimmune

disease and infections (particularly Neisseria meningitidis, due to the role that the C56789 complex plays

in attacking Gram-negative bacteria).

Mutations in the complement regulators factor H and membrane cofactor protein have been associated

with atypical haemolytic uraemic syndrome. Moreover, a common single nucleotide polymorphism in

factor H (Y402H) has been associated with the common eye disease age-related macular

degeneration. Both of these disorders are currently thought to be due to aberrant complement activation

on the surface of host cells.

Mutations in the C1 inhibitor gene can cause hereditary angioedema, an autoimmune condition resulting

from reduced regulation of the complement pathway.

Mutations in the MAC components of complement, especially C8, are often implicated in recurrent

Neisserial infection.

4th Unit

Antigen ReceptorsBoth B cells and T cells have surface receptors for antigen. Each cell has thousands of receptors of a single specificity; that is, with a binding site for a particularepitope.

T-cell receptors (TCRs) enable the cell to bind to and, if additional signals are present, to be activated by and respond to an epitope presented by another cell called the antigen-presenting cell or APC.

B-cell receptors (BCRs) enable the cell to bind to and, if additional signals are present, to be activated by and respond to an epitope on molecules of a soluble antigen. The response ends with descendants of the B cell secreting vast numbers of a soluble form of its receptors. These are antibodies.

Here we discuse about TCR only.

T cell receptorThe T cell receptor or TCR is a molecule found on the surface of T lymphocytes (or T cells) that is, in

general, responsible for recognizing antigens bound to major histocompatibility complex (MHC)

molecules.

The TCR is composed of two different protein chains (that is, it is a hetero dimer ). In 95% of T cells, this

consists of an alpha (α) and beta (β) chain, whereas in 5% of T cells this consists of gamma and

delta (γ/δ) chains.

When the TCR engages with antigen and MHC, this activates the T lymphocyte through a series of

biochemical events mediated by associated enzymes, co-receptors, specialized accessory molecules and

activated or released transcription factors.

The two chains of the T cell receptor

T cell receptor alpha locus

Identifiers

Symbol(s) TRA@ TCRA

Entrez 6955

OMIM 186880

T cell receptor beta locus

Identifiers

Symbol(s) TRB@ TCRB

Entrez 6957

OMIM 186930

Structural characteristics of the TCR

The TCR, which is anchored in the cell membrane, consists of two halves which form a pair (or dimer) of

protein chains. The halves are called the alpha (α) and beta (β) fragments (in γ/δ T cells, the halves are

gamma (γ) and delta (δ) fragments). Each fragment is divided in turn into a constant (C) and variable (V)

region. The constant region has an end which is anchored in the cell membrane. The variable region

faces outward and binds to the HLA molecule and the antigen it presents. On the α chain, the variable

region is called Vα and the constant region is called Cα; on the β chain they are called Vβ and Cβ

respectively.

The structure of TCR is very similar to immunoglobulin Fab fragments, which are regions defined as the

combined light and heavy chain of an antibody arm. Each chain of the TCR is a member of

the immunoglobulin superfamily and possesses one N-terminal immunoglobulin (Ig)-variable (V) domain,

one Ig-constant (C) domain, a transmembrane/cell membrane-spanning region, and a short cytoplasmic

tail at the C-terminal end.

The variable domain of both the TCR α-chain and β-chain have three hypervariable or complementarity

determining regions (CDRs), whereas the variable region of the β-chain has an additional area of

hypervariability (HV4) that does not normally contact antigen and therefore is not considered a CDR.

The residues are located in two regions of the TCR, at the interface of the α- and β-chains and in the β-

chain framework region that is thought to be in proximity to the CD3 signal-transduction complex . CDR3

is the main CDR responsible for recognizing processed antigen, although CDR1 of the alpha chain has

also been shown to interact with the N-terminal part of the antigenic peptide, whereas CDR1 of the β-

chain interacts with the C-terminal part of the peptide.

CDR2 is thought to recognize the MHC. CDR4 of the β-chain is not thought to participate in antigen

recognition, but has been shown to interact with superantigens.

The constant domain of the TCR domain consists of short connecting sequences in which a cysteine

residue forms disulfide bonds, which forms a link between the two chains.

Or

The TCR is a heterodimer composed of one α and one β chain of approximately equal length. Each chain has a short cytoplasmic tail but it is to small to be able to transduce an activation signal to the cell. Both chains have a transmembrane region comprised of hydrophobic amino acids by which the molecule is anchored in the cell membrane. Both chains have a constant region and a variable region similar to the immunoglobulin chains. The variable region of both chains contains hypervariable regions that determine the specificity for antigen. Each T cell bears a TCR of only one specificity (i.e. there is allelic exclusion).The genetic basis for the generation of the vast array of antigen receptors on B cells has been discussed previously (see lecture on Ig genetics). The generation of a vast array of TCRs is accomplished by similar mechanism. The germline genes for the TCR β genes are composed of V, D and J gene segments that rearrange during T cell development to produce many different TCR β chains. The germline genes for the

TCR α genes are composed of V and J gene segments which rearrange to produce α chains. The specificity of the TCR is determined by the combination of α and β chains.

There is a small population of T cells that express TCRs that have γ and δ chains instead of α and β chains. These gamma/delta T cells predominate in the mucosal epithelium and have a repertoire biased toward certain bacterial and viral antigens. The genes for the δ chains have V, D and J gene segments whereas the genes for the γ chains have only V and J gene segments but the repertoire is considerably smaller that than that of the alpha/beta T cells. The gamma/delta T cells recognize antigen in an MHC-independent manner unlike the alpha/beta T cells.

Antigen presentation stimulates T cells to become either "cytotoxic" CD8+ cells or "helper" CD4+ cells.

Generation of the TCR

Processes for TCR formation are similar to those described for B cell antigen receptors, otherwise known

as immunoglobulins.

The TCR alpha chain is generated by VJ recombination, whereas the beta chain is generated

by V(D)J recombination (both involve a somewhat random joining of gene segments to generate the

complete TCR chain).

Similarly, generation of the TCR gamma chain involves VJ recombination, whereas generation of

the TCR delta chain occurs by V(D)J recombination.

The intersection of these specific regions (V and J for the alpha or gamma chain, V D and J for the beta

or delta chain) corresponds to the CDR3 region that is important for antigen-MHC recognition (see

above).

It is the unique combination of the segments at this region, along with palindromic and random N- and P-

nucleotide additions, which accounts for the great diversity in specificity of the T cell receptor for

processed antigen.

The TCR Complex

The T-cell receptor complex with TCR-α and TCR-β chains, CD3 and ζ-chain accessory molecules.

The transmembrane region of the TCR is composed of positively charged amino acids.

It is thought that such structure allows the TCR to associate with other molecules like CD3 which possess

three distinct chains (γ, δ, and ε) in mammals and either a ζ2 complex or a ζ/η complex.

These accessory molecules have negatively charged transmembrane regions and are vital to

propagating the signal from the TCR into the cell; the cytoplasmic tail of the TCR is extremely short,

making it unlikely to participate in signaling.

The CD3- and ζ-chains, together with the TCR, form what is known as the T cell receptor complex.

TCR Co-Receptors

The signal from the T cell complex is enhanced by simultaneous binding of the MHC molecules by a

specific co-receptor.

On helper T cells, this co-receptor is CD4 that exclusively binds the class II MHC.

On cytotoxic T cells, this co-receptor is CD8 that is specific for class I MHC.

The co-receptor not only ensures the specificity of the TCR for an antigen, but also allows prolonged

engagement between the antigen presenting cell and the T cell and recruits essential molecules

(e.g., LCK) inside the cell involved in the signaling of the activated T lymphocyte.

Associated Molecules of the TCR complex involved in T-cell Activation

The essential function of the TCR complex is to identify specific bound antigen and elicit a distinct and

critical response. The mechanism by which a T-cell elicits this response upon contact with its unique

antigen is termed T-cell activation. There are a myriad of molecules involved in the complex biochemical

process by which this occurs which, in a wider context, is generally termed trans-membrane signalling.

The most common mechanism for activation and regulation of molecules beneath the lipid bilayer is via

phosphorylation/dephosphorylation by protein kinases. T-cells largely utilise the SRC family of kinases in

transmembrane signalling to phosphorylate tyrosines that are part of immunoreceptor tyrosine/based

activation motifs (ITAM).

Early signally steps implicate the following kinases in TCR associated reactions.

Lck - Associated with the transmembrane tail of CD4

Fyn - Associated with ITAMs of the IgAlpha and Igbeta regions of the TCR complex

CD45 - The transmembrane tail of which functions as a Tyrosine phosphatase)

Zap70 - Binds to ITAM sequences upon phosphorylation by Lck and Fyn

5th Unit

Major histocompatibility complexThe major histocompatibility complex (MHC) is a large genomic region or gene family found in

most vertebrates that encodes MHC molecules. MHC molecules play an important role in the immune

system and autoimmunity.

Proteins are continually synthesized in the cell. These include normal proteins (self) and microbial

invaders (nonself). A MHC molecule inside the cell takes a fragment of those proteins and displays it on

the cell surface. (The protein fragment is sometimes compared to a hot dog, and the MHC protein to the

bun.) When the MHC-protein complex is displayed on the surface of the cell, it can be presented to a

nearby immune cell, usually a T cell or natural killer (NK) cell. If the immune cell recognizes the protein as

nonself, it can kill the infected cell, and other infected cells displaying the same protein.

Because MHC genes must defend against a great diversity of microbes in the environment, with a great

diversity of proteins, the MHC genes themselves must be diverse. The MHC is the most gene-dense

region of the mammalian genome. MHC genes vary greatly from individual to individual, that is,

MHC alleles have polymorphisms (diversity). This polymorphism is adaptive in evolution because it

increases the likelihood that at least some individuals of a population will survive an epidemic.

