HLA system (MHC glycoproteins)

MHC glycoproteins class I (Major histocompatibility complex) The function of MHCgpI is presentation of peptide fragments from inside the cell (which are produced by cell, including viral peptides if are present)on the cell surface so as to be recognized by T lymphocytes (cytotoxic, CD8) Present on all nucleated cells of the organism 3 isotypes classical human MHC gp. (HLA - A,-B,-C) 3 isotypes non-classical MHC gp. (HLA - E,-F,-G; molecule CD1)

Antigen presentation to cytotoxic T cells

MHC gp I structure MHC gp class I consists of transmembrane chain a and non-covalently associated b2mikroglobulin a chain has 3 domains, 2 N-terminal (a1, a2 - binding site for peptides) and 1 C-terminal domain (a3 - anchored in the cytoplasmic membrane, a structure similar to imunoglobulin domain)

MHC gpI peptide binding MHC gp I bind peptides with a length of 8 to 10 aminoacides Certain MHC gp molecule binds peptides sharing common structural features - coupling motif (critical are aminoacides near the end of peptide)

The binding of endogenous peptides occurs in the endoplasmic reticulum during biosynthesis of MHC gp I After a chain a and b2mikroglobulin create in the ER, folding into the correct conformation and the mutual association and the association of an appropriate peptide, the complex is further processed in the Golgi apparatus and then is presented on the cell surface Linked peptides are derived from proteins degraded by proteasome, proteasom degradate unneeded or damaged cytoplasmic proteins (labeled with ubiquitin), peptide fragments are transported into the ER by specific membrane pump TAP (transporter associated with antigen processing

MHC gpI peptide binding

Binding the peptide to MHCgpI

Non-classical MHC gp I HLA - E,-F,-G; CD1 molecules Structurally similar to classical MHC gp Are less polymorphic There are only on some cells They specialize in binding of specific ligands

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

MHC glycoproteins class II The function of MHC gpII is the presentation of peptide fragments from protein whitch are ingested by cell on the cell surface so as to be recognized by T lymphocytes (helper, CD4) Occur on the APC (dendritic cells, monocytes, macrophages, B lymphocytes) 3 isotypes of MHC gpII (DR, DQ, DP)

MHC gp II structure MHC gp II consist of 2 non-covalently associated transmembrane subunits a and b The peptide binding site consists of N-terminal domains a1 and b1 Binding of peptide is necessary for a stable MHC gp conformation and thus ensure its long presentation on the cell surface

MHC gp II peptide binding MHC gpII bind peptides with a length of 15 to 35 aminoacides (but possibly longer - because the peptide binding site is open at both ends) Certain MHC gp molecule binds peptides sharing common structural features - coupling motif After a string a and b are created in ER, fold into the correct conformation and the mutual associated are connected with another transmembrane chain called invariant chain, which blocks the binding site for the peptide, this complex is further processed in the Golgi apparatus, secretory vesicles isolated from GA merge with endosomes, then split the invariant chain and peptide fragments from cell absorbed proteins bind into binding site of MHC gp and the complex is then presented on cell surface

MHC gp II peptide binding

Antigen prezentation

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

MHC glycoproteins polymorphism HLA complex is located on chromosome 6 For MHC gp is typical high polymorphism, there are up to hundreds of different forms of alelic isotypes (except the non-classical MHC gp, and DR a chain) Codominant inheritance of alelic forms (Individual has 3 cell surface isotypes of HLA molecules (HLA-A,-B,-C) mostly in 2 different alelic forms) Polymorphism has a protective significance at individual and population level MHC gp polymorphism causes complications in transplantation

HLA typing = determmination of HLA antigens on the surface of lymphocytes Carry out during the testing before transplantation and in determination of paternity 1) Serotyping Microlymfocytotoxic test Allospecific serums (obtained from multiple natal to 6 weeks after birth, obtained by vaccination of volunteers, or commercially prepared sets of typing serums (monoclonal antibodies)) Principle - the incubation of lymphocytes with typing serums in the presence of rabbit complement, then is added the vital dye which stained dead cells - cells carrying specific HLA are killed by cytotoxic Ab against the Ag, the percentage of dead cells is a measure of serum toxicity (forces and antileukocyte antibody titre) Positive reaction is considered more than 10% dead cells (serological typing can be done also by flow cytometry

