Download NK cells Interferons J. Ochotná

NK cells
Interferons
J. Ochotná
NK cells
 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 - IFNa, IFNb
NK cells receptors
 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)
 C-type lektin family - eg CD94/NKG2
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
 TNFa
Interferons
 Belongs to the humoral component of non-specific
mechanisms
 IFNa - produced by virus infected lymphocytes,
monocytes and macrophages
 IFNb - produced by virus-infected fibroblasts and epithelial
cells
 IFNa and IFNb - 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)
 IFNg - produced by TH1 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, participate in
parasitosis and allergy
 Connective tissue mast cells - the
connective tissue, 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)
 Apply during inflammation, in angiogenesis, in tissue
remodeling
 Regulation of immune response
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, C4a, C5a)
Mast cell activation by cross-linking of
IgE Fc receptors
 Establishing of multivalent antigen (multicellular parasite)
to IgE linked 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, TGFb, 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 b, 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 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)
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 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
Binding the peptide to MHCgpI
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
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 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

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 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
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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 - k, l
 Types of H chains - m, d, g (g1-4) and (a1, a2), 
 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
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