Download No Slide Title

Topic 6
The T Cell Antigen
Receptor Complex
©Dr. Colin R.A. Hewitt
[email protected]
What you should know by the end of this lecture
• Each clone of T cells expresses a single TcR specificity
• How the TcR was discovered
• The similarities and differences between TcR and antibodies
• The structure and organisation of the TcR genes
• Somatic recombination in TcR genes
• Generation of diversity in TcR
• Structure function relationship of TcR
• Why TcR do not undergo somatic mutation
Discovery of the T cell antigen receptor (TcR)
Polyclonal T cells
from an immunised
strain A mouse
Grow and clone a single antigenspecific T cell in-vitro with antigen,
IL-2 and antigen presenting cells
Monoclonal (cloned) T cells
In vitro “clonal selection” means each daughter cell has the same
antigen specificity as the parent cell
Most molecules present on the monoclonal T cells will be
identical to the polyclonal T cells EXCEPT for the antigen
combining site of the T cell antigen receptor
Making anti- clonotypic TcR antibodies
T cell clone
from a strain A mouse
Naïve strain A mouse
Make monoclonal antibodies by hybridisation of the spleen cells
with a myeloma cell line
The strain A mouse will not make antibodies to the hundreds of different molecules
associated with strain A T cells due to self tolerance
BUT
The naïve mouse has never raised T cells with the specificity of the T cell clone,
SO
the only antigen in the immunisation that the A strain mouse has never seen will be
the antigen receptor of the monoclonal T cells
Making anti- clonotypic TcR antibodies
Screen the supernatant of each cloned hybridoma against a panel of T
cell clones of different specificity
(i.e.cells with subtly different antigen-binding structures)
Monoclonal antibodies
YYY
Clone used for
immunisation
T cell clones
Y Y Y
Anti-TcR Abs that recognise only one clone of T cells are CLONOTYPIC
Hypothesise that anti-clonotype Abs recognise the antigen receptor
Discovery of the T cell antigen receptor (TcR)
Y
Y
YYYYYYY
Lyse cells and add anti-clonotype Ab
that binds to unique T cell structures
YYYY
Capture anti-clonotype Ab-Ag
complex on insoluble support
IMMUNOPRECIPITATION
Wash away unbound protein
YYYY
Elute Ag from Ab and analyse the
clonotypically-expresssed
proteins biochemically
Principal component was a heterodimeric 90kDa protein
composed of a 40kDa and a 50kDa molecule ( and  chains)
Several other molecules were co-immunoprecipitated.
Structure of the TcR polypeptides
T cell clone A
T cell clone B
T cell clone C
Intact TcR chain polypeptides
Cyanogen bromide digestion of the  and  proteins
Biochemical analysis of digestion products
C
V
C
V
C
V
Polypeptides contain a variable, clone-dependent pattern of digestion
fragments and a fragment common to all TcR
Cloning of the TcR genes
T
B
The experimental strategy
•
The majority of genes expressed by T and B lymphocytes will be
similar
•
Genes that greatly differ in their expression are most likely to be
directly related to the specialised function of each cell
•
Subtract the genes expressed by B cells from the genes expressed by
T cells leaving only the genes directly related to T cell function
Cloning of TcR genes by subtractive hybridisation
T
B
mRNA
AAAAA
AAAAA
T cell single
stranded cDNA
Hybridise the
AAAAA cDNA and mRNA shared
between T and B cells
Digest unhybridised B cell mRNA
AAAAA
Discard hybrids
AAAAA
AAAAA
Isolate non-hybridising
material specific to T cells
Clone and
sequence T cellspecific genes
Analysis of T cell-specific genes
Of the T cell-specific genes cloned, which cDNA encoded the TcR?
