Generation of Ig Receptors

and TCRs
Lecture 8
02/09/2017
Haley Tucker
 Overview
How is all of the variability encoded
in antibody and T cell receptor (TCR)
molecules?
VDJ recombination
Class Switching for Antibodies
 Antibody hyper-variable regions are
encoded in separate V, J and D exons

Germlin
eV
segment

The antibody repertoire

is the number of
specificities available to
an individual.
In humans ~ 1011
unique antibody
specificities.
But the number available
at any time is limited by
How do we get the variability seen at CD3
the total number of B cells
(HVR3) the N terminus in the variable regions?
 CDR1 and CR2 are encoded in the V gene
segment, CDR3 is encoded at a junction of gene
segments
 Gene segments encoding Ig variable regions are
very far apart, but are brought closer together in
lymphocytes
Nobel prize in 1987 for this
discovery In 1976, using newly discovered
restriction enzymes (enzymes that
cleave DNA at a very specific nucleotide
sequenceusually 4 to 8 bp in length),
Susumu Tonegawa discovered that the
V and C gene segments were far from
each other (total length of heavy
chain locus is 2 megabases!) in most
somatic cells, but in B lymphocytes
(or in B cell tumors in the actual
paper), the V and C genes were on the
same DNA fragment
This is a Southern Blot with DNA from
nonlymphoid cells of normal person or B
cells from a CLL patient. The V and C
region
This isprobes are The
heretical! on different
DNA is restriction
C region probe V region probe fragments the non-lymphoid cells, but
mixed up in lymphocytes
are on a single fragment in the B cells.
 Whats going on?
Overview of VDJ recombination
*Combinatorial use of V region gene segments
drives much of antibody diversity
# of possible light chains:
40 V x 5 J= 200

# possible light chains:

30 V x 4 J = 120

# possible heavy chains:

40 V x 25 D x 6 J = 6000

TOTAL number of Igs from

combination of random
heavy chains with light
chains:
6000 x 200 ()+ 6000 x 120
()= 1.9 x 106 (all heavy
chains cannot pair with all
light chains).
*Human heavy and light chain loci

Ig H and L chains
are encoded by V,
(D,) and J gene
segments

What is the
advantage of this
unusual process of
rearrangement of
gene segments to
encode antibodies?

DIVERSITY of
antigen
 *Recombination Signal Sequences (RSS) mark
sites of recombination for V,D, J gene segments

Recombination signal sequences are at the ends of gene segments

(5 or 3) where recombination occurs
Heptamer- spacer- nonamer
12/23 rule: In general recombination machinery only pairs RSSs of
12 bp spacer with RSSs of 23 bp spacers
Prevents V to J in heavy chain.
*VDJ recombination results in excision or
inversion of intervening sequence

If genes are transcribed in same

direction, excision of signal joint
occurs (common)

If genes are transcribed in

opposite directions, inversion
occurs (less common)

extrachromosomal circle
containing the signal joint
identifies lymphocytes that
recently underwent
rearrangement of Ig (or TCR)
genes. After division, the circle is
lost.
 *Enzymatic steps in VDJ
recombination
1. Rag1/2 complex binds
RSSs
2. The segments to be
joined are synapsed
3. The RSS is cleaved
from the gene
segment, yielding a
closed hairpin on the
coding side, and a
4. Ku70:Ku80
double stranded are break
recruited
on the signal to theside
breaks: these are
ubiquitous proteins
that are part of DSBR
Rag1/2 only expressed in lymphocytes at the time of recombination
in all
Transcriptional activity is thought to contribute to accessibility cells
of the RSS sites.
This limits recombination of genes, which could otherwise be disastrous for cells, to the V, D, J
gene segments at the right stages of lymphocyte development.
* 5. For the signal joint, DNA ligase
IV: XRCC4 repair enzymes join
the break
6. For the coding end, DNA -*PKcs
is recruited to Ku70/80. DNA-
PKcs phosphorylates Artemis,
which makes single-strand nick
to open hairpin
7. TdT is recruited, which
randomly adds nucleotides to
the nicked ends. Additionally,
repair enzymes (exonucleases)
remove nucleotides. Together
these generate diverse ends
8. DNA ligase IV:XRCC4 then joins
the processed ends.
SCID results from a failure of any
of these enzymes along the
pathway: without Ku70:80,
RAG1/2, DNA-PKcs, or Artemis B
and T cells do not develop. IR-SCID
is due to defects in DNA repair
enzymes. *DNA-PKc is original
Structure of Rag1:Rag2
complex

Required for
endonucleas
e
activity

Nonomer
Binding
Domains
 RAG-1 and RAG-2 act as a dimer
RAG-1 contains Zn2+ dependent endonuclease activity, RAG-2 is
a cofactor
RAG complex makes a single strand break 5 of heptamer. This
free 3OH then performs a nucleophilic attack on the other
 *Closer look at the types of
diversity generated at coding joint
heptamer

heptamer RAG-1/RAG-2 makes a nick between the

heptamers and the D or J gene segments.

