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