Assumptions made after the analysis of Ig genes:
TcR genes rearrange from germline configuration
Ig gene probes can be used as TcR genes will be homologous to Ig genes
Restriction
enzyme sites
32P
V
D
J
C
GERMLINE
DNA
C
REARRANGED
DNA
32P
V DJ
Find two restriction sites that flank the TcR region
Cut the T cell cDNA and placental (i.e. germline) DNA
and Southern blot the fragments
The TcR genes rearrange, but are not
immunoglobulin genes
Gel electrophoresis followed by Southern blot using a TcR probe
Placenta
Size of
digested
genomic
DNA
B
T
Rearranged
allele
The T cell antigen receptor
Antigen
combining site
Resembles an Ig Fab fragment
VL
VH V
L
VH
CL
CH
CH
CH CH
V V
CL
Fab
Fc
CH CH
Domain structure: Ig gene superfamily
Monovalent
Carbohydrates
No alternative constant regions
C C
Hinge
+
+
Cytoplasmic tail
+
Transmembrane region
Never secreted
Heterodimeric, chains are disuphidebonded
Very short intracytoplasmic tail
Positively charged amino acids in the
TM region
Antigen combining site made of
juxtaposed V and V regions
30,000 identical specificity TcR per cell
CH CL VH VLof Ig
C C V V of the TcR
View structures
T cell antigen receptor diversity
•
Unlike MHC molecules TcR are highly variable in the individual
•
Diversity focused on small changes in the charge & shape
presented at the end of the T cell receptor.
•
TcR diversity to the peptide antigens that bind to MHC molecules
•
Mechanisms of diversity closely related to T cell development
•
Random aspects of TcR construction ensures maximum diversity
•
Mechanisms of diversity generation similar to immunoglobulin
genes
Generation of diversity in the TcR
COMBINATORIAL DIVERSITY
Multiple germline segments
In the human TcR
Variable (V) segments: ~70, 52
Diversity (D) segments: 0, 2
Joining (J) segments: 61, 13
The need to pair  and  chains to form a binding site
doubles the potential for diversity
JUNCTIONAL DIVERSITY
Addition of non-template encoded (N) and palindromic (P) nucleotides at
imprecise joints made between V-D-J elements
SOMATIC MUTATION IS NOT USED TO GENERATE DIVERSITY IN TcR
Organisation of TcR genes
L&V
x70-80
C
J x 61
TcR 
L&V
x52 D1 J1 x 6
C1 D2
J2 x 7
TcR 
TcR genes segmented into V, (D), J & C elements
(VARIABLE, DIVERSITY, JOINING & CONSTANT)
Closely resemble Ig genes (~IgL and ~IgH)
This example shows the mouse TcR locus
C2
TcR  gene rearrangement by
SOMATIC RECOMBINATION
Vn V2 V1
J
Germline TcR 
Rearranged TcR
1° transcript
Spliced TcR  mRNA
Rearrangement very similar to the IgL chains
C
TcR  gene rearrangement RESCUE PATHWAY
There is only a 1:3 chance of the join between the V and J region being in frame
Vn+1 Vn V2 V1
J
C
 chain tries for a second time to make a productive join using new V and J elements
Productively
rearranged TcR
1° transcript
TcR  gene rearrangement
SOMATIC RECOMBINATION
L & V
x52
D1
J
C1
D2
J
C2
Germline TcR 
D-J Joining
V-DJ joining
Rearranged TcR  1° transcript
C-VDJ joining
Spliced TcR  mRNA
TcR  gene rearrangement RESCUE PATHWAY
There is a 1:3 chance of productive D-J rearrangement and a 1:3 chance of
productive D-J rearrangement
(i.e only a 1:9 chance of a productive  chain rearrangement)
V
D1
J
C1
D2
J
C2
Germline TcR 
D-J Joining
V-DJ joining
2nd chance at
V-DJ joining
Need to remove
non productive
rearrangement
Use (DJC)2
elements
V, D, J flanking sequences
Sequencing upstream and downstream of V, D and J elements revealed
conserved sequences of 7, 23, 9 and 12 nucleotides.