There are two general classes of MHC molecules: Class I and Class II. Class I MHC molecules are found

on almost all cells and present proteins to cytotoxic T cells. Class II MHC molecules are found on certain

immune cells themselves, chieflymacrophages and B cells, also known as antigen-presenting cells

(APCs). These APCs ingest microbes, destroy them, and digest them into fragments. The Class II MHC

molecules on the APCs present the fragments to helper T cells, which stimulate an immune reaction from

other cells.

Classification

In humans, the 3.6-Mb (3 600 000 base pairs) MHC region on chromosome 6 contains 140 genes

between flanking genetic markers MOG and COL11A2. About half have known immune functions

(see human leukocyte antigen). The same markers in the marsupial Monodelphis domestica (gray short-

tailed opossum) span 3.95 Mb and contain 114 genes, 87 shared with humans.

SubgroupsThe MHC region is divided into three subgroups, class I, class II, and class III.

Name Function Expression

MHC class I

Encodes non-identical pairs (heterodimers) of peptide-binding proteins, as well as antigen-

All nucleated cells. MHC class I proteins contain an α chain & β2-micro-globulin(not part of the MHC encoded by chromosome

processing molecules such as TAP and Tapasin.

15). They present antigen fragments to cytotoxic T-cells via theCD8 receptor on the cytotoxic T-cells and also bind inhibitory receptors on NK cells.

MHC class II

Encodes heterodimeric peptide-binding proteins and proteins that modulate antigen loading onto MHC class II proteins in the lysosomal compartment such as MHC II DM, MHC II DQ, MHC II DR, and MHC II DP.

On most immune system cells, specifically on antigen-presenting cells. MHC class II proteins contain α & β chains and they present antigen fragments to T-helper cells by binding to the CD4receptor on the T-helper cells.

MHC class IIIregion

Encodes for other immune components, such as complement components (e.g., C2, C4, factor B) and some that encode cytokines (e.g., TNF-α) and also hsp.

Variable (see below).

Class III has a function very different from that of class I and class II, but, since it has a locus between the

other two (on chromosome 6 in humans), they are frequently discussed together.

ResponsesThe MHC proteins act as "signposts" that serve to alert the immune system if foreign material is present

inside a cell. They achieve this by displaying fragmented pieces of antigens on the host cell's surface.

These antigens may be self or nonself. If they are nonself, there are two ways by which the foreign

protein can be processed and recognized as being "nonself".

Phagocytic cells such as macrophages, neutrophils, and monocytes degrade foreign particles

that are engulfed during a process known as phagocytosis. Degraded particles are then presented on

MHC Class II molecules.

On the other hand, if a host cell was infected by a bacterium or virus, or was cancerous, it may

have displayed the antigens on its surface with a Class I MHC molecule. In particular, cancerous cells

and cells infected by a virus have a tendency to display unusual, nonself antigens on their surface.

These nonself antigens, regardless of which type of MHC molecule they are displayed on, will initiate

the specific immunity of the host's body.

Cells constantly process endogenous proteins and present them within the context of MHC I. Immune

effector cells are trained not to react to self peptides within MHC, and as such are able to recognize when

foreign peptides are being presented during an infection/cancer.

MHC class I

MHC class I molecules are one of two primary classes of major histocompatibility complex (MHC)

molecules (the other one being simplyMHC class II) and are found on every nucleated cell of the body

(and thus not on red blood cells though paradoxically are found on platelets). Their function is to display

fragments of proteins from within the cell to T cells; healthy cells will be ignored while cells containing

foreign proteins will be attacked by the immune system. Because MHC class I molecules present

peptides derived from cytosolic proteins, the pathway of MHC class I presentation is often called

the cytosolic or endogenous pathway.

MHC class II

MHC (major histocompatibility complex) Class II molecules are found only on a few specialized cell

types, including macrophages,dendritic cells and B cells, all of which are professional antigen-presenting

cells (APCs).

The peptides presented by class II molecules are derived from extracellular proteins (not cytosolic as

in class I); hence, the MHC class II-dependent pathway of antigen presentation is called

the endocytic or exogenous pathway.

Loading of class II molecules must still occur inside the cell; extracellular proteins are endocytosed,

digested in lysosomes, and bound by the class II MHC molecule prior to the molecule's migration to the

plasma membrane.

STRUCTURE OF CLASS I MHC MOLECULES  

MHC molecules are anchored in the cell membrane at the bottom of the illustration; they can then bind to immune cells at the top of the illustration. The MHC Class I molecule (left) on most cells binds to the T-cell receptor (TCR) and CD8 receptor (top). The MHC Class II molecule (right) on immune cells binds to the TCR and CD4 receptor on other immune cells (top).

MHC Class-I

MHC Class-I genes (MHC-I) code glucoproteins, with immunoglobulin structure: they present one heavy

chain type α, subdivided in three regions: α1, α2 y α3. These three regions are exposed to the

extracellular space, and they are linked to the cellular membrane through a transmembrane region. The α

chain is always associated to a molecule of β2 microglobulin, which is coded by an independent region

on chromosome 15. These molecules are present in the surface of all nucleated cells.

The most important function of the gene products for the Class-I genes is the presentation of intracellular

antigenic peptides to the cytotoxic T lymphocytes (CD8+). The antigenic peptide is located in a cleft

existing between the α1 and α2 regions in the heavy chain.

In humans, there are many different isotypes (different genes) for the Class-I molecules, which can be

grouped as:

"classic molecules", whose function consist in antigen presentation to the T8 lymphocytes: inside

this group we find HLA-A, HLA-B y HLA-C.

"non classic molecules" (named also MHC class IB), with specialized functions: they do not

present antigens to T lymphocytes, but they interact with inhibitory receptors in NK cells; inside this

group we find HLA-E, HLA-F, HLA-G.

Structure of a molecule of MHC Class-I.

MHC Class-II

These genes code glucoproteins with immunoglobulin structure, but in this case the functional complex is

formed by two chains, one α and one β (each one with two domains: α1 and α2, β1 and β2). Each chain

is linked to the cellular membrane through a transmembrane region, and both chains are confronted, with

domains 1 and 2 consecutives, in the extracellular espace.

These molecules are present mostly in the membrane of the antigen presenting cells (dendritic and

phagocytic cells), where they present processed extracellular antigenic peptides to the helper T

lymphocytes (CD4+). The antigenic peptide is located in a cleft formed by α1 and β1 peptides.

MHC-II molecules in humans present 5-6 isotypes, and can be grouped in:

"classic molecules", presenting peptides to T4 lymphocytes; inside this group we find HLA-DP,

HLA-DQ, HLA-DR;

"non classic molecules", accessories, with intracellular functions (they are not exposed in the

cellular membrane, but in internal membrares inlysosomes); normally, they load the antigenic

peptides on the classic MHC-II molecules; in this group are included HLA-DM and HLA-DO.

On top of the MHC-II molecules, in the Class-II region are located genes coding for antigen processing

molecules, such as TAP (transporter associated with antigen processing) or Tapasin.

Structure of a molecule of MHC Class-II.

Function of MHC class I

MHC class I molecules bind peptides mainly generated from degradation of cytosolic proteins. The MHC-I

peptide complex is then inserted into the plasma membrane of the cell. The peptide is bound to the

extracellular part of the MHC-I molecule. The function of the MHC-I is thus to display to the environment,

specifically cytotoxic T cells (CTLs), the proteins that are being produced within the cell.

A normal cell will display peptides from normal cellular proteins on its MHC-I, and CTLs will not be

activated in response to them. When a cell expresses peptides that are not normally present in cells, such

as after viral infection, these foreign peptides will be recognized by CTLs, which will become activated

and kill the cell. Additionally, reduction in the normal levels of expression of MHC-I, which can occur in

some virally infected cells as well as some cancer cells, will activate Natural killer cells to destroy the cell.

Effect of viruses

MHC class I molecules are loaded with peptides generated from the degradation of ubiquitinated cytosolic

proteins in proteasomes. As viruses induce cellular expression of viral proteins, some of these products

are tagged for degradation, with the resulting peptide fragments entering the endoplasmic reticulum and

binding to MHC I molecules. In this way, the MHC class I-dependent pathway of antigen presentation is

the primary way for a virus-infected cell to signal T cells that abnormal proteins are being synthesized as

a result of infection.

The fate of the virus-infected cell is almost always induction of apoptosis through cell-mediated immunity,

reducing the risk of infecting neighboring cells. As an evolutionary response to this method of immune

surveillance, many viruses are able to down-regulate or otherwise prevent the presentation of MHC class

I molecules on the cell surface. In contrast to cytotoxic T lymphocytes, Natural killer (NK) cells are

normally inactivated upon recognizing MHC I molecules on the surface of cells. Therefore, in the absence

of MHC I molecules, NK cells are activated and recognize the cell as aberrant, suggesting they may be

infected by viruses attempting to evade immune destruction. Several human cancers also show down-

regulation of MHC I, giving transformed cells the same survival advantage of being able to avoid normal

immune surveillance designed to destroy any infected or transformed cells

Reaction of MHC class II to bacteria

Because class II MHC is loaded with extracellular proteins, it is mainly concerned with presentation of

extracellular pathogens (for example, bacteria that might be infecting a wound or the blood). Class II

molecules interact exclusively with CD4+ ("helper") T cells (THC). The helper T cells then help to trigger an

appropriate immune response which may include localizedinflammation and swelling due to recruitment of

phagocytes or may lead to a full-force antibody immune response due to activation of B cells.

IMPORTANT ASPECTS OF MHC 

Although there is a high degree of polymorphism for a species, an individual has maximum of six different class I MHC products and only slightly more class II MHC products (considering only the major loci).  

Each MHC molecule has only one binding site.  The different peptides a given MHC molecule can bind all bind to the same site, but only one at a time. 

Because each MHC molecule can bind many different peptides, binding is termed degenerate. 