2) Molecular genetic methods For typing are used hypervariable sections in the II. exon genes coding for HLA class II; to determine HLA class I is used polymorphism in II. and III. exon coding genes 2a) PCR-SSP = Polymerase chain reaction with sequential specific primers Extracted DNA is used as a substrate in a set of PCR reactions Each PCR reaction contains primers pair specific for a certain allele (or group of alleles) Positive and negative reactions are evaluated by electrophoresis, each combination of alleles has a specific electrophoretic painting

2b) PCR-SSO PCR reaction with sequence-specific oligonucleotides Multiplication of hypervariable sections of genes coding HLA Hybridization with enzyme or radiolabeled DNA probes specific for individual alleles 2c) PCR-SBT Sequencing based typing The most accurate method of HLA typing We get the exact sequence of nucleotides, which compares with a database of known sequences of HLA alleles

T – lymphocytes J. Ochotná

T lymphocytes cellular component of antigen-specific mechanisms several different subsets of T lymphocytes regulation of immune processes, the destruction of virus-infected cells or tumor cells recognize antigen processed and presented by the APC T cells are after activation stimulated to multiplication and differentiation into effector cells and part of them differentiate into the memory cells

T-lymphocytes development T cells originate in bone marrow and then migrate to the thymus where they mature (abT lymphocytes), the final differentiation is after activation by antigen processed and presented by APC gdT cells can develop outside the thymus (the minority population) Pluripotent hematopoietic stem cells Pro-thymocytes – double negative T cells - are coming from the bone marrow to the thymus, where they begin to rearrange TCRb genes, expressing on their surface, called pre-TCR (Composed of b chain, pre-TCRa and CD3 complex), then begin TCRa genes rearrangement Cortical thymocytes – double positive T cells - express on their surface TCR (composed of chains a, b and CD3) and CD4 and CD8 co-receptor (double positive T lymphocyte), at this stage occurs the selection of autoreactive cells and cells with dysfunctional TCR Medullary thymocytes (mature T cell) - retain the expression of CD4 or CD8, then migrate to secondary lymphoid organs

T-lymphocytes selection Negative selection - the elimination of autoreactive cells, when thymocytes binds enough strongly by their TCR complex of MHCgp with normal peptides (from autoantigens)which are presented on surface of thymic cells thymocyte receives signals leading to apoptotic cell death PAE cells (peripherial antigen expressing cells) Positive selection - the elimination of cells with dysfunctional TCR, positively are selected thymocytes that recognize MHC gp with low affinity, then maintain the expression of CD4 or CD8 (depending what class of MHC gp binds to the TCR). These mature T cells (Medullary thymocytes) leave the thymus and migrate to secondary lymphoid organs 98% of pro-thymocytes in the thymus during its development dies

T cell development

T-lymphocytes surface markers TCR - recognizes Ag peptide complexed with MHC gp CD3 - TCR component, participation in signal transduction CD4 or CD8 - co-receptors, binding to MHC gp CD28 - costimulatory receptor, binds to CD80, CD86 on APC CTLA-4 (CD152) - inhibitory receptor, binds to CD80, CD86

Interaction between APC and T cell

T-lymphocytes subpopulations ab-T lymphocytes - have TCRab, major type (95%), thymus need in development, recognize antigens in the complex MHC-peptide gp gd-T lymphocytes - (5%) may develop outside the thymus, some are able to recognize native Ag, apply in defense of the skin and mucous membranes

ab T-lymphocytes Expressing the CD4 co-receptor (co-receptor for MHC class II gp), precursors of helper T cells (TH), they can be classified according to the production of cytokines TH0 - produce a mixture of cytokines such as TH1 and TH2 TH1 - IL-2, IFNg (help macrophages ) TH2 - IL-4, IL-5, IL-6, IL-10 (B lymphocytes assistance) TH3 - TGFb Treg - regulatory T cells arise in the thymus from a part of autoreactive lymphocytes, suppress the activity of TH1 and partly function as TS, suppression of autoreactive T cell clones

ab T-lymphocytes Expressing the CD8 co-receptor (co-receptor for MHC gp class I), precursors of cytotoxic T cells (TC), or suppressor T cells (TS) TC - recognize cells infected by viruses or other intracellular parasites and some cancer cells TS - inhibit the function of other lymphocytes

