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)

Szomatikus hipermutáció

FR1 FR2 FR3 FR4CDR2 CDR3CDR1

Aminsav szám

Variabilitás

80

100

60

40

20

20 40 60 80 100 120

A különböző specificitású ellenanyagokban található pont mutációk összehasonlítása

Wu - Kabat analízissel

Mik a következményei az immunválasz során végbemenő mutációknakegy adott epitóp ellen irányuló ellenanyagban?

Hogyan befolyásolja az ellenanyag specificitását és affinitását?

NH2

COOH

0 10

10

20

20

30

30

40

40

50

50

60

60

70

70

80

80

90

90

100

100

110 120

0 10

10

20

20

30

30

40

40

50

50

60

60

70

70

80

80

90

90

100

100

110 120

CDR1 CDR2 CDR3

Light chain

Heavy chain

CDR1 CDR2 CDR3

VL CL

LIGHT CHAIN

Disulphide bridges

FR1 FR2 FR3 FR4

FR1 FR2 FR3 FR4

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.

CDR1 and CDR2 regions are encoded by the V-geneThe CDR3 of L-chain is encoded by V and J The CDR3 of H-cain is encoded by V, D and J genes

CDR1CDR2

CDR3

CDR1CDR2

CDR3

Antigén determináns

H-CHAIN

L-CHAINCDR1CDR2

CDR3

CDR1CDR2

CDR3

Antigén determináns

H-CHAIN

L-CHAIN

Antigén determinánsAntigén determináns

H-CHAIN

L-CHAIN

FR1 FR2 FR3 FR4CDR2 CDR3CDR1

Amino acid No.

Variability80

100

60

40

20

20 40 60 80 100 120

Wu - Kabat analysis compared point mutations in Ig of different specificity.

FR1 FR2 FR3 FR4CDR2 CDR3CDR1

Amino acid No.

Variability80

100

60

40

20

20 40 60 80 100 120

Amino acid No.

Variability80

100

60

40

20

20 40 60 80 100 120

Wu - Kabat analysis compared point mutations in Ig of different specificity.

•The framework supports the hypervariable loops

•The framework forms a compact barrel/sandwich with a

hydrophobic core

•The hypervariable loops join, and are more flexible than, the

strands

•The sequences of the hypervariable loops are highly variable

amongst antibodies of different specificities

•The variable sequences of the hypervariable loops influences

the shape, hydrophobicity and charge at the tip of the

antibody

•Variable amino acid sequence in the hypervariable loops

accounts for the diversity of antigens that can be recognised by

a repertoire of antibodies

Hypervariable loops and framework: Summary

B – CELL ACTIVATION

Where and how do all these things take place?

B-cell recycling in the absence of antigen (lymph node)

B cells in blood

Efferentlymph

T cell area

B cell area

Antigen entersnode in afferent

lymphatic

Y

Y

Y

Y

Y

YY

Y

Y

Y

Y

Y

Y

Y

YY

Y

YB cells leave blood & enter lymph node via

high endothelial venulesB cellsproliferate

rapidly

GERMINAL CENTRETransient structure ofIntense proliferation

Germinal centrereleases B cellsthat differentiateinto plasma cells

Recirculating B cells are trapped by foreign antigens in lymphoid organs

Germinal Center Reaction

T CELL DEPENDENT B CELL ACTIVATION IN LYMPHOID T CELL DEPENDENT B CELL ACTIVATION IN LYMPHOID ORGANSORGANS

IgM

IgGIgAIgE

„Dating” in the peripheral lymphoid organs

Antigen-stimulated B cells become trapped in the T-cell zone

The primary foci and secondary follicle formation

The structure of the germinal centre

Somatic hypermutation

FDC

Somatic hypermutation

DZ

LZ

LZ: light zoneDZ: dark zoneFDC: follicular dendritic cell

Antigen is bound on the surface of follicular dendritic cells (FDC)

FDC FDC-s bind immune complexes (Ag-Ab ) Ag detectable for 12 months following immunization A single cell binds various antigens

B cells recognize Ag on the surface of FDC

Fig. 9.15. On the surface of FDC-s immune complexes form the so-called iccosomes, that can be released and taken up later by the surrounding germinal center B cells

Ig-Ig-/CD79a/CD79a Ig-Ig-/CD79b/CD79b

ITAM: ITAM: IImmunoreceptor mmunoreceptor TTyrosineyrosine--based based AActivation ctivation MMotifotif

Y

Y

Y

YITAMITAM ITAMITAM

Ig domain + CHOIg domain + CHO

SIGNALING UNITS OF THE B-CELL RECEPTOR

ITAM:ITAM: Y YxxxxLL x7x7 YYxxxxII

Main steps of B-cell signal transductionMain steps of B-cell signal transduction

CONSEQUENCES OF B-CELL RECEPTOR CROSS LINKINGCONSEQUENCES OF B-CELL RECEPTOR CROSS LINKING

Ag binding, cross-linking of

surface IgLymphocyte

activationPhenotypic/

Functional change

KINETICS OF LYMPHOCYTE ACTIVATION

ANTIGEN SIGNAL1.

Ko-receptorAdhesion molecule

Cytokines SIGNAL2.

Resting lymphocyte G0PTK activation RNA synthesis Free Ca++ Protein synthesis Protein phosphorylation DNA synthesis

Lymphoblast

0 10sec 1min 5min 1hr 6 hrs 12 hrs 24 hrs

Nyugvó limfocita G0

G1

G2

M

Ssejtosztódás

DNA synthesis

Effector cell Memory cell

Transport Membrane changeRNA and protein synthesis

Resting lymphocyte G0

AntigAntigenicenicdeterminantdeterminant

C3C3dd

THE THE CO-STIMULATORYCO-STIMULATORY ROLE OF ROLE OF CR2 (CD21) CR2 (CD21) COMPLEMENT RECEPTOR IN B – LYMPHOCYTESCOMPLEMENT RECEPTOR IN B – LYMPHOCYTES

ANTIGÉN

CD21CD21/CR2/CR2

CD19CD19

YY

TAPA=CD81TAPA=CD81

Enhanced B-cell activation

BB-CELL-CELL

THE NEURAMINIC ACID RECEPTOR CD22 INHIBITS THE NEURAMINIC ACID RECEPTOR CD22 INHIBITS ACTIVATION THROUGH THE A B-CELL RECEPTOR ACTIVATION THROUGH THE A B-CELL RECEPTOR

B B CellCellAntigAntigeenn

Tissue cells

BaBacctteeriumrium

MannMannoseose

CD22CD22

Neuraminic acid

Inhibited B cell activation

EFFECTOR FUNCTIONS OF ANTIBODIES

PLASMA 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

SIZESHAPE

HYDROPHOBICHYDROPHYLIC

POSITIVELY CHARGEDNEGATIVELY CHARGED

FEATURES OF THE BINDING SITE

ANTIGEN BINDING IS MEDIATED BY

NON-COVALENT INTERACTIONS

One binding site is able to interact with more than

one antigen

The strength of interaction (affinity/avidity) varies in a

broad range

10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -2 M

K =D [AB]

[A] [B]

C D2/LFA-3

C D28/B7 LFA-1/IC AM -1

AffinityANTIBODIES

Growth factors

MHC – peptid - TCR

Adhesion molecules