Transcript Switch regions

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.
SOMATIC HYPERMUTATION
VL
J gene product
V gene product
CDR1
CDR2
CDR3
Complementary Determining Region = hypervariable region
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 APE1
Base excised and replaced with any nucleotide
MUTATION!!!!
The almost random variation produced by somatic hypermutation
allows selection of variant immunoglobulins with improved antigenbinding sites
Day 0.
Ag
Day7 7nap
CDR1 CDR2 CDR3
CDR1 CDR2
IgM
PRIMARY
Day
21 21
nap
1
2
3
4
5
6
7
8
Plasma cell
clones
AFFINITY
MATURATION
immune response
Day 14.
14 Ag
nap
CDR3
IgM/IgG
IgG
SECONDARY
Immune response
Hypervariable regions
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
ISOTYPE SWITCH
embrionális
Embryonal
L1 V1 L2 V2 Ln Vn D1 - 12
DNS
DNA
5'
szomatikus
rekombináció
Somatic
recombination
D–
D-J kapcsolódás
átrendeződött
Rearranged
DNA
DNS
L1
V1
Ln
Vn
C Cδ C3
CM CD CG3
J1-4
J
CA1Cε2
C1
CG1
CE2C1
CE1 C2
CG
2 CG
4 Cε1
CA2
C
1
C4
D1D2 J1J2-4 C
CM Cδ
CD
5'
3'
szomatikus rekombináció
Somatic
recombination
V
-D-J kapcsolódás
CM Cδ
CD
D2J1 J2-4 C
3'
L1 V1
5'
Primerprimer
RNA transcript
RNS-átirat
mRNA
mRNS
3’
Transcription
transzkripció
5'
L1 V1D2 J1 J2-4 C
CM
Cδ
CD
IgM
Cγ1
IgG
Cγ2
IgG
Cγ3
IgG
Modification Cγ4
IgG
AAAA
transzláció
Translation
L V DJ C
C
polipeptid
Ig ISOTYPES
Cµ
3'
Processing
átalakítás
C
L1 V1 D2 J1 CM
naszcens
Nascent
polypeptide
módosítás
V
C
V–D–J
NEHÉZL
ÁNC (M
)
Heavy
chain
IgM
Cα
IgA
Cε
IgE
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.
Organisation of the functional human heavy chain C region genes
J regions
C
Cd
C3
C1 C1 C2
C4
Ce
C2
Switch regions
C
S
Cd
C3
S3
C1
S1
C1
S1
C2
S2
C4
S4
Ce
Se
C2
S2
• Upstream of C regions are repetitive regions of DNA called
switch regions. (The exception is the Cd region that has no
switch region).
• 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.
Switch recombination
C
Cd
C3
C1
C1
C2
C4
Cd
Ce
Cd
C2
S3
C3
C3
C
S1
C
C1
V23D5J4
C3
V23D5J4
C1
V23D5J4
C1
V23D5J4
C3
V23D5J4
C1
V23D5J4
C1
IgG3 produced.
Switch from IgM
IgA1 produced.
Switch from IgG3
IgA1 produced.
Switch from IgM
At each recombination constant regions are deleted from the genome
An IgE - secreting B cell will never be able to switch to IgM, IgD, IgG1-4 or IgA1
C
L VDJ
Cδ
Rearranged DNA in
C2 C4 Ce C
3'
5'
S
S
S
S
IgM-producing cell
S
Switch regions
C
Cδ, C2, C4
ISOTYPE SWITCH
L VDJ
3'
5'
Ce
C
L VDJ
5'
3'
Rearranged DNA in
IgE-producing cell
L
VDJ
Primary RNA
3'
transcript
5'
AAAA
e-Heavy chain
Ce mRNA
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 Deaminase
NO 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 (FcR)
receptors
• Cytophylic property - antibody isotypes have
distinct complement activating and FcR binding
activity
SECRETED ANTIBODIES BIND TO THE ANTIGEN
COMPLEMENT ACTIVATION
IMMUNE COMPLEX
OPSONIZATION
Ig Fc region
Conformational change?
Association?
FcR
CR
PHAGOCYTOSIS
Macrophage
COMPLEMENT ACTIVATION – classical
pathway
BINDING TO CELLS – cytophilic property
DEGRADATION
ISOTYPE DEPENDENT
IgG1 and IgG3 >> IgG2 és IgG4
EFFECTOR FUNCTIONS OF ANTIBODIES
INHIBITION
Binding of bacteria to
epithelial cells
Binding of viruses to
receptor
Binding of bacterial
toxins to target cells
NEUTRALIZATION
Small proportion of
antibodies
OPSONIZATION
COMPLEMENT
ACTIVATION
Binding of antibody
increases phagocytosis
PLAZMA CELL
FcR
Opsonization by C3b
Complement
C3b
FcR
FcR CR1
PHAGOCYTES
ENGULFMENT, DEGRADATION
T – CELLS PROMOTE B – CELL DIFFERENTIATION
T-CELL
ANTIGEN
CYTOKINES
B -CELL
PLASMA CELL
ISOTYPE SWITCH AND AFFINITY MATURATION OCCURS IN
COLLABORATION WITH T – CELLS ONLY
HOW T – CELLS RECOGNIZE ANTIGENS?
T-cell development and
differentiation
A T-cell precursors migrate from the
bone marrow to thymus
T-cell markers are induced after thymocytes interact with
the thymic epithelial cells
A Notch-1receptor and its cytoplasmic region, acting as a
transcription factor is required for the development
of the T-cell lineage
THE RESULT OF SOMATIC GENE REARRANGEMENTS
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
INDEPENDENT OF ANTIGEN
OCCURS DURING B-CELL DEVELOPMENT IN THE BONE
MARROW
The αβ and γδ T-cell lineages develop from
a common precursor
A TCR-rearrangement—similar to BCR
A β-chain rearrangement (like Ig heavy chain)
Efficiency of beta chain
rearrangement is about
80%
RAG-1 RAG-2 genes
become are inactivated
Timocyte proliferation
CD4, CD8 expressionó
TCR-rearrangement is similar to that of the BCR
The α-Chain rearrangement (Ig light chain)
B- AND T-CELL RECEPTORS SHARE BASIC
STRUCTURE
mIg H
mIg L