TCR TCR (T cell receptor) is heterodimer consisting of a and b (g,d) chain and associated CD3 complex, which is necessary for signal transfer (is connected with PTK) "N-terminal parts of a and b (g,d) chain form the binding site for Ag

TCR cooperation with co-receptors CD4, CD8

TCR development The analogy with the formation of BCR Chains b and d - correspond to IgH gene complex of immunoglobulins - V, D, J, C segments Chains a and g - correspond to genes for L chains of immunoglobulins - V, J, C segments Rearrangement of genes is similar to the BCR and performed by the same recombinases

Antigen-specific mechanisms

TH1 based immune response

TH1 immune response - inflammatory reaction TH1 cells cooperate with macrophages and transform them in activated (NO production - destroy intracellular parasites) Activated macrophages secrete some cytokines (IL-1, TNF, ...) that help to stimulate T cells and stimulate local inflammation, which helps suppress infection Interaction between TH1 cells and macrophages is a fundamental mechanism of delayed-type immunopathological reactions (DTH Delayed-type hypersensitivity)

The infected macrophage produces protein fragments derived from intracellular parasites, some of which are presented on the surface by MHC gp class II Macrophages and dendritic cells stimulated by certain microorganisms produce IL-12 TH precursor, which detects the infected macrophage and receives signals via the TCR, CD 28 and receptor for IL-12 and other adhesion and signaling molecules proliferates and differentiates to the effector TH1 cells that produce IFNg and IL-2. IFNg activates macrophage NO synthase IL-2 is an autocrine growth factor for TH1 cells

Interaction between APC and TH precursor

TH2 based immune response

TH2 immune response – help to B-lymphocytes TH2 cells cooperate with B lymphocytes (which were stimulated by Ag) by cytokine production (IL-4, IL-5, IL-6) and direct intercellular contact For stimulation of B lymphocytes is usually necessary cooperation between APC → TH2 cell → B lymphocyte In minimal model, where the B cell becomes a good APC (CD80, CD86) is sufficient cooperation between TH2 cell → B lymphocyte

TH precursor, which detects the infected macrophage and receives signals through the TCR, CD 28 receptor for IL-4 receptor and IL-2 and other adhesion and signaling molecules proliferates and differentiates in the effector TH2, which provide B lymphocytes auxiliary signals via cytokines secreted by IL-4, IL-5, IL-6 and adhesion molecules through CD 40L, which bind to the costimulatory receptor on B lymphocytes CD 40 Interaction between CD40 (B lymphocytes) and CD40L (TH2 cells) is essential for the initiation of somatic mutations, izotype switching and formation of memory cells IL-4, IL-5, IL-6: stimulation of B lymphocytes

Assistance to B lymphocytes Specific direct assistance to B lymphocytes: TH2 lymphocytes assisting B lymphocytes that were stimulated by the same Ag, which caused the rise of TH2 To stimulate the secretion of cytokines by TH2 cell is sufficient signal via the TCR (signal through a costimulatory receptor CD28 is no longer necessary) One clone of TH2 cells can provide specific assistance to B lymphocytes of different specificities (must present the relevant Ag peptides by MHC gp II, which are recognized by TCR)

Assistance to B lymphocytes Indirect assistance to B cells ("bystander help"): TH2 lymphocytes assisting B lymphocytes that were stimulated by another Ag than that which caused the rise of TH2 Contact between TH2 cell → B lymphocytes via adhesion molecules, cytokine secretion, binding CD40-CD40L Danger of activation autoreactive B lymphocytes