MHC polymorphism is determined only in the germline.  There are no recombinational mechanisms for generating diversity. 

MHC molecules are membrane-bound; recognition by T cells requires cell-cell contact. 

Alleles for MHC genes are co-dominant.  Each MHC gene product is expressed on the cell surface of an individual nucleated cell. 

A peptide must associate with a given MHC of that individual, otherwise no immune response can occur.  That is one level of control.

Mature T cells must have a T cell receptor that recognizes the peptide associated with MHC.  This is the second level of control. 

Cytokines (especially interferon-γ) increase level of expression of MHC. 

Peptides from the cytosol associate with class I MHC and are recognized by Tc cells.  Peptides from within vesicles associate with class II MHC and are recognized by Th cells. 

Polymorphism in MHC is important for survival of the species.

Antigen processing pathway

Both types of molecules present antigenic peptides to T lymphocytes, which are responsible for the

specific immune response to destroy the pathogen producing those antigens. However, Class-I and II

molecules correspond to two different pathways of antigen processing, and are associated to two different

systems of immune defense:

Table 2. Characteristics of the antigen processing pathways

Characteristic MHC-II pathway MHC-I pathway

Composition of the stable peptide-MHC

complex

Polymorphic chains α and β, peptide binds to both

Polymorphic chain α and β2 microglobulin, peptide bound to α

chain

Types of antigen presenting cells (APC)

Dendritic cells, mononuclear phagocytes, B lymphocytes, some endothelial cells,

epithelium of thymusAll nucleated cells

T lymphocytes able to respond

Helper T lymphocytes (CD4+) Cytotoxic T lymphocytes (CD8+)

Origin of antigenic proteins

Proteins present in endosomes or lysosomes (mostly

internalized from extracellular medium)

cytosolic proteins (mostly synthetized by the cell; may also

enter from the extracellular medium via phagosomes)

Enzymes responsible Proteases from endosomes and lysosomes Cytosolic proteasome

for peptide generation (for instance,cathepsin)

Location of loading the peptide on the MHC

moleculeSpecialized vesicular compartment Endoplasmic reticulum

Molecules implicated in transporting the

peptides and loading them on the MHC

molecules

DM, invariant chainTAP (transporter associated with

antigen processing)

MHC class I Ag processing pathway  

MHC class II Ag processing pathway

Human leukocyte antigen(HLA)The human leukocyte antigen system (HLA) is the name of the major histocompatibility complex (MHC)

in humans. The super locuscontains a large number of genes related to immune system function in

humans. This group of genes reside on chromosome 6, and encode cell-surface antigen-presenting

proteins and many other genes. The HLA genes are the human versions of the MHC genes that are found

in most vertebrates (and thus are the most studied of the MHC genes). The proteins encoded by certain

genes are also known as antigens, as a result of their historic discovery as factors in organ

transplantations. The major HLA antigens are essential elements for immune function. Different classes

have different functions:

HLA antigens corresponding to MHC class I (A, B & C) present peptides from inside the cell (including

viral peptides if present). These peptides are produced from digested proteins that are broken down in

the proteasomes. The peptides are generally small polymers, about 9amino acids in length. Foreign

antigens attract killer T-cells (also called CD8 positive- or cytotoxic T-cells) that destroy cells.

HLA antigens corresponding to MHC class II (DP,DM, DOA,DOB,DQ, & DR) present antigens from

outside of the cell to T-lymphocytes. These particular antigens stimulate T-helper cells to multiply, and

these T-helper cells then stimulate antibody-producing B-cells to produce Antibodies to that specific

antigen. Self-antigens are suppressed by suppressor T-cells.

HLA antigens corresponding to MHC class III encode components of the complement system.

HLA have other roles. They are important in disease defense. They may be the cause of organ transplant

rejections. They may protect against or fail to protect (if down regulated by an infection) cancers. They

may mediate autoimmune disease (examples: type I diabetes,coeliac disease). Also, in reproduction, HLA

may be related to the individual smell of people and may be involved in mate selection .

Aside from the genes encoding the 6 major antigens, there are a large number of other genes, many

involved in immune function, located on the HLA complex. Diversity of HLA in human population is one

aspect of disease defense, and, as a result, the chance of two unrelated individuals having identical HLA

molecules on all loci is very low. Historically, HLA genes were identified as a result of the ability to

successfully transplant organs between HLA similar individuals.

HLA region of Chromosome 6

Classification

Schematic representation of MHC class I

MHC class I proteins form a functional receptor on most nucleated cells of the body.

There are 3 major and 3 minor MHC class I genes in HLA:

HLA-A

HLA-B

HLA-C

minor genes are HLA-E, HLA-F and HLA-G

β2-microglobulin binds with major and minor gene subunits to produce a heterodimer

Illustration of an HLA-DQ molecule (magenta and blue) with a bound ligand (yellow) floating on the plasma membrane of the

cell.

There are 3 major and 2 minor MHC class II proteins encoded by the HLA. The genes of the class II

combine to form heterodimeric (αβ) protein receptors that are typically expressed on the surface ofantigen

presenting cells.

Major MHC class II

HLA-DP

α-chain encoded by HLA-DPA1 locus

β-chain encoded by HLA-DPB1 locus

HLA-DQ

α-chain encoded by HLA-DQA1 locus

β-chain encoded by HLA-DQB1 locus

HLA-DR

α-chain encoded by HLA-DRA locus

4 β-chains (only 3 possible per person), encoded by HLA-

DRB1, DRB3, DRB4, DRB5 loci

The other MHC class II proteins, DM and DO, are used in the internal processing of antigens, loading the

antigenic peptides generated from pathogens onto the HLA molecules ofantigen-presenting cell.

Functions

The proteins encoded by HLAs are those on the outer part of body cells that are (effectively) unique to

that person. The immune system uses the HLAs to differentiate self cells and non-self cells. Any cell

displaying that person's HLA type belongs to that person (and therefore is not an invader).

DR protein (DRA:DRB1*0101 gene products) with bound Staphylococcal enterotoxin ligand (subunit I-C), view is top down

showing all DR amino acid residues within 5 Angstroms of the SEI peptide.

In infectious disease. When a foreign pathogen enters the body, specific cells called antigen-presenting

cells (APCs) engulf the pathogen through a process called phagocytosis. Proteins from the pathogen are

digested into small pieces (peptides) and loaded onto HLA antigens (specifically MHC class II). They are

then displayed by the antigen presenting cells for certain cells of the immune system calledT cells, which

then produce a variety of effects to eliminate the pathogen.

Through a similar process, proteins (both native and foreign, such as the proteins of viruses) produced

inside most cells are displayed on HLA antigens (specifically MHC class I) on the cell surface. Infected

cells can be recognized and destroyed by components of the immune system (specifically CD8+ T cells).

The image off to the side shows a piece of a poisonous bacterial protein (SEI peptide) bound within the

binding cleft portion of the HLA-DR1 molecule. In the illustration far below, a different view, one can see

an entire DQ with a bound peptide in a similar cleft, as viewed from the side. Disease-related peptides fit

into these 'slots' much like a hand fits into a glove or a key fits into a lock. In these configurations peptides

are presented to T-cells. The T-cells are restricted by the HLA molecules when certain peptides are within

the binding cleft. These cells have receptors that are like antibodies and each cell only recognizes a few

class II-peptide combinations. Once a T-cell recognizes a peptide within an MHC class II molecule it can

stimulate B-cells that also recognize the same molecule in their sIgM antibodies. Therefore these T-cells

help B-cells make antibodies to proteins they both recognize. There are billions of different T-cells in each

person that can be made to recognize antigens, many are removed because they recognize self antigens.

Each HLA can bind many peptides, and each person has 3 HLA types and can have 4 isoforms of DP, 4

isoforms of DQ and 4 Isoforms of DR (2 of DRB1, and 2 of DRB3,DRB4, or DRB5) for a total of 12

isoforms. In such heterozygotes it is difficult for disease related proteins to escape detection.

In graft rejection. Any cell displaying some other HLA type is "non-self" and is an invader, resulting in the

rejection of the tissue bearing those cells. Because of the importance of HLA in transplantation, the HLA

loci are among of the most frequently typed by serology or PCR relative to any other autosomal alleles.

HLA and autoimmune diseases

HLA allele Diseases with increased risk Relative risk

HLA-B27

Ankylosing spondylitis 12

Postgonococcal arthritis 14

Acute anterior uveitis 15

HLA-DR3

Autoimmune hepatitis 14

Primary Sjögren syndrome 10

Diabetes mellitus type 1 5

HLA-DR4

Rheumatoid arthritis 4

Diabetes mellitus type 1 6

HLA-DR3 and-DR4 combined Diabetes mellitus type 1 15

HLA-B47 21-hydroxylase deficiency 15

Unless else specified in boxes, then ref is: 

In autoimmunity. HLA types are inherited, and some of them are connected with autoimmune

disordersand other diseases. People with certain HLA antigens are more likely to develop certain

autoimmune diseases, such as Type I Diabetes, Ankylosing spondylitis, Celiac Disease, SLE (Systemic

Lupus Erythematosus), Myasthenia Gravis, inclusion body myositis and Sjögren's syndrome. HLA typing

has led to some improvement and acceleration in the diagnosis of Celiac Disease and Type 1 diabetes;

however for DQ2 typing to be useful it requires either high resolution B1*typing (resolving *0201 from

*0202), DQA1*typing, or DR serotyping. Current serotyping can resolve, in one step, DQ8. HLA typing in

autoimmunity is being increasingly used as a tool in diagnosis. In Celiac disease it is the only effective

means of discriminating between 1st degree relatives who are at risk from those who are not at risk, prior

to the appearance of sometimes irreversible symptoms such as allergies and secondary autoimmune

disease.