TCR
T cell receptor
TCR
V
TCR
C
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
T-CELL
Single binding
C site
No somatic
mutation
ESTIMATED VARIABILITY OF IMMUNOGLOBULIN AND T-CELL RECEPTOR
GENES
GENES/
KAPCSOLÓDÁS
IMMUNOGLOBULIN
T CELL RECEPTOR
H
/


VARIABLE (V)
65
70
52
~70
DIVERZITY (D)
27
0
2
0
rare
-
OFTEN
-
JOINING (J)
6
5/4
13
61
JOINING + P + N
2
1 50%
2
1
D (3 frame)
V GENE PAIRS
3.4x106
5.8x106
JOINING
~3x107
~2x1011
TOTAL
~1014
SOMATIC HYPERMUTATON
1018
NO
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
ACCESSORY CELL
ANTIGEN BINDING
NO INTERACTION
V
T-CELL
ACTIVATION
C
Antigen receptor
B-CELL
/
T-CELL
Shaping the T-cell repertoire.
Positive and negative selection Thymus
Few TCR reacts with
the MHC (about 2%) most
T-cells die of neglect.
( no survival signals)
α-chain rearrangement can
continue until the assembly of
a functional αβ receptor
has been assembled.
Selection of developing T-cells in the thymus
Bare lymphocyte syndrome
MHCI vagy MHCII deficiency
Lack of CD8+ or CD4+ cells
Role of co-receptors in the
development 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
Multiple stages of T-cell development in the thymus
CD25+ FoxP3+ cells
FoxP3-deficiency:
autoimmune disease
IPEX: immune
dysregulation
polyendocrinopathy,
enteropathy, X-linked
syndrome