HLA systemHLA system

(MHC glycoproteins) (MHC glycoproteins)

MHC glycoproteins class I MHC glycoproteins class I (Major histocompatibility complex)(Major histocompatibility complex)

The function of MHCgpI is presentation of peptide The function of MHCgpI is presentation of peptide fragments from inside the cell (which are produced fragments from inside the cell (which are produced by cell, including viral peptides if are present)on by cell, including viral peptides if are present)on the cell surface so as to be recognized by T the cell surface so as to be recognized by T lymphocytes (cytotoxic, CD8) lymphocytes (cytotoxic, CD8)

Present on all nucleated cells of the organism Present on all nucleated cells of the organism

3 isotypes classical human MHC gp.3 isotypes classical human MHC gp. (HLA - A,-B,-C)(HLA - A,-B,-C)

3 isotypes non-classical MHC gp.3 isotypes non-classical MHC gp. (HLA - E,-F,-G; (HLA - E,-F,-G; molecule CD1) molecule CD1)

Antigen presentation to cytotoxic T cellsAntigen presentation to cytotoxic T cells

MHC gp I structure MHC gp I structure

MHC gp class I consists of transmembrane MHC gp class I consists of transmembrane chain chain and non-covalently associated and non-covalently associated 22mikroglobulin mikroglobulin

chain has 3 domains, 2 N-terminal (chain has 3 domains, 2 N-terminal (1, 1, 2 - 2 - binding site for peptides) and 1 C-terminal binding site for peptides) and 1 C-terminal domain (domain (3 - anchored in the cytoplasmic 3 - anchored in the cytoplasmic membrane, a structure similar to imunoglobulin membrane, a structure similar to imunoglobulin domain)domain)

MHC gpI peptide binding MHC gpI peptide binding

MHC gp I bind peptides with a length of 8 to 10 MHC gp I bind peptides with a length of 8 to 10 aminoacidesaminoacides

Certain MHC gp molecule binds peptides sharing Certain MHC gp molecule binds peptides sharing common structural features - common structural features - coupling motifcoupling motif (critical are aminoacides near the end of peptide) (critical are aminoacides near the end of peptide)

The binding of endogenous peptides occurs in the The binding of endogenous peptides occurs in the endoplasmic reticulum during biosynthesis of MHC gp endoplasmic reticulum during biosynthesis of MHC gp II

After a chain After a chain and and 22mikroglobulin create in the ER, mikroglobulin create in the ER,

folding into the correct conformation and the mutual folding into the correct conformation and the mutual association and the association of an appropriate association and the association of an appropriate peptide, the complex is further processed in the Golgi peptide, the complex is further processed in the Golgi apparatus and then is presented on the cell surface apparatus and then is presented on the cell surface

Linked peptides are derived from proteins degraded Linked peptides are derived from proteins degraded by proteasome, proteasom degradate unneeded or by proteasome, proteasom degradate unneeded or damaged cytoplasmic proteins (labeled with damaged cytoplasmic proteins (labeled with ubiquitin), peptide fragments are transported into the ubiquitin), peptide fragments are transported into the ER by specific membrane pump TAP (transporter ER by specific membrane pump TAP (transporter associated with antigen processingassociated with antigen processing

MHC gpI peptide bindingMHC gpI peptide binding

Binding the peptide to MHCgpIBinding the peptide to MHCgpI

Non-classical MHC gpNon-classical MHC gp II

HLA - E,-F,-G; CD1 moleculesHLA - E,-F,-G; CD1 molecules

Structurally similar to classical MHC gp Structurally similar to classical MHC gp

Are less polymorphic Are less polymorphic

There are only on some cells There are only on some cells

They specialize in binding of specific ligands They specialize in binding of specific ligands

HLA-E and HLA-G - occurs on the trophoblast cells HLA-E and HLA-G - occurs on the trophoblast cells

Complexes of HLA-E and HLA-G with peptides are Complexes of HLA-E and HLA-G with peptides are recognized by inhibiting receptors of NK cells and recognized by inhibiting receptors of NK cells and contribute to the tolerance of the fetus in utero contribute to the tolerance of the fetus in utero

MHC glycoproteins class IIMHC glycoproteins class II

The function of MHC gpII is the presentation of The function of MHC gpII is the presentation of peptide fragments from protein whitch are peptide fragments from protein whitch are ingested by cell on the cell surface so as to be ingested by cell on the cell surface so as to be recognized by T lymphocytes (helper, CD4) recognized by T lymphocytes (helper, CD4)

Occur on the APC (dendritic cells, monocytes, Occur on the APC (dendritic cells, monocytes, macrophages, B lymphocytes) macrophages, B lymphocytes)

3 isotypes of MHC gpII (DR, DQ, DP) 3 isotypes of MHC gpII (DR, DQ, DP)

MHC gp IIMHC gp II structure structure

MHC gp II consist of 2 non-covalently associated MHC gp II consist of 2 non-covalently associated transmembrane subunits transmembrane subunits and and

The peptide binding site consists of N-terminal The peptide binding site consists of N-terminal domains domains 1 and 1 and 1 1

Binding of peptide is necessary for a stable MHC Binding of peptide is necessary for a stable MHC gp conformation and thus ensure its long gp conformation and thus ensure its long

presentation on the cell surfacepresentation on the cell surface

MHC gp II peptide bindingMHC gp II peptide binding

MHC gpII bind peptides with a length of 15 to 35 MHC gpII bind peptides with a length of 15 to 35 aminoacides (but possibly longer - because the peptide aminoacides (but possibly longer - because the peptide binding site is open at both ends) binding site is open at both ends)

Certain MHC gp molecule binds peptides sharing common Certain MHC gp molecule binds peptides sharing common structural features - structural features - coupling motif coupling motif

After a string After a string and and are created in ER, fold into the correct are created in ER, fold into the correct conformation and the mutual associated are connected with conformation and the mutual associated are connected with another transmembrane chain called invariant chain, which another transmembrane chain called invariant chain, which blocks the binding site for the peptide, this complex is blocks the binding site for the peptide, this complex is further processed in the Golgi apparatus, secretory vesicles further processed in the Golgi apparatus, secretory vesicles isolated from GA merge with endosomes, then split the isolated from GA merge with endosomes, then split the invariant chain and peptide fragments from cell absorbed invariant chain and peptide fragments from cell absorbed proteins bind into binding site of MHC gp and the complex is proteins bind into binding site of MHC gp and the complex is then presented on cell surfacethen presented on cell surface