In cancer. Some HLA mediated diseases are directly involved in the promotion of cancer. Gluten

sensitive enteropathy is associated with increased prevalence of Enteropathy-associated T-cell

Lymphoma, and DR3-DQ2 homozygotes are within the highest risk group with close to 80% of gluten

sensitive EATL cases. More often, however, HLA molecules play a protective role, recognizing the

increase in antigens that were not tolerated because of low levels in the normal state. Abnormal cells may

be targeted for apoptosis mediating many cancers before clinical diagnosis. Prevention of cancer may be

a portion of heterozygous selection acting on HLA.

6th Unit AntigenAn antigen is a molecule recognized by the immune system. Originally the term came

from antibody generator and was a molecule that binds specifically to an antibody, but the term now also

refers to any molecule or molecular fragment that can be bound by a major histocompatibility

complex (MHC) and presented to a T-cell receptor. "Self" antigens are usually tolerated by the immune

system; whereas "Non-self" antigens are identified as intruders and attacked by the immune

system. Autoimmune disorders arise from the immune system reacting to its own antigens.

Similarly, an immunogen is a specific type of antigen. An immunogen is defined as a substance that is

able to provoke an adaptive immune response if injected on its own. Said another way, an immunogen is

able to induce an immune response, while an antigen is able to combine with the products of an immune

response once they are made. The overlapping concepts of immunogenicity andantigenicity are

thereby subtly different. According to a current text book:

Immunogenicity is the ability to induce a humoral and/or cell-mediated immune response

Antigenicity is the ability to combine specifically with the final products of the [immune response] (i.e.

secreted antibodies and/or surface receptors on T-cells). Although all molecules that have the property of

immunogenicity also have the property of antigenicity, the reverse is not true."

At the molecular level, an antigen is characterized by its ability to be "bound" at the antigen-binding site of

an antibody. Note also that antibodies tend to discriminate between the specific molecular

structures presented on the surface of the antigen (as illustrated in the Figure). Antigens are

usually proteins or polysaccharides. This includes parts (coats, capsules, cell walls, flagella, fimbrae, and

toxins) of bacteria, viruses, and other microorganisms. Lipids and nucleic acids are antigenic only when

combined with proteins and polysaccharides. Non-microbial exogenous (non-self) antigens can include

pollen, egg white, and proteins from transplanted tissues and organs or on the surface of transfused

blood cells. Vaccines are examples of immunogenic antigens intentionally administered to induceacquired

immunity in the recipient.

Cells present their immunogenic-antigens to the immune system via a histocompatibility molecule.

Depending on the antigen presented and the type of the histocompatibility molecule, several types

of immune cells can become activated.

Each antibody binds to a specific antigen; an interaction similar to a lock and key.

Related concepts

Epitope - The distinct molecular surface features of an antigen capable of being bound by an

antibody (a.k.a. antigenic determinant). Antigenic molecules, normally being "large" biological

polymers, usually present several surface features that can act as points of interaction for specific

antibodies. Any such distinct molecular feature constitutes an epitope. Most antigens therefore have

the potential to be bound by several distinct antibodies, each of which is specific to a particular

epitope. Using the "lock and key" metaphor, the antigen itself can be seen as a string of keys - any

epitope being a "key" - each of which can match a different lock. Different antibody idiotypes, each

having distinctly formedcomplementarity determining regions, correspond to the various "locks" that

can match "the keys" (epitopes) presented on the antigen molecule.

Allergen - A substance capable of causing an allergic reaction. The (detrimental) reaction may

result after exposure via ingestion, inhalation, injection, or contact with skin.

Superantigen - A class of antigens which cause non-specific activation of T-cells resulting in

polyclonal T cell activation and massive cytokine release.

Tolerogen - A substance that invokes a specific immune non-responsiveness due to

its molecular form. If its molecular form is changed, a tolerogen can become an immunogen.

Immunoglobulin binding protein - These proteins are capable of binding to antibodies at

positions outside of the antigen-binding site. That is, whereas antigens are the "target" of antibodies,

immunoglobulin binding proteins "attack" antibodies. Protein A, protein G and protein L are examples

of proteins that strongly bind to various antibody isotypes.

Antigens can be classified in order of their class.Exogenous antigens

Exogenous antigens are antigens that have entered the body from the outside, for example

by inhalation, ingestion, or injection. The immune system's response to exogenous antigens is

often subclinical. By endocytosis or phagocytosis, exogenous antigens are taken into the antigen-

presenting cells (APCs) and processed into fragments. APCs then present the fragments to T

helper cells (CD4+) by the use of class II histocompatibility molecules on their surface. Some T

cells are specific for the peptide:MHC complex. They become activated and start to

secrete cytokines. Cytokines are substances that can activate cytotoxic T lymphocytes (CTL),

antibody-secreting B cells, macrophages, and other particles.

Some antigens start out as exogenous antigens, and later become endogenous (for example,

intracellular viruses). Intracellular antigens can be released back into circulation upon the

destruction of the infected cell, again.

Endogenous antigens

Endogenous antigens are antigens that have been generated within previously normal cells as a

result of normal cell metabolism, or because of viral or intracellular bacterial infection. The

fragments are then presented on the cell surface in the complex with MHC class I molecules. If

activated cytotoxic CD8+ T cells recognize them, the T cells begin to secrete varioustoxins that

cause the lysis or apoptosis of the infected cell. In order to keep the cytotoxic cells from killing

cells just for presenting self-proteins, self-reactive T cells are deleted from the repertoire as a

result of tolerance (also known as negative selection). Endogenous antigens

include xenogenic (heterologous), autologous and idiotypic or allogenic (homologous) antigens.

Autoantigens

An autoantigen is usually a normal protein or complex of proteins (and sometimes DNA or RNA)

that is recognized by the immune system of patients suffering from a specificautoimmune

disease. These antigens should, under normal conditions, not be the target of the immune

system, but, due to mainly genetic and environmental factors, the normalimmunological

tolerance for such an antigen has been lost in these patients.

Tumor antigens

Tumor antigens or neoantigens are[citation needed] those antigens that are presented by MHC I or MHC

II molecules on the surface of tumor cells. These antigens can sometimes be presented by tumor cells

and never by the normal ones. In this case, they are called tumor-specific antigens (TSAs) and, in

general, result from a tumor-specific mutation. More common are antigens that are presented by tumor

cells and normal cells, and they are called tumor-associated antigens (TAAs). Cytotoxic T

lymphocytes that recognize these antigens may be able to destroy the tumor cells before they proliferate

or metastasize.

Tumor antigens can also be on the surface of the tumor in the form of, for example, a mutated receptor, in

which case they will be recognized by B cells.

Nativity

A native antigen is an antigen that is not yet processed by an APC to smaller parts. T cells cannot bind

native antigens, but require that they be processed by APCs, whereas B cellscan be activated by native

ones.

Antigenic Specificity

Antigen(ic) specificity is the ability of the host cells to recognize an antigen specifically as a unique

molecular entity and distinguish it from another with exquisite precision. Antigen specificity is due primarily

to the side-chain conformations of the antigen. It is a measurement, although the degree of specificity

may not be easy to measure, and need not be linear or of the nature of a rate-limited step or equation.[

ANTIGEN-ANTIBODY REACTIONS AND SELECTED TESTS

A reaction that occurs when an antigen combines with a corresponding antibody to produce an immune complex. A substance that induces the immune system to form a corresponding antibody is called an immunogen. All immunogens are also antigens because they react with corresponding antibodies; however, an antigen may not be able to induce the formation of an antibody and therefore may not be an immunogen. For instance, lipids and all low-molecular-weight substances are not immunogenic. However, many such substances, termed haptens, can be attached to immunogens, called carriers, and the complex then acts as a new immunogen.

A molecule of antibody has two identical binding sites for one antigen or more, depending on its class. Each site is quite small and can bind only a comparably small portion of the surface of the antigen, which is termed an epitope. The specificity of an antibody for an antigen depends entirely upon the possession of the appropriate epitope by an antigen. The binding site on the antibody and the epitope on the antigen are complementary regions on the surface of the respective molecules which interlock in the antigen-antibody reaction. The intensity with which

an antibody binds to the antigen depends on the exactitude of the fit between the respective binding site and epitope, as well as some inherent characteristics of the reacting molecules and factors in the environment. The epitope must be continuous spatially, but not structurally: in other words, if the molecule of the antigen consists of several chains, then an epitope may be formed by adjacent regions on two different chains, as well as by adjacent regions on the same chain. If the epitope is now modified either chemically (for example, by altering the hapten) or physically (for example, by causing the chains to separate), then its fit in the binding site will be altered or abolished, and the antigen will react with the antibody either less strongly or not at all.

The immune complex formed in the reaction consists of closely apposed, but still discrete, molecules of antigen and antibody. Therefore, the immune complex can dissociate into the original molecules. The proportion of the dissociated, individual molecules of antigen and antibody to those of the immune complex clearly depends on the intensity of the binding. These proportions can be measured in a standardized procedure, so that the concentration of antigen [Ag], antibody [Ab], and the immune complex [AgAb] becomes known. A fraction is then calculated and called either the dissociation constant or the association constant. The magnitude of either of these constants can be used subsequently to assess the intensity of the antigen-antibody reaction.

Only one epitope of its kind generally occurs on each molecule of antigen, other than that which consists of multiple, identical units, though many epitopes of different configuration are possible. Particles, however, either natural ones such as cells or suitably treated artificial ones made of, for example, latex or glass, typically carry multiple identical epitopes, as well as nonidentical ones, because their surfaces contain many molecules of the same antigen. Immune complexes comprising many molecules eventually reach sufficient size to scatter light, at which point they can be detected by nephelometry or turbidimetry; if their growth continues, they become visible as precipitates, which can also be assayed by such methods as immunodiffusion. Since particles typically carry many molecules of antigen, they can be, in principle, aggregated and the reaction can be detected by inspection. Antigen-antibody reactions can also be detected at very low concentration of reactants through special techniques such as immunofluorescence and radioimmunoassay.

