LECOM-PharmacyImmunology 5 & 6

Adaptive (acquired) immune response

Antigen & Antibody

Dr. Saber Hussein

Objectives• Define antigenicity and immunogenicity• Four characteristics of immuogenic molecule

(requirements for immunogenicity)• Foreignness• High molecular weight• Chemical complexity• Degradability

• Define haptens and their functions• Define antigenic determinants• Capture of protein antigens by antigen presenting cells• Antigen recognized by T lymphocytes• Antigens recognized by B lymphocytes

Learning Objectives1. Basic structure of Abs in relation to specificity &

diversity2. Variable & constant regions of light & heavy

chain3. Biological & chemical characteristics of the 5

classes of Ab4. Compare polyclonal & monoclonal Ab5. Three characteristics of primary Ag-Ab reaction6. The forces that foster the primary Ag-Ab reaction7. Affinity & avidity of Abs8. Secondary Ag-Ab reaction; lattice formation

Objectives9. Know the role of receptors in the recognition of

antigen in the adaptive immune system:– Antibodies as receptors of B lymphocytes– T cell receptor (TCR) for antigens

10. Development of immune repertoires1. Maturation of lymphocytes

2. Production of diverse antigen receptors

3. Maturation and selection of B lymphocytes

4. Maturation and selection of T lymphocytes

Antigenicity & Immunogenicity• Antigenicity:

– The ability to bind an Ab or an activated T cell– Every immunogen is an antigen, BUT not every

antigen is an immunogen

• Immunogenicity: – Ability to elicit immune response– Only proteins can induce cellular immunity– Humoral immunity can be induced by:

• Proteins• Lipopolysaccharides• Nucleic acids• Other substances

Features of an Immunogen

1. High molecular weight

2. Chemical complexity

3. Solubility or biodegradability

4. Foreignness or nonself

Ab cross-reactions with different Ags• Abs react most strongly with homologous Ag• Sometimes they cross-react with other Ags• Cross-reaction

– The reaction between an Ag and an Ab that was generated against a different Ag but with some similarity with the cross-reacting Ag

• Cross-reactions are related to chemical structure of Ags:– i. Chemical nature of hapten’s groups– ii. Position of substitutions– iii. Size of substituted groups– iv. Charge– v. Stereoisomerism

Haptens, Antigenic Determinants (Epitopes)

• Ag has 2 functional regions:1. Hapten2. Carrier

• Epitopes are immunologically active portions of Ag• Epitopes on an Ag are recognized by B cells and T cells• Antigenic determinants serve as fingerprint of

macromolecules• Size of an epitope is determined by the size of the Ab’s

Ag-binding site• Size of recognizable epitope by an Ab:

– 6 sugar residues– 15-20 amino acids (Some linear epitopes are as small as 5 aas)

Ab

Carrier

Hapten

Haptens &Antigenic DeterminantsHaptens

1. Usually small molecules

2. Not immunogenic by themselves

3. Always antigenic with a specific Ab

4. Immunogenic when combined with a carrier molecule (large)

5. Simple hapten: only 1 antigenic determinant

6. Complex hapten: > 2 antigenic determinants

Antigenic Determinants

1. Small part of the molecule

– Few amino acids

– A short carbohydrate moiety- few sugars

2. Must be accessible to be functional

3. Charge & polarity

4. Conformation dependent

Ag recognized by T lymphocytes

• T lymphocytes recognize only protein antigens

• Proteins must be presented in the form of short peptides

• They must be presented by an APC with the appropriate MHC molecule:– MHC I presents antigen to the cytotoxic T cell– MHC II presents antigen to the helper T cell

Antigens recognized by B lymphocytes

• T-cell independent route of antigen recognition: – B lymphocytes recognize certain antigens without

the help of the TH cell

– These include:• Lipopolysaccharides• Nucleic acids: DNA & RNA

– No long term immunity results through this route– Only IgM is produced– No memory cells

Antigens recognized by B lymphocytes

• T-cell dependent route:– Recognizes protein antigens– Long term immunity– IgG is produced by class switching– Memory cells