Mutual regulation of activities TH1versus TH2 Whether the TH precursor cell will develop into TH1 or TH2 decides cytokine ratio of IL-12 and IL-4 IL-12 is produced by macrophages and dendritic cells stimulated by certain microorganisms IL-4 is produced by activated basophils and mast cells TH1 cytokines (mainly IFNg) inhibit the development of TH2 and stimulate the development of TH1 (IL-2 stimulates also TH2) Cytokines produced by TH2 (IL-4, IL-10) inhibit the development of TH1 and stimulate the development of TH2 TH3 development is stimulated by a specific cytokine environment (IL-4, IL-10, TGFb); TH3 produce TGFb and cooperate with B cells in MALT

TC based immune response

Cytotoxic T lymphocytes stimulation TC recognize cells infected with viruses or other intracellular parasites, and some tumor cells Precursor of TC, which recognizes a complex of MHC gp I- antigenic peptide on the surface of APC via TCR and receives signals via CD 28 proliferates and differentiates to clone mature effector cytotoxic cells (CTL); TH1 cells help to TC by production IL-2 Effector TC are spread by bloodstream into tissues; for activation of cytotoxic mechanisms is sufficient signal through the TCR (signal through a costimulatory receptor CD28 is no longer necessary)

Professional APC are dendritic cells or macrophages that are infected with virus, or swallowed antigens from dead infected, tumor or stressed cells In order APC could activate the TC precursor, APC must be stimulated by contact with TH cells via CD 40, then the dendritic cell begins to express CD 80, CD86 and secrete cytokines (IL-1, IL-12) = change of resting APC in activated

Tc effector functions Cytotoxic granules containing perforin and granzymes (perforin creates pores in the cytoplasmic membrane of target cell, in some cases may lead to osmotic lysis of the target cell, formed pores in the cell receiving granzymes that cause the target cell to die by apoptosis. Fas ligand (FasL) - which binds to the apoptotic receptor Fas (CD95) presented on the surface of many different cells (also on the surface of TC) TNFb

Antibody-based immune response

Antibody responses induced by T-independent antigens Cause predominantly IgM production Bacterial polysaccharides, lipopolysaccharides, and polymeric forms of protein T-dependent antigens Reaction to these Ag occurs in two phases - primary and secondary response Initiate the development of memory cells and formation of high-affinity antibodies

T-independent and T-dependent immune response

Antibody responses induced by T-dependent antigen Primary phase of antibody response The first contact with Ag Takes place in secondary lymphoid organs Stimulation of B cells by Ag binding to BCR Ag absorption by APC and its presentation via MHC gp class II to precursors of TH cell → formation of clone of antigen-specific TH2 cells, which provide assistance to competent B lymphocytes, leading to their proliferation, differentiation into plasma (produce Ab) and memory cells

Plasma cells are spread by bloodstream into the organism (particularly bone marrow) Antibodies produced in the primary stage (3-4 days) are IgM and have a low affinity for Ag, create with Ag immune complexes Immune complexes are captured in the secondary lymphoid organs on the surface of FDC (follicular dendritic cells) - Ag presenting cells to B lymphocytes

Secondary phase of antibodies response Recognition of Ag on FDC (If is sufficient amount of immune complexes on FDCs) Under the influence of signals from the FDC (Ag) and TH2 cells (CD40L, cytokines) is again started the proliferation and differentiation of B cells accompanied with somatic mutations → formation of clones of B cells with new BCR → survive only B cells with a BCR with the highest affinity for Ag = affinity maturation of antibodies There is also isotype switching, which isotypes arise determines cytokine environment

In the secondary phase of the immune response generate antibodies with higher affinity for Ag and other effector characteristics dependent on isotype, also formed a memory cells for next meeting with the Ag Antibodies in the body after primary infection persist for a long time Contact between CD40 (B lymphocytes) and CD40L (TH2 lymphocytes) is essential for the initiation of somatic mutations, isotype switching and formation of memory cells

Primary and secondary immune reaction Primary immune reaction – occurs on the firsth exposure to antigen Secondary immune reaction –occurs after subsequent encounter with the same antigen and is more rapid leading to the activation of previously generated memory cells

Primary and secondary immune reaction

B lymphocytes

B-lymphocytes B-lymphocytes (B cells) are cells responsible especially for specific, antibody-mediated immune response. They also have great importance for the immune memory (which is used for vaccination). B-cells recognize native antigen through BCR (B cell receptor) B-lymphocyte whitch bind Ag through BCR are stimulated to proliferate and differentiate to effector plasma cells which produce large quantities of antibodies of the same specificity as the BCR (it is actually the same protein in soluble form). Part of stimulated B-cells differentiate to memory cells.