The reaction between antigen and antibody is followed by a structural change in the remainder of the antibody molecule. The change results in the appearance of previously hidden regions of the molecule. Some of these hidden regions have specific functions, such as binding complement. Fixation of complement by immune complexes has been used to detect and measure antigen-antibody reactions.

The chief use of antigen-antibody reactions has been in the determination of blood groups for transfusion, serological ascertainment of exposure to infectious agents, and development of immunoassays for the quantification of various substances.

NATURE OF ANTIGEN-ANTIBODY REACTIONS

Lock and Key Concept

The combining site of an antibody is located in the Fab portion of the molecule and is constructed from thehypervariable regions of the heavy and light chains. X-Ray crystallography studies of antigen-antibody

interactions show that the antigenic determinant nestles in a cleft formed by the combining site of the antibody(see the below figure). Thus, our concept of antigen-antibody reactions is one of a key (i.e. the antigen) which fits into a lock (i.e. the antibody).

Non-covalent Bonds

The bonds that hold the antigen to the antibody combining site are all non-covalent in nature. These includehydrogen bonds, electrostatic bonds, Van der Waals forces and hydrophobic bonds. Multiple bonding between the antigen and the antibody ensures that the antigen will be bound tightly to the

antibody.

Reversibility

 Since antigen-antibody reactions occur via non-covalent bonds, they are by their nature reversible.

AFFINITY AND AVIDITY

Affinity

Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody. It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site of the antibody as illustrated in Figure a.

Affinity is the equilibrium constant that describes the antigen-antibody reaction as illustrated in Figure b. Most antibodies have a high affinity for their antigens.

 

Figure a

Figure b

Avidity

Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies. Avidity is influenced by both the valence of the antibody and the valence of the antigen. Avidity is more than the sum of the individual affinities. This is illustrated in Figure c.

To repeat, affinity refers to the strength of binding between a single antigenic determinant and an individual antibody combining site whereas avidity refers to the overall strength of binding between multivalent antigens and antibodies.

Figure c

SPECIFICITY AND CROSS REACTIVITY

Specificity

Specificity refers to the ability of an individual antibody combining site to react with only one antigenic determinant or the ability of a population of antibody molecules to react with only one antigen. In general, there is a high degree of specificity in antigen-antibody reactions. Antibodies can distinguish differences in:

The primary structure of an antigen Isomeric forms of an antigen

Secondary and tertiary structure of an antigen

Cross reactivity

Cross reactivity refers to the ability of an individual antibody combining site to react with more than one antigenic determinant or the ability of a population of antibody molecules to react with more than one antigen. Figure d illustrates how cross reactions can arise. Cross reactions arise because the cross reacting antigen shares anepitope in common with the immunizing antigen or because it has an epitope which is structurally similar to one on the immunizing antigen (multispecificity).

Figure d

TESTS FOR ANTIGEN-ANTIBODY REACTIONS

Factors affecting measurement of antigen-antibody reactions

The only way that one knows that an antigen-antibody reaction has occurred is to have some means of directly or indirectly detecting the complexes formed between the antigen and antibody. The ease with which one can detect antigen-antibody reactions will depend on a number of factors.

Affinity The higher the affinity of the antibody for the antigen, the more stable will be the interaction. Thus, the

ease with which one can detect the interaction is enhanced.

Avidity Reactions between multivalent antigens and multivalent antibodies are more stable and thus easier to detect.

Antigen to antibody ratio The ratio between the antigen and antibody influences the detection of antigen-antibody complexes because the size of the complexes formed is related to the concentration of the antigen and antibody. This is depicted in Figure e.

Figure e

Physical form of the antigen The physical form of the antigen influences how one detects its reaction with an antibody. If the antigen is a particulate, one generally looks for agglutination of the antigen by the antibody. If the antigen is soluble one generally looks for the precipitation of the antigen after the production of large insoluble antigen-antibody complexes.

Agglutination TestsAgglutination/Hemagglutination When the antigen is particulate, the reaction of an antibody with the antigen can be detected by

agglutination (clumping) of the antigen. The general term agglutinin is used to describe antibodies that

agglutinate particulate antigens. When the antigen is an erythrocyte the term hemagglutination is used. All

antibodies can theoretically agglutinate particulate antigens but IgM, due to its high valence, is particularly

good agglutinin and one sometimes infers that an antibody may be of the IgM class if it is a good

agglutinating antibody.

Qualitative agglutination test Agglutination tests can be used in a qualitative manner to assay for the presence of an antigen or an antibody. The antibody is mixed with the particulate antigen and a positive test is indicated by the agglutination of the particulate antigen. (Figure f).

For example, a patient's red blood cells can be mixed with antibody to a blood group antigen to determine a person's blood type. In a second example, a patient's serum is mixed with red blood cells of a known blood type to assay for the presence of antibodies to that blood type in the patient's serum.

Figure f

Quantitative agglutination test Agglutination tests can also be used to measure the level of antibodies to particulate antigens. In this test, serial dilutions are made of a sample to be tested for antibody and then a fixed number of red blood cells or bacteria or other such particulate antigen is added. Then the maximum dilution that gives agglutination is determined. The maximum dilution that gives visible agglutination is called the titer. The results are reported as the reciprocal of the maximal dilution that gives visible agglutination. Figure h illustrates a quantitative hemagglutination test.

Prozone effect - Occasionally, it is observed that when the concentration of antibody is high (i.e. lower dilutions), there is no agglutination and then, as the sample is diluted, agglutination occurs (See Patient 6 in Figure g). The lack of agglutination at high concentrations of antibodies is called the prozone effect. Lack of agglutination in the prozone is due to antibody excess resulting in very small complexes that do not clump to form visible agglutination.

Figure g

Applications of agglutination tests

i. Determination of blood types or antibodies to blood group antigens.

ii. To assess bacterial infections

e.g. A rise in titer of an antibody to a particular bacterium indicates an infection with that bacterial type. N.B. a fourfold rise in titer is generally taken as a significant rise in antibody titer.

Practical considerations Although the test is easy to perform, it is only semi-quantitative.

Passive hemagglutination The agglutination test only works with particulate antigens. However, it is possible to coat erythrocytes

with a soluble antigen (e.g. viral antigen, a polysaccharide or a hapten) and use the coated red blood cells

in an agglutination test for antibody to the soluble antigen (Figure h). This is called passive

hemagglutination. The test is performed just like the agglutination test. Applications include detection of

antibodies to soluble antigens and detection of antibodies to viral antigens.

Figure h

Coomb's Test (Antiglobulin Test)Direct Coomb's Test When antibodies bind to erythrocytes, they do not always result in agglutination. This can result from the

antigen/antibody ratio being in antigen excess or antibody excess or in some cases electrical charges on

the red blood cells preventing the effective cross linking of the cells. These antibodies that bind to but do

not cause agglutination of red blood cells are sometimes referred to as incomplete antibodies. In no way

is this meant to indicate that the antibodies are different in their structure, although this was once thought

to be the case. Rather, it is a functional definition only. In order to detect the presence of non-

agglutinating antibodies on red blood cells, one simply adds a second antibody directed against the

immunoglobulin (antibody) coating the red cells. This anti-immunoglobulin can now cross link the red

blood cells and result in agglutination. This test is illustrated in Figure k and is known as the Direct

Coomb's test. 

Figure i

Indirect Coomb's Test If it is necessary to know whether a serum sample has antibodies directed against a particular red blood

cell and you want to be sure that you also detect potential non- agglutinating antibodies in the sample,

an Indirect Coomb's test is performed (Figure j). This test is done by incubating the red blood cells with

the serum sample, washing out any unbound antibodies and then adding a second anti-immunoglobulin

reagent to cross link the cells.

Figure j

Applications These include detection of anti-rhesus factor (Rh) antibodies. Antibodies to the Rh factor generally do not

agglutinate red blood cells. Thus, red cells from Rh+ children born to Rh- mothers, who have anti-Rh

antibodies, may be coated with these antibodies. To check for this, a direct Coombs test is performed. To

see if the mother has anti-Rh antibodies in her serum an Indirect Coombs test is performed. 

Hemagglutination Inhibition 

The agglutination test can be modified to be used for the measurement of soluble antigens. This test is

called hemagglutination inhibition. It is called hemagglutination inhibition because one measures the

ability of soluble antigen to inhibit the agglutination of antigen-coated red blood cells by antibodies. In this

test, a fixed amount of antibodies to the antigen in question is mixed with a fixed amount of red blood cells

coated with the antigen (see passive hemagglutination above). Also included in the mixture are different

amounts of the sample to be analyzed for the presence of the antigen. If the sample contains the antigen,

the soluble antigen will compete with the antigen coated on the red blood cells for binding to the

antibodies, thereby inhibiting the agglutination of the red blood cells. as illustrated in Figure k.

By serially diluting the sample, you can quantitate the amount of antigen in your unknown sample by its

titer. This test is generally used to quantitate soluble antigens and is subject to the same practical considerations as the agglutination test.

Figure k

Precipitation tests

Radial Immunodiffusion (Mancini) In radial immunodiffusion antibody is incorporated into the agar gel as it is poured and different dilutions of the antigen are placed in holes punched into the agar. As the antigen diffuses into the gel, it reacts with the antibody and when the equivalence point is reached a ring of precipitation is formed as illustrated in Figure l.

The diameter of the ring is proportional to the log of the concentration of antigen since the amount of antibody is constant. Thus, by running different concentrations of a standard antigen one can generate a standard cure from which one can quantitate the amount of an antigen in an unknown sample. Thus, this is a quantitative test. If more than one ring appears in the test, more than one antigen/antibody reaction has occurred. This could be due to a mixture of antigens or antibodies. This test is commonly used in the clinical laboratory for the determination of immunoglobulin levels in patient samples.

