Generation of diversity in lymphocyte antigen receptors Jan. 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: ~ Number of genes in humans: ~ 2.5 x 10 5 How can there be so many Ig and TCR variants??? –Germline theory –Somatic diversification theory

Healthy control Nonlymphoid cells Patient with chronic lymphocytic leukemia Single 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 chains 40 x 25 x 6 = 6000 Light chains 40 x 5 = 200  30 x 4 = 120 Total possible: 320 x 6000 = 1.9x10 6

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 Silent Neutral Deleterious Positive

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