Generation of diversity in lymphocyte antigen receptors Jan. 31, Feb. 2 & 5 Chapter 4.
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Transcript of Generation of diversity in lymphocyte antigen receptors Jan. 31, Feb. 2 & 5 Chapter 4.
Generation of diversity in lymphocyte antigen
receptorsJan. 31, Feb. 2 & 5
Chapter 4
Objectives
• Discuss molecular mechanisms of generating diversity in immunoglobulins and TCRs:– Somatic recombination
• Combinatorial diversity• Junctional diversity
– Somatic hypermutation– Isotype switching
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
Healthy controlNonlymphoid cells
Patient with chronic lymphocytic leukemiaSingle B cell clone
Does the Ig gene locus differ in nonlymphoid cells vs B cells?
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
Multiple gene segments increase Ig diversity
•Pseudogenes•Gene duplications and diversification•Total length of heavy chain locus > 2 Mb
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
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
• See gene recombination animation on CD
Marker of cells that have undergone V(D)J recombination
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
Lymphocyte-specific and ubiquitous enzymes are
required• Ubiquitous DNA repair enzymes are
also required– DNA ligases– DNA-dependent protein kinase– Artemis– Many others
Junctional diversity•Nucleotide deletion can also occur
•Occurs in HV3 (CDR3) region
•What problem could these events cause??
Diversity in the TCR gene locus
The TCR gene is most variable in the CDR3 region
CDR3
Summary: Ig vs TCR
Further Ig diversity arises through affinity maturation
Affinity maturation is due to somatic hypermutation
SilentNeutralDeleteriousPositive
Somatic hypermutation
• Mechanism is not well defined
• Requires enhancers and an active promoter
• Requires activation-induced cytidine deaminase (AID)
Diversity of antibodies due to constant region
differences
Diversity of antibodies• Abs with the
same idiotype (Ag specificity) can be of different isotypes
Diversity of antibodies: isotypes
Alternative RNA processing generates transmembrane or
secreted Ig
IgM and IgD are coexpressed in mature
naïve B cells
Isotype switching occurs in activated B cells
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)
Summary: mechanisms that generate diversity in lymphocyte
receptors