NK cellsNK cells

InterferonsInterferons

J. J. OchotnáOchotná

NK cellsNK cells Part of antigen non-specific mechanisms Part of antigen non-specific mechanisms (innate) (innate)

They do not have antigen-specific receptors They do not have antigen-specific receptors

Recognize cells that have abnormally lowRecognize cells that have abnormally low MHCgpI (some tumor and virus infected cells) MHCgpI (some tumor and virus infected cells)

They are able to kill quickly - without prior They are able to kill quickly - without prior stimulation, proliferation and differentiation stimulation, proliferation and differentiation

Activators of NK cells - IFNActivators of NK cells - IFN, IFN, IFN

Activating receptorsActivating receptors - Some surface lectins, - Some surface lectins, Fc receptor CD16 Fc receptor CD16 ADCCADCC (antibody-dependent cellular cytotoxicity) NK cells (antibody-dependent cellular cytotoxicity) NK cells recognize cell opsonized IgG antibody through the Fc recognize cell opsonized IgG antibody through the Fc receptor CD16, this leads to the activation of cytotoxic receptor CD16, this leads to the activation of cytotoxic mechanisms (NK degranulation) mechanisms (NK degranulation)

Inhibitory receptorsInhibitory receptors - Signals provided through these - Signals provided through these receptors inhibit the cytotoxic mechanisms receptors inhibit the cytotoxic mechanisms

Imunoglobulin familyImunoglobulin family - KIR (killer - KIR (killer

inhibitor receptors)inhibitor receptors) C type lektin familyC type lektin family - eg CD94/NKG2 - eg CD94/NKG2

Receptors of NK cellsReceptors of NK cells

Cytotoxic mechanisms of NK cellsCytotoxic mechanisms of NK cells The resulting reaction of NK cell after meeting The resulting reaction of NK cell after meeting

with another cell depends on which signal prevail, with another cell depends on which signal prevail, whether activating or inhibitory signals whether activating or inhibitory signals

Cytotoxic granulesCytotoxic granules contain perforin and contain perforin and granzyme (perforin creates pores in the granzyme (perforin creates pores in the cytoplasmic membrane of target cells, in some cytoplasmic membrane of target cells, in some cases may cause osmotic lysis of the target cell, cases may cause osmotic lysis of the target cell, formed pores in the cell receiving granzymes, formed pores in the cell receiving granzymes, that cause the target cell to die by that cause the target cell to die by apoptosisapoptosis. .

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

TNFTNF

InterferonsInterferons Belongs to the humoral component of non-specific Belongs to the humoral component of non-specific

mechanismsmechanisms IFNIFN - produced by virus infected lymphocytes, - produced by virus infected lymphocytes,

monocytes and macrophagesmonocytes and macrophages IFNIFN - produced by virus-infected fibroblasts and - produced by virus-infected fibroblasts and

epithelial cellsepithelial cells IFNIFN and IFN and IFN- bind to receptors on the surface of - bind to receptors on the surface of

infected and healthy cells and induce in them an infected and healthy cells and induce in them an antiviral state (synthesis of enzymes that block antiviral state (synthesis of enzymes that block viral replication in the cell) viral replication in the cell)

IFNIFN - produced by T - produced by THH1 cells, has regulatory 1 cells, has regulatory function, activates macrophages and stimulates the function, activates macrophages and stimulates the expression of MHCgpexpression of MHCgp

Basophils and mast cells Basophils and mast cells

and their importance in and their importance in

immune responsesimmune responses

Mast cellsMast cells

Mucosal mast cells - in the mucous membranes of respiratory and gastrointestinal tract, produce histamine, serotonin, heparin, tryptase, leukotriene C4 ..., participate in parasitosis and allergy

Connective tissue mast cells - the connective tissue, producing tryptase, chymase, prostaglandinD2 ..., are multiplicated in fibrosis, in parasitosis and allergy are not participating

Functions of the mast cellsFunctions of the mast cells Defense against parasitic infectionsDefense against parasitic infections

In pathological circumstances, responsible for the early type In pathological circumstances, responsible for the early type of hypersensitivity (immunopathological reaction typeI)of hypersensitivity (immunopathological reaction typeI)

