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

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

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 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

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

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

The αβ and γδ T-cell lineages develop from a common precursor

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

The α-Chain rearrangement (Ig light chain)

TCR-rearrangement is similar to that of the BCR

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

Shaping the T-cell repertoire.Positive and negative selection Thymus

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

Multiple stages of T-cell development in the thymus

CD25+ FoxP3+ cells

FoxP3-deficiency: autoimmune disease

IPEX: immune dysregulation polyendocrinopathy, enteropathy, X-linked syndrome