V
7
23
9
V
7
23
9
12
9
7
9
D
7
12
9
12
7
J
9
23
7
J
Recombination signal sequences (RSS)
HEPTAMER - Always contiguous with
coding sequence
9
V 7
23
√
V
9
7
12
23
7 D 7
9
9
12
9
9
12
7
D
NONAMER - Separated from
the heptamer by a 12 or 23
nucleotide spacer
7 J
23
7
12
9
√
9
23
7
J
12-23 RULE – A gene segment flanked by a 23mer RSS can only be linked to a segment
flanked by a 12mer RSS
Molecular explanation of the 12-23 rule
12-mer = one turn
23-mer = two turns
23
V
7
Intervening DNA
of any length
9
12
9
7 DJ
Molecular explanation of the 12-23 rule
V4
V1
V8
V9
V3
V2
V7
V6
V3
V4
V2
V5
9
9
23-mer
• Heptamers and nonamers
align back-to-back
7
7
V7
V8
V9
12-mer
V1
V6
Loop of
intervening
DNA is
excised
DJ
• The shape generated by the
RSS’s acts as a target for
recombinases
V5
DJ
• An appropriate shape can not be formed if two 23-mer flanked elements
attempted to join (i.e. the 12-23 rule)
Junctional diversity
Mini-circle of DNA is
permanently lost from the
genome
9
7
V
7
12
23
9
9
23
Coding joint
7
7
12
9
Signal joint
DJ
VDJ
Imprecise and random events that occur when the DNA breaks and
rejoins allows new nucleotides to be inserted or lost from the sequence at
and around the coding joint.
Non-deletional recombination
V1
V1
V1
V3
V2
7
V2
V3
9
23
V4
23
V4
7
V9
DJ
9
V9
V4
Looping out works if all V
genes are in the same
transcriptional orientation
9
DJ
12
7
D J
How does recombination occur
when a V gene is in opposite
orientation to the DJ region?
9
12
7
DJ
Non-deletional recombination
9
23
7 V4
9 12 7 D J
1.
2.
9
7 V4
23
9
3.
V4 and DJ in opposite
transcriptional orientations
23
9
23
7 V4
7 V4
4.
9
23
7 V4
9 12 7 D J
2.
1.
9
9
23
7
12
9
23
9
V4
12
7
7
V4
D J
Heptamer ligation - signal
joint formation
7 D J
3.
V4
9
23
9
4.
9
12
23
7
7
D J
7
7
12
9
V to DJ ligation coding joint
formation
V4 D J
Fully recombined VDJ regions in same transcriptional orientation
No DNA is deleted
Steps of TcR gene recombination
V
7 23
V
7 23
D J
9 12 7
7 23
9 12 7
9
7 23
D J
The two RAG1/RAG 2 complexes
bind to each other and bring the V
region adjacent to the DJ region
9
9 12 7 9 12 7
V
9
Recombination activating
gene products, (RAG1 & RAG
2) and ‘high mobility group
proteins’ bind to the RSS
9
• The recombinase complex makes single
stranded nicks in the DNA, the ends of
each broken strand.
• The nicks are ‘sealed’ to form a hairpin
structure at the end of the V and D
regions and a flush double strand break
at the ends of the heptamers.
• The recombinase complex remains
associated with the break
D J
Steps of TcR gene recombination
V
7
23
9
D J
9 12 7
V D J
D
J
9 12 7 7 23 9
V
A number of other proteins, (Ku70:Ku80,
XRCC4 and DNA dependent protein
kinases) bind to the hairpins and the
heptamer ends.
The hairpins at the end of the V and D
regions are opened, and exonucleases
and transferases remove or add
random nucleotides to the gap between
the V and D region
DNA ligase IV joins the ends of the V
and D region to form the coding joint
and the two heptamers to form the
signal joint.
Junctional diversity: P nucleotide additions
7 23
V
AT GTGACAC
J D TA CACTGTG
9
9 12 7
V
TC CACAGTG
AG GTGTCAC
7
7
9
23
12
9
The recombinase complex makes single
stranded nicks at random sites close to the
ends of the V and D region DNA.
TC
AG
TC CACAGTG
AG GTGTCAC
7
GTGACAC
CACTGTG
7
V V
AT
AT
J JDTA DTA
9
23
12
9
The 2nd strand is cleaved and hairpins form between
the complimentary bases at ends of the V and D
region.
D J
V3
V2
V4
CACAGTG
GTGTCAC
7
GTGACAC
CACTGTG
7
9
23
12
V5
9
V9
Heptamers are ligated by
DNA ligase IV
TC
AG
V
AT
J DTA
V
V8
V7
TC
AG
V and D regions juxtaposed
AT
TA
V6
D J
Generation of the palindromic sequence
V
V
V
TC
AG
TC
AG
TC~GA
AG
AT
TA
D J
AT
TA
D J
Regions to be joined are juxtaposed
Endonuclease cleaves single strand at
random sites in V and D segment
The nicked strand ‘flips’ out
AT
TA~TA
D J
The nucleotides that flip out, become
part of the complementary DNA strand
In terms of G to C and T to A pairing, the ‘new’ nucleotides are palindromic.
