generation of diversity in lymphocyte antigen receptors jan. 31, feb. 2 & 5 chapter 4
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Generation of diversity in lymphocyte antigen
receptorsJan. 31, Feb. 2 & 5
Chapter 4
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Objectives
• Discuss molecular mechanisms of generating diversity in immunoglobulins and TCRs:– Somatic recombination
• Combinatorial diversity• Junctional diversity
– Somatic hypermutation– Isotype switching
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The problem of Ig and TCR diversity:
• Human immunoglobulin repertoire: ~ 1011
• Number of genes in humans: ~ 2.5 x 105
• How can there be so many Ig and TCR variants???– Germline theory– Somatic diversification theory
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Healthy controlNonlymphoid cells
Patient with chronic lymphocytic leukemiaSingle B cell clone
Does the Ig gene locus differ in nonlymphoid cells vs B cells?
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The Ig gene is nonfunctional in germline
DNA• The genes encoding each Ig chain are
actually a family of gene segments located in one region of DNA (locus)
• V gene segments (~100 AA of V region)• J gene segments (remainder of V region)• D gene segments (between V and J
segments)
• Must be physically rearranged to become a functional gene– Somatic recombination– Lymphocytes die during development if Ig/TCR
recombination does not occur
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Multiple gene segments increase Ig diversity
•Pseudogenes•Gene duplications and diversification•Total length of heavy chain locus > 2 Mb
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Multiple gene segments increase Ig diversity
Combinatorial diversity:
Heavy chains40 x 25 x 6 = 6000
Light chains40 x 5 = 200 30 x 4 = 120
Total possible:320 x 6000 = 1.9x106
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Recombination occurs at specific sites
• Recombination signal sequences (RSS) occur adjacent to coding sequences in V, D, and J segments– Heptamer-spacer-nonamer– 12/23 rule
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• See gene recombination animation on CD
Marker of cells that have undergone V(D)J recombination
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Lymphocyte-specific and ubiquitous enzymes are
required• RAG-1 and RAG-2 are
lymphocyte-specific– Fibroblasts transfected
with RAG-1 + RAG-2 undergo somatic recombination of Ig genes
– RAG-KO mice have no B or T cells
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Lymphocyte-specific and ubiquitous enzymes are
required• Ubiquitous DNA repair enzymes are
also required– DNA ligases– DNA-dependent protein kinase– Artemis– Many others
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Junctional diversity•Nucleotide deletion can also occur
•Occurs in HV3 (CDR3) region
•What problem could these events cause??
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Diversity in the TCR gene locus
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The TCR gene is most variable in the CDR3 region
CDR3
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Summary: Ig vs TCR
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Further Ig diversity arises through affinity maturation
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Affinity maturation is due to somatic hypermutation
SilentNeutralDeleteriousPositive
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Somatic hypermutation
• Mechanism is not well defined
• Requires enhancers and an active promoter
• Requires activation-induced cytidine deaminase (AID)
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Diversity of antibodies due to constant region
differences
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Diversity of antibodies• Abs with the
same idiotype (Ag specificity) can be of different isotypes
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Diversity of antibodies: isotypes
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Alternative RNA processing generates transmembrane or
secreted Ig
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IgM and IgD are coexpressed in mature
naïve B cells
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Isotype switching occurs in activated B cells
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Isotype switching
• Irreversible
• Only occurs after a given B cell has encountered antigen
• Mechanism not fully understood– Requires AID– Requires DNA repair enzymes– Requires external signals (helper T cells)
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Summary: mechanisms that generate diversity in lymphocyte
receptors