MHC gp II peptide bindingMHC gp II peptide binding

Antigen prezentation

Antigen presentation to T lymphocyte1. Signal: TCR – MHC gp I(II)+Ag peptid (APC)2. Co-stimulating signal: CD 28 (T lymphocyte) – CD 80, CD 86 (APC)

MHC glycoproteinsMHC glycoproteins polymorphismpolymorphism

HLA complex is located on chromosome 6 HLA complex is located on chromosome 6

For MHC gp is typical high polymorphism, there are up to For MHC gp is typical high polymorphism, there are up to hundreds of different forms of alelic isotypes (except the hundreds of different forms of alelic isotypes (except the non-classical MHC gp, and DR non-classical MHC gp, and DR chain) chain)

Codominant inheritance of alelic forms (IndividualCodominant inheritance of alelic forms (Individualhas 3 cell surface isotypes of HLA molecules has 3 cell surface isotypes of HLA molecules (HLA-A,-B,-C) mostly in 2 different alelic forms) (HLA-A,-B,-C) mostly in 2 different alelic forms)

Polymorphism has a protective significance at individual Polymorphism has a protective significance at individual and population level and population level

MHC gp polymorphism causes complications in MHC gp polymorphism causes complications in transplantation transplantation

HLA typing = determmination of HLA HLA typing = determmination of HLA antigens on the surface of lymphocytesantigens on the surface of lymphocytes

Carry out during the testing before transplantation Carry out during the testing before transplantation and in determination of paternityand in determination of paternity

1) Serotyping1) Serotyping Microlymfocytotoxic test Microlymfocytotoxic test Allospecific serumsAllospecific serums (obtained from multiple natal to 6 weeks (obtained from multiple natal to 6 weeks

after birth, obtained by vaccination of volunteers, or after birth, obtained by vaccination of volunteers, or commercially prepared sets of typing serums (monoclonal commercially prepared sets of typing serums (monoclonal antibodies)) antibodies))

PrinciplePrinciple - the incubation of lymphocytes with typing serums in - the incubation of lymphocytes with typing serums in the the presence of rabbit complement, then is added the vital presence of rabbit complement, then is added the vital dye which stained dead cells dye which stained dead cells

- cells carrying specific HLA are killed by cytotoxic Ab - cells carrying specific HLA are killed by cytotoxic Ab against the Ag, the percentage of dead cells is a against the Ag, the percentage of dead cells is a measure measure of serum toxicity (forces and antileukocyte antibody of serum toxicity (forces and antileukocyte antibody titre)titre)

Positive reaction is considered more than 10% dead cells Positive reaction is considered more than 10% dead cells

(serological typing can be done also by flow cytometry (serological typing can be done also by flow cytometry

2) Molecular genetic methods2) Molecular genetic methods For typing are used hypervariable sections in the II. exon For typing are used hypervariable sections in the II. exon

genes coding for HLA class II; to determine HLA class I is genes coding for HLA class II; to determine HLA class I is used polymorphism in II. and III. exon coding genesused polymorphism in II. and III. exon coding genes

2a) PCR-SSP2a) PCR-SSP = Polymerase chain reaction with sequential specific = Polymerase chain reaction with sequential specific primers primers

Extracted DNA is used as a substrate in a set of PCR Extracted DNA is used as a substrate in a set of PCR reactionsreactions

Each PCR reaction contains primers pair specific for a Each PCR reaction contains primers pair specific for a certain allele (or group of alleles) certain allele (or group of alleles)

Positive and negative reactions are evaluated by Positive and negative reactions are evaluated by electrophoresis, each combination of alleles has a electrophoresis, each combination of alleles has a specific electrophoretic painting specific electrophoretic painting

2b) PCR-SSO2b) PCR-SSO PCR reaction with sequence-specific PCR reaction with sequence-specific

oligonucleotides oligonucleotides Multiplication of hypervariable sections of genes Multiplication of hypervariable sections of genes coding HLA coding HLA

Hybridization with enzyme or radiolabeled DNA Hybridization with enzyme or radiolabeled DNA probes specific for individual alleles probes specific for individual alleles 2c) PCR-SBT2c) PCR-SBT

Sequencing based typingSequencing based typing The most accurate method of HLA typingThe most accurate method of HLA typing We get the exact sequence of nucleotides, which We get the exact sequence of nucleotides, which

compares with a database of known sequences of compares with a database of known sequences of HLA alleles HLA alleles

T – lymphocytesT – lymphocytes

J. J. OchotnáOchotná

T lymphocytesT lymphocytes cellular component of antigen-specific cellular component of antigen-specific

mechanismsmechanisms

several different several different subsetssubsets of T lymphocytes of T lymphocytes

regulation of immune processes, the destruction regulation of immune processes, the destruction of virus-infected cells or tumor cells of virus-infected cells or tumor cells

recognize antigen processed and presented by recognize antigen processed and presented by the APCthe APC

T cells are after activation stimulated to T cells are after activation stimulated to multiplication and differentiation into multiplication and differentiation into effector effector cellscells and part of them differentiate into the and part of them differentiate into the memory cellsmemory cells

T-lymphocytes developmentT-lymphocytes development T cells originate in bone marrow and then migrate to the thymus T cells originate in bone marrow and then migrate to the thymus

where they mature (where they mature (T lymphocytes), the final differentiation is T lymphocytes), the final differentiation is after activation by antigen processed and presented by APC after activation by antigen processed and presented by APC

T cells can develop outside the thymus (the minority population) T cells can develop outside the thymus (the minority population)

Pluripotent hematopoietic stem cellsPluripotent hematopoietic stem cells

Pro-thymocytesPro-thymocytes – – double negative T cellsdouble negative T cells - are coming from the - are coming from the bone marrow to the thymus, where they begin to rearrange TCRbone marrow to the thymus, where they begin to rearrange TCR genes, expressing on their surface, called genes, expressing on their surface, called pre-TCRpre-TCR (Composed of (Composed of chain, pre-TCRchain, pre-TCRand CD3 complex), then begin TCRand CD3 complex), then begin TCR genes genes rearrangement rearrangement