Figure l

Immunoelectrophoresis In immunoelectrophoresis, a complex mixture of antigens is placed in a well punched out of an agar gel and the antigens are electrophoresed so that the antigen are separated according to their charge. After electrophoresis, a trough is cut in the gel and antibodies are added. As the antibodies diffuse into the agar, precipitin lines are produced in the equivalence zone when an antigen/antibody reaction occurs as illustrated in Figure m.

This tests is used for the qualitative analysis of complex mixtures of antigens, although a crude measure of quantity (thickness of the line) can be obtained. This test is commonly used for the analysis of components in a patient' serum. Serum is placed in the well and antibody to whole serum in the trough. By comparisons to normal serum, one can determine whether there are deficiencies on one or more serum components or whether there is an overabundance of some serum component (thickness of the line). This test can also be used to evaluate purity of isolated serum proteins.

Figure m

Countercurrent electrophoresis In this test the antigen and antibody are placed in wells punched out of an agar gel and the antigen and antibody are electrophoresed into each other where they form a precipitation line as illustrated in Figure n. This test only works if conditions can be found where the antigen and antibody have opposite charges. This test is primarily qualitative, although from the thickness of the band you can get some measure of quantity. Its major advantage is its speed.

Figure n

Radioimmunoassay (RIA)/Enzyme Linked Immunosorbent Assay (ELISA)

Radioimmunoassays (RIA) are assays that are based on the measurement of radioactivity associated

with immune complexes. In any particular test, the label may be on either the antigen or the antibody.

Enzyme Linked Immunosorbent Assays (ELISA) are those that are based on the measurement of an

enzymatic reaction associated with immune complexes. In any particular assay, the enzyme may be

linked to either the antigen or the antibody.

Competitive RIA/ELISA for Ag Detection The method and principle of RIA and ELISA for the measurement of antigen is shown in Figure p. By using known amounts of a standard unlabeled antigen, one can generate a standard curve relating radioactivity (cpm) (Enzyme) bound versus amount of antigen. From this standard curve, one can determine the amount of an antigen in an unknown sample.

The key to the assay is the separation of the immune complexes from the remainder of the components. This has been accomplished in many different ways and serves as the basis for the names given to the assay:

Figure p

Precipitation with ammonium sulphate Ammonium sulphate (33 - 50% final concentration) will precipitate immunoglobulins but not many

antigens. Thus, this can be used to separate the immune complexes from free antigen. This has been called the Farr Technique

Anti-immunoglobulin antibody The addition of a second antibody directed against the first antibody can result in the precipitation of the immune complexes and thus the separation of the complexes from free antigen.

Immobilization of the Antibody The antibody can be immobilized onto the surface of a plastic bead or coated onto the surface of a plastic plate and thus the immune complexes can easily be separated from the other components by simply washing the beads or plate (Figure q). This is the most common method used today and is referred to as Solid phase RIA or ELISA. In the clinical laboratory, competitive RIA and ELISA are commonly used to quantitate serum proteins, hormones, drugs metabolites.

Figure q

Non-competitive RIA/ELISA for Ag or Ab Non-competitive RIA and ELISAs are also used for the measurement of antigens and antibodies. In

Figure r, the bead is coated with the antigen and is used for the detection of antibody in the unknown

sample. The amount of labeled second antibody bound is related to the amount of antibody in the

unknown sample. This assay is commonly employed for the measurement of antibodies of the IgE class

directed against particular allergens by using a known allergen as antigen and anti-IgE antibodies as the

labeled reagent. It is called the RAST test (radioallergosorbent test). In Figure s, the bead is coated with

antibody and is used to measure an unknown antigen. The amount of labeled second antibody that binds

is proportional to the amount of antigen that bound to the first antibody.

Figure r

Figure s

Tests for Cell Associated Antigens

Immunofluorescence Immunofluorescence is a technique whereby an antibody labeled with a fluorescent molecule (fluorescein or rhodamine or one of many other fluorescent dyes) is used to detect the presence of an antigen in or on a cell or tissue by the fluorescence emitted by the bound antibody.

Direct Immunofluorescence In direct immunofluorescence, the antibody specific to the antigen is directly tagged with the fluorochrome (Figure t)

Figure t

Indirect Immunofluorescence In indirect immunofluorescence, the antibody specific for the antigen is unlabeled and a second anti-immunoglobulin antibody directed toward the first antibody is tagged with the fluorochrome (Figure u). Indirect fluorescence is more sensitive than direct immunofluorescence since there is amplification of the signal..

Figure u

Flow CytometryFlow cytometry is commonly used in the clinical laboratory to identify and enumerate cells bearing a particular antigen. Cells in suspension are labeled with a fluorescent tag by either direct or indirect immunofluorescence. The cells are then analyzed on the flow cytometer.

Figure w. illustrates the principle of flow cytometry. In a flow cytometer, the cells exit a flow cell and are illuminated with a laser beam. The amount of laser light that is scattered off the cells as they passes through the laser can be measured, which gives information concerning the size of the cells. In addition, the laser can excite the fluorochrome on the cells and the fluorescent light emitted by the cells can be measured by one or more detectors.

The type of data that is obtained from the flow cytometer is shown in Figure w. In a one parameter

histogram, increasing amount of fluorescence (e.g. green fluorescence) is plotted on the x axis and the

number of cells exhibiting that amount of fluorescence is plotted on the y axis. The fraction of cells that

are fluorescent can be determined by integrating the area under the curve. In a two parameter histogram,

the x axis is one parameter (e.g. red fluorescence) and the y axis is the second parameter (e.g. green

fluorescence). The number of cells is indicated by the contour and the intensity of the color.

Figure v

Figure w

Complement FixationAntigen/antibody complexes can also be measured by their ability to fix complement because an antigen/antibody complex will "consume" complement if it is present, whereas free antigens or antibodies do not. Tests for antigen/antibody complexes that rely on the consumption of complement are termed complement fixation tests and are used to quantitate antigen/antibody reactions. This test will only work with complement fixing antibodies (IgG and IgM are best).

The principle of the complement fixation test is illustrated in Figure x. Antigen is mixed with the test serum to be assayed for antibody and antigen/antibody complexes are allowed to form. A control tube in which no antigen is added is also prepared. If no antigen/antibody complexes are present in the tube, none of the complement will be fixed. However, if antigen/antibody complexes are present, they will fix complement and thereby reduce the amount of complement in the tube. After allowing complement fixation by any antigen/antibody complexes, a standard amount of red blood cells, which have been pre-coated with anti-erythrocyte antibodies is added. The amount of antibody-coated red blood cells is predetermined to be just enough to completely use up all the complement initially added, if it were still there. If all the complement was still present (i.e. no antigen/antibody complexes formed between the

antigen and antibody in question), all the red cells will be lysed. If antigen/antibody complexes are formed between the antigen and antibody in question, some of the complement will be consumed and, thus, when the antibody-coated red cells are added not all of them will lyse. By simply measuring the amount of red cell lysis by measuring the release of hemoglobin into the medium, one can indirectly quantitate antigen/antibody complexes in the tube. Complement fixation tests are most commonly used to assay for antibody in a test sample but they can be modified to measure antigen.

Figure x

7th Unit ImmunizationImmunization, or immunisation, is the process by which an individual's immune system becomes

fortified against an agent (known as theimmunogen).

When an immune system is exposed to molecules that are foreign to the body (non-self), it will

orchestrate an immune response, but it can also develop the ability to quickly respond to a subsequent

encounter (through immunological memory). This is a function of the adaptive immune system. Therefore,

by exposing an animal to an immunogen in a controlled way, its body can learn to protect itself: this is

called active immunization.

The most important elements of the immune system that are improved by immunization are the B

cells (and the antibodies they produce) andT cells. Memory B cell and memory T cells are responsible for

a swift response to a second encounter with a foreign molecule. Passive immunization is when these

elements are introduced directly into the body, instead of when the body itself has to make these

elements.

Immunization can be done through various techniques, most commonly vaccination. Vaccines

against microorganisms that cause diseasescan prepare the body's immune system, thus helping to fight

or prevent an infection. The fact that mutations can cause cancer cells to produce proteins or other

molecules that are unknown to the body forms the theoretical basis for therapeutic cancer vaccines. Other

molecules can be used for immunization as well, for example in experimental vaccines

against nicotine (NicVAX) or the hormone ghrelin (in experiments to create an obesity vaccine).

Passive and active immunization

Immunization can be achieved in an active or passive fashion: vaccination is an active form of

immunization.

Passive immunization

Passive immunization is where pre-synthesized elements of the immune system are transferred to a

person so that the body does not need to produce these elements itself. Currently,antibodies can be used

for passive immunization. This method of immunization begins to work very quickly, but it is short lasting,

because the antibodies are naturally broken down, and if there are no B cells to produce more antibodies,

they will disappear.

Passive immunization occurs physiologically, when antibodies are transferred from mother

to fetus during pregnancy, to protect the fetus before and shortly after birth.

Artificial passive immunization is normally administered by injection and is used if there has been a recent

outbreak of a particular disease or as an emergency treatment for toxicity (for example, for tetanus). The

antibodies can be produced in animals ("serum therapy") although there is a high chance of anaphylactic

shock because of immunity against animal serum itself. Thus, humanized antibodies produced in

vitro by cell culture are used instead if available.

Immunity can be acquired, without the immune system being challenged with an antigen. This is done by transfer of serum or gamma-globulins from an immune donor to a non-immune individual. Alternatively, immune cells from an immunized individual may be used to transfer immunity. Passive immunity may be acquired naturally or artificially.

Naturally acquired passive immunityImmunity is transferred from mother to fetus through placental transfer of IgG or colostral transfer of IgA.