The nucleotides GA and TA were not in the genomic sequence and
introduce diversity of sequence at the V to D join.
Junctional Diversity – N nucleotide additions
V
TC~GA CACTCCTTA
AT
AG
TTCTTGCAA
TA~TA
D J
Terminal deoxynucleotidyl transferase
(TdT) adds nucleotides randomly to
the P nucleotide ends of the singlestranded V and D segment DNA
V
TC~GA CACACCTTA
AT
AG
TTCTTGCAA TA~TA
D J
Complementary bases anneal
V
TC~GACACACCTTA
D J
Exonucleases nibble back free ends
V
TC
CACACCTTA
TC~GA
GTT ATAT
AT
AGC
TTCTTGCAA
TA
TA~TA
AG
D J
DNA polymerases fill in the gaps
with complementary nucleotides
and DNA ligase IV joins the strands
TTCTTGCAA TA~TA
Junctional Diversity
V
TCGACGTTATAT
AGCTGCAATATA D
J
TTTTT Germline-encoded nucleotides
TTTTT Palindromic (P) nucleotides - not in the germline
TTTTT Non-template (N) encoded nucleotides - not in
the germline
Creates an essentially random sequence between the V region, D region
and J region in beta chains and the V region and J region in alpha chains.
How does somatic recombination work?
1. How is an infinite diversity of specificity generated from finite
amounts of DNA?
Combinatorial diversity and junctional diversity
2. How do V region find J regions and why don’t they join to C regions?
12-23 rule
3. How does the DNA break and rejoin?
Imprecisely, with the random removal and addition of nucleotides to
generate sequence diversity.
Why do V regions not join to J or C regions?
V
D
J
C
IF the elements of the TcR did not assemble in the correct order, diversity of
specificity would be severely compromised
2x
DIVERSITY
Full potential of the beta
chain for diversity needs
V-D-J-C joining - in the
correct order
1x
DIVERSITY
Were V-J joins allowed in
the beta chain, diversity
would be reduced due to
loss of the imprecise join
between the V and D
regions
Location of junctional diversity
TcR  chain
TcR  chain
CDR3
CDR
1 CDR2
V-D
Join
Variability
V-J
Join
Amino acid No.
of TcR chain
CDR = Complemantarity determining region
D-J
join
Location of junctional diversity in TcR
2
1
3
2
3
1
CDR’s
TcRV monomer
TcR chain
The trimolecular complex
MHC class I and TcR V/V
MHC class II TcR /
V and V of TcR recognising
a peptide from MHC class I
ribbon plot
 TcR recognising a peptide
from MHC class II
ribbon plot
Turn through 90º
V and V of TcR recognising
a peptide from MHC class I wire
plot showing amino acid sidechains
 TcR recognising a peptide
from MHC class II wire plot showing
amino acid sidechains
TcR contact and anchor residue side chains
interact with side chains of TcR
Hypervariable loops - CDRs
1/2 /3 1/2
1/2
/3
1/2
The most variable loops of the TcR - the CDR3 interact with the most variable
part of the MHC-peptide complex CDR’s 1 and 2 interact largely with the MHC
molecule
View structures
T cell co-receptor molecules
Lck PTK
TcR
3
CD8
Lck PTK TcR
CD4
2
 
MHC Class I
MHC Class II
CD4 and CD8 can increase the sensitivity of T cells to peptide antigen MHC
complexes by ~100 fold
CD8 and CD4 contact points on MHC
class I and class II
CD8 binding site
MHC class I
CD8 binding site
MHC class II
 TcR 
TcR-CD3 complex
CD3

CD3



The intracytoplasmic region
of the TcR chain is too short
to transduce a signal
  

  
The CD3   or  (zeta) chains
are required for cell surface
expression of the TcR-CD3
complex and signalling
through the TcR
 
Signalling is initiated by aggregation of TcR by MHC-peptide complexes on APC
Transduction of signals by the TcR
CD3
     
ITAMs
The cytoplasmic domains of the CD3 complex contain 10 Immunoreceptor
Tyrosine -based Activation Motifs (ITAMS) - 2 tyrosine residues separated
by 9-12 amino acids - YXX[L/V]X6-9YXX[L/V]
As with B cell receptors, immunoreceptor tyrosine-based activation motifs