Cortical thymocytesCortical thymocytes – – double positive T cellsdouble positive T cells - express on their - express on their surface TCR (composed of chains surface TCR (composed of chains and CD3) and CD4 and CD8 and CD3) and CD4 and CD8 co-receptor (double positive T lymphocyte), at this stage occurs co-receptor (double positive T lymphocyte), at this stage occurs the selection of autoreactive cells and cells with dysfunctional TCR the selection of autoreactive cells and cells with dysfunctional TCR

Medullary thymocytesMedullary thymocytes (mature T cell) - retain the expression of (mature T cell) - retain the expression of CD4 or CD8, then migrate to secondary lymphoid organs CD4 or CD8, then migrate to secondary lymphoid organs

T-lymphocytes selectionT-lymphocytes selection Negative selectionNegative selection - the elimination of - the elimination of autoreactiveautoreactive cells, cells,

when thymocytes binds enough strongly by their TCR when thymocytes binds enough strongly by their TCR complex of MHCgp with normal peptides (from complex of MHCgp with normal peptides (from autoantigens)which are presented on surface of thymic cells autoantigens)which are presented on surface of thymic cells thymocyte receives signals leading to apoptotic cell death thymocyte receives signals leading to apoptotic cell death PAE cellsPAE cells (peripherial antigen expressing cells) (peripherial antigen expressing cells)

Positive selectionPositive selection - the elimination of cells with - the elimination of cells with dysfunctional TCRdysfunctional TCR, , positively are selected thymocytes that recognize MHC gp positively are selected thymocytes that recognize MHC gp with low affinity, then maintain the expression of CD4 or CD8 with low affinity, then maintain the expression of CD4 or CD8 (depending what class of MHC gp binds to the TCR). These (depending what class of MHC gp binds to the TCR). These mature T cellsmature T cells (Medullary thymocytes) leave the thymus and (Medullary thymocytes) leave the thymus and migrate to secondary lymphoid organs migrate to secondary lymphoid organs

98% of pro-thymocytes in the thymus during its development 98% of pro-thymocytes in the thymus during its development dies dies

T cell developmentT cell development

T-lymphocytes surface markersT-lymphocytes surface markers

TCRTCR - recognizes Ag peptide complexed with MHC gp - recognizes Ag peptide complexed with MHC gp

CD3CD3 - TCR component, participation in signal transduction - TCR component, participation in signal transduction

CD4CD4 or or CD8CD8 - co-receptors, binding to MHC gp - co-receptors, binding to MHC gp

CD28CD28 - costimulatory receptor, binds to CD80, CD86 on - costimulatory receptor, binds to CD80, CD86 on APCAPC

CTLA-4CTLA-4 (CD152) - inhibitory receptor, binds to CD80, CD86 (CD152) - inhibitory receptor, binds to CD80, CD86

Interaction between APC and T cellInteraction between APC and T cell

T-lymphocytes subpopulationsT-lymphocytes subpopulations

-T lymphocytes-T lymphocytes - have TCR - have TCR, major , major type (95%), thymus need in development, type (95%), thymus need in development, recognize antigens in the complex MHC-recognize antigens in the complex MHC-peptide gp peptide gp

-T lymphocytes-T lymphocytes - (5%) may develop - (5%) may develop outside the thymus, some are able to outside the thymus, some are able to recognize native Ag, apply in defense of recognize native Ag, apply in defense of the skin and mucous membranes the skin and mucous membranes

T-lymphocytesT-lymphocytes

Expressing the CD4 co-receptorExpressing the CD4 co-receptor (co-receptor for MHC (co-receptor for MHC class II gp), precursors of class II gp), precursors of helper T cells (Thelper T cells (THH)),, they can they can be classified according to the production of cytokines be classified according to the production of cytokines

TTHH00 - produce a mixture of cytokines such as T - produce a mixture of cytokines such as THH1 and T1 and THH22 TTHH11 - IL-2, IFN - IL-2, IFN (help macrophages ) (help macrophages ) TTHH22 - IL-4, IL-5, IL-6, IL-10 (B lymphocytes assistance) - IL-4, IL-5, IL-6, IL-10 (B lymphocytes assistance)

TTHH33 - TGF - TGF

TregTreg - regulatory T cells arise in the thymus from a part - regulatory T cells arise in the thymus from a part of autoreactive lymphocytes, suppress the activity of Tof autoreactive lymphocytes, suppress the activity of THH1 1 and partly function as Tand partly function as TSS, suppression of autoreactive , suppression of autoreactive T cell clonesT cell clones

T-lymphocytesT-lymphocytes

Expressing the CD8 co-receptorExpressing the CD8 co-receptor (co-receptor (co-receptor for MHC gp class I), for MHC gp class I), precursors of cytotoxic T precursors of cytotoxic T cells (TC)cells (TC), or , or suppressor T cells (TS)suppressor T cells (TS)

TTCC - recognize cells infected by viruses or other - recognize cells infected by viruses or other intracellular parasites and some cancer cells intracellular parasites and some cancer cells

TTSS - inhibit the function of other lymphocytes - inhibit the function of other lymphocytes

TCRTCR

TCR (T cell receptor) is heterodimer TCR (T cell receptor) is heterodimer consisting of consisting of and and ( () chain) chain and associated and associated CD3 complexCD3 complex,,which is necessary for signal which is necessary for signal transfer (is connected with PTK) transfer (is connected with PTK)

"N-terminal parts of "N-terminal parts of and and ( () chain form the ) chain form the

binding site for Agbinding site for Ag

TCR cooperation with co-receptors CD4, CD8TCR cooperation with co-receptors CD4, CD8

TCR developmentTCR development

The analogy with the formation of BCR The analogy with the formation of BCR

Chains Chains and and - correspond to IgH gene complex - correspond to IgH gene complex of of immunoglobulins immunoglobulins

- V, D, J, C segments - V, D, J, C segments

Chains Chains and and - correspond to genes for L - correspond to genes for L chains ofchains of immunoglobulins immunoglobulins - V, J, C segments - V, J, C segments