Surface characteristics of B lymphocytes CD 10 - immature B lymphocyte CD 19 - characteristic surface sign of B cells CD 20 - on the surface of Ig-positive B lymphocytes IgM, IgD - BCR MHC gp II - Ag presenting molecules CD 40 – costimulating receptor

B cell development Development of B lymphocytes takes place in the bone marrow and completes after activation with Ag in secondary lymphoid organs. Pluripotent hematopoietic stem cell Progenitor B cell - begin recombination processes which lead to a large number of clones B lymphocytes with individual specific BCR Pre - B cell - expression of pre-B receptor (composed of H (m) chain and alternate L chain) Immature - B lymphocyte - expression of surface IgM (BCR) at this stage elimination of autoreactive clones Mature B lymphocyte - expression of surface IgM and IgD (BCR)

Critical moments in the B cell development Completion of the rearrangment of genes for H chain and surface expression of pre-BCR Successful rearrangement of genes for L chain and surface expression of IgM (BCR) Testing of immature B cells, whether they are autoreactive Another critical stage are somatic mutations and affinity maturation, when survive only B cells with the highest affinity for antigen.

BCR BCR is composed from surface immunoglobulin (IgM, IgD - H chains are transmembrane, recognizes Ag) and associated signaling molecules (Iga and IGb), which are associated with the cytoplasmic protein-tyrosine kinases (PTK) Src Group After binding of Ag to 2 or more BCR will approximate PTK, mutual phosphorylation and phosphorylation of other cytoplasmic proteins, leading to changes in gene transcription, proliferation, differentiation and secretion of antibodies The signal by binding Ag to the BCR can be amplified by cooperation with CR2, which binds C3dg (opsonin)

Elimination of autoreactive B lymphocytes By random rearrangement of genes, connecting inaccuracy, H-L pairing and somatic mutations may also arise clones of B cells bearing autoreactive receptors and produce autoreactive antibodies. Majority of autoreactive B lymphocytes are eliminated as the immature B lymphocytes in the bone marrow, if its BCR bind autoantigen with sufficient affinity, receives a signal leading to apoptotic death (clonal deletion). If some of the autoreactive clones pass this elimination, their autoreaktivity usually do not come because lack of TH lymphocytes for their activation, many autoantigens are cryptic, or occur in low concentrations and are ignored by the immune system. Tolerance to self-antigens is critical in preventing autoimmunity in the organism.

Antigen recognition by B cell in secondary lymphoid organs

Ontogenesis of antibodies Synthesis of specific antibodies begins around the 20.-24. week of gestation, the total concentration of IgA and IgM remains undetectable until birth, IgG begin to form after birth B lymphocytes respond to immunization predominantly by IgM formation, switching to other isotype is slower Slow growth of own IgG decline in maternal IgG (about 3. to 6.month)

The IgM concentration reaches values comparable to adults in the 1- 3 year of life, IgG and IgA between 10.-15. year Antibody response to polysaccharide antigens appears until around 2. year of life In old age is a lower antibody response to new stimuli and increased autoantibodies production

Immunoglobulins

Immunoglobulin structure 2 heavy (H) chains covalently linked by disulfide bonds, each H chain is connected to a light (L) chain by disulfide bonds H chain consists of 4 to 5 domains (1 variable, 3-4 constant) L chain consists of 2 immunoglobulin domains (1 variable, 1 constant) Types of L chains - k, l Types of H chains - m, d, g (g1-4) and (a1, a2), e

Variable domains of L and H chain form the binding site for Ag Hinge region where the heavy chain linked by disulfide bonds Immunoglobulins are glykoproteins (glycosilated Fc part) J chain Secretory component