(ITAMs) are involved in the transmission of the signals from the receptor
and require clustering of TcR/CD3 and the CD4 or CD8 co-receptors
Phosphorylation by Src kinases
Kinase domain
Enzyme domain that
phosphorylates tyrosine
residues (to give phosphotyrosine)
SH2 domain SH3 domain Unique region
Phosphotyrosine
receptor domain
Adaptor
protein
recruitment
domain
ITAM
binding
domain
• Phosphorylation changes the properties of a protein, by changing its conformation
• Changes in conformation can activate or inhibit a biochemical activity, or create a
binding site for other proteins
• Phosphorylation is rapid and requires no protein synthesis or degradation to
change the biochemical activity of a target protein
• It is reversible via the action of phosphatases that remove phosphate
Regulation of Src kinases
Kinase domain
Inhibitory tyrosine residue
SH2 domain SH3 domain Unique region
Activating tyrosine residue
Phosphorylation of ‘Activating Tyrosine’ stimulates kinase activity
Kinase domain
SH2 domain SH3 domain Unique region
Phosphorylation of ‘Inhibitory Tyrosine’ inhibits kinase activity
by blocking access to the Activating Tyrosine Residue
Early T cell activation
MHC II
MHC II
CD4
CD45
Receptor associated kinases accumulate
under the membrane in close proximity
to the cytoplasmic domains of the TcR CD3 complex
As the T cell antigen receptor binds
the MHC-peptide antigen, the
phosphatase CD45 activates kinases
such as Fyn
P
Fyn
Lck
Zap-70
This mechanism of activation is
similar to the used to activate Syk in
B cells
CD4
CD45
MHC II
T cell activation
Fyn phosphorylates the ITAMs of
CD3, ,  and  ITAMS
The tyrosine kinase ZAP-70 binds
to the phosphorylated ITAMs of
CD3 - further activation requires
ligation of the co-receptor, CD4
P
Fyn
Lck
Zap-70
T cell activation
Binding of CD4 co-receptor to MHC
class II brings Lck into the complex,
which then phosphorylates and
activates ZAP-70
MHC II
ZAP-70 phosphorylates LAT and
SLP-76
P
SLP-76
P
Fyn
P
LAT
P
P
Lck
Zap-70
Activated ZAP-70 phosphorylates LAT & SLP-76
Tyrosine rich cell membrane
associated Linker of Activation
in T cells (LAT) and SLP-76
associate with cholesterol-rich
lipid rafts
T cell activation
MHC II
Activated ZAP-70 phosphorylates
Guanine-nucleotide exchange factors
(GEFS) that in turn activate the small GTP
binding protein Ras
Ras activates the MAP
kinase cascade
P
SLP-76
P
Fyn
Lck
P
Tec Tec
LAT
P
P
SLP76 binds Tec kinases
and activates
phospholipase C-  (PLC-)
Zap-70
PLC- cleaves phosphotidylinositol
bisphosphate (PIP2) to yield diacylglycerol
(DAG) and inositol trisphosphate (IP3)
Transmission of signals from the cell
surface to the nucleus
Almost identical to transmission in B cells
• T cell-specific parts of the signalling cascade are associated with receptors
unique to T cells - TcR, CD3 etc.
• Subsequent signals that transmit signals to the nucleus are common to
many different types of cell.
• The ultimate goal is to activate the transcription of genes, the products of
which mediate host defence, proliferation, differentiation etc.
Once the T cell-specific parts of the cascade are complete, signalling to
the nucleus continues via three common signalling pathways via:
1.The mitogen-activated protein kinase (MAP kinase) pathway
2.An increase in intracellular calcium ion concentration mediated by IP3
3.The activation of Protein Kinase C mediated by DAG
Simplified scheme linking antigen recognition
with transcription of T cell-specific genes
• MAP Kinase cascade
Small G-protein-activated MAP kinases found in all multicellular animals activation of MAP kinases ultimately leads to phosphorylation of transcription
factors from the AP-1 family such as Fos and Jun.