When the 3OH group left on the coding

strand attacks the phosphodiester bond
on the other strand, a hairpin is formed

Artemis:DNA-PK opens the hairpins at

random locations: if this occurs anywhere
other than right at the hairpin, P
(palindromic) nucleotides are created

N nucleotides are added by TdT to

generate more diversity at the CDR3
locus (particularly in heavy chains, since
TdT isnt generally expressed when light
chains are being rearranged)
*

Random complementarity
between the processed strands
allows them to pair

Exonucleases remove unpaired

nucleotides

Repair enzymes fill in gaps

DNA ligase IV:XRCC4 then joins

the processed ends

This process can lead to frame-

shifts, so 2/3 rearrangements will
be non-productive
 *VDJ recombination summary
Heavy and light chains are encoded by gene segments that are
joined by recombination of DNA
Heavy chain is encoded by a V (variable) + D (diversity) + J
(joining)
Light
The chain is encoded
hypervariable by V
regions + J (this
CDR1 can happen
and CDR2 either for
are encoded on the V
gene light chains)
orsegment. BUT the hypervariable region CDR3 is encoded by
the
Onlyjunction of V(D)J
1 Immunoglobulin is expressed by any one B cell (Allelic
exclusionmore
The combinatorial later)
diversity generated by pairing of different V, D, J
segments + different heavy and light chains to for the Ig
heterodimer, has the potential to yield ~2x106 different
immunoglobulin
Its estimated thatmolecules
there are ~ 1011 different possible immunoglobulin
specificities
Additional diversity comes from the imprecise nature of the VDJ
recombination machinery
Recombination occurs between gene segments with RSSs following
the 12/23 rule recombinases initiate the single-stranded nick that breaks
RAG-1/RAG-2
the DNA between the coding and signal joints. DNA-PK + Artemis open the
hairpin
DSBR enzymes repair the break, bringing the V next to the J, for example, in
the genome
Exonucleases randomly remove nucleotides at the join, while TdT
adds random nucleotides, greatly enhancing variability at the CDR3
region
Only 1/3 rearrangements is in-frame, so this Is an inherently wasteful
process, but necessary for recognition of all of the pathogens we encounter
 *Cloning of the TCR eluded
researchers for years
TCR is only membrane bound, has low
affinity for antigen, and binds peptide in the
context of MHC. Isolation and
characterization of TCR was a challenge
In 1980s, researchers generated mAbs
against TCRs and determined it was a
heterodimer of and chains.
Using the mAbs, researchers attempted to
purify, sequence and clone the TCR.but
Hedrick and Davis (1984) succeeded in cloning
the TCR using subtractive hybridization

3 important
assumptions:

1.Because TCR is
membrane-bound, the
RNA would be in
membrane-bound
polyribosomes

2. B cell and T cell

transcripts are very
similar, but differ in
the TCR

3.TCR would
rearrange like BCR, so
probes from TCR
would hybridize to
Cloning of TCR part 2
 *Human TCR loci

Altogether, combinatorial diversity yields at max:

TCR: 80 x 61= 4880
TCR: 52 x 2 X 13 = 1352
TCR: 4880 X 1352= ~7 X 106
Junctional diversity is key for TCRs too, yielding ~1018
 Generation of TCRs

Rearranged V(D)J region encodes CDR3s: critical for peptide

12/23 rule applies to TCR as well
*Enzymatic steps in VDJ
recombination
1. Rag1/2 complex binds
RSSs
2. The segments to be
joined are synapsed

3. The RSS is cleaved

from the gene
segment, yielding a
closed hairpin on the
coding side, and a
double stranded break
4. Ku70:Ku80 areside
on the signal
recruited to the
breaks: these are
ubiquitous proteins
that are part of DSBR
*
5. For the signal joint, DNA ligase
IV: XRCC4 repair enzymes join
the break
6. For the coding end, DNA PKcs is
recruited to Ku70/80. PKcs
phosphorylates Artemis, which
makes single-strand nick to
open hairpin
7. TdT is recruited, which
randomly adds nucleotides to
the nicked ends. Additionally,
repair enzymes (exonucleases)
remove nucleotides. Together
these generate diverse ends
8. DNA ligase IV:XRCC4 then joins
the processed ends.

SCID results from a failure of any

of these enzymes along the
pathway: without Ku70:80,
RAG1/2, DNA-PKcs, B and T cells
do not develop
TCR variability is focused on the
CDR3 regions

CDR3 is encoded by
V(D)J join, and it
binds to the peptide.

CDRs 1 and 2 bind

primarily to the MHC
molecules
Comparison of Ig and TCR diversity

More D in Ig
than TCR

Many more
J segments
in TCR:
increased
CDR3
diversity

P and N are
important
for both
 T cells: TCR locus is imbedded in
the TCR locus
TCR is excised if TCR is
successfully rearranged
 *Summary
Combinatorial association of V(D)J regions in Ig and
TCR chains yields diversity of variable regions the
join of V(D)J generates CDR3 in both chains
Imprecise V(D)J joining enables even more diversity
VDJ recombination happens during B and T cell
development
Ig secondary diversification mechanisms enable
1. diversification of antigen binding region- somatic
hypermutation
2. class switching- different effector mechanisms
associated with same antigen recognition
Secondary diversification occurs after B cell is activated
by antigen

Midterm 1: introductory chapters, innate immunity, Antibody

structure and antigen recognition, TCR structure and antigen
recognition, Generation of Ig and TCR diversity (VDJ