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Activated B -cell

a. a. Ag. Ag. Ag. B-CELL DIFFERENTIATION IN THE PERIPHERY. Memory B -cell. Activated B -cell. SOMATIC HYPERMUTATION. M ature na i ve B-cell. ISOTYPE SWITCH. Self recognition Clonal deletion. Self structure. PERIPHERAL LYMPHOID ORGANS. Available B-cell repertoire.

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Activated B -cell

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

  2. Self recognition Clonal deletion Self structure PERIPHERAL LYMPHOID ORGANS Available B-cell repertoire Antigen dependent Clonal division Antigen – non-self Memory cell repertoire Effector cell repertoire Potential B-cell repertoire BONE MARROW

  3. The molecular genetics of immunoglobulins • 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

  4. MEMBRANE BOUND AND SECRETED IMMUNOGLOBULIN

  5. Cm Primary transcript RNA AAAAA Each domain of the H chain is encoded by a separate exon Secretioncodingsequence Polyadenylation site (secreted) pAs Polyadenylation site (membrane) pAm Cm1 Cm2 Cm3 Cm4 Membranecodingsequence The constant region has additional optional exons

  6. Cm1 Cm2 Cm3 Cm4 DNA Transcription pAm Cm1 Cm2 Cm3 Cm4 1° transcript AAAAA Cleavage & polyadenylation at pAm and RNA splicing Protein Membrane coding sequence encodes transmembrane region that retains IgM in the cell membrane Fc Membrane IgM constant region Cm1 Cm2 Cm3 Cm4 AAAAA mRNA

  7. Cm1 Cm2 Cm3 Cm4 DNA Transcription pAs Cm1 Cm2 Cm3 Cm4 1° transcript AAAAA Cleavage polyadenylation at pAs and RNA splicing mRNA Protein Secretion coding sequence encodes the C terminus of soluble, secreted IgM Fc Secreted IgM constant region Cm1 Cm2 Cm3 Cm4 AAAAA

  8. ISOTYPE SWITCH

  9. Organisation of the functional human heavy chain C region genes Cm Cd Cg3 Cg1 Ca1 Cg2 Cg4 Ce Ca2 J regions 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.

  10. C Cδ C3 C1 Cε2 C1 C 1 C4Cε1 C2 C Cδ Embryonal DNA Somatic recombination D – J Rearranged DNA Somatic recombination V – D – J C Cδ Primer RNA transcript Transcription Ig ISOTYPES Cµ IgM Cγ1 IgG Cγ2 IgG Cγ3 IgG Cγ4 IgG Cα IgA Cε IgE C Cδ Processing C mRNA Translation Nascent polypeptide C Modification Heavy chain IgM

  11. Cm Cd Cg3 Cg1 Ca1 Cg2 Cg4 Ce Ca2 Sm Sg3 Sg1 Sa1 Sg2 Sg4 Se Sa2 Switch regions • Upstream of C regions are repetitive regions of DNA called switch regions. (The exception is the Cd region that has no switch region). • The Sm consists of 150 repeats of [(GAGCT)n(GGGGGT)] where n is between 3 and 7. • 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

  12. Cm Cd Cg3 Cg1 Ca1 Cg2 Cg4 Ce Ca2 Sg3 Cd Cd Cg3 Cm Cm Sg1 Cg3 Cg1 V23D5J4 V23D5J4 Cg3 V23D5J4 Ca1 Ca1 IgG3 produced. Switch from IgM IgA1 produced. Switch from IgM IgA1 produced. Switch from IgG3 V23D5J4 V23D5J4 V23D5J4 Cg3 Ca1 Ca1 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

  13. 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)

  14. 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

  15. Lack of germinal centers in lymph nodes of X-linked Hyper-IgM syndrome patients

  16. SOMATIC HYPERMUTATION

  17. VL J2 gene product V35 gene product CDR1 CDR2 CDR3 Complementary Determining Region = hypervariable region

  18. STRUCTURE OF THE VARIABLE REGION Hypervariable (HVR) or complimentarity determining regions (CDR) HVR3 150 Variability Index 100 HVR2 HVR1 50 FR2 FR1 FR4 FR3 0 25 75 100 50 Amino acid residue • Framework regions (FR)

  19. Light chain LIGHT CHAIN Disulphide bridges FR1 FR2 FR3 FR4 CDR1 CDR2 CDR3 Heavy chain VL CL FR1 FR2 FR3 FR4 CDR1 CDR2 CDR3

  20. Hypervariable loops and framework: Summary • The framework supports the hypervariable loops • The framework forms a compact b barrel/sandwich with a hydrophobic core • The hypervariable loops join, and are more flexible than, the b 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

  21. SOMATIC HYPERMUTATION Day 0. Ag Plasma cell clones 1 2 3 4 5 6 7 8 Day 7 PRIMARY immune response AFFINITYMATURATION 9 1011 12 13 14 15 16 Day 14 Day 14. Ag 17 1819 20 21 22 23 24 Day 21 SECONDARY Immune response Hypervariable regions

  22. Day 6 Day 12 Day 8 Day 18 Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 6 Clone 7 Clone 8 Clone 9 Clone 10 Deleterious mutation CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 Beneficial mutation Neutral mutation Somatic hypermutation leads to affinity maturation Lower affinity - Not clonally selected Higher affinity - Clonally selected Identical affinity - No influence on clonal selection 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.

  23. 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

  24. 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)

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