Regulation of immune response Regulation of immune response

Apply during inflammation, in angiogenesis, in tissue Apply during inflammation, in angiogenesis, in tissue remodeling remodeling

Involved in the maintenance of physiological functions Involved in the maintenance of physiological functions of mucosal of mucosal

Contribute to the normal metabolism of connective tissueContribute to the normal metabolism of connective tissue

Communication between the immune and nervous system Communication between the immune and nervous system

Activatio schema of the mast cellActivatio schema of the mast cell

Mast cells can be stimulatedMast cells can be stimulated to degranulate by direct injury to degranulate by direct injury (opioids, alcohols, and certain antibiotics), cross-linking of IgE Fc (opioids, alcohols, and certain antibiotics), cross-linking of IgE Fc receptors, or by anafylatoxins (C3a, C5a) receptors, or by anafylatoxins (C3a, C5a)

Establishing of multivalent antigen (multicellular parasite) Establishing of multivalent antigen (multicellular parasite) to highaffinnity to highaffinnity Fc receptor for IgEFc receptor for IgE (Fc (FcRI) RI)

Aggregation of several molecules FcAggregation of several molecules FcRI RI

Initiate mast cell degranulation (cytoplasmic granules mergers Initiate mast cell degranulation (cytoplasmic granules mergers with the surface membrane and release their contents) with the surface membrane and release their contents)

Activation of arachidonic acid metabolism (leukotriene C4, Activation of arachidonic acid metabolism (leukotriene C4, prostaglandin D2) prostaglandin D2)

Start of production of cytokines (TNF, TGFStart of production of cytokines (TNF, TGF, IL-4, 5,6 ...) , IL-4, 5,6 ...)

After binding After binding C3aC3a or or C5aC5a to its receptor on mast cell (activate to its receptor on mast cell (activate degranulation independent on IgE) degranulation independent on IgE)

Activation schema of mast cellActivation schema of mast cell

Secretory products of mast cellsSecretory products of mast cells

Cytoplasmatic granules: hydrolytic enzymes, proteoglycans (heparin, chondroitin sulphate), biogenic amines (histamine, serotonin)

Histamine causes vasodilation, increased vascular permeability, erythema, edema, itching, contraction of bronchial smooth muscle, increases intestinal peristalsis, increased mucus secretion of mucosal glands in the respiratory tract and GIT (helps eliminate the parasite)

Arachidonic acid metabolites (leukotriene C4, prostaglandin D2)

Cytokines (TNF, TGF , IL-4, 5,6 ...)

The role of mast cells in development of The role of mast cells in development of allergyallergy

BasophilsBasophils

Differentiate from myeloid precursor Differentiate from myeloid precursor

They are considered to be the circulating form of They are considered to be the circulating form of mast mast

Receptor equipment, containing granules, the Receptor equipment, containing granules, the mechanisms of stimulation and functions are very mechanisms of stimulation and functions are very similar to mast cellssimilar to mast cells

They are responsible for the emergence of They are responsible for the emergence of anaphylactic shock anaphylactic shock

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 nuclear cells of the organism Present on all nuclear cells of the organism

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

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

Structure of MHC gp IStructure of MHC gp I MHC gp class I consists of transmembrane MHC gp class I consists of transmembrane

chain chain and non-covalently associated and non-covalently associated 22mikrotubulin mikrotubulin

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

Binding site for the peptide is structurally eminent Binding site for the peptide is structurally eminent groove whose bottom is made up of groove whose bottom is made up of structure and structure and sides are bounded by 2 sides are bounded by 2 helix helix

Binding of peptide is necessary for a stable conformation Binding of peptide is necessary for a stable conformation of MHCgp and thus ensure its long presentation on the of MHCgp and thus ensure its long presentation on the cell surface cell surface

Binding of peptides to MHCgpIBinding of peptides to MHCgpI 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 (critical are aminoacides near the end of peptide) 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

After a string After a string and and 22mikrotubulin create in the ER, mikrotubulin 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 derived from proteins degraded Linked peptides derived from proteins degraded proteasome, which cleaves cytoplasmic proteins for proteasome, which cleaves cytoplasmic proteins for destruction (labeled with ubiquitin), peptide fragments destruction (labeled with ubiquitin), peptide fragments are transported into the ER by specific membrane are transported into the ER by specific membrane pump pump

