B – CELL ACTIVATION

LigandLigand

SIGNALSIGNAL

Cross - linkingCross - linking

LigandLigand

CConformational changeonformational change

SIGNALSIGNAL

RECEPTOR MEDIATED CELL ACTIVATION

ligandligand

kinase activationkinase activation

phosphorylationphosphorylation

rrecruitmentecruitment oof adaptorsf adaptors

CROSS – LINKING OF THE RECEPTOR INITIATES A SIGNALING CASCADE

SIGNALSIGNAL

Gene transcriptionActivation of

transcription factors

(review) BCR signaling

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

KINETICS OF LYMPHOCYTE ACTIVATION

ANTIGEN SIGNAL1.

Co-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

Sproliferation

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

ANTIGEN

CD21CD21

CD19CD19

YY

TAPA=CD81TAPA=CD81

Enhanced B-cell activation

BB-CELL-CELL

THE NEURAMIC ACID RECEPTOR CD22 INHIBITS THE NEURAMIC 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 (sialic) acid

Inhibited B cell activation

ANTIBODY DIVERSITY

s

s

s

s

s

s

s

s

s ss s

CH2

CH3

s

s

s

s

s

s

s

s

ss

VL

VH

CL

CH1 ss

ss

s

s

s

s

ss

effektor funkciók

konstans domének

antigénkötés

variábilis domének

STRUCTURE OF IMMUNOGLOBULINS/ANTIBODIES

SS

COMPLEMENT ACTIVATION

BINDING TO CELLS

DEGRADATION

TRANSPORT

Light chain (L)

Heavy chain (H)

VL

CL

VH

CH

Antigen binding

Variable domains

Antigen

Constans domains

Effector functions

MMultiple myeloma (MM)ultiple myeloma (MM)PPlasmlasmaa cell tumors – tumor cells reside in cell tumors – tumor cells reside in tthe bone marrowhe bone marrowProduce immunoglobulins of monoclonal origin,Produce immunoglobulins of monoclonal origin, serum concentration 50-100mg/mlserum concentration 50-100mg/mlRodney Porter & Gerald Edelman 1959 – 1960Rodney Porter & Gerald Edelman 1959 – 1960 myeloma protein purification myeloma protein purification

AMINO ACID SEQUENCE OF IMMUNOGLOBULINS

50 kDa50 kDaHeavy chainHeavy chain

25 kDa25 kDaLight chain Light chain

Gel electrophoresisGel electrophoresis

Variable Constant

123456789101112131415161718

Reduction

L H

GENETIC BACKGROUND OF ANTIBODY DIVERSITY

VLVLVHVH

Mechanism of the generation of variability?Different rules for encoding the variable and constant regions?

Symmetric molecule two identical VH and VL both chromosomes encode for the same sequence?

S – S S – S

MOLECULAR GENETICS OF IMMUNOGLOUBLINS

• A single C region gene encoded in the GERMLINE and separate from the V region genes

• Multiple choices of V region genes available• A mechanism to rearrange V and C genes in the genome so that

they can fuse to form a complete Immunoglobulin gene.

In 1965, Dreyer & Bennett proposed that for a single isotype of antibody there may be:

How can the bifunctional nature of antibodies be explained genetically?

This was genetic heresy as it violated the then accepted notion that DNA was identical in every cell of an

individual

Proof of the Dreyer - Bennett hypothesis

VV

VV

V

V

VV

V

V

VV

V

A mechanism to rearrange V and C genes in the genome exists so that they can fuse to form a complete

Immunoglobulin gene

CV

C

A single C region gene is encoded in the germline and separated from the multiple V region genes

Find a way to show the existence of multiple V genes and rearrangement to the C gene

Approach

Tools:

• A set of cDNA probes to specifically distinguish V regions from C regions

• DNA restriction enzymes to fragment DNA

• Examples of germline (e.g. placenta) and mature B cell DNA (e.g. a plasmacytoma/myeloma)

C

VV

VV

V

V

VV

V

Germline DNA

CV

V

VV

V

Rearranged DNA

MMany GENEany GENESS (10 (10 000 – 100000 – 100 000)000)

VV22 CC VVnn CCVV11 CC

1 GEN1 GEN

HHigh rate of somatic mutations in igh rate of somatic mutations in tthe he VV-region-region

VV CC

GGeenn

ProteinProtein

1 G1 GENEN = 1 = 1 PROTEIN PROTEIN

DOGMA OF MOLECULAR BIOLOGY

CHARACTERISTICS OF IMMUNOGLOBULIN SEQUENCE

THEORIES

Liver cellLiver cell BB-cell-cell

1.5. Kb1.5. Kb

BB-cell-cellV C

6.0 Kb6.0 Kb

V C

4.0 Kb4.0 Kb

DNA-extractionDigestion by restriction enzyme

Gel electrophoresis Southern blot

VCVC

KbKb

6,06,0

1,51,5

V-V-probeprobe

4,04,0 CC

VVC-C-probeprobe

Experiment of Susumi Tonegawa 1975 Basel

There are many vThere are many vaariable genesriable genes but only one constant genebut only one constant gene

V CV V V

GERM LINEGERM LINE

V aV and nd CC g geenes nes gget close to each other in B-ceet close to each other in B-cellls onlyls only

CV V V

BB-CELL-CELL

CONCLUSION

Ig gene sequencing complicated the model

The structures of germline VL genes were similar for V, and V,However there was an anomaly between germline and rearranged DNA:

Where do the extra 13 amino acids come from?