Antigens recognized by B lymphocytesT-cell dependent

1. Recognizes protein antigens

2. Long term immunity

3. IgG is produced by class switching

4. Memory cells

T-cell independent

1. Recognizable Ags:• Lipopolysaccharides• Nucleic acids: DNA &

RNA

2. No long term immunity results through this route

3. Only IgM is produced

4. No memory cells

Antigen Capture and Presentation to Lymphocytes

• A model of how a T cell receptor (TCR) recognizes a complex of a peptide antigen displayed by a major histocompatibility (MHC) molecule

– MHC molecules are expressed on antigen-presenting cells and function to display peptides derived from protein antigens

– Peptides bind to the MHC molecules by anchor residues, which attach the peptides to pockets in the MHC molecules

– The TCR of every T cell recognizes some residues of the peptide and some (polymorphic) residues of the MHC molecule

Fig. 3-1

The capture and display of microbial antigens• Microbes enter the body

– through an epithelium and are captured by antigen-presenting cells resident in the epithelium

– enter lymphatic vessels or – blood vessels

• The microbes and their antigens are transported to peripheral lymphoid organs – the lymph nodes, – the spleen,

• where protein antigens are displayed for recognition by T lymphocytes

Fig. 3-2

The capture and presentation of protein

antigens by dendritic cells

• Immature dendritic cells in the epithelium capture microbial antigens by surface receptors and leave the epithelium

• The dendritic cells migrate to draining lymph nodes, being attracted there by chemokines produced in the nodes

• During their migration, and in response to the microbe, the dendritic cells mature

• Mature DC express high level MHC & costimulators

• In the lymph nodes, the dendritic cells present antigens to naive T cells Fig 3-4

Langerhans cells

Properties of MHC

molecules and genes

• Some of the important features of MHC molecules are listed, with their significance for immune responses

Fig 3-8

Role of MHC in Antigen Presentation to T Cells

• Ag processing– The event whereby the Ag is prepared to be presented to

lymphocytes in a form they can recognize

– It includes fragmentation of the protein Ag into small peptides in the macrophage and the presentation to T cells

• Ag-presenting cells (APCs) bind peptide Ags to their MHC II and present it to the CD4+ helper T cells

• APCs present peptides to CD8+ cytotoxic T cells with their MHC I

• Ag-presenting cells (APCs) include macrophages and other cells

Major Histocompatibility Complex (MHC)

m

MHC Restriction of T Cells

• The process by which the MHC controls interactions between immune cells

• It involves the recognition of foreign antigens in association with class I or II molecules

• The following reactions are MHC-restricted:1. Antigen presentation

2. T- and B-cell cooperation

3. Cytotoxic T-cell interaction with target cells• Malignant cells

• Viral infected cell

What Regions of HLA Complex Encode MHC I & MHC II?

Coding Regions:• MHC-I coding region:

– HLA-A, HLA-B and HLA-C

• MHC-II coding region:– HLA-D [DN, DO, DP, DQ & DR]

Antigen-MHC Class II Complex

What kind of T cell do we see here?

Fig 4-1: Properties of antibodies and T cell antigen receptors (TCRs)

• Antibodies may be expressed as membrane receptors or secreted proteins

• TCRs only function as membrane receptors • When Ig or TCR molecules recognize antigens, signals are

delivered to the lymphocytes by proteins associated with the antigen receptors

• The antigen receptors and attached signaling proteins form the B cell receptor (BCR) & TCR complexes

• Single antigen receptors are shown recognizing antigens, • Signaling requires the cross-linking of two or more

receptors by binding to adjacent antigen molecules

Fig 4-1

CD4,8

Fig 4-1

T cellB cell

Fig 4-1

:zeta)

Fig 4-2: The structure of antibodies

• Schematic diagrams of a secreted IgG (A) and a membrane form of IgM (B) illustrate the domains of the heavy and light chains and the regions of the proteins that participate in antigen recognition and effector functions

• N and C refer to the amino-terminal and carboxy-terminal ends of the polypeptide chains, respectively

Fig 4-2

Fig 4-3: Features of the major isotypes (classes) of antibodies

• The table summarizes some important features of the major antibody isotypes of humans.

