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B-CELL DIFFERENTIATION IN THE PERIPHERY Ag Ag Ag Memory B-cell Activated B-cell Mature naive B-cell ISOTYPE SWITCH SOMATIC HYPERMUTATION BONE MARROW 3 2 1 4 n 5 Potential B-cell repertoire Self recognition Clonal deletion Self structure PERIPHERAL LYMPHOID ORGANS 3 2 Available B-cell repertoire n 4 Antigen dependent Clonal division Antigen – non-self 3 3 3 3 3 3 3 3 3 3 3 3 3 Memory cell repertoire 3 3 3 3 3 3 3 3 3 Effector cell repertoire 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 MEMBRANE BOUND AND SECRETED IMMUNOGLOBULIN The constant region has additional optional exons Cm Primary transcript RNA Each domain of the H chain is encoded by a separate exon Cm1 Cm2 AAAAA Secretion coding sequence Cm3 Polyadenylation site (secreted) pAs Polyadenylation site (membrane) pAm Cm4 Membrane coding sequence Membrane IgM constant region DNA Cm1 Cm2 Cm3 Cm4 Transcription 1° transcript pAm Cm1 Cleavage & polyadenylation at pAm and RNA splicing mRNA Cm2 Cm3 Cm1 Cm2 Cm3 Cm4 Cm4 AAAAA AAAAA Membrane coding sequence encodes transmembrane region that retains IgM in the cell membrane Protein Fc Secreted IgM constant region DNA Cm1 Cm2 Cm3 Cm4 Transcription 1° transcript pAs Cm1 Cm2 Cm3 Cm4 AAAAA Cleavage polyadenylation at pAs and RNA splicing mRNA Cm1 Cm2 Cm3 Cm4 Protein AAAAA Secretion coding sequence encodes the C terminus of soluble, secreted IgM Fc 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. Organisation of the functional human heavy chain C region genes J regions Cm Cd Cg3 Cg1 Ca1 Cg2 Cg4 Ce Ca2 embrionális Embryonal L1 V1 L2 V2 Ln Vn D1 - 12 DNS DNA 5' szomatikus rekombináció Somatic recombination D– D-J kapcsolódás átrendeződött Rearranged DNA DNS L1 V1 Ln Vn Cm Cδ Cg3 CM CD CG3 J1-4 J CA1Cε2 Ca1 CG1 CE2Cg1 CE1 Ca2 CG 2 CG 4 Cε1 CA2 C g1 Cg4 D1D2 J1J2-4 Cm CM Cδ CD 5' 3' szomatikus rekombináció Somatic recombination V -D-J kapcsolódás CM Cδ CD D2J1 J2-4 Cm 3' L1 V1 5' Primerprimer RNA transcript RNS-átirat mRNA mRNS 3’ Transcription transzkripció 5' L1 V1D2 J1 J2-4 Cm CM Cδ CD IgM Cγ1 IgG Cγ2 IgG Cγ3 IgG Modification Cγ4 IgG AAAA transzláció Translation L V DJ Cm C polipeptid Ig ISOTYPES Cµ 3' Processing átalakítás Cm L1 V1 D2 J1 CM naszcens Nascent polypeptide módosítás V C V–D–J NEHÉZL ÁNC (M ) Heavy chain IgM Cα IgA Cε IgE Switch regions Cm Sm Cd Cg3 Sg3 Cg1 Sg1 Ca1 Sa1 Cg2 Sg2 Cg4 Sg4 Ce Se Ca2 Sa2 • 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 Switch recombination Cm Cd Cg3 Cg1 Ca1 Cg2 Cg4 Cd Ce Cd Ca2 Sg3 Cg3 Cg3 Cm Sg1 Cm Cg1 V23D5J4 Cg3 V23D5J4 Ca1 V23D5J4 Ca1 V23D5J4 Cg3 V23D5J4 Ca1 V23D5J4 Ca1 IgG3 produced. Switch from IgM IgA1 produced. Switch from IgG3 IgA1 produced. Switch from IgM 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 VL J2 gene product V35 gene product CDR1 CDR2 CDR3 Complementary Determining Region = hypervariable region STRUCTURE OF THE VARIABLE REGION • Hypervariable (HVR) or complimentarity determining regions (CDR) HVR 3 Variability Index 15 0 10 0 5 0 0 HVR HVR1 2 FR1 25 FR 2 FR 3 7 50 Amino acid 5 residue(FR) • Framework regions FR 4 10 0 100 Light chain 90 80 70 60 50 40 LIGHT CHAIN 30 20 10 NH2 0 10 FR1 20 30 40 FR2 CDR1 50 60 70 FR3 80 CDR2 90 100 110 120 FR4 Disulphide bridges CDR3 COOH 100 90 Heavy chain 80 70 60 50 VL 40 30 20 10 0 10 20 FR1 30 40 50 FR2 CDR1 60 70 80 FR3 CDR2 90 100 110 120 FR4 CDR3 CL 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 SOMATIC HYPERMUTATION Day 0. Ag Day7 7nap CDR1 CDR2 CDR3 CDR1 CDR2 IgM PRIMARY immune response Day 14 14 nap IgM/IgG Day 14. Ag Day 21 21 nap CDR3 1 2 3 4 5 6 7 8 AFFINITY 9 MATURATI 10 11 ON 12 13 14 15 16 IgG SECONDARY Immune Plasma cell clones Hypervariable regions 17 18 19 20 21 22 23 24 Somatic hypermutation leads to affinity maturation Day 6 Day 8 Day 12 Day 18 CDR3 CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 CDR1 CDR2 CDR3 CDR1 CDR2 Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 6 Clone 7 Clone 8 Clone 9 Clone 10 Deleterious mutationLower affinity - Not clonally selected Beneficial mutation Higher affinity - Clonally selected Neutral mutation 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. FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 H-CHAIN CDR2 CDR1 100 CDR3 Variability 80 60 40 Antigén determináns 20 CDR1 CDR2 CDR3 L-CHAIN 20 40 60 80 100 120 Amino acid No. Wu - Kabat analysis compared point mutations in Ig of different specificity. CDR1 and CDR2 regions are encoded by the V-gene The CDR3 of L-chain is encoded by V and J 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 Bcells Very much site specific, CDR regions of the BCR (some other genes too, but limited (CD95)