Artificially acquired passive immunityImmunity is often artificially transferred by injection with gamma-globulins from other individuals or gamma-globulin from an immune animal. Passive transfer of immunity with immune globulins or gamma-globulins is practiced in numerous acute situations of infections (diphtheria, tetanus, measles, rabies, etc.), poisoning (insects, reptiles, botulism), and as a prophylactic measure (hypogammaglobulinemia). In

these situations, gamma-globulins of human origin are preferable although specific antibodies raised in other species are effective and used in some cases (poisoning, diphtheria, tetanus, gas gangrene, botulism). While this form of immunization has the advantage of providing immediate protection, heterologous gamma-globulins are effective for only a short duration and often result in pathological complications (serum sickness) and anaphylaxis. Homologous immunoglobulins carry the risk of transmitting hepatitis and HIV.

Passive transfer of cell-mediated immunity can also be accomplished in certain diseases (cancer, immunodeficiency). However, it is difficult to find histocompatible (matched) donors and there is severe risk of graft versus host disease.

Active immunizationActive immunization entails the introduction of a foreign molecule into the body, which causes the body

itself to generate immunity against the target. This immunity comes from the T cells and the B cells with

their antibodies.

Active immunization can occur naturally when a person comes in contact with, for example, a microbe. If

the person has not yet come into contact with the microbe and has no pre-made antibodies for defense

(like in passive immunization), the person becomes immunized. The immune system will eventually

create antibodies and other defenses against the microbe. The next time, the immune response against

this microbe can be very efficient; this is the case in many of the childhood infections that a person only

contracts once, but then is immune.

Artificial active immunization is where the microbe, or parts of it, are injected into the person before they

are able to take it in naturally. If whole microbes are used, they are pre-treated,Attenuated vaccine.

Naturally acquired active immunityExposure to different pathogens leads to sub-clinical or clinical infections which result in a protective immune response against these pathogens.

Artificially acquired active immunityImmunization may be achieved by administering live or dead pathogens or their components. Vaccines used for active immunization consist of live (attenuated) organisms, killed whole organisms, microbial components or secreted toxins (which have been detoxified).  

Live vaccinesThe first live vaccine was cowpox virus introduced by Edward Jenner as a vaccine for smallpox (see vaccine section); however, variolation,  innoculation using pus from a patient with a mild case of smallpox has been in use for over a thousand years.

Live vaccines are used against a number of viral infections (polio (Sabin vaccine), measles, mumps, rubella, chicken pox, hepatitis A, yellow fever, etc.). The only example of live bacterial vaccine is one against tuberculosis (Mycobacterium bovis: Bacille Calmette-Guerin vaccine: BCG). Whereas many studies have shown the efficacy of BCG vaccine, a number of studies also cast doubt on its benefits.

Live vaccines normally produce self-limiting non-clinical infections and lead to subsequent immunity, both humoral and cell-mediated, the latter being essential for intracellular pathogens. However, they carry a serious risk of causing overt disease in immunocompromised individuals. Furthermore, since live vaccines are often attenuated (made less pathogenic) by passage in animal or thermal mutation, they can revert to their pathogenic form and cause serious illness. It is for this reason, polio live (Sabin) vaccine, which was used for many years, has been replaced in many countries by the inactivated (Salk) vaccine.

Killed vaccinesWhile live vaccines normally produce only self-limiting non-clinical infections and subsequent immunity, they carry a serious risk of causing overt disease in immunocompromised individuals. Killed (heat, chemical or UV irradiation) viral vaccines include those for polio (Salk vaccine), influenza, rabies, influenza, rabies, etc. Most bacterial vaccines are killed organisms ( typhoid, cholera, plague, pertussis, etc.). Other bacterial vaccines utilize their cell wall components (haemophilus, pertussis, meningococcus, pneumococcus, etc.). Some viral vaccines (hepatitis-B, rabies, etc.) consist of antigenic proteins cloned into a suitable vector (e.g., yeast). When the pathogenic mechanism of an agent involves a toxin, a modified form of the toxin (toxoid) is used as a vaccine (e.g., diphtheria, tetanus, cholera). These subunit vaccines are designed to reduce the toxicity problems. Each type of vaccine has its own advantages and disadvantages.

Sub-unit vaccinesSome vaccines consist of subcomponents of the pathogenic organisms, usually proteins or polysaccharides. Since polysaccharides are relatively weak T-independent antigens, and produce only IgM responses without immunologic memory, they are made more immunogenic and T-dependent by conjugation with proteins (e.g., haemophilus, meningococcus, pneumococcus, etc.). Hepatitis-B, rabies vaccines consist of antigenic proteins cloned into a suitable vector (e.g., yeast). These subunit vaccines are designed to reduce the problems of toxicity and risk of infection. When the pathogenic mechanism of an agent involves a toxin, a modified form of the toxin (toxoid) is used as vaccine (e.g., diphtheria, tetanus, etc.). Toxoids, although lose their toxicity, they remains immunogenic.

Other novel vaccinesA number of novel approaches to active immunization are in the investigative stage and are used only experimentally. These include anti-idiotype antibodies, DNA vaccines and immunodominant peptides (recognized by the MHC molecules) and may be available in the future. Anti-idiotype antibodies against polysaccharide antibody produce long lasting immune responses with immunologic memory. Viral peptide genes cloned into vectors, when injected transfect host cells and consequently produce a response similar to that produced against live-attenuated viruses (both cell-mediated and humoral). Immunodominant peptides are simple and easy to prepare and, when incorporated into MHC polymers,

can provoke both humoral and cell mediated responses.

Adjuvants Vaccines typically contain one or more adjuvants, used to boost the immune response.Weaker antigens may be rendered more immunogenic by the addition of other chemicals. Such chemicals are known as adjuvants. There are many biological and chemical substances that have been used in experimental conditions (Table 1). However, only Aluminum salts (alum) are approved for human use and it is

incorporated in DTP vaccine. Furthermore, pertussis itself has adjuvant effects. Adjuvants used experimentally include mixtures of oil and detergents, with (Freund’s complete adjuvant) or without certain bacteria (Freund’s incomplete adjuvant). Bacteria most often used in an adjuvant are Mycobacteria (BCG) and Nocardia. In some instance sub-cellular fractions of these bacteria can also be used effectively as adjuvants. Newer adjuvant formulations include synthetic polymers and oligonucleotides. Most adjuvants recognize TOLL-like receptors thus activating mononuclear phagocytes and inducing selective cytokines that can enhance Th1 or Th2 responses, depending on the nature of the adjuvant.

The protective immunity conferred by a vaccine may be life-long (measles, mumps, rubella, small pox, tuberculosis, yellow fever, etc.) or may last as little as a few months (cholera). The primary immunization may be given at the age of 2-3 months (diphtheria, pertussis, tetanus, polio), or 13-15 months (mumps, measles, rubella).  The currently recommended schedule of routine immunization in the USA (recommended by CDC and AIP) is summarized in Table 2.  This schedule is revised on yearly basis or as need by the CDC Advisory Committee on Immunization Practice (AICP). 

Table 1.  Selected adjuvants in clinical or experimental useAdjuvant type human use Experimental only

Salts:

aluminum hydroxide, aluminum phosphate-calcium phosphate

YesYes Slow release of antigen,

TLR interaction and cytokine induction

Beryllium hydroxide 

No 

Synthetic particles:

Liposomes, ISCOMs, polylactates

 

 

NoNo

 

Slow release of antigen

Polynucleotides:

CpG and others 

No*TLR interaction and cytokine induction

Bacterial products:

B.pertussisYes TLR interaction and

cytokine inductionM. bovis (BCG and others) NoMineral oils No Antigen depot

Cytokines:

IL-1, IL-2, IL12, IFN-γ, etc.

 No*

Activation and differentiation of T- and B cells and APC.

Prophylactic versus therapeutic immunizationMost vaccines are given prophylactic ally, i.e., prior to exposure to the pathogen. However, some vaccines can be administered therapeutically , i.e., post exposure (e.g., rabies virus). The effectiveness of this mode of immunization depends on the rate of replication of the pathogen, incubation period and pathogenic mechanism. For this reason, only a booster shot with tetanus is sufficient if the exposure to the pathogen is within less than 10 years and if the exposure is minimal (wounds are relative superficial). In a situation where pathogen has a short incubation period, the pathogenic mechanism is such that only a small amount of pathogenic molecules could be fatal (e.g., tetanus and diphtheria) and/or bolus of

infection is relatively large, both passive and active post exposure immunization are essential. Passive prophylactic immunization is also normal in cases of defects in the immune system, such as hypogammaglobulinemias.

Adverse effects of immunizationActive immunization may cause fever, malaise and discomfort. Some vaccine may also cause joint pains or arthritis (rubella), convulsions, sometimes fatal (pertussis), or neurological disorders (influenza). Allergies to egg may develop as a consequence of viral vaccines produced in egg (measles, mumps, influenza, yellow fever). Booster shots result in more pronounced inflammatory effects than the primary immunization. The noticeable and serious side effects documented have been those following the DTP vaccine (Table 3). Most of these were attributable to the whole pertussis component of the vaccine and have been eliminated since the use of the acellular pertussis preparation.

Table 3. Approximate rates of adverse event occurring within 48 hours DTP vaccination

Event Frequency

Local

redness, swelling, pain 1 in 2-3 doses

Mild/moderate systemic

fever, drowsiness, fretfulness 1 in 2-3 doses

vomiting, anorexia 1 in 5-15 doses

More serious systemic

persistent crying, fever 1 in 100-300 doses

collapse, convulsions 1 in 1750 doses

acute encephalopathy 1 in 100,000 doses

permanent neurological deficit 1 in 300,000 doses

8th Unit Hybridoma technology

Hybridoma technology is a technology of forming hybrid cell lines (called hybridomas) by fusing a specific antibody-producing B cell with a myeloma (B cell cancer) cell that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis. The antibodies produced by the hybridoma are all of a single specificity and are thereforemonoclonal antibodies (in contrast to polyclonal antibodies). The production of monoclonal antibodies was invented by Cesar Milstein, Georges J. F. Köhler and Niels Kaj Jerne in 1975.