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

CD1 molecules - bind glycolipids or other highly CD1 molecules - bind glycolipids or other highly hydrophobic compounds, these complexes are hydrophobic compounds, these complexes are recognized by specialized recognized by specialized T lymphocytes (NK-T T lymphocytes (NK-T lymphocytes) lymphocytes)

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 (auxiliary CD4) recognized by T lymphocytes (auxiliary 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)

Structure of MHC gp IIStructure of MHC gp II 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

Binding of peptides to MHC gp IIBinding of peptides to MHC gp II

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

Diagram of the biosynthesis of complex MHC Diagram of the biosynthesis of complex MHC

gpII with peptides from ingested proteingpII with peptides from ingested protein

Polymorphism of MHC glycoproteinsPolymorphism of MHC glycoproteins 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 (Individual has 3 cell Codominant inheritance of alelic forms (Individual has 3 cell surface isotypes of HLA molecules (HLA-A,-B,-C) mostly in 2 surface isotypes of HLA molecules (HLA-A,-B,-C) mostly in 2 different alelic forms) different alelic forms)

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

Ppolymorphism MHC gp causes complications in Ppolymorphism MHC gp 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 a specific HLA are killed by cytotoxic Ab - cells carrying a 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

ImmunoglobulinsImmunoglobulins

Structure of immunoglobulinsStructure of immunoglobulins

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

Antibody cleavage by papain Antibody cleavage by pepsin

Functions of immunoglobulinesFunctions of immunoglobulines

Neutralization of AgNeutralization of Ag

Agglutinate AgAgglutinate Ag Complement activation (IgM, IgG) Complement activation (IgM, IgG)

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

Activation of mast cells using IgEActivation of mast cells 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) - Several hundred (variable) - Several hundred DD (Diversity) - about 50 (Diversity) - about 50 J J (joining)- 9 (joining)- 9 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 signal sequences At the ends of V, D, J segments that are signal sequences which are recognized enzyme VDJ recombinase that carry which are recognized enzyme VDJ recombinase that carry out the rearrangement of these genesout the rearrangement of these genes

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

The genetic basis for the development The genetic basis for the development of immunoglobulinof immunoglobulin

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.

Variability of immunoglobulinsVariability of immunoglobulins

Variability of immunoglobulins is determined by: Variability of immunoglobulins is determined by:

1)1) Diversity of the combination V(D) J Diversity of the combination V(D) J segmentssegments

2)2) Connecting variabilityConnecting variability - after excision of gene - after excision of gene sequences the end are not cut off exactly sequences the end are not cut off exactly

3)3) The enzyme terminal transferaseThe enzyme terminal transferase - prolonge - prolonge cut off ends with the short random sequences cut off ends with the short random sequences

4)4) Somatic mutationsSomatic mutations of V segmentsof V segments of the of the rearranged genes after contact with Ag on the rearranged genes after contact with Ag on the surface of FDC surface of FDC

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 what isotype occurs: regulate what isotype occurs:

IL-4IL-4 stimulates switching to IgG1 and IgE, IgG4 stimulates switching to IgG1 and IgE, IgG4 TGFTGF stimulates switching to IgG2 and IgA stimulates switching to IgG2 and IgA

Regulation, whether it will be Regulation, whether it will be secreted or secreted or membrane formmembrane form is at the level of mRNA (at 3´end is at the level of mRNA (at 3´end of C segment are after the sequences encoding of C segment are after the sequences encoding secreted form a sequences of membrane-form) secreted form a sequences of membrane-form)

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

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

Development of B lymphocytesDevelopment of B lymphocytes 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 development Critical moments in the development

of B lymphocytesof B lymphocytes 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.

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.

Recognition of Ag by B cell Recognition of Ag 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 concentration of IgM reaches values The concentration of IgM reaches values comparable comparable to adults in the 1- 3 year of life, IgG and Ig to adults in the 1- 3 year of life, IgG and Ig A between 10.-15. yearA between 10.-15. year

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

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