The Generation of BCR/TCR Diversity

Questions (outlines):
1. Do yyou reallyy understand V(D)J
( ) recombination?
Functions of RAG?

2 Wh
2. Whatt are the
th differences
diff bbetween
t th
the IIg and
d TCR
gene recombination?

3. What is class switch? Functions of AID?

Repertoire: a group of diverse molecules,

segments, or motifs
 1011 Ab in humans

Dryer and Bennett 2-gene model, 1965 Tonegawa gene recombination model, 1976
Ig genes are rearranged
in B cells

A population of normal B cells

h many diff
has differentt rearranged
d
genes, so they yield a smear of
DNA fragment sizes.

(different probes)
Probe: mRNA encoding light chain ( ___ )
 The germline organization of the Ig heavy
heavy-
and light-chain loci in the human genome
(Ch #22)

(8 families or clans)
(Ch #2)

(7 families or clans)
(Ch #14)

(3 families or clans)
V-region genes are constructed from gene segments
(95-101 aa) (< 13 aa) (90-95 aa) (2-3 aa) (10-15 aa)

CDR 3
V region sequences are constructed from gene segments
V-region

Vl 90-95 aa 10-15 aa VH 90-95 aa 2-3 aa 10-15 aa

()
Light-chain production in a mouse

page 809-811
but naive
Conserved heptamer and nonamer sequences (recombination
signal sequences,
sequences RSSs) flank the gene segments encoding
the V regions of heavy (H) and light ( and ) chains
(recombination signal sequences)

2 1

1 2
 RAG: recombination-activating
genes
Ku70:Ku80 + DNA-PKcs
DNA PKcs to form
DNA-PK
Artemis (with nuclease activity)
TdT: terminal deoxynucleotidyl
y y
transferase
DNA ligase IV (form complex with
XRCC4) as DSBR: double-
strand
t d break
b k repairi
3OH

P-nucleotide addition 5 P
(palindrome)

N-nucleotide addition
TdT: terminal deoxynucleotidyl
transferase

SCID: severe combined immune

deficiency ( RAG-1/2 )
 The introduction of P-and N-nucleotides at the joints
between gene segments during Ig gene rearrangement
Rearrangement of V,
V D,
D or J gene segments produces
a functional heavy-chain gene

IgM
allelic
exclusion
T-cell receptor gene rearrangement
The germline organization of the human T-cell receptor and loci

C1 and C2 are highly homologous.

TCR - and -chain gene rearrangement and expression

CDR1CDR2CDR3
The organization of the human T-cell receptor and loci

-chain locus is located

within h chain
i hi the h i locus
l
TCR Ig

(all TCR molecules)

-gene is within -gene

fragments more Js of and chain
fewer V fragments,
use RAG1/2 enzymes
follow the one-turn/two turn joining rule
allelic exclusion is less stringent
The numbers of human TCR gene segments and the sources for
TCR diversity compared with those of Igs
 Structural variation in immunoglobulin constant regions
The Ig isotypes are encoded by a cluster of Ig heavy-chain
heavy chain C-region
C region genes

: pseudogene
2 forms of Igs
Transmembrane and secreted forms of Ig are derived from the same
heavy-chain sequence by alternative RNA processing
Coexpression of IgD and IgM is regulated by RNA processing
Synthesis, assembly, and
secretion of the Igs

IgM: H+L
HL
(HL)2

IgG: 2H
H2+L
H2L+L
H2L2

(rough
endoplasmic
reticulum)
Immunoglobulin isotype (class) switch

IgM IgG
The formation of a gene for the heavy chain of an Ab molecule
page
p g 809-811

(recombination)

(Ab class switching)

Isotype switching involves recombination between specific switch signals

150 X (GAGCT)n(GGGGGT),
(GAGCT)n(GGGGGT) n=3-7

Enzyme: unknown
E k
AID (activation-induced cytidine deaminase)
UNG, APE1, Ku70:Ku80
AID (activation-induced cytidine deaminase) in class
switching and somatic hypermutation

Normal IgM, little IgG in AID-/- mice.

AIDhyper-IgM syndrome (type II)
AID (activation-induced cytidine deaminase) in class
switching and somatic hypermutation

No somatic hypermutation in AID-/- mice.

Somatic hypermutation introduces variation into the rearranged Ig variable region that is
subject to negative and positive selection to yield improved Ag binding (affinity maturation)
Activation-induced cytidine
deaminase (AID) is the initiator
off mutations
t ti in
i somatic
ti
hypermutation gene conversion,
and class switching

The sequence
seq ence of AID is related to that of a
protein known as APOBEC1 (apolipoprotein
B mRNA editing catalytic polypeptide 1),
which convert cytosine to uracil by
deamination in apolipoprotein B mRNA.
Generation of single
strand nicks in DAN by
sequential
ti l action
ti off AID,
AID
UNG, and APE1

UNG: uracil-DNA-glycosylase
(to form abasic site in the DNA)

APE1: apurinic/apyrimidinic
endonuclease
d l 1
(to generate a single-strand nick)
 AID initiates processes that lead to
somatic hypermutation, gene conversion
and
d class
l switch
it h recombination
bi ti

(synthesize DNA over the abasic

sites and have a random
nucleotide insertion )

(
(through
g homologous
g
recombination with pseudogenes)
The primary antibody
repertoire is diversified
b th
by three processes that
th t
modify the rearranged
Ig gene
Class switching involves
recombination between
specific switch signals

S : 150 repeats of
(GAGCT)3-7(GGGGGT)
- form R-loop

V(D)J recombination
(page 186)