• Isotypes are classified on the basis of their heavy chains• Each isotype may contain either or light chain• Each of the 5 classes differ in their locations in our body

and how they stimulate the innate system to remove antigen• The schematic diagrams illustrate the distinct shapes of the

secreted forms of these antibodies• IgA consists of two subclasses: IgA1 and IgA2• IgG consists of 4 subclasses: IgG1, IgG2, IgG3, & IgG4 • The serum concentrations are average values in normal

individuals

Fig 4-3: Features of the major isotypes of Abs

Breast-fedneonates getit with the

mother’s milk

Antiparasitic activity

with mother’s milk

Fig 4-3: Features of the major isotypes of Abs

Diagnostic for acute infections

                                                                  

       

            

Polyclonal & Monoclonal Abs• Polyclonal Abs1. Heterogeneous mix of

Abs2. With specificity against

the same Ag3. Produced by variety of

Ab-producing cells4. They are many clones of

cells5. Polyclonal Abs recognize

& react against different epitopes on the Ag

6. Avidity

• Monoclonal Abs1. Produced by a single

clone of cells2. Resultant Abs are

identical in all aspects3. Same affinity4. Same binding specificity5. Recognize the same

epitope6. They are produced in

hybridoma between activated B cells and malignant plasma cells (fusion)

Three dimensional representation of the IgG molecule

IgG molecule

IgG• IgG digestion with papain

produces 3 fragments– 2 identical Fab fragments

• Fab fragments, are capable of binding Ag because they contain the Ag-binding site

– Fc fragment: a fragment composed of H chain only.

• It crystallizes in the cold

Pentameric structure of IgM

• The structure of IgM is similar to that of IgG except the IgM heavy chain has an extra domain.

• A small, cysteine-rich protein called J chain initiates cross linking of C3 and C4 of

– five IgM monomers to make the circulating, pentameric form of IgM

Dimeric structure of IgA

• Dimeric IgA held together by – J chain and

– secretory component J chain

Secretory component

Secretory IgA • IgA represents 15-20% of serum immunoglobulin

• It constitutes the majority of Ab found in secretions

• Humans have 2 types of IgA:– IgA1 and IgA2– IgA1is the prominent

subclass in serum and is found mainly as monomer

– IgA2 is the prominent Ig in secretions (saliva, gut, respiratory mucus) and occurs as a dimer with two Fc ends of the Abs bound together by a J chain

• Secretion across the mucosa is mediated by a specific secretory component which binds to a cell receptor

IgE• IgE is similar to IgG

except – it has an extra constant

region domain on the H-chain

• Functions:– Type I hypersensitivity

– Anti-parasitic

– Degranulation of mast cells

IgD

• IgD is similar to the structure of IgG.

• Its only known function is as part of the signaling complex of B cells

Primary Ag-Ab reaction

1. The first interaction between Ag & Ab

2. Key-lock principle

3. Ag-Ab interaction is precise = specific

4. Characteristics of Ag-Ab reaction:

i. Rapid, in seconds

ii. Independent of electrolytes, salt, buffer

iii. Not visible

Fig 4-4: Binding of an Ag by an Ab • This model of a protein antigen bound to an antibody molecule shows how the antigen-

binding site can accommodate soluble macromolecules in their native (folded) conformation.

• The heavy chains (H) of the antibody are red• L chains: yellow• Ag is blue

Chemical forces foster Ab-Ag

Four noncovalent interactions hold antigenic determinants w/in Ab-binding site:

1. Coulombic (electrostatic, ionic) interactions

2. Van der Waals forces

3. Hydrogen bonds

4. Hydrophobic interactions

Secondary Ag-Ab reaction & Secondary response

• Secondary Ag-Ab reaction:

The conversion of the invisible primary reactions macroscopically visible ones as in the case of precipitation and agglutination

• Secondary response:The immune response which follows a second encounter with a particular AgIt is usually stronger (affinity maturity)

Lattice formation

• Occurs wheni. Ag-Ab complexes

aggregate in form of precipitation in liquid medium -

ii. Agglutination, including particulate components, other than Ag and Ab, such as cells

