a a Activated B-cell Mature naive B-cell Memory B-cell B-CELL DIFFERENTIATION IN THE PERIPHERY SOMATIC HYPERMUTATION ISOTYPE SWITCH Ag

PERIPHERAL LYMPHOID ORGANS Available B-cell repertoire BONE MARROW Potential B-cell repertoire Self structure Self recognition Clonal deletion Antigen – non-self Antigen dependent Clonal 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 RNAAAAAA CC Polyadenylation site (secreted) pAs Polyadenylation site (membrane) pAm The constant region has additional optional exons C1C1C2C2C3C3C4C4 Each domain of the H chain is encoded by a separate exon Secretion coding sequence Membrane coding sequence

mRNA C1C1C2C2C3C3C4C4 AAAAA Transcription Membrane IgM constant region C1C1C2C2C3C3C4C4 1° transcript pAm AAAAA C1C1C2C2C3C3C4C4 DNA Membrane coding sequence encodes transmembrane region that retains IgM in the cell membrane Fc Protein Cleavage & polyadenylation at pAm and RNA splicing

mRNA Secreted IgM constant region C1C1C2C2C3C3C4C4 AAAAA C1C1C2C2C3C3C4C4 DNA Cleavage polyadenylation at pAs and RNA splicing 1° transcript pAs C1C1C2C2C3C3C4C4 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 C2C2CC C4C4C2C2C1C1C1C1C3C3CC CC Organisation of the functional human heavy chain C region genes

C  Cδ C  3 C  1 Cε2 C  1 C  1 C  4 Cε1 C  2 C  Cδ IgM CC 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 CµIgM Cγ1IgG Cγ2IgG Cγ3IgG Cγ4IgG CαIgA CεIgE Heavy chain

C2C2CC C4C4C2C2C1C1C1C1C3C3CC CC 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 S3S3S1S1S1S1S2S2S4S4SS S2S2 SS 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.

C2C2CC C4C4C2C2C1C1C1C1C3C3CC CC CC CC C3C3 V 23 D 5 J 4 S3S3 CC CC C3C3 C1C1 S1S1 C1C1 C3C3 C1C1 C3C3 IgG3 produced. Switch from IgM V 23 D 5 J 4 C1C1 IgA1 produced. Switch from IgG3 V 23 D 5 J 4 C1C1 IgA1 produced. Switch from IgM Switch recombination At each recombination constant regions are deleted from the genome An 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 germinal centers in lymph nodes of X-linked Hyper-IgM syndrome patients

SOMATIC HYPERMUTATION

CDR1CDR2CDR3 VL Complementary Determining Region = hypervariable region V35 gene product J2 gene product

STRUCTURE OF THE VARIABLE REGION Hypervariable (HVR) or complimentarity determining regions (CDR) HVR 3 FR1 FR 2 FR 3 FR 4 HVR1 HVR 2 Variability Index Amino acid residue Framework regions (FR)

CDR1 CDR2 CDR3 Light chain Heavy chain CDR1 CDR2 CDR3 VLCL LIGHT CHAIN Disulphide bridges FR1 FR2 FR3 FR4

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

SOMATIC HYPERMUTATION Day 0. Ag Day 14. Ag PRIMARY immune response SECONDARY Immune response AFFINITY MATURATI ON Day 21 Day 14 Day 7 Hypervariable regions Plasma cell clones

Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 6 Clone 7 Clone 8 Clone 9 Clone 10 CDR1CDR2CDR3 Day 6 CDR1CDR2CDR3CDR1CDR2CDR3CDR1CDR2CDR3 Day 8 Day 12 Day 18 Deleterious mutation Beneficial mutation Neutral mutation Lower affinity - Not clonally selected Higher affinity - Clonally selected Identical affinity - No influence on clonal selection Somatic hypermutation leads to affinity maturation Hypermutation occurs under the influence of activated T cells Mutations 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-gene The CDR3 of L-chain is encoded by V and J The CDR3 of H-cain is encoded by V, D and J genes

SOME CHARACTERISTICS OF SOMATIC HYPERMUTATIONS  Mutations made by AID (same enzyme as for class switching)  Both CSR and SHM requires strand brakes  10-3 / Bp mutation rate (a million times more than expected)  Both CSR and SHM occurs in germinal center B- cells  Very much site specific, CDR regions of the BCR (some other genes too, but limited (CD95)