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B-CELL DIFFERENTIATION IN THE PERIPHERY SOMATIC HYPERMUTATION
Ag Ag Ag Memory B-cell Activated B-cell SOMATIC HYPERMUTATION Mature naive B-cell ISOTYPE SWITCH
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Potential B-cell repertoire
BONE MARROW Self structure Self recognition Clonal deletion PERIPHERAL LYMPHOID ORGANS Available B-cell repertoire Antigen – non-self Antigen dependent Clonal division Effector cell repertoire Memory cell repertoire
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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
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MEMBRANE BOUND AND SECRETED IMMUNOGLOBULIN
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The constant region has additional optional exons
Primary transcript RNA AAAAA Cm Cm1 Cm2 Cm3 Cm4 Each domain of the H chain is encoded by a separate exon Secretion coding sequence Polyadenylation site (secreted) pAs Polyadenylation site (membrane) pAm Membrane coding sequence
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Membrane IgM constant region
Cm1 Cm2 Cm3 Cm4 DNA Transcription Cm1 Cm2 Cm3 Cm4 1° transcript pAm AAAAA Cleavage & polyadenylation at pAm and RNA splicing Cm1 Cm2 Cm3 Cm4 AAAAA mRNA Membrane coding sequence encodes transmembrane region that retains IgM in the cell membrane Fc Protein
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Secreted IgM constant region
Cm1 Cm2 Cm3 Cm4 DNA Cleavage polyadenylation at pAs and RNA splicing 1° transcript pAs Cm1 Cm2 Cm3 Cm4 Transcription AAAAA mRNA Cm1 Cm2 Cm3 Cm4 AAAAA Secretion coding sequence encodes the C terminus of soluble, secreted IgM Fc Protein
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ISOTYPE SWITCH
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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 Ca2 Ce Cg4 Cg2 Ca1 Cg1 Cg3 Cd Cm Organisation of the functional human heavy chain C region genes
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Somatic recombination D – J
C Cδ C3 C1 Cε2 C1 C 1 C4 Cε1 C2 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
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Switch regions Ca2 Ce Cg4 Cg2 Ca1 Cg1 Cg3 Cd Cm
Sg3 Sg1 Sa1 Sg2 Sg4 Se Sa2 Sm 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
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At each recombination constant regions are deleted from the genome
Switch recombination Ca2 Ce Cg4 Cg2 Ca1 Cg1 Cg3 Cd Cm Sg3 Cm Cd Cg3 V23D5J4 Cg1 Sg1 Ca1 Cm Cd Cg3 V23D5J4 Cg3 V23D5J4 Ca1 Cg3 V23D5J4 IgG3 produced. Switch from IgM V23D5J4 Ca1 IgA1 produced. Switch from IgG3 V23D5J4 Ca1 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
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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)
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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
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SOMATIC HYPERMUTATION
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CDR1 CDR2 CDR3 VL J2 gene product V35 gene product
Complementary Determining Region = hypervariable region
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STRUCTURE OF THE VARIABLE REGION
Hypervariable (HVR) or complimentarity determining regions (CDR) HVR3 FR1 FR2 FR3 FR4 HVR1 HVR2 Variability Index 25 75 50 100 Amino acid residue 150 Framework regions (FR)
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LIGHT CHAIN Light chain Disulphide bridges Heavy chain VL CL
FR FR FR FR4 CDR1 CDR CDR3 Heavy chain VL CL FR FR FR FR4 CDR1 CDR CDR3
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SOMATIC HYPERMUTATION Hypervariable regions
Day 0. Ag Plasma cell clones 1 2 3 4 5 6 7 8 Day 7 PRIMARY immune response AFFINITY MATURATION 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
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Hypermutation occurs under the influence of activated T cells
Somatic hypermutation leads to affinity maturation Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 6 Clone 7 Clone 8 Clone 9 Clone 10 CDR1 CDR2 CDR3 Day 6 CDR1 CDR2 CDR3 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 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.
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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
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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
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FEATURES OF THE BINDING SITE
SIZE SHAPE HYDROPHOBIC HYDROPHYLIC POSITIVELY CHARGED NEGATIVELY CHARGED 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
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Growth factors MHC – peptid - TCR Adhesion molecules ANTIBODIES Affinity
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