Rearrangement of genes is similar to the BCR and Rearrangement of genes is similar to the BCR and performed by the same performed by the same recombinasesrecombinases

Antigen-specific mechanismsAntigen-specific mechanisms

TTHH1 based immune 1 based immune responseresponse

TTHH1 immune response 1 immune response - inflammatory - inflammatory

reactionreaction TTHH1 cells 1 cells cooperate with macrophagescooperate with macrophages and transform and transform

them in activatedthem in activated (NO production - destroy intracellular (NO production - destroy intracellular parasites)parasites)

Activated macrophages secrete some cytokines (IL-1, Activated macrophages secrete some cytokines (IL-1, TNF, ...) that help to stimulate T cells and TNF, ...) that help to stimulate T cells and stimulate local stimulate local inflammationinflammation, which helps suppress infection , which helps suppress infection

Interaction between TInteraction between THH1 cells and macrophages is a 1 cells and macrophages is a fundamental mechanism of delayed-type fundamental mechanism of delayed-type immunopathological reactions (DTH Delayed-type immunopathological reactions (DTH Delayed-type hypersensitivity) hypersensitivity)

The infected macrophage produces protein The infected macrophage produces protein fragments derived from intracellular parasites, fragments derived from intracellular parasites, some of which are presented on the surface by some of which are presented on the surface by MHC gp class IIMHC gp class II

Macrophages and dendritic cells stimulated by Macrophages and dendritic cells stimulated by certain microorganisms produce certain microorganisms produce IL-12IL-12

TTHH precursor precursor, which detects the infected , which detects the infected macrophage and receives signals via the macrophage and receives signals via the TCR, CD TCR, CD 2828 and and receptor for IL-12receptor for IL-12 and other adhesion and and other adhesion and signaling molecules proliferates and differentiates signaling molecules proliferates and differentiates to the to the effector Teffector THH1 cells1 cells that produce that produce IFNIFN and and IL-2 IL-2. .

IFNIFN activates macrophage NO synthase activates macrophage NO synthase IL-2IL-2 is an autocrine growth factor for T is an autocrine growth factor for THH1 cells 1 cells

Interaction between APC and TInteraction between APC and THH precursor precursor

TTHH2 based immune 2 based immune responseresponse

TTHH2 immune response – help to B-2 immune response – help to B-lymphocyteslymphocytes

TTHH2 cells 2 cells cooperate with B lymphocytescooperate with B lymphocytes (which (which were stimulated by Ag) by cytokine production (IL-were stimulated by Ag) by cytokine production (IL-4, IL-5, 4, IL-5, IL-6) and direct intercellular contact IL-6) and direct intercellular contact

For stimulation of B lymphocytes is usually For stimulation of B lymphocytes is usually necessary cooperation between necessary cooperation between APC → TAPC → THH2 cell → B 2 cell → B lymphocytelymphocyte

In In minimal modelminimal model, where the B cell becomes a good , where the B cell becomes a good APC (CD80, CD86) is sufficient cooperation APC (CD80, CD86) is sufficient cooperation between between TTHH2 cell → B lymphocyte2 cell → B lymphocyte

TTHH precursor precursor, which detects the infected , which detects the infected macrophage and receives signals through the macrophage and receives signals through the TCR, CD 28 receptor for IL-4TCR, CD 28 receptor for IL-4 receptor and IL-2 and receptor and IL-2 and other adhesion and signaling molecules other adhesion and signaling molecules proliferates and differentiates in the proliferates and differentiates in the effector Teffector THH22, , which provide B lymphocytes auxiliary signals via which provide B lymphocytes auxiliary signals via cytokines secreted by cytokines secreted by IL-4, IL-5, IL-6IL-4, IL-5, IL-6 and adhesion and adhesion molecules through molecules through CD 40LCD 40L, which bind to the , which bind to the costimulatory receptor on B lymphocytes CD 40 costimulatory receptor on B lymphocytes CD 40

Interaction between Interaction between CD40 (B lymphocytes) and CD40 (B lymphocytes) and CD40L (TH2 cells)CD40L (TH2 cells) is essential for the initiation of is essential for the initiation of somatic mutations, izotype switching and somatic mutations, izotype switching and formation of memory cells formation of memory cells

IL-4, IL-5, IL-6IL-4, IL-5, IL-6: stimulation of B lymphocytes : stimulation of B lymphocytes

Assistance to B lymphocytesAssistance to B lymphocytes

Specific direct assistance to B lymphocytes:Specific direct assistance to B lymphocytes:

TTHH2 lymphocytes assisting B lymphocytes that were 2 lymphocytes assisting B lymphocytes that were stimulated by the same Ag, which caused the rise of stimulated by the same Ag, which caused the rise of TTHH2 2

To stimulate the secretion of cytokines by TTo stimulate the secretion of cytokines by THH2 cell is 2 cell is sufficient signal via the TCR (signal through a sufficient signal via the TCR (signal through a costimulatory receptor CD28 is no longer necessary) costimulatory receptor CD28 is no longer necessary)

One clone of TOne clone of THH2 cells can provide specific assistance 2 cells can provide specific assistance to B lymphocytes of different specificities (must to B lymphocytes of different specificities (must present the relevant Ag peptides by MHC gp II, which present the relevant Ag peptides by MHC gp II, which are recognized by TCR) are recognized by TCR)

Assistance to B lymphocytesAssistance to B lymphocytes

Indirect assistance to B cells ("bystander help"):Indirect assistance to B cells ("bystander help"):

TTHH2 lymphocytes assisting B lymphocytes that 2 lymphocytes assisting B lymphocytes that were stimulated by another Ag than that which were stimulated by another Ag than that which caused the rise of Tcaused the rise of THH2 2

Contact between TContact between THH2 cell → B lymphocytes2 cell → B lymphocytes via via adhesion molecules, cytokine secretion, binding adhesion molecules, cytokine secretion, binding CD40-CD40L CD40-CD40L

Danger of activation autoreactive B lymphocytes Danger of activation autoreactive B lymphocytes

Mutual regulation of activities TMutual regulation of activities THH1versus 1versus TTHH22

Whether the TWhether the THH precursor cell will develop into T precursor cell will develop into THH1 or T1 or THH2 2 decides decides cytokine ratio of IL-12 and IL-4cytokine ratio of IL-12 and IL-4

