Download Generation of diversity in lymphocyte antigen receptors

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: ~ 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
Does the Ig gene locus differ in nonlymphoid
cells vs B cells?
Patient with chronic
Healthy control
lymphocytic leukemia
Nonlymphoid cells Single B cell clone
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 k
30 x 4 = 120 l
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
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