CLVL

~ 95 ~ 100

L CLVL

~ 95 ~ 100

JL

Some of the extra amino acids are provided by

one of a small set of J or JOINING regions

L

CLVL

~ 208

L

Further diversity in the Ig heavy chain

VL JL CLL

CHVH JHDHL

The heavy chain was found to have further amino acids (0 – 8) between the JH és CH genes

D (DIVERSITY) region

Each light chain requires 2 recombination events

VL to JL and VLJL to CL

Each heavy chain requires 3 recombination events JH to DH , VH to JHDH, and VHJHDH to CH

HOW MANY IMMUNOGLOBULIN GENE SEGMENTS

Variable (V) 132/40 105/30 123/65

Diversity (D) 0 0 27

Joining (J) 5 4 9

Gene segments Light chain Heavy chain

kappa lambda

Chromosome 2 kappa light chain gene segments

Chromosome 22 lambda light chain gene segments

Chromosome 14 heavy chain gene segments

IMMUNOGLOBULIN CHAINS ARE ENCODED BY MULTIPLE GENE SEGMENTS

ORGANIZATION OF IMMUNOGLOBULIN GENE SEGMENTS

DDuuring ring BB-lymphocyte -lymphocyte developmentdevelopment

Jk Jκ Jκ JκVκ Vκ VκB-cell 1

JκVκB-cell 2

80 V80 Vκκ 4 J4 Jκκ

Vκ Vκ Vκ Vκ Jκ Jκ Jκ JκGerm lineGerm line

SOMATIC REARRANGEMENT OF KAPPA (κ) CHAIN GENE SEGMENTS

DNA

pACCκEJJ

Vκ-Jκ

VκVκ P

CCκJVκ ProteinProtein

mRNAmRNACCκJVκ AAAA

TransTranslationlation

EXPRESSION OF THE KAPPA CHAIN

PrimaPrimary ry RNRNAA transcripttranscript

CCκEJJVκLeader

Efficiency of somatic gene rearrangement?

During B-cell developmentDuring B-cell development

VH2 JH JH

120 VH120 VH 4 JH4 JH

VH1 VH3 D JH JH

12 D12 D

D DD

JH JH

JH JHD D

SOMATIC REARRANGMENT OF THE HEAVY CHAIN GENE SEGMENTS

D DVH1 VH2 VH3

VH1 VH2

VHVH DD JHJH

VLVL JLJL

V-DV-Domainsomains

C-DC-Domainsomains

VHVH--DD--JHJH VLVL--JLJL

VARIABILITY OF B-CELL ANTIGEN RECEPTORS AND ANTIBODIES

B cells of one individual 1 2 3 4

D – J recombination

V – DJ recombination

VDJ – δ transcription

δ translation

Surrogate light chain

V – J recombination

VJ – (or VJ - ) transcription

or translation

B-sejt

mIgD mIgM

ORDER OF REARRANGEMENTS OF IMMUNOGLOBULIN GENE SEGMENTS

Secreted IgM

Estimates of combinatorial diversity

Taking account of functional V D and J genes:

40 VH x 27 DH x 6JH = 6,480 combinationsD can be read in 3 frames: 6,480 x 3 = 19,440 combinations

29 Vx 5 J = 145 combinations30 Vx 4 J = 120 combinations

= 265 different light chains

If H and L chains pair randomly as H2L2 i.e. 19,440 x 265 = 5,151,600 possibilities Due only to COMBINATORIAL diversity

In practice, some H + L combinations do not occur as they are unstableCertain V and J genes are also used more frequently than others.

There are other mechanisms that add diversity at the junctions between genes - JUNCTIONAL diversity

GENERATES A POTENTIAL B-CELL REPERTOIRE

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

Evidence for allelic exclusion

Allotypes can be identified by staining B cell surface Ig with antibodies

a/a b/b a/b

YBb YBa YBb

Y

YB ab

YBa AND

ALLOTYPE- a polymorphism in the Heavy chain C region of Ig

Suppression of H chain rearrangement by pre-B cell receptor prevents expression of two

specificities of antibody per cell

Allelic exclusion is needed for efficient clonal selection

All daughter cells must express the same Ig specificityotherwise the efficiency of the response would be compromised

Suppression of H chain gene rearrangement helps to prevent the emergence ofnew daughter specificities during proliferation after clonal selection

S. typhi

Antibody

S. typhi

YY

Y Y

Suppression of H chain gene rearrangementensures only one specificty of Ab expressed per cell.

Allelic exclusion prevents unwanted responses

BSelf antigenexpressed bye.g. brain cells

S. aureusY Y

YYYB

S. aureus

YY

Y

YY

Y

Y

AntiS. aureus

Antibodies

Y

Y Y

Y Y

YAntibrainAbs

One Ag receptor per cell IF there were two Ag receptors per cell

Y

Y Y

Y

YY Y

AntiS. aureus

Antibodies

Prevents induction of unwanted responses by pathogens

Allelic exclusion is needed to prevent holes in the repertoire

Exclusion of anti-brain B cells i.e. self tolerance

YYBB

One specificity of Agreceptor per cell

S. aureus

Anti-brain IgAND

anti-S. Aureus IgYYYBB

IF there were two specificitiesof Ag receptor per cell

Anti-brain Ig

BB

Deletion Anergy

OR

anti S.Aureus B cells will be excluded leaving a “hole in the repertoire”

BUT

YYYBB

SYNTHESIS OF IMMUNOGLOBULINS

ER

Golgi

mRNA

Ribosome

Leader sequence

Membrane Ig Secreted Ig

H and L chains are synthesized on separated

ribosomes

CHAPERONES