IL-12IL-12 is produced by macrophages and dendritic cells is produced by macrophages and dendritic cells stimulated by certain microorganismsstimulated by certain microorganisms

IL-4 IL-4 is produced by activated basophils and mast cells is produced by activated basophils and mast cells

TTHH1 cytokines (mainly IFN1 cytokines (mainly IFN) inhibit the development of T) inhibit the development of THH2 2 and stimulate the development of Tand stimulate the development of THH1 (IL-2 stimulates also 1 (IL-2 stimulates also TTHH2)2)

Cytokines produced by TCytokines produced by THH2 (IL-4, IL-10) inhibit the 2 (IL-4, IL-10) inhibit the development of Tdevelopment of THH1 and stimulate the development of T1 and stimulate the development of THH2 2

TTHH3 development is stimulated by a specific cytokine 3 development is stimulated by a specific cytokine environment (IL-4, IL-10, TGFenvironment (IL-4, IL-10, TGF); T); THH3 produce TGF3 produce TGF and and cooperate with B cells in MALT cooperate with B cells in MALT

TTCC based immune based immune responseresponse

Cytotoxic T lymphocytes stimulationCytotoxic T lymphocytes stimulation

TTCC recognize cells infected with viruses or other recognize cells infected with viruses or other intracellular parasites, and some tumor cells intracellular parasites, and some tumor cells

Precursor of TPrecursor of TCC, which recognizes a complex , which recognizes a complex of of MHC gp I- antigenic peptideMHC gp I- antigenic peptide on the surface of on the surface of APC via APC via TCRTCR and receives signals via and receives signals via CD 28CD 28 proliferates and differentiates to clone mature proliferates and differentiates to clone mature effector cytotoxic cells (CTL)effector cytotoxic cells (CTL); T; THH1 cells help to T1 cells help to TCC by by production production IL-2IL-2

Effector TEffector TCC are spread by bloodstream into tissues; are spread by bloodstream into tissues; for activation of cytotoxic mechanisms is sufficient for activation of cytotoxic mechanisms is sufficient signal through the TCR (signal through a signal through the TCR (signal through a costimulatory receptor CD28 is no longer costimulatory receptor CD28 is no longer necessary) necessary)

Professional APC are dendritic cells or Professional APC are dendritic cells or

macrophages that are infected with virus, or macrophages that are infected with virus, or

swallowed antigens from dead infected, tumor or swallowed antigens from dead infected, tumor or

stressed cellsstressed cells

In order APC could activate the TIn order APC could activate the TCC precursor, APC precursor, APC

must be stimulated by contact with Tmust be stimulated by contact with THH cells via cells via

CD 40, then the dendritic cell begins to express CD 40, then the dendritic cell begins to express

CD 80, CD86 and secrete cytokines (IL-1, IL-12) = CD 80, CD86 and secrete cytokines (IL-1, IL-12) =

change of resting APC in activated change of resting APC in activated

Tc effector functionsTc effector functions Cytotoxic granulesCytotoxic granules containing containing perforinperforin and and

granzymes granzymes (perforin creates pores in the (perforin creates pores in the cytoplasmic membrane of target cell, in some cytoplasmic membrane of target cell, in some cases may lead to osmotic lysis of the target cell, cases may lead to osmotic lysis of the target cell, formed pores in the cell receiving granzymes that formed pores in the cell receiving granzymes that cause the target cell to die by cause the target cell to die by apoptosisapoptosis. .

Fas ligand (FasL)Fas ligand (FasL) - which binds to the apoptotic - which binds to the apoptotic receptor Fas (CD95) presented on the surface of receptor Fas (CD95) presented on the surface of many different cells (also on the surface of Tmany different cells (also on the surface of TCC) )

TNFTNF

Antibody-based immune Antibody-based immune responseresponse

Antibody responses induced byAntibody responses induced by

T-independent antigensT-independent antigens Cause predominantly IgM production Cause predominantly IgM production Bacterial polysaccharides, Bacterial polysaccharides,

lipopolysaccharides, and polymeric forms of lipopolysaccharides, and polymeric forms of protein protein

T-dependent antigensT-dependent antigens Reaction to these Ag occurs in two phases - Reaction to these Ag occurs in two phases -

primary and secondary responseprimary and secondary response Initiate the development of Initiate the development of memory cellsmemory cells

and formation of high-affinity antibodiesand formation of high-affinity antibodies

T-independent and T-independent and T-dependent immune T-dependent immune responseresponse

Antibody responses induced Antibody responses induced by T-dependent antigenby T-dependent antigen

Primary phase of antibody responsePrimary phase of antibody response

The first contact with Ag The first contact with Ag

Takes place in secondary lymphoid organsTakes place in secondary lymphoid organs

Stimulation of B cells by Stimulation of B cells by Ag binding to BCRAg binding to BCR

Ag absorption by APCAg absorption by APC and its presentation via MHC gp and its presentation via MHC gp class II to precursors of Tclass II to precursors of THH cell → formation of clone of cell → formation of clone of

antigen-specific Tantigen-specific THH2 cells, which provide assistance to 2 cells, which provide assistance to

competent B lymphocytes, leading to their proliferation, competent B lymphocytes, leading to their proliferation, differentiation into plasma (produce Ab) and memory differentiation into plasma (produce Ab) and memory cellscells

Plasma cellsPlasma cells are spread by bloodstream into the are spread by bloodstream into the organism (particularly bone marrow)organism (particularly bone marrow)

Antibodies produced in the primary stage (3-4 Antibodies produced in the primary stage (3-4 days) are days) are IgMIgM and have a low affinity for Ag, and have a low affinity for Ag, create with Ag immune complexescreate with Ag immune complexes

Immune complexesImmune complexes are captured in the are captured in the secondary lymphoid organs on the surface of FDC secondary lymphoid organs on the surface of FDC (follicular dendritic cells) - Ag presenting cells to (follicular dendritic cells) - Ag presenting cells to B lymphocytes B lymphocytes

Secondary phase of antibodies responseSecondary phase of antibodies response

Recognition of Ag on FDCRecognition of Ag on FDC

(If is sufficient amount of immune complexes on (If is sufficient amount of immune complexes on FDCs) FDCs)