• Increases in intracellular calcium via IP3
IP3, produced by PLC-, binds to calcium channels in the ER and releases
intracellular stores of Ca++ into the cytosol. Increased intracellular [Ca++]
activate a phospatase, calcineurin, which in turn activates the transcription
factor NFAT.
• Activation of Protein Kinase C family members via DAG
DAG stays associated with the membrane and recruits protein kinase C
family members. The PKC, serine/threonine protein kinases, ultimately
activate the transcription factor NFkB
The activated transcription factors AP-1, NFAT and NFkB induce B cell
proliferation, differentiation and effector mechanisms
Estimate of the number of human TcR and Ig
Excluding somatic hypermutation
Element
Immunoglobulin
H
k
 TcR


Variable segments
40
59
52
~70
Diversity segments
27
2
D segments in
all 3 frames
Yes
0
-
Yes
0
-
Joining segments
6
2
9
(1)*
13
2
Joints with N & P
nucleotides
No. of V gene pairs
2360
Junctional diversity
~1013
~1016**
Total diversity
61
1
3640
~1013
~1016
* Only half of human k chains have N & P regions
**No of distinct receptors increased further by somatic hypermutation
Why do TcR not undergo somatic mutation?
Antigen presentation
Foreign antigen
APC
Y
T cell help
Y
B
T
Self Antigen
Anergy or deletion
of anti-self cells
Y
T
No
T cell help
Y
B
Antibody
Affinity maturation
due to somatic
mutation
Why do TcR not undergo somatic mutation?
Y
Y
B
B
Y
B
Y
B
Y
B
Affinity maturation due to somatic mutation
Y Y
B
B
Occasional B cell
that somatically mutates
to become self reactive
The lack of somatic mutation in TcR helps
to prevent autoimmunity
T cell help
Y
No T cell help
X
B
Occasional B cell
that somatically mutates
to become self reactive
Y
T
Y
T
T cell that doesn’t mutate T cell that mutates can
can not help the
may help the self reactive
self reactive B cell
B cell
Autoantibody production
If TcR did undergo somatic mutation:
TcR interacts with entire top surface of MHC-peptide antigen complex
Somatic mutation in the TcR could mutate amino acids that interact with
the MHC molecule causing a complete loss of peptide-MHC recognition
If TcR did undergo somatic mutation:
TcR-MHC interaction is one of many between the T cell and APC
On-off rate of TcR determines rate of ‘firing’ to give qualitatively different
outcomes
Must be of relatively low affinity as cells with high affinity TcR are
deleted to prevent self reactivity.
If TcR underwent affinity maturation, they would be deleted
Why do B cell receptors need to mutate?
Neutralisation of
bacterial toxins
Ab-Ag interaction must be of high affinity
to capture and neutralise toxins in
extracellular fluids
`
Toxin binding
blocked
Prevents
toxicity
`
Y
There is a powerful selective advantage
to B cells that can somatically mutate
their receptors to increase affinity
SOMATIC MUTATION
An alternative TcR: 
Discovered as Ig-homologous, rearranging genes in non  TcR T cells
V
V
V
V V
J
C
Human  locus
3x D 3x J 1x C
12x V1 3x J
C1
2x J
C2
The  locus is located between the V and J regions
V to J rearrangement deletes D, J and C TcR cells can not express  TcR
Few V regions, but considerable junctional diversity as  chain can use 2 D regions
 T cells
Distinct lineage of cells with unknown functions
1-5% of peripheral blood T cells
In the gut and epidermis of mice, most T cells express  TcR
Ligands of  TcR are unknown
Possibly recognise:
Antigens without involvement of MHC antigens - CD1
Class IB genes
Summary
• The TcR was discovered using clonotypic antibodies
• Antibodies and TcR share many similarities, but there are
significant differences in structure and function
• The structure and organisation of the TcR genes is similar to the Ig
genes
• Somatic recombination in TcR genes is similar to that in Ig genes
• The molecular mechanisms that account for the diversity of TcR
include combinatorial and junctional diversity
• TcR do not somatically mutate
• The highly variable CDR loops map to the distal end of the TcR
• The most variable part of the TcR interacts with the peptide