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Diversification of antibodies after B-cells encounter antigen
Alternative splicing
Somatic hypermutation
Ig. class switch
The surface and secreted forms of an immunoglobulin are derived from the same heavy-chain gene by
alternative RNA processing.
CDR1CDR1 CDR2CDR2 CDR3CDR3
VL
Complementary Determining Region = hypervariable region
V gene product J gene product
STRUCTURE OF THE VARIABLE REGION
Hypervariable (HVR)
or complimentarity
determining regions (CDR)
Framework regions (FR)
Somatic hypermutation is targeted to the rearranged gene segments that encode
immunoglobulin V regions.
AID: activation induced cytidine deaminase AID
Cytosine to uracil change….. UNG Uracil-DNA Glycosilase, abasic nucleotide is excised by the nuclease APE1Base excised and replaced with any nucleotide MUTATION!!!!
CDR1 CDR2 CDR3 CDR1 CDR2 CDR3
7 nap
14 nap
21 napIgG
IgM/IgG
IgM
The almost random variation produced by somatic hypermutation allows selection of variant immunoglobulins with improved antigen-
binding sites Day 0.Ag
Day 14. Ag
PRIMARY
immune response
SECONDARY
Immune response
AFFINITY MATURATION
Day 21
Day 7
Hypervariable regions
Plasma cell clones
12345678
910111213141516
1718192021222324
átrendeződöttDNS
primerRNS-átirat
mRNS
naszcenspolipeptid
átalakítás
transzláció
módosítás
5'
AAAA
3'L1 V1D2J1J2-4 CM CD
transzkripció
V C
L V DJ C
L1 V1D2J1CM
5' 3'
szomatikus rekombinációV-D-J kapcsolódás
5' 3'
L1 V1 Ln Vn D1D2J1J2-4 CM CD
L1 V1 D2J1 J2-4 CM CD
NEHÉZLÁNC (M)
szomatikus rekombinációD-J kapcsolódás
embrionálisDNS
L1 V1 Ln Vn J1-4 CM CDD1 - 125'
3’
L2 V2 C 3G
CE2 C 1G
CG2
CA1
CG4 CE1 CA2
C Cδ C3
C1 Cε2 C1 C 1 C4 Cε1 C2
C Cδ
C Cδ
C Cδ
IgM
C
C
Embryonal DNA
Rearranged DNA
Primer RNA transcript
mRNA
Nascent polypeptide
Somatic recombination D – J
Somatic recombination V – D – J
Transcription
Processing
Translation
Modification
Ig ISOTYPES
Cµ IgM
Cγ1 IgG
Cγ2 IgG
Cγ3 IgG
Cγ4 IgG
Cα IgA
Cε IgEHeavy chain
Antibody isotype switching
Throughout the immune response the specificity of an antibody will be essentially the same (notwithstanding affinity maturation)
The effector function of antibodies throughout a response needs to change drastically as the response progresses.
Antibodies are able to retain Variable regions whilst exchanging Constant regions that contain the structures that interact with cells.
J regions C2CC4C2C1C1C3CC
Organisation of the functional human heavy chain C region genes
C2CC4C2C1C1C3CC
Switch regions
• The S consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n
is between 3 and 7.
• Switching is mechanistically similar in many ways to V(D)J
recombination.
• Isotype switching does not take place in the bone marrow, however,
and it will only occur after B cell activation by antigen and
interactions with T cells.
S3 S1 S1 S2 S4 S S2S
• Upstream of C regions are repetitive regions of DNA called switch regions. (The exception is the C region that has no switch region).
C2CC4C2C1C1C3CC
C
C
C3V23D5J4
S3
C
C
C3
V23D5J4
C1
S1
C1
C3
V23D5J4 C1
C3V23D5J4
IgG3 produced.Switch from IgM
V23D5J4 C1
IgA1 produced.Switch from IgG3
V23D5J4 C1
IgA1 produced.Switch from IgM
Switch recombination
At each recombination constant regions are deleted from the genomeAn IgE - secreting B cell will never be able to switch to IgM, IgD, IgG1-4 or IgA1
AAAA
5' 3'L VDJ
5' 3'
VDJL
5' 3'VDJL
5' 3'VDJL
SS SSS S SS
Rearranged DNA in
IgM-producing cell
Rearranged DNA in
IgE-producing cell
Primary RNAtranscript
C mRNA
-Heavy chain
C Cδ C2 C4 C C
C Cδ, C2, C4
C C
Switch regions
ISOTYPE SWITCH
All isotype switch recombination is productive
Different recombination signal sequences and
enzymes from VDJ rearrangement
Happens after antigenic stimulation
Regulated by external signals, not random
Hyper IgM syndrome Type 2. Activation Induced Cytidine DeaminaseNO HYPERMUTATION AND ISOTYPE SWITCH
ANTIBODY MEDIATED EFFECTOR FUNCTIONS
• Neutralization – binding of the antibody inhibits the binding of the pathogen to the cell surface, entry to the cell or multiplication
• Opsonization – binding of the antibody triggers complement activation and binding to the cell surface by complement (CR1) and IgG (FcR) receptors
• Cytophylic property - antibody isotypes have distinct complement activating and FcR binding activity
IMMUNE COMPLEX
SECRETED ANTIBODIES BIND TO THE ANTIGEN
Macrophage
FcR CR
COMPLEMENT ACTIVATION
OPSONIZATION
PHAGOCYTOSIS
DEGRADATION
Ig Fc regionConformational change?Association?