AgAb

Affinity & Avidity• Affinity is the strength of Ag-Ab bonds between a single

epitope and an individual Ab’s binding site• Avidity: The binding strength between a multivalent Ab

(polyclonal Ab) and a multivalent Ag• Ag + Ab Ag..Ab

K = [Ag..Ab]/[Ag][Ab]• The higher [Ag..Ab], the larger is K (the associated Ab

and Ag), the higher is affinity of the Ab to the Ag.• K = Equilibrium constant

= Association constant

= Ab affinity

Affinity maturity

• Ag + Ab Ag..Ab

KD = [Ag][Ab] / [Ag..Ab]

• The lower the KD (dissociation constant) the higher the affinity

• Affinity maturity: after repeated exposure to the Ag the affinity increases

Monoclonal Ab production• Immunize animals, rats or mice, with Ag• When the animals start to make a good Ab response remove their

spleens and prepare a cell suspension• Fuse spleen cells with a myeloma cell line by the addition of

polyethylene glycol (PEG), which promotes membrane fusion– Only a small proportion of the cells fuse successfully

• The fusion mixture is then set up in culture with medium containing “HAT”

– HAT = Mixture of • Hypoxanthine• Aminopterin (powerful toxin that blocks a metabolic

pathway)• Thymidine (H & T intermediate metabolites help the cell

bypass the pathway when added)– Spleen cells can grow/survive in HAT– Myeloma cells are sensitive to HAT because of metabolic

defect that prevents them from using the bypass– HAT culture contains:

• Spleen cells: die naturally in 1-2 weeks• Myeloma cells: Killed by HAT• Fused cells (hybridoma): Survive because of

immortality of myeloma and HAT-resistance of the spleen cells

– Some produce antibody– Any wells containing growing cells are tested for

the production of the desired Ab (often by solid phase immunoassay)

– Positive ones are cloned by plating out so that there is only one cell in each well

– This produces a clone derived from a single progenitor, which is both:

» Immortal» Producer of monoclonal Ab

Humoral Immune Response

B cells produce Abs• B cells are specialized white blood cells produced

in the bone marrow. • Each B cell contains multiple copies of one kind

of antibody as a surface receptor for antigen. • The entire population of B cells has the ability

to specifically bind to millions of different antigens

• When the antibody on the surface of a B cell binds to an antigen, the cell can be stimulated to undergo proliferation and differentiation.

• This process is called clonal selection.

Clonal selection• The cells produced make the same Ab, but become

memory cells and plasma cells– Memory cells insure that subsequent infections by the

pathogen receive a more rapid response. – Plasma cells secrete large amounts of the Ag-specific

Ab • T helper cells are required for the clonal selection of B

cells• Ab secreted by plasma cells forms complexes with free

pathogens and their toxic products• The complexes can:

– inactivate pathogens & – stimulate other innate systems including

• phagocytes and complement to eliminate the danger from our extracellular fluids

Abs and their diversity

• An Ab immunoglobulin is a "Y" shaped molecule made up of two identical "light" and "heavy" chains of amino acids.

• The variable region includes the N-terminal 110-130 amino acids of the light and heavy chains, and is responsible for binding to antigen.

• The constant region is the C-terminal end and contains similar amino acids for each class of Ab.

Abs Diversity (con)• When a stem cell changes to become a B cell,

DNA segments for both heavy (VDJ) and light (VJ) chains are randomly combined.

• Each B cell ends up with functional genes for making one light and one heavy chain coding for an Ab as a membrane-bound receptor.

• Ab specificity depends on the gene fragments used.

• Abs are produced that can react with almost any chemical structure in nature.

The immune system creates billions of different Abs with a limited number of genes by rearranging DNA segments during B cell development, prior to Ag exposure. Mutation can also increase genetic variation in Abs

Heavy chain

Light chain

Abs Diversity

Ab Class switching• At first, B cells contain IgM molecules only.

• Class switching occurs after Ag binding, when plasma cells are produced.

• Class switching refers to a DNA rearrangement changing the heavy chain constant gene in memory cells.

• Loss of coding regions for the constant part of the heavy chain causes IgG, IgA, and IgE to be produced.

Ab Class switching to produce IgA