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ISOTYPE SWITCH
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ISOTYPE SWITCH

Jan 03, 2016

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ISOTYPE SWITCH. C  C δ C3. C 1 C ε 2 C1. 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 Presentation
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Page 1: ISOTYPE SWITCH

ISOTYPE SWITCH

Page 2: 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

Page 3: ISOTYPE SWITCH

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).

Page 4: ISOTYPE SWITCH

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

Page 5: ISOTYPE SWITCH

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

Page 6: ISOTYPE SWITCH

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

Page 7: ISOTYPE SWITCH

SOMATIC HYPERMUTATION

Page 8: ISOTYPE SWITCH

CDR1CDR1 CDR2CDR2 CDR3CDR3

VL

Complementary Determining Region = hypervariable region

V35 gene product J2 gene product

Page 9: ISOTYPE SWITCH

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)

Page 10: ISOTYPE SWITCH

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

Page 11: ISOTYPE SWITCH

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.

Page 12: 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

Page 13: ISOTYPE SWITCH

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

Page 14: ISOTYPE SWITCH

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

Page 15: ISOTYPE SWITCH

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

Page 16: ISOTYPE SWITCH

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

Page 17: ISOTYPE SWITCH

T CELL ACTIVATION

Page 18: ISOTYPE SWITCH

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

Page 19: ISOTYPE SWITCH

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)

Page 20: ISOTYPE SWITCH

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

Page 21: ISOTYPE SWITCH

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

Page 22: ISOTYPE SWITCH

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

Page 23: ISOTYPE SWITCH

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

Page 24: ISOTYPE SWITCH

APC T

THE IMMUNOLOGICAL SYNAPSE

Page 25: ISOTYPE SWITCH

T CELL

APC

Page 26: ISOTYPE SWITCH

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

Page 27: ISOTYPE SWITCH

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

Page 28: ISOTYPE SWITCH

?

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

Page 29: ISOTYPE SWITCH

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

Page 30: ISOTYPE SWITCH

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

Page 31: ISOTYPE SWITCH

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.

Page 32: ISOTYPE SWITCH

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

Page 33: ISOTYPE SWITCH

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

Page 34: ISOTYPE SWITCH

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

Page 35: ISOTYPE SWITCH

Gene transcriptionProliferation

IL-2

CYTOSKELETON

β γα

THE HIGH AFFINITY IL-2 RECEPTOR

Ligand bindingNo signaling

STATSignal Transducer and

Activatior of Transcription

JAKJanus kinase

Page 36: ISOTYPE SWITCH

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

Page 37: ISOTYPE SWITCH

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

Page 38: ISOTYPE SWITCH

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

Page 39: ISOTYPE SWITCH

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

Page 40: ISOTYPE SWITCH

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

Page 41: ISOTYPE SWITCH

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

Page 42: ISOTYPE SWITCH

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

Page 43: ISOTYPE SWITCH

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

Page 44: ISOTYPE SWITCH

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

Page 45: ISOTYPE SWITCH

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

Page 46: ISOTYPE SWITCH

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

Page 47: ISOTYPE SWITCH

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