Under the influence of signals from the FDC (Ag) and Under the influence of signals from the FDC (Ag) and TTHH2 cells (CD40L, cytokines) is again started the 2 cells (CD40L, cytokines) is again started the proliferation and differentiation of B cells proliferation and differentiation of B cells accompanied with accompanied with somatic mutationssomatic mutations → formation of → formation of clones of B cells with new BCR → survive only B cells clones of B cells with new BCR → survive only B cells with a BCR with the highest affinity for Ag = with a BCR with the highest affinity for Ag = affinity affinity maturation of antibodiesmaturation of antibodies

There is also There is also isotype switchingisotype switching, which isotypes arise , which isotypes arise determines cytokine environment determines cytokine environment

In the secondary phase of the immune response In the secondary phase of the immune response generate generate antibodies with higher affinity for Agantibodies with higher affinity for Ag and other effector characteristics dependent on and other effector characteristics dependent on isotypeisotype, also formed a , also formed a memory cellsmemory cells for next for next meeting with the Ag meeting with the Ag

Antibodies in the body after primary infection Antibodies in the body after primary infection persist for a long time persist for a long time

Contact between Contact between CD40CD40 (B lymphocytes) and (B lymphocytes) and CD40LCD40L (T (THH2 lymphocytes) is essential for the 2 lymphocytes) is essential for the initiation of initiation of somatic mutations, isotype switching somatic mutations, isotype switching and formation of memory cells and formation of memory cells

Primary and secondary immune reactionPrimary and secondary immune reaction

• Primary immune reactionPrimary immune reaction – occurs on the firsth – occurs on the firsth exposure to antigenexposure to antigen

• Secondary immune reactionSecondary immune reaction –occurs after –occurs after subsequent encounter with the same antigen and is subsequent encounter with the same antigen and is more rapid leading to the activation of previously more rapid leading to the activation of previously generated memory cellsgenerated memory cells

Primary and secondary immune reactionPrimary and secondary immune reaction

B lymphocytesB lymphocytes

B-lymphocytesB-lymphocytes

B-lymphocytes (B cells)B-lymphocytes (B cells) are cells responsible are cells responsible especially for specific, especially for specific, antibodyantibody-mediated immune -mediated immune response. They also have great importance for the response. They also have great importance for the immune memoryimmune memory (which is used for vaccination). (which is used for vaccination).

B-cells recognize native antigen through BCR (B cell B-cells recognize native antigen through BCR (B cell receptor) receptor)

B-lymphocyte whitch bind Ag through BCR are B-lymphocyte whitch bind Ag through BCR are stimulated to proliferate and differentiate to stimulated to proliferate and differentiate to effector effector plasma cellsplasma cells which produce large quantities which produce large quantities of antibodies of the same specificity as the BCR (it of antibodies of the same specificity as the BCR (it is actually the same protein in soluble form). Part of is actually the same protein in soluble form). Part of stimulated B-cells differentiate to stimulated B-cells differentiate to memory cells.memory cells.

Surface characteristics of B Surface characteristics of B

lymphocyteslymphocytes CD 10CD 10 - immature B lymphocyte - immature B lymphocyte

CD 19CD 19 - characteristic surface sign of B cells - characteristic surface sign of B cells

CD 20CD 20 - on the surface of Ig-positive B - on the surface of Ig-positive B lymphocytes lymphocytes

IgM, IgDIgM, IgD - BCR - BCR

MHC gp IIMHC gp II - Ag presenting molecules - Ag presenting molecules

CD 40CD 40 – costimulating receptor – costimulating receptor

B cell developmentB cell development Development of B lymphocytes takes place in the bone marrow Development of B lymphocytes takes place in the bone marrow

and completes after activation with Ag in secondary lymphoid and completes after activation with Ag in secondary lymphoid organs. organs.

Pluripotent hematopoietic stem cell Pluripotent hematopoietic stem cell

Progenitor B cellProgenitor B cell - begin recombination processes which lead - begin recombination processes which lead to a large number of clones B lymphocytes to a large number of clones B lymphocytes with individual specific BCR with individual specific BCR

Pre - B cellPre - B cell - expression of pre-B receptor (composed of H ( - expression of pre-B receptor (composed of H () ) chain and alternate L chain) chain and alternate L chain)

Immature - B lymphocyteImmature - B lymphocyte - expression of surface IgM (BCR) - expression of surface IgM (BCR) at this stage elimination at this stage elimination of autoreactive clones of autoreactive clones

Mature B lymphocyte -Mature B lymphocyte - expression of surface IgM and IgD (BCR) expression of surface IgM and IgD (BCR)

Critical moments in the B cell Critical moments in the B cell developmentdevelopment

Completion of the rearrangment of genes for H Completion of the rearrangment of genes for H chain and surface expression of pre-BCRchain and surface expression of pre-BCR

Successful rearrangement of genes for L chain Successful rearrangement of genes for L chain and surface expression of IgM (BCR) and surface expression of IgM (BCR)

Testing of immature B cells, whether they are Testing of immature B cells, whether they are autoreactive autoreactive

Another critical stage are somatic mutations and Another critical stage are somatic mutations and affinity maturation, when survive only B cells with affinity maturation, when survive only B cells with the highest affinity for antigen. the highest affinity for antigen.

BCRBCR BCR is composed from BCR is composed from surface surface

immunoglobulin (IgM, IgDimmunoglobulin (IgM, IgD - H chains - H chains are transmembrane, recognizes Ag) are transmembrane, recognizes Ag) and associated and associated signaling moleculessignaling molecules (Ig (Ig and IGand IG), which are associated with the ), which are associated with the cytoplasmic protein-tyrosine kinases cytoplasmic protein-tyrosine kinases (PTK) Src Group (PTK) Src Group

After binding of Ag to 2 or more BCR After binding of Ag to 2 or more BCR will approximate PTK, mutual will approximate PTK, mutual phosphorylation and phosphorylation of phosphorylation and phosphorylation of other cytoplasmic proteins, leading to other cytoplasmic proteins, leading to changes in gene transcription, changes in gene transcription, proliferation, differentiation and proliferation, differentiation and secretion of antibodiessecretion of antibodies

The signal by binding Ag to the BCR The signal by binding Ag to the BCR can be amplified by cooperation with can be amplified by cooperation with CR2, which binds C3dg (opsonin) CR2, which binds C3dg (opsonin)

Elimination of autoreactive B Elimination of autoreactive B

lymphocyteslymphocytes By random rearrangement of genes, connecting inaccuracy, By random rearrangement of genes, connecting inaccuracy,

H-L pairing and somatic mutations may also arise clones of B H-L pairing and somatic mutations may also arise clones of B cells bearing autoreactive receptors and produce autoreactive cells bearing autoreactive receptors and produce autoreactive antibodies. antibodies.

