NK cells Interferons J. Ochotná

NK cells Part of antigen non-specific mechanisms (innate)  Part of antigen non-specific mechanisms (innate)  They do not have antigen-specific receptors  Recognize cells that have abnormally low MHCgpI expression (some tumor and virus infected cells)  They are able to kill quickly - without prior stimulation, proliferation and differentiation  Activators of NK cells - IFN , IFN 

 Activating receptors - Some surface lectins, Fc receptor CD16 ADCC (antibody-dependent cellular cytotoxicity) NK cells recognize cell opsonized IgG antibody through the Fc receptor CD16, this leads to the activation of cytotoxic mechanisms (NK degranulation)  Inhibitory receptors - Signals provided through these receptors inhibit the cytotoxic mechanisms (recognize MHC gpI)  Imunoglobulin family - KIR (killer inhibitor receptors) inhibitor receptors)  C-type lektin family - eg CD94/NKG2 NK cells receptors

NK cell cytotoxic mechanisms  The resulting reaction of NK cell after meeting with another cell depends on which signal prevail, whether activating or inhibitory signals  Cytotoxic granules contain perforin and granzyme (perforin creates pores in the cytoplasmic membrane of target cells, in some cases may cause 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  TNF 

Interferons  Belongs to the humoral component of non-specific mechanisms  IFN  - produced by virus infected lymphocytes, monocytes and macrophages  IFN  - produced by virus-infected fibroblasts and epithelial cells  IFN  and IFN  - bind to receptors on the surface of infected and healthy cells and induce in them an antiviral state (synthesis of enzymes that block viral replication in the cell)  IFN  - produced by T H 1 cells, has regulatory function, activates macrophages and stimulates the expression of MHCgp

Basophils and mast cells and their importance in immune responses

Mast 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

Mast cell functions  Defense against parasitic infections  In pathological circumstances, responsible for the early type of hypersensitivity (immunopathological reaction typeI)  Regulation of immune response  Apply during inflammation, in angiogenesis, in tissue remodeling  Involved in the maintenance of physiological functions of mucosal  Contribute to the normal metabolism of connective tissue  Communication between the immune and nervous system

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

Mast cell activation by cross-linking of IgE Fc receptors  Establishing of multivalent antigen (multicellular parasite) to highaffinnity Fc receptor for IgE (Fc  RI)  Aggregation of several molecules Fc  RI  Initiate mast cell degranulation (cytoplasmic granules mergers with the surface membrane and release their contents)  Activation of arachidonic acid metabolism (leukotriene C4, prostaglandin D2)  Start of production of cytokines (TNF, TGF , IL-4, 5,6...)

Activation schema of mast cell

Secretory 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 allergy

Basophils  Differentiate from myeloid precursor  They are considered to be the circulating form of mast  Receptor equipment, containing granules, the mechanisms of stimulation and functions are very similar to mast cells  They are responsible for the emergence of anaphylactic shock

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)

MHC gp I structure  MHC gp class I consists of transmembrane chain  and non-covalently associated  2 mikrotubulin   chain has 3 domains, 2 N-terminal (  1,  2 - binding site for peptides) and 1 C-terminal domain (  3 - anchored in the cytoplasmic membrane, a structure similar to imunoglobulin domain)  Binding site for the peptide is structurally eminent groove whose bottom is made up of  structure and sides are bounded by 2  helix  Binding of peptide is necessary for a stable conformation of MHCgp and thus ensure its long presentation on the cell surface

Peptide binding to MHCgpI  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  After a chain  and  2 mikrotubulin 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 derived from proteins degraded by proteasome, which cleaves cytoplasmic proteins for destruction (labeled with ubiquitin), peptide fragments are transported into the ER by specific membrane pump

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

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 IIstructure MHC gp II structure  MHC gp II consist of 2 non-covalently associated transmembrane subunits  and   The peptide binding site consists of N-terminal domains  1 and  1  Binding of peptide is necessary for a stable MHC gp conformation and thus ensure its long presentation on the cell surface

Peptides binding to MHC gp II  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  and  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 

Binding the peptide to MHCgpII

Antigen prezentation

Antigen presentation to T lymphocyte 1.Signal: TCR – MHC gp I(II)+Ag peptid (APC) 2.Co-stimulating signal: CD 28 (T lymphocyte) – CD 80, CD 86 (APC)

MHC glycoproteinspolymorphism 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  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) - 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 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

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 - ,  Types of L chains - ,  Types of H chains - , ,  (  1-4) and (  1,  2), 

 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

Immunoglobulins functions

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

 Gene segments for H chains – on chromosome 14 V (variable) - Several hundred D (Diversity) - about 50 J (joining)- 9 C constant domains of H chain  Gene segments for L chains -  on chromosome 2 - 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 genetic basis of the immunoglobulins development

The rearrangement of genes coding H chain 1) DJ rearrangement - excision a section IgH between D and J segment (runs on both chromosomes) 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) 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 . If rearrangement is unsuccessful, B lymphocyte die. Transcript of rearranged IgH gene into mRNA, splicing of the primary transcript. The first form H chain . If rearrangement is unsuccessful, B lymphocyte die.

The rearrangement of genes coding L chain 1) First, rearrange the genes encoding the L chain , 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  genes is unsuccessful, start the regrouping genes. 3) Not all H and L chain can form together a stable dimmers. If regrouping unsuccessful, B lymphocyte die. 1) First, rearrange the genes encoding the L chain , 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  genes is unsuccessful, start the regrouping genes. 3) Not all H and L chain can form together a stable dimmers. If regrouping unsuccessful, B lymphocyte die.

Variability of immunoglobulins  Variability of immunoglobulins is determined by: 1) Diversity of the combination V(D) J segments 2) Connecting variability - after excision of gene sequences the end are not cut off exactly 3) The enzyme terminal transferase - prolonge cut off ends with the short random sequences 4) Somatic mutations of V segments of the rearranged genes after contact with Ag on the surface of FDC

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

 Cytokines regulate which isotype occurs: IL-4 stimulates switching to IgG1 and IgE, IgG4 TGF  stimulates switching to IgG2 and IgA  Regulation, whether it will be secreted or membrane form is at the level of mRNA (at 3´end of C segment are after the sequences encoding secreted form a sequences of membrane-form)

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

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 (  ) 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 (Ig  and IG  ), 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 T H 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 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 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