COMPLEMENT ACTIVATION – classical pathway
BINDING TO CELLS – cytophilic property
ISOTYPE DEPENDENT
IgG1 and IgG3 >> IgG2 és IgG4
EFFECTOR FUNCTIONS OF ANTIBODIES
PLAZMA CELL
NEUTRALIZATION
Small proportion of antibodies
INHIBITIONBinding of bacteria to
epithelial cellsBinding of viruses to
receptorBinding of bacterial toxins to target cells
OPSONIZATION
Binding of antibody increases phagocytosis
FcR
FcR
FcR CR1
ComplementC3b
COMPLEMENT ACTIVATION
Opsonization by C3b
PHAGOCYTES
ENGULFMENT, DEGRADATION
PLASMA CELL
ANTIGEN
B -CELL
T – CELLS PROMOTE B – CELL DIFFERENTIATION
ISOTYPE SWITCH AND AFFINITY MATURATION OCCURS IN COLLABORATION WITH T – CELLS ONLY
HOW T – CELLS RECOGNIZE ANTIGENS?
CYTOKINES
T-CELL
A Notch-1receptor and its cytoplasmic region, acting as a transcription factor is required for the development
of the T-cell lineage
1. Combination of gene segments results in a huge number of various variable regions of the heavy and light chains expressed by different B-cells
SOMATIC GENE REARRANGEMENT
2. Successful somatic rearrangement in one chromosome inhibits gene rearrangement in the other chromosome
ALLELIC EXCLUSION
3. One B-cell produces only one type of heavy and one type of light chain
COMMITMENT TO ONE TYPE OF ANTIGEN BINDING SITE
4. The B-cell pool consist of B-cells with differently rearranged immunoglobulin genes
THE RESULT OF SOMATIC GENE REARRANGEMENTS
INDEPENDENT OF ANTIGEN
OCCURS DURING B-CELL DEVELOPMENT IN THE BONE MARROW
A β-chain rearrangement (like Ig heavy chain)
A TCR-rearrangement—similar to BCR
Efficiency of beta chain rearrangement is about 80%
RAG-1 RAG-2 genes become are inactivated
Timocyte proliferation CD4, CD8 expressionó
C
mIg H
mIg L
TCR
TCR
TT-CELL-CELL
C
VT cell receptor T cell receptor TCRTCR
B- AND T-CELL RECEPTORS SHARE BASIC STRUCTURE
TCR = +
The variable region of the -chain is generated by gene rearrangements of the V – D – J gene segments
analogous to the generation of IgH diversity
The variable region of the -chain is generated by the recombination of V and J analogous to IgL
Single binding siteNo somatic
mutation
GENES/KAPCSOLÓDÁS
IMMUNOGLOBULIN
H /
VARIABLE (V) 65 70
DIVERZITY (D) 27 0
D (3 frame) rare -
JOINING (J) 6 5/4
JOINING + P + N 2 1 50%
V GENE PAIRS 3.4x106
JOINING ~3x107
TOTAL ~1014
SOMATIC HYPERMUTATON
ESTIMATED VARIABILITY OF IMMUNOGLOBULIN AND T-CELL RECEPTOR GENES
T CELL RECEPTOR
52 ~70
2 0
OFTEN -
13 61
2 1
5.8x106
~2x1011
1018
NO
ANTIGEN BINDING
NO INTERACTION
ACCESSORY CELL
T-CELL ACTIVATION
AntigeAntigen receptorn receptor
TT-CELL-CELL B-CELLB-CELL
CHARACTERISTICS OF T-CELL ANTIGEN RECOGNITION
1. The TCR is not able to interact directly with soluble or cell-bound antigen
2. T-cell activation can be induced by antigen in the presence of acessory cells, only
3. T-cells recognize virus-infected cells
VV
CC
Few TCR reacts with the MHC (about 2%) mostT-cells die of neglect. ( no survival signals)
α-chain rearrangement can continue until the assembly ofa functional αβ receptor has been assembled.
Selection of developing T-cells in the thymus
Bare lymphocyte syndrome
MHCI vagy MHCII deficiencyLack of CD8+ or CD4+ cells
Role of co-receptors in thedevelopment of single + T-cells
DC Macrophage in medulla of Thymus. Special transcription factor expressed… AIRE.Tissue spec. Antigens expressed
AIRE mutaton: Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy