isotype switch
DESCRIPTION
ISOTYPE SWITCH. C C δ C3. C 1 C ε 2 C1. C 1 C 4 C ε 1 C 2. C C δ. Embryonal DNA. Somatic recombination D – J. Rearranged DNA. Somatic recombination V – D – J. C C δ. Primer RNA transcript. Transcription. Ig ISOTYPES C µ IgM C γ 1IgG C γ 2IgG - PowerPoint PPT PresentationTRANSCRIPT
ISOTYPE SWITCH
átrendeződöttDNS
primerRNS-átirat
mRNS
naszcenspolipeptid
átalakítás
transzláció
módosítás
5'
AAAA
3'L1 V1D2J1J2-4 CM CD
transzkripció
V C
L V DJ C
L1 V1D2J1 CM
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
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 may 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
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
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 DeaminaseRNS editing enzymeNO HYPERMUTATION AND ISOTYPE SWITCH
SOMATIC HYPERMUTATION
CDR1CDR1 CDR2CDR2 CDR3CDR3
VL
Complementary Determining Region = hypervariable region
V35 gene product J2 gene product
STRUCTURE OF THE VARIABLE REGION
• Hypervariable (HVR) or complimentarity determining regions (CDR)
HVR3
FR1 FR2 FR3 FR4
HVR1HVR2
Var
iabi
lity
Ind
ex
25 7550 100Amino acid residue
150
100
50
0
• Framework regions (FR)
CDR1 CDR2 CDR3 CDR1 CDR2 CDR3
7 nap
14 nap
21 napIgG
IgM/IgG
IgM
SOMATIC HYPERMUTATIONDay 0.
Ag
Day 14. Ag
PRIMARY
immune response
SECONDARY
Immune response
AFFINITY MATURATION
Day 21
Day 14
Day 7
Hypervariable regions
Plasma cell clones
12345678
910111213141516
1718192021222324
Clone 1Clone 2Clone 3Clone 4Clone 5Clone 6Clone 7Clone 8Clone 9Clone 10
CD
R1
CD
R2
CD
R3
Day 6
CD
R1
CD
R2
CD
R3
CD
R1
CD
R2
CD
R3
CD
R1
CD
R2
CD
R3
Day 8 Day 12 Day 18
Deleterious mutationBeneficial mutationNeutral mutation
Lower affinity - Not clonally selectedHigher affinity - Clonally selectedIdentical affinity - No influence on clonal selection
Somatic hypermutation leads to affinity maturation
Hypermutation occurs under the influence of activated T cellsMutations are focussed on ‘hot spots’ (i.e. the CDRs) and are due to double stranded
breaks repaired by an error prone DNA repair enzyme.
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
ANTIBODY MEDIATED EFFECTOR FUNCTIONS
SPECIFIC ANTIBODY
Bacterial toxin
Toxin receptor
Neutralization
Internalization
Bacterium in the interstitium
Bacterium in the plasma
Opsonization Complement activation
Phagocytosis Phagocytosis and lysis
COMPLEMENT
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
T CELL ACTIVATION
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
TT-CELL-CELLAntigAntigen receptoren receptor
TCRTCR
VV
CC
The VARIABLE REGIONS OF - AND -CHAINS ARE GENERATED BY SOMATIC RECOMBINATION
mRNS
not functional
Recombination of V and J genes can occur after multiple unsuccessful recombination
next funcional
further funkcional (no allelic exclusion)
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
Similar but not identical signaling elements in B and T Similar but not identical signaling elements in B and T cellscells
BCRBCR TCRTCR
Ly
n
KinasesKinasesSykSyk
BtkBtk
fyn
ZAP70ZAP70ItkItk
SLP-65/BLNKSLP-65/BLNK
PLCPLC22
Adaptors +Adaptors +substratessubstrates
PLCPLC11
SLP-76SLP-76
Antigen
BCR
Antigen
MHC
TCR
CD3
APC
s s
ss
ss
s
ss
V V
C C
s
α βss
ss
ss
ss
CD3
s s
ε δ ε γζ ζ
D/E X7 D/E X2 X7 YXXL/I YXXL/IP P
ITAMImmunoreceptor Tyrosine-based
Activation Motif
ACTIVATION
T CELL RECEPTOR MEDIATED SIGNALING
Multisubunit Immune Recognition Receptors
MIRR
APC T
THE IMMUNOLOGICAL SYNAPSE
T CELL
APC
interaction recognition
1 2 3 4
5 6 7 8
stabilization separation
Negulescu P.A. et. al. Immunity 4: 421-430, 1996
THE INTERACTION OF T CELLS AND ANTIGEN PRESENTING CELLS
THE IMMUNOLOGICAL SYNAPSE
T CELL
ANTIGEN PRESENTING CELL
CD48
CD2
ICAM-1
LFA-1
B7
CD28CD4
SIGNALING COMPLEX
adaptor
ACTIVATEDT CELL
ICAM – Intercellular Adhesion Molecule
?
C D 4 5 R O
C D 5
C D 4
C D 3
C D 2
C D 2 8
C D 1 5 2 ( C T L A - 4 )
C D 4 9 d / 2 9 ( V L A - 4 )
C D 1 5 4 ( C D 4 0 L )
C D 4 0
C D 2 2
C D 7 2
M H C I I
C D 5 8 ( L F A - 3 )
C D 5 9
C D 8 0 ( B 7 - 1 )
C D 8 6 ( B 7 - 2 )
C D 2 0
C D 4 3
C D 1 0 6 ( V C A M )
C D 5 4 ( I C A M - 1 )C D 1 1 a / 1 8 ( L F A - 1 )
C D 1 0 2 ( I C A M - 2 )
C D 5 0 ( I C A M - 3 )
C D 1 9
C d 7 9bC d 7 9a
s I g M
C D 2 1 ( C R 2 )
C D 8 1 ( T A P A )
C D 2 3 ( F c R I I )e
M H C I
C D 8
BCR
TCR
THE CONTACT OF APC AND T CELLS IS STABILIZED BY ADHESION MOLECULES
*
*
*
*
**
B CELL T CELL
B-lymphocyte
Cytotoxic T-limfocyta (Tc)
Helper T-lymphocyte (Th)
RECOGNITION EFFECTOR CELL
Plasma cell
Cell killing
Macrophage activation
Lymphocyte activation
Inflammation
Antibody production
cytokines
cytokinesBCR + antigen
TCR + peptide + MHC-I
TCR + peptide + MHC-II
Effector cell retains specific receptor
Effector cells secrete cytokines
Peptides of endogenous proteins (virus, tumor) bind to class I MHC
molecules
Tc
Endogenous Ag
RECOGNITION OF EXOGENOUS AND ENDOGENOUS ANTIGENES BY T-LYMPHOCYTES
Exogenous Ag
Th
Peptides of exogenous proteins (toxin, bacteria, allergen) bind to class II MHC
molecules
Antigen presentation - T cells are co-stimulated
APC Th
Signal 1 antigen & antigenreceptor
Signal 2
B7 family members (CD80 & CD86) CD28
ACTIVATION
Costimulatory molecules are expressed by professional APC including dendritic cells, monocytes, macrophages, and B cells, but not by cells that have no
immunoregulatory functions such as muscle, nerves, hepatocytes, epithelial cells etc.
Th
ROLE OF CO-STIMULATION IN THE ACTIVATION OF HELPER T CELLS
Th
CD40
CD40L
B7B7
CD28
Th
NORMAL TISSUE CELLS DO NOT EXPRESS CD40 OR B7 CO-STIMULATORY MOLECULES
APC APC APC
IL-2
IL-2R
Express a low affinity IL-2 receptor-
and chains andproduce no IL-2
Mechanism of co-stimulation in T cells
Signal 1
NFAT binds to the promoter of of the chain gene of the IL-2 receptor.
The chain converts the IL-2Rto a high affinity form
IL-2
IL-2R
1
Antigen
Resting T cells
IL-2
IL-2R
1
Antigen
2
Costimulation
Signal 2Activates AP-1 and NF-B to increase IL-2 gene transcription by 3 fold
Stabilises and thus increases the half-life of IL-2 mRNA by 20-30 fold
IL-2 production increased by 100 fold overall
Mechanism of co-stimulation in T cells
Immunosuppressive drugs illustrate the importance of IL-2 in immune responses
Cyclosporin & FK506 inhibit IL-2 by disrupting TcR signalling
Rapamycin inhibits IL-2R signalling
Gene transcriptionProliferation
IL-2
CYTOSKELETON
β γα
THE HIGH AFFINITY IL-2 RECEPTOR
Ligand bindingNo signaling
STATSignal Transducer and
Activatior of Transcription
JAKJanus kinase
adhesion
costimulation
recognition
INITIATION OF T CELL PROLIFERATION
IL-2R
G1
S
M
G0
G2
IL-1R
IL-2R
IL-2Rα
IL-2Rlow affinity
IL-2Rhigh affinity
IL-1
IL-2
transferrin
insulin PROLIFERATION
IL-2
IL-2
AUTOCRINE GROTH FACTOR
calcineurin
NF-ATn
NF-ATc NF-AT
Ca2+
NF-ATn
TF
calcineurinPTP-ase
calcineurinPTP-ase
calcineurinPTP-ase
P
PLC PLC
FK506CSA
Cytokines, activation molecules IL-2, IL-2R TGF
Cycliphilin A isomerase FKBP2 isomerase
Inactive phosphataseAktive phosphatase
Dephosphorylation of cytoplasmic NF-AT induces translocation to the nucleus
MECHANISM OF THE ACTION OF THE IMMUNE SUPPRESSIVE DRUGY CYCLOSPORIN A AND FK506/PROGRAPH
TCR-CD3Other
receptors
NOT ANTIGEN SPECIFIC
CO-STIMULATION IS ESSENTIAL FOR PRIMING OF NAIVE T LYMPHOCYTES
The antigen-specific and the co-stimulatory signals have to be induced in concert to induce T lymphocyte activation
The antigen-specific and co-stimulatory signals can be delivered simultaneously by professional antigen presenting
cells, only
The antigen-specific and the co-stimulatory singnals has to be delivered by the same professional antigen presenting cell
Activated APC Resting APCAPC not presenting
antigen
T-cell activation T-cell anergy No effect
CD4CD4 CD28CD28CD28 CD4
B7 B7
2 1
1
2
T CELLS REQUIRE TWO SIGNALS TO GET ACTIVATED
ANTIGEN SPECIFIC ACTIVATION, ANERGY OR NEGLECTION
IL-2
IL-2R
1
Antigen
Epithelialcell
NaïveT cell
Signal 1 only
Anergy
The T cell is unable to produce IL-2 and therefore is unable to proliferate or be clonally
selected.
Unlike immunosupressive drugs that inhibit ALL specificities of T cell, Signal 1 in the absence of
signal 2 causes T cell unresponsiveness to a specific antigen
Self peptide epitopes presentedby a non-classical APC e.g. an
epithelial cell
PROFESSIONAL ANTIGEN PRESENTING CELLS
Express MHC class I and class II molecules
Express co-stimulatory molecules (B7, CD40)
Take up extracellular antigens
B cells – soluble proteins, toxins (ADAPTIVE)
Macrophages – extracellular pathogens (bacteria, yeast)
INNATE – particles
Dendritic cells – viruses, apoptotic cells
CHARACTERISTICS OF PROFESSIONAL ANTIGEN PRESENTING CELLS
Macrophage Dendritic cell B - lymphocyte
Ag uptake phagocytosis +++ phagocytosis +++ Ag-specific mIg virus infection ++++ ++++
MHC expression induced +/+++ constitutive ++++ constitutive +++ bacteria, cytokine immature/mature +++/++++ activation ++++
Pesented Ag particulate Ag protein soluble protein intra/extracellular virus protein, allergen toxin pathogens apoptotic cell
Co-stimulation induced +/++ constitutive ++++ induced +/+++ éretlen/érett +++/++++
Localization lymphoid tissue lymphoid tissue lymphoid tissue connective tissue connective tissue peripheral blood body cavities epithelium
Lymph node evenly immature – tissue follicles mature – T cell area
Macrophage
Dendritic cell
Activated macrophage
Phagocytosis of bacteria, degradation (LPS, TLR)
DANGER SIGNAL
Activated dendritic cell
Virus, extracellular pathogenes, inflammatory cytokines (LPS, TLR)
DANGER SIGNAL
Monocyte
CHANGES IN TISSUE ENVIRONMENT RESULTS IN MACROPHAGE AND DENDRITIC CELL ACTIVATION
LYMPHBLOOD TISSUE
Tissue DC
Activated DC
INTERACTION OG DC AND T CELL
T CELL ACTIVATION
BLOOD
Naive T cells
Effector and memory T cells
TISSUE LYMPH NODE TISSUE
LYMPH
InflammationPathogen
ACTIVATION AND MIGRATION OF DENDRITIC CELLS
ANTIGEN
Activated dendritic cells in the lymphatic tissues act as antigen
presenting cells
Tight contact with specific T cells
DENDRITIC CELL T - LYMPHOCITE
INTERACTION IN THE LYMPHOID ORGANS
The danger hypothesis & co-stimulation
Fuchs & Matzinger 1995
Full expression of T cell function and self tolerance depends upon when and where co-stimulatory molecules are expressed.
Apoptotic cell death.A natural, often usefulcell death.
APC
APC
No danger
No dangerCell containing onlyself antigens
Innocuous challenge to the immune system fails to activate APC and failsto activate the immune system
The danger hypothesis
APC
APC
Necrotic cell deathe.g. tissue damage,virus infection etc
Pathogens recognisedby microbial patterns
DANGER
APC that detect ‘danger’ signals express costimulatorymolecules, activate T cells and the immune response