lim 210: immunology ii lecture 2: antibody diversity
TRANSCRIPT
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LIM 210: Immunology II
Lecture 2: Antibody Diversity
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Mechanisms contributing to generation of antibody diversity
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Mechanisms contributing to generation of antibody diversity
1. Genesis of antibody diversity2. Combinatorial diversity (somatic recombination)3. Junctional diversity4. Somatic hypermutations5. Random assortment of H and L chains6. Class switching in the C-region
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Genesis of antibody diversity
• Presence of multiple V genes in the germ line contributes to Ig diversity
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Immunogenetics
• Multi germ line gene segments:
Antibody genes are composed of segments
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B Cell: humoral or antibody mediated immunity
• Humoral Immunity: ANTIBODIES– Matures in bone marrow– Moves to lymph nodes (& other
locations)– Secretes antibodies “as plasma
cells”– Also creates “memory” B cells– B cells secrete soluble antibodies:
humoral immunity.– T cells interact directly with their
targets: cellular immunity
B-lymphocyte
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B cell Development: gene rearrangement and RNA processing
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Genesis of antibody diversity
• A mature B-cell which has undergone V(D)J recombination is distinct from all others at 3 levels:
– The choice of V, D and J segments from germ line DNA
– The combination of re-arranged heavy and light chains– The junctional insertion and deletion of nucleotides which
occurs during rearrangement
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CLONAL SELECTION
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Mechanisms contributing to generation of antibody diversity
• How is Ig diversity specified genetically?– Multiple germ line gene segments– Combinatorial diversity (or V-J & V-D-J recombination)– Junctional diversity – Somatic hypermutations – Random assortment of H and L chains– Class switching in C region gene
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Functional gene segments for the V regions of human heavy and light chains
Presence of multiple V genes in the germ line contributes to Ig diversity
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Mechanisms contributing to generation of antibody diversity
Germ line genes H k λV segments 65 40 30J segments 6 5 4D segments 27 0 0
Combinatorial joiningV × J (×D) 11,000 200 120
H-L chain associationsH × k 2.2 x 106 From 177 segmentsH × λ 1.3 x 106
Note: Junctional diversity is also estimated to add substantially to overall diversity
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Immature B cell: gene rearrangement and RNA processing
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Mature B cell: Antigen stimulation, Activation & differentiation, Class switching
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Somatic recombination of gene segments
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Light Chain Splicing• There are two different L chain genes, called kappa and
lambda. Immunoglobulins use one or the other. • At the kappa L chain gene, there is a single region of
DNA that codes for the C domain. Upstream from the C domain is a group of about 250 V domains and another group of 5 J (for "joining") regions.– Each V region has a 5’UTR segment, a separate
exon, attached to it: the leader.• During the development of the B cell, a randomly
chosen V domain joins with a randomly chosen J domain to form a VJ domain. This occurs by splicing out the DNA between them. – Note that this is a very different mechanism from
intron spicing that occurs in the RNA of most genes!
• Once the VJ domain is formed, it can be transcribed into RNA along with the C domain and any DNA that lies between the VJ and C. All the intervening RNA is spliced out as an intron, so the final messenger RNA has VJC all together; this is then translated into a L chain protein.
• Lambda light chain genes are slightly different: fewer V regions, and four different C regions each of which has its own J. Lambda chains use only a single DNA splice, to join a randomly selected V region to one of the 4 J-C regions.
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Heavy Chains• The process of producing a heavy chain is very similar to L
chains, except that there is an additional group of 5 D regions between the V regions and the J regions.
• Two DNA splices occur for H chains: first a random J joins with a random D to form a DJ region, then a random V region is added to form a VDJ domain.
• Just as in the L chain, this VDJ is transcribed along with the C region, and any intervening RNA is spliced out as an intron. The final product has VDJC all joined together and gets translated into an H chain protein.
• All of the C regions are at the heavy chain locus. Initially, the CM region is transcribed. CD is also transcribed and expressed using an alternative RNA splicing mechanism.
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Heavy Chain Rearrangement
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Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display
22-20
DNA rearrangements bring together segments of a gene for expression.
Fig. 22.24
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Mechanism of junctional diversity
• Imprecise V(D)J joining– Only appropriate segments get joined (V always joins to J
in a light chain, not V to V). It is site specific.– Catalyzed by RAG-1 and RAG-2 (recombination activating
genes)– During recombination, nucleotides can be lost or inserted
(may shift reading frame)– Can have a second round of V(D)J rearrangement (receptor
editing)
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Mechanism of junctional diversity
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Somatic Hypermutation
• Somatic Hypermutation– is a mechanism by which random base change mutations occurs in the V regions in B
cells. – This mechanism doesn't work in other cells and doesn't affect other genes: only a region
of about 1.5 kb is affected. – It only occurs as the B cell is maturing: after it has been stimulated to divide by an
antigen, somatic hypermutation occurs to modify the antigen binding region. – Those cells that bind the antigen most tightly survive and divide more than the others.
This process is called “affinity maturation”. – It is triggered by the enzyme “activation-induced cytidine deaminase” (AID), which
deaminates cytidine to uracil. This base mismatch can be incorrectly repaired by several different mechanisms to generate mutations.
– Occurs randomly after antigenic stimulation and principally in CDR1, CDR2, CDR3 regions (more frequent in CDR3).
– Introduces point mutations in V genes at a higher rate than for normal mammalian genes.
– Mutation rate of V genes is 1 base pair change per 103 base pairs/cell division; it is 10-7 in other mammalian genes.
– Can give rise to Ig with different (new) antigen specificities leading to high or low affinity Abs. High affinity B cell clones are selectively expanded (Affinity Maturation)
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Class Switching• Heavy chains fall into 5 classes, based
on their C regions. • Each H gene has C regions for all 5
classes arranged on the chromosome, with the IgM C region nearest to the V regions.
• There are several different C regions for some of the classes.
• IgM is the initial Ab made by each B cell.
• However, after a while the B cell switches to a different class.
• This is done using a third DNA splice, in which the DNA between the VDJ and the constant region for the new class is spliced out.
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Mature B cell: Antigen stimulation, Activation & differentiation, Class switching
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Random Assortment of H and L chains
• Random Assortment of H and L chains.
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Changes in immunoglobulin and TCR genes that occur during B-cell and T-cell development and differentiation