Activated B-cellMature naive B-cell

Memory B-cell

B-CELL DIFFERENTIATION IN THE PERIPHERY

SOMATIC HYPERMUTATION

ISOTYPE SWITCH

Ag

AgAg

n42 3

PERIPHERAL LYMPHOID ORGANS

Available B-cell repertoire

n3 42 51BONE

MARROW

Potential B-cell repertoire

Self structure

Self recognitionClonal deletion

Antigen – non-self

Antigen dependentClonal division

Effector cell repertoire

Memory cell repertoire

• If the BCR and the soluble antibodies are identical, by what mechanism switch from one to the other is controlled? MEMBRANE VS SECRETED IMMUNOGLOBULIN

• By what mechanism are antibodies with the same specificity but with different isotypes generated?ISOTYPE SWITCH

• How could antibodies increase their affinity in the course of the immune response?SOMATIC HYPERMUTATION

The molecular genetics of immunoglobulins

MEMBRANE BOUND AND SECRETED IMMUNOGLOBULIN

Primary transcript RNA AAAAA

C

Polyadenylation site (secreted)

pAs

Polyadenylation site (membrane)

pAm

The constant region has additional optional exons

C1 C2 C3 C4

Each domain of the H chain is encoded by a

separate exonSecretion

codingsequence

Membranecoding

sequence

mRNAC1 C2 C3 C4 AAAAA

Transcription

Membrane IgM constant region

C1 C2 C3 C41° transcriptpAm

AAAAA

C1 C2 C3 C4DNA

Membrane coding sequence encodes

transmembrane regionthat retains IgM in the

cell membrane

Fc

Protein

Cleavage & polyadenylation at pAm and RNA splicing

mRNA

Secreted IgM constant region

C1 C2 C3 C4 AAAAA

C1 C2 C3 C4DNA

Cleavage polyadenylation at pAs and RNA splicing

1° transcriptpAs

C1 C2 C3 C4

Transcription

AAAAA

Secretion coding sequence encodes the C terminus of soluble,

secreted IgM

Fc

Protein

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

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

• Switching is mechanistically similar in many ways to V(D)J

recombination, but

• All recombination events are productive

• Different recombination signal sequences and enzymes are involved

• Requires antigen stimulation of B cell

• Not a random event, but regulated by external signals such as T cell

derived cytokines

• Isotype switching does not take place in the bone marrow, but occurs

after B cell activation in the peripheral lymphoid organs

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).

• The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7.

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

Model for Class Switch Recombination (CSR)

AID (Activation Induced (citidin) Deaminase C →U, RNA editing enzyme)UNG excises U → abasic sites, AP-endonuclease/lyase activity → ss nicks Class switch defects - Hiper IgM syndrome type 2 in humans (autosomal)

•HYPER IgM SYNDROME (Autosomal)

-Intrinsic B cell defect, activation induced deaiminase (AID) deficiency. Cytidine uridine conversion. -The enyme is involved in affinity maturation and Ig. class switch - Lack of opportunistic infections

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)

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

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