Majority of autoreactive B lymphocytes are eliminated as the Majority of autoreactive B lymphocytes are eliminated as the immature B lymphocytes in the bone marrow, if its BCR bind immature B lymphocytes in the bone marrow, if its BCR bind autoantigen with sufficient affinity, receives a signal leading autoantigen with sufficient affinity, receives a signal leading to apoptotic death (clonal deletion). to apoptotic death (clonal deletion).

If some of the autoreactive clones pass this elimination, their If some of the autoreactive clones pass this elimination, their autoreaktivity usually do not come because lack of Tautoreaktivity usually do not come because lack of THH lymphocytes for their activation, many autoantigens are lymphocytes for their activation, many autoantigens are cryptic, or occur in low concentrations and are ignored by the cryptic, or occur in low concentrations and are ignored by the immune system. immune system. Tolerance to self-antigens is critical in preventing Tolerance to self-antigens is critical in preventing autoimmunity in the organism. autoimmunity in the organism.

Antigen recognition by B cell Antigen recognition by B cell

in secondary lymphoid organsin secondary lymphoid organs

Ontogenesis of antibodiesOntogenesis of antibodies Synthesis of specific antibodies begins around the 20.-Synthesis of specific antibodies begins around the 20.-

24. week of gestation, the total concentration of IgA 24. week of gestation, the total concentration of IgA

and IgM remains undetectable until birth, IgG begin to and IgM remains undetectable until birth, IgG begin to

form after birth form after birth

B lymphocytes respond to immunization predominantly B lymphocytes respond to immunization predominantly

by IgM formation, switching to other isotype is slower by IgM formation, switching to other isotype is slower

Slow growth of own IgG decline in maternal IgG (about Slow growth of own IgG decline in maternal IgG (about

3. to 6.month) 3. to 6.month)

The IgM concentration reaches values The IgM concentration reaches values comparable comparable to adults in the 1- 3 year of life, IgG and to adults in the 1- 3 year of life, IgG and IgA between 10.-15. yearIgA between 10.-15. year

Antibody response to polysaccharide Antibody response to polysaccharide antigens appears until around 2. year of antigens appears until around 2. year of life life

In old age is a lower antibody response to In old age is a lower antibody response to new stimuli and increased autoantibodies new stimuli and increased autoantibodies productionproduction

ImmunoglobulinsImmunoglobulins

Immunoglobulin structure Immunoglobulin structure

2 heavy (H) chains covalently linked by disulfide 2 heavy (H) chains covalently linked by disulfide bonds, each H chain is connected to a light (L) bonds, each H chain is connected to a light (L) chain by disulfide bondschain by disulfide bonds

H chain H chain consists of 4 to 5 domainsconsists of 4 to 5 domains (1 variable, 3-4 (1 variable, 3-4 constant) constant)

L chain consists of 2 immunoglobulin domains L chain consists of 2 immunoglobulin domains (1 variable, 1 constant)(1 variable, 1 constant)

Types of L chains - Types of L chains - , ,

Types of H chains - Types of H chains - , , , , ( (1-4) and (1-4) and (1, 1, 2), 2),

Variable domains of L and H chain form the binding Variable domains of L and H chain form the binding site for Ag site for Ag

Hinge region where the heavy chain linked by Hinge region where the heavy chain linked by disulfide bondsdisulfide bonds

Immunoglobulins are glykoproteins (glycosilated Fc Immunoglobulins are glykoproteins (glycosilated Fc part)part)

J chainJ chain

Secretory component Secretory component

Immunoglobulins functions Immunoglobulins functions

Antigen neutralizationAntigen neutralization

Antigen agglutinationAntigen agglutination Complement activation (IgM, IgG) Complement activation (IgM, IgG)

Opsonization (IgA, IgG, IgE) Opsonization (IgA, IgG, IgE)

Mast cell activation using IgEMast cell activation using IgE

ADCC ADCC

Classes of immunoglobulins and their Classes of immunoglobulins and their

functionsfunctions Distinguished by the constant part of H chain toDistinguished by the constant part of H chain to

IgM, IgD, IgG (IgG1 - IgG4), IgA (IgA1, IgA2), IgE IgM, IgD, IgG (IgG1 - IgG4), IgA (IgA1, IgA2), IgE

IgM IgM - as a monomer form BCR - as a monomer form BCR - secreted as pentamer (10 binding sites) - secreted as pentamer (10 binding sites) - first isotype that forms after the meeting with - first isotype that forms after the meeting with Ag Ag - neutralization of Ag, activates complement, - neutralization of Ag, activates complement, do not bind to Fc receptors on phagocytes do not bind to Fc receptors on phagocytes - (concentration of 0.9 to 2.5 g / l; biol. half-life - (concentration of 0.9 to 2.5 g / l; biol. half-life 6 days) 6 days)

IgDIgD - monomer form a BCR - monomer form a BCR - in serum is in a very low concentration - in serum is in a very low concentration        - (0.1 g / l; biol. half-life 3 days)        - (0.1 g / l; biol. half-life 3 days)

IgGIgG - isotypes IgG1-IgG4 different ability of complement - isotypes IgG1-IgG4 different ability of complement

activation and binding to Fc receptors of activation and binding to Fc receptors of

phagocytes (opsonization) phagocytes (opsonization)

       - function: neutralization, opsonization, complement        - function: neutralization, opsonization, complement

activation activation

       - passes the placenta (passive imunization from the        - passes the placenta (passive imunization from the

mother) mother)

       - formed in secondary immune response       - formed in secondary immune response

- (concentration of 8 to 18 g / l; biol. half-life of 21 - (concentration of 8 to 18 g / l; biol. half-life of 21

days) days)