Monoclonal antibody

Monoclonal antibodies (mAb or moAb) are monospecific antibodies that are the same because they

are made by identical immune cells that are all clones of a unique parent cell.

Given almost any substance, it is possible to create monoclonal antibodies that specifically bind to that

substance; they can then serve to detect or purify that substance. This has become an important tool

in biochemistry, molecular biology and medicine. When used as medications, the non-proprietary drug

name ends in -mab (see "Nomenclature of monoclonal antibodies").

A general representation of the methods used to produce monoclonal antibodies.

Production method of monoclonal antibody

(1) Immunisation of a mouse

(2) Isolation of B cells from the spleen

(3) Cultivation of myeloma cells

(4) Fusion of myeloma and B cells

(5) Separation of cell lines

(6) Screening of suitable cell lines

(7) in vitro (a) or in vivo (b) multiplication

(8) Harvesting

Laboratory animals (mammals, e.g. mice) are first exposed to an antigen to which we are interested in

isolating an antibody against. Usually this is done by a series of injections of the antigen in question, over

the course of several weeks. Once splenocytes are isolated from the mammal's spleen, the B cells are

fused with immortalized myeloma cells. The myeloma cells are selected beforehand to ensure they are

not secreting antibody themselves and that they lack the hypoxanthine-guanine

phosphoribosyltransferase (HGPRT) gene, making them sensitive to the HAT medium (see below). The

fusion is accomplished using polyethylene glycol or the Sendai virus. It is performed by making the cell

membranes more permeable.

Fused cells are incubated in HAT medium (hypoxanthine-aminopterin-thymidine medium) for roughly 10

to 14 days. Aminopterin blocks the pathway that allows for nucleotide synthesis. Hence, unfused

myeloma cells die, as they cannot produce nucleotides by the de novoor salvage pathways because they

lack HGPRT. Removal of the unfused myeloma cells is necessary because they have the potential to

outgrow other cells, especially weakly established hybridomas. Unfused B cells die as they have a short

life span. In this way, only the B cell-myeloma hybrids survive, since the HGPRT gene coming from the B

cells is functional. These cells produce antibodies (a property of B cells) and are immortal (a property of

myeloma cells). The incubated medium is then diluted into multiwell plates to such an extent that each

well contains only one cell. Since the antibodies in a well are produced by the same B cell, they will be

directed towards the same epitope, and are thus monoclonal antibodies.

The next stage is a rapid primary screening process, which identifies and selects only those hybridomas

that produce antibodies of appropriate specificity. The hybridoma culture supernatant, secondary enzyme

labelled conjugate, and chromogenic substrate, are then incubated, and the formation of a coloured

product indicates a positive hybridoma. Alternatively, immunocytochemical screening can also be used.

The B cell that produces the desired antibodies can be cloned to produce many identical daughter clones.

Supplemental media containing interleukin-6 (such as briclone) are essential for this step. Once a

hybridoma colony is established, it will continually grow in culture medium like RPMI-1640 (with antibiotics

and foetal bovine serum) and produce antibodies.

Multiwell plates are used initially to grow the hybridomas, and after selection, are changed to larger tissue

culture flasks. This maintains the well-being of the hybridomas and provides enough cells for

cryopreservation and supernatant for subsequent investigations. The culture supernatant can yield 1 to

60 µg/ml of monoclonal antibody, which is maintained at 20 °C or lower until required.

By using culture supernatant or a purified immunoglobulin preparation, further analysis of a potential

monoclonal antibody producing hybridoma can be made in terms of reactivity, specificity, and cross-

reactivity.

Summary

Principle steps in the production of a hybridoma. Spleen cells are prepared from animals, usually mice, which have been immunized with a selected antigen. These cells are then fused with myeloma cells maintained in culture in the laboratory. The product of this fusion is referred to as a hybridoma. Surprisingly, a hybrid of two cells can survive and also continue to divide. In this particular hybrid the myeloma cells contribute the capacity for survival, whereas the spleen cells direct the synthesis of antibodies with the preselected specificity. By special arrangements it is possible to achieve a multiplication of hybridoma cells but not of isolated myeloma cells. The hybrids obtained are propagated in a highly diluted state so that colonies deriving from single hybrid cells can be isolated. By use of a sensitive method the clones which produce the specific antibodies are identified. A particular hybridoma can then be used for future, unlimited production of a highly specific antibody.

Application of Monoclonal antibodyThe availability of monoclonal antibodies has opened completely new possibilities for basic as well as applied biomedical research. The following examples of the use of monoclonal antibodies can be given.

1. Detailed studies of the distribution of different functions in different parts of antigen molecules. These studies may concern building elements of infectious agents; cell products such as enzymes and hormones; surface structures of cells etc. The mapping of variations in the surface components of influenza virus which explain the occurrence of repeated infections is one example.

2. High degree purification of substances, e.g. interferon, by taking advantage of the unique capacity displayed by a particular monoclonal antibody to bind to a certain antigen. In this case one uses a technique referred to as affinity chromatography.

3. Diagnostic characterization of diseases by identification of special structures on the surface or on the inside of cells. Hereby it is possible to distinguish between different forms of tumours and follow the

development of tumours. Furthermore, it is possible to distinguish between different kinds of normal white blood cells. This is of importance for the characterization of certain immune deficiency conditions as seen e.g. in connection with the disease AIDS (acquired immune deficiency syndrome).Diseases caused by infectious agents can also be diagnosed by use of monoclonal antibodies. Thus, virus infected cells and bacteria or parasites inside or outside cells can be identified with a unique degree of specificity.

4. Treatment of diseases. Monoclonal antibodies against specialized white blood cells have been used with some success in connection with transplantation. There may also be possibilities to use monoclonal antibodies for treatment of tumours.

Polyclonal antibodies

Polyclonal antibodies (or antisera) are antibodies that are obtained from different B cell resources. They are a combination of immunoglobulin molecules secreted against a specificantigen, each identifying a different epitope.

Production

These antibodies are typically produced by immunization of a suitable mammal, such as a mouse, rabbit

or goat. Larger mammals are often preferred as the amount of serum that can be collected is greater.

An antigen is injected into the mammal. This induces the B-lymphocytes to

produce IgG immunoglobulins specific for the antigen. This polyclonal IgG is purified from the

mammal’s serum.

By contrast, monoclonal antibodies are derived from a single cell line.

Many methodologies exist for polyclonal antibody production in laboratory animals. Institutional guidelines

governing animal use and procedures relating to these methodologies are generally oriented around

humane considerations and appropriate conduct for adjuvant (agents which modify the effect of other

agents while having few if any direct effects when given by themselves) use. This includes adjuvant

selection, routes and sites of administration, injection volumes per site and number of sites per animal.

Institutional policies generally include allowable volumes of blood per collection and safety precautions

including appropriate restraint and sedation or anesthesia of animals for injury prevention to animals or

personnel.

The primary goal of antibody production in laboratory animals is to obtain high titer, high

affinity antisera for use in experimentation or diagnostic tests. Adjuvants are used to improve or enhance

an immune response to antigens. Most adjuvants provide for an injection site, antigen depot which allows

for a slow release of antigen into draining lymph nodes.

Many adjuvants also contain or act directly as:

1. surfactants which promote concentration of protein antigens molecules over a large surface area,

and

2. immunostimulatory molecules or properties. Adjuvants are generally used with soluble protein

antigens to increase antibody titers and induce a prolonged response with accompanying

memory.

Such antigens by themselves are generally poor immunogens. Most complex protein antigens induce

multiple B-cell clones during the immune response, thus, the response is polyclonal. Immune responses

to non-protein antigens are generally poorly or enhanced by adjuvants and there is no system memory.

Animal selection

Animals frequently used for polyclonal antibody production include chickens, goats, guinea pigs,

hamsters, horses, mice, rats, and sheep. However, the rabbit is the most commonly used laboratory

animal for this purpose. Animal selection should be based upon:

1. the amount of antibody needed,

2. the relationship between the donor of the antigen and the recipient antibody producer (generally

the more distant the phylogenetic relationship, the greater the potential for high titer antibody

response) and

3. the necessary characteristics [e.g., class, subclass (isotype), complement fixing nature] of the

antibodies to be made. Immunization and phlebotomies are stress associated and, at least when

using rabbits and rodents, specific pathogen free (SPF) animals are preferred. Use of such

animals can dramatically reduce morbidity and mortality due to pathogenic organisms,

especially Pasteurella multocida in rabbits.

Goats or horses are generally used when large quantities of antisera are required. Many investigators

favor chickens because of their phylogenetic distance from mammals. Chickens transfer high quantities of

IgY (IgG) into the egg yolk and harvesting antibodies from eggs eliminates the need for the invasive

bleeding procedure. One week’s eggs can contain 10 times more antibodies than the volume of rabbit

blood obtained from one weekly bleeding. However, there are some disadvantages when using certain

chicken derived antibodies in immunoassays. Chicken IgY does not fix mammalian complement

component C1 and it does not perform as a precipitating antibody using standard solutions.

Although mice are used most frequently for monoclonal antibody production, their small size usually

prevents their use for sufficient quantities of polyclonal, serum antibodies. However, polyclonal antibodies

in mice can be collected from ascites fluid using any one of a number of ascites producing methodologies.

When using rabbits, young adult animals (2.5–3.0 kg or 5.5-6.5lbs) should be used for primary

immunization because of the vigorous antibody response. Immune function peaks atpuberty and primary

responses to new antigens decline with age. Female rabbits are generally preferred because they are

more docile and are reported to mount a more vigorous immune response than males. At least two

animals per antigen should be used when using outbred animals. This principle reduces potential total

failure resulting from non-responsiveness to antigens of individual animals.

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