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