Immunoglobulins functions Antigen neutralization Antigen agglutination Complement activation (IgM, IgG) Opsonization (IgA, IgG, IgE) Mast cell activation using IgE ADCC

Classes of immunoglobulins and their functions Distinguished by the constant part of H chain to IgM, IgD, IgG (IgG1 - IgG4), IgA (IgA1, IgA2), IgE IgM - as a monomer form BCR - secreted as pentamer (10 binding sites) - first isotype that forms after the meeting with Ag - neutralization of Ag, activates complement, do not bind to Fc receptors on phagocytes - (concentration of 0.9 to 2.5 g / l; biol. half-life 6 days) IgD - monomer form a BCR - in serum is in a very low concentration        - (0.1 g / l; biol. half-life 3 days)

IgG - isotypes IgG1-IgG4 different ability of complement activation and binding to Fc receptors of phagocytes (opsonization)        - function: neutralization, opsonization, complement activation        - passes the placenta (passive imunization from the mother)        - formed in secondary immune response - (concentration of 8 to 18 g / l; biol. half-life of 21 days)

IgA - mucosal IgA - protection of mucous membranes, neutralization, opsonization, do not activate complement - dimer, the secretory component - saliva, tears, breast milk - serum IgA - monomer, dimer or trimer - (Concentration of 0.9 to 3.5 g / l; biol. half-life of 6 days) IgE - applies in defense against multicellular parasites - is the main cause of allergic reactions       - (concentration of 3x10-4 g / l; biol. half-life 2 days)

The genetic basis for the development of immunoglobulin

The genetic basis of the immunoglobulins development Gene segments for H chains – on chromosome 14 V (variable) D (Diversity) J (joining) C constant domains of H chain Gene segments for L chains - k on chromosome 2                                          - l on chromosome 22 V (variable) J (joining) C constant domain of L chain At the ends of V, D, J segments that are signal sequences which are recognized enzyme VDJ recombinase that carry out the rearrangement of these genes On the sides of C segments are so-called switch sequences, which are recognized by enzyme recombinase that carry out isotype switching

The rearrangement of genes coding H chain 1) DJ rearrangement - excision a section IgH between D and J segment (runs on both chromosomes) 2) VD rearrangement - excision section between some V segment and DJ, if is rearrangement on some chromosome successfull, stops the regrouping on the second chromosome – it is called allelic exclusion (this is also true for L chain) Transcript of rearranged IgH gene into mRNA , splicing of the primary transcript. The first form H chain m.    If rearrangement is unsuccessful, B lymphocyte die.

The rearrangement of genes coding L chain 1) First, rearrange the genes encoding the L chain k, there is excision of sections between a V and J segment (simultaneously on both chromosomes), if the rearrangement is successful on one chromosomes, regrouping on the second chromosome stops – it is called allelic exclusion. 2) If regrouping of the k genes is unsuccessful, start the regrouping genes l. 3) Not all H and L chain can form together a stable dimmers. If regrouping unsuccessful, B lymphocyte die.

Isotype (class) switching Occurs during the terminal differentiation of B lymphocyte after activation with Ag on the surface of FDC (require costimulating signal through CD40) Enzymes recombinases recognize the switch sequences located on the sides of C segments (this sequence is not between Cm and Cd segments - B cell can produce before isotype class switching IgM and IgD simultaneously) and excise gene segments   After elimination of the C domain part is transcribed into mRNA that segment, which is the closest to VDJ segment and after splicing and translation arise corresponding isotype of the H chain

Isotype switching

Isotype switching Cytokines regulate which isotype occurs: IL-4 stimulates switching to IgE and IgG1, IgG4 TGFb stimulates switching to IgG2 and IgA

Anti-idiotypic antibodies IDIOTYP = summary of identical binding structures for Ag on antibodies the same specificity Idiotypic structures of 1st generation antibodies can be recognized by some B lymphocytes as antigens and can form against them anti-idiotypic antibodies (2nd generation antibodies; some binding sites may remind Ag, which caused formation of 1st generation antibodies) Against the 2nd generation antibodies formate 3rd generation antibodies (anti-antiidiotypic antibodies) The idiotypic network may play a role in regulation of antibody response