IgAIgA - - mucosal IgAmucosal IgA - protection of mucous membranes, - protection of mucous membranes, neutralization, opsonization, neutralization, opsonization, do not activate complement do not activate complement - dimer, the secretory component - dimer, the secretory component - saliva, tears, breast milk - saliva, tears, breast milk - - serum IgAserum IgA - monomer, dimer or trimer - monomer, dimer or trimer - (Concentration of 0.9 to 3.5 g / l; - (Concentration of 0.9 to 3.5 g / l; biol. half-life of 6 days) biol. half-life of 6 days)

IgEIgE - applies in defense against multicellular parasites - applies in defense against multicellular parasites - is the main cause of allergic reactions - is the main cause of allergic reactions       - (concentration of 3x10-4 g / l; biol. half-life 2       - (concentration of 3x10-4 g / l; biol. half-life 2 days) days)

The genetic basis for the development of The genetic basis for the development of

immunoglobulinimmunoglobulin

Gene segments for H chainsGene segments for H chains – on chromosome 14 – on chromosome 14 VV (variable) (variable) DD (Diversity) (Diversity) J J (joining) (joining) CC constant domains of H chain constant domains of H chain

Gene segments for L chainsGene segments for L chains - - on chromosome 2 on chromosome 2

                                         -                                          - on chromosome 22 on chromosome 22 VV (variable) (variable) JJ (joining) (joining) CC constant domain of L chain constant domain of L chain

At the ends of V, D, J segments that are At the ends of V, D, J segments that are signal sequencessignal sequences which are recognized enzyme VDJ recombinase that carry which are recognized enzyme VDJ recombinase that carry out the out the rearrangement of these genesrearrangement of these genes

On the sides of C segments are so-called On the sides of C segments are so-called switch sequencesswitch sequences, , which are recognized by enzyme recombinase that carry which are recognized by enzyme recombinase that carry out out isotype switching isotype switching

The genetic basis of the immunoglobulins The genetic basis of the immunoglobulins development development

The rearrangement of genes coding H The rearrangement of genes coding H

chainchain 1) DJ rearrangement1) DJ rearrangement - excision a section IgH - excision a section IgH

between between DD and and JJ segment (runs on both segment (runs on both chromosomes)chromosomes)

2) VD rearrangement2) VD rearrangement - excision section between - excision section between some some VV segment and segment and DJ DJ, if is rearrangement on some , if is rearrangement on some chromosome successfull, stops the regrouping on the chromosome successfull, stops the regrouping on the second chromosome – it is called second chromosome – it is called allelic exclusionallelic exclusion (this (this is also true for L chain)is also true for L chain)

Transcript of rearranged IgH gene into mRNA , Transcript of rearranged IgH gene into mRNA , splicing of the primary transcript. The first form H splicing of the primary transcript. The first form H chain chain . .       If rearrangement is unsuccessful, B lymphocyte die. If rearrangement is unsuccessful, B lymphocyte die.

The rearrangement of genes coding L The rearrangement of genes coding L chainchain

1)1) First, rearrange the genes encoding the First, rearrange the genes encoding the L L chain chain , there is excision of sections between a , there is excision of sections between a VV and and JJ segment (simultaneously on both segment (simultaneously on both chromosomes), if the rearrangement is successful chromosomes), if the rearrangement is successful on one chromosomes, regrouping on the second on one chromosomes, regrouping on the second chromosome stops – it is called chromosome stops – it is called allelic exclusionallelic exclusion. .

2) 2) If regrouping of the If regrouping of the genes is unsuccessful, genes is unsuccessful, start the regrouping genes start the regrouping genes . .

3) 3) Not all H and L chain can form together a Not all H and L chain can form together a stable dimmers. stable dimmers.

If regrouping unsuccessful, B lymphocyte die. If regrouping unsuccessful, B lymphocyte die.

Isotype (class) switchingIsotype (class) switching Occurs during the terminal differentiation of B Occurs during the terminal differentiation of B

lymphocyte after activation with Ag on the surface lymphocyte after activation with Ag on the surface of FDC (require costimulating signal through CD40)of FDC (require costimulating signal through CD40)

Enzymes recombinases recognize the switch Enzymes recombinases recognize the switch sequences located on the sides of C segments (this sequences located on the sides of C segments (this sequence is not between Csequence is not between C and C and C segments - B segments - B cell can produce before isotype class switching IgM cell can produce before isotype class switching IgM and IgD simultaneously) and excise gene segments and IgD simultaneously) and excise gene segments    

After elimination of the C domain part is transcribed After elimination of the C domain part is transcribed into mRNA that segment, which is the closest to VDJ into mRNA that segment, which is the closest to VDJ segment and after splicing and translation arise segment and after splicing and translation arise correspondingcorresponding isotype of the H chainisotype of the H chain

Isotype switchingIsotype switching

Isotype switchingIsotype switching

CytokinesCytokines regulate which isotype occurs: regulate which isotype occurs:

IL-4IL-4 stimulates switching to stimulates switching to IgEIgE and IgG1, and IgG1, IgG4 IgG4 TGFTGF stimulates switching to IgG2 and stimulates switching to IgG2 and IgAIgA

Anti-idiotypic antibodiesAnti-idiotypic antibodies IDIOTYPIDIOTYP = summary of identical = summary of identical

binding structures for Ag on binding structures for Ag on antibodies the same specificity antibodies the same specificity

Idiotypic structures of Idiotypic structures of 1st generation antibodies1st generation antibodies can be recognized by some B lymphocytes as can be recognized by some B lymphocytes as antigens and can form against them antigens and can form against them anti-idiotypic anti-idiotypic antibodies (2nd generation antibodiesantibodies (2nd generation antibodies; some ; some binding sites may remind Ag, which caused binding sites may remind Ag, which caused formation of 1st generation antibodies)formation of 1st generation antibodies)

Against the 2nd generation antibodies formate Against the 2nd generation antibodies formate 3rd generation antibodies (anti-antiidiotypic 3rd generation antibodies (anti-antiidiotypic antibodies)antibodies)

The The idiotypic networkidiotypic network may play a role in may play a role in regulation of antibody response regulation of antibody response