1. The T Cell Antigen
Receptor Complex

2. Generating Evidence for and isolating the TCR
Generation of cytotoxic T lymphocytes (CTLs)
Zinkernagel and Doherty – 1974 demonstrated role of another molecule, MHC got Nobel Prize 1996
Isolation of the TCR using monclonal antibodies that were clonotypic i.e recognized single T cell clone
Identification of genes of the TCR

5. Discovery of the T cell antigen receptor (TcR)
Polyclonal T cells
from an immunised strain A mouse
Grow and clone a single antigen-specific 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

6. 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

7. 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) Y
Monoclonal antibodies Y Y
Clone used for
immunisation Y Y Y Y Y Y
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

8. Discovery of the T cell antigen receptor (TcR)
Lyse cells and add anti-clonotype Ab
that binds to unique T cell structures Y
Capture anti-clonotype Ab-Ag
complex on insoluble support
IMMUNOPRECIPITATION
Wash away unbound protein Y
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.

9. 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
Polypeptides contain a variable, clone-dependent pattern of digestion fragments and a fragment common to all TcR

10. Cloning of the TcR genesB T
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

11. Cloning of TcR genes by subtractive hybridisation
AAAAA
mRNA
Hybridise the
cDNA and mRNA shared
between T and B cells
AAAAA
T cell single
stranded cDNA
AAAAA
Discard hybrids
AAAAA
Digest unhybridised B cell mRNA
Clone and
sequence T cell-
specific genes
Isolate non-hybridising material specific to T cells

12. 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
GERMLINE
DNA V D J C
32P V D J C
REARRANGED
DNA
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

14. TCR
Each a and b chain consists of one ‘Ig-like’ N-terminal variable region (V), one Ig-like constant (C) domain, a hydrophobic transmembrane region, and a short cytoplasmic region.
Thus the extracellular portion of the ab heterodimer is structurally similar to the antigen-binding fragment (Fab) of an Ig, which is made up of the V and C regions of a light chain and the V region and one C region of a heavy chain.

15. The T cell antigen receptor
combining site
Resembles an Ig Fab fragment
Fab VH VL Fc CL CH Va Vb
Domain structure: Ig gene superfamily
Carbohydrates
Monovalent Ca Cb
No alternative constant regions
Never secreted
Hinge
Heterodimeric, chains are disuphide-bonded
Transmembrane region + + +
Very short intracytoplasmic tail
Positively charged amino acids in the TM region
Cytoplasmic tail
Antigen combining site made of juxtaposed Va and Vb regions
30,000 identical specificity TcR per cell

16. T cell antigen receptor diversity
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

17. 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

18. Estimate of the number of human TcR and Ig
Excluding somatic hypermutation
Immunoglobulin
 TcR
Element H
  
Variable segments 40 59 52
~70
Diversity segments 27 2
D segments in
all 3 frames - -
Yes
Yes
Joining segments 6 9 13 61
Joints with N & P
nucleotides 2
(1)* 2 1
2360
3640
No. of V gene pairs
Junctional diversity
~1013
~1013
Total diversity
~1016**
~1016
* Only half of human k chains have N & P regions
**No of distinct receptors increased further by somatic hypermutation

19. Organisation of TcR genes
L & V
x70-80
J x 61 C
TcR 
L & V
x52
D1
Jb1 x 6
C1
D2
Jb2 x 7
C2
TcR 
TcR genes segmented into V, (D), J & C elements
(VARIABLE, DIVERSITY, JOINING & CONSTANT)
Closely resemble Ig genes (a~IgL and b~IgH)
This example shows the mouse TcR locus

20. TcR a gene rearrangement by SOMATIC RECOMBINATION
Germline TcR 
Vn J C
V2
V1
Rearranged TcR
1° transcript
Spliced TcR mRNA
Rearrangement very similar to the IgL chains

21. TcR a 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 J C
V2
V1
Vn+1
a chain tries for a second time to make a productive join using new V and J elements
Productively
rearranged TcR
1° transcript

22. TcR b gene rearrangement SOMATIC RECOMBINATION
L & V
x52
D1 J
C1
D2
C2
Germline TcR 
D-J Joining
V-DJ joining
Rearranged TcR 1° transcript
C-VDJ joining
Spliced TcR mRNA

23. TcR b 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 b chain rearrangement)
D1 J
C1
D2
C2
Germline TcR 
D-J Joining
V-DJ joining V
2nd chance at
V-DJ joining
Need to remove non productive
rearrangement
Use (DJC)b2 elements

24. 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. Va 7 23 9 Ja 7 12 9 Db 7 12 9 Vb 7 23 9 Jb

25. Recombination signal sequences (RSS)
HEPTAMER - Always contiguous with coding sequence
NONAMER - Separated from the heptamer by a 12 or 23 nucleotide spacer Vb 7 23 9 Db 12 Jb √ √ Vb 7 23 9 Db 12 Jb
12-23 RULE – A gene segment flanked by a 23mer RSS can only be linked to a segment flanked by a 12mer RSS

26. Molecular explanation of the 12-23 rule
23-mer = two turns
12-mer = one turn
Intervening DNA
of any length 23 Vb 9 7 12 Db Jb 7 9

27. Molecular explanation of the 12-23 ruleV1 D J V2 V3 V4 V8 V7 V6 V5 V9 V1 V2 V3 V4
Loop of intervening
DNA is excised V8 V7 V6 V5
Heptamers and nonamers align back-to-back
The shape generated by the RSS’s acts as a target for recombinases 7 9
23-mer
12-mer V9 D J
An appropriate shape can not be formed if two 23-mer flanked elements attempted to join (i.e. the rule)

28. Mini-circle of DNA is permanently lost from the genome
Junctional diversity 7 12 9 23 7 12 9 23
Mini-circle of DNA is permanently lost from the genome
Signal joint
Coding joint V D J V D J
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.

29. Non-deletional recombinationV1 V2 V3 V4 V9 D J
Looping out works if all V genes are in the same transcriptional orientation V1 7 23 9 D 12 J
How does recombination occur when a V gene is in opposite orientation to the DJ region? V4 V1 V2 V3 V9 D J D J 7 12 9 V4 23

30. Non-deletional recombinationJ 7 12 9 V4 23
V4 and DJ in opposite transcriptional orientations D J 7 12 9 V4 23 1. D J 7 12 9 V4 23 2. D J 7 12 9 V4 23 3. D J 7 12 9 V4 23 4.

31. Fully recombined VDJ regions in same transcriptional orientation7 12 9 V4 23 1. D J 7 12 9 V4 23 2.
Heptamer ligation - signal joint formation D J V4 7 12 9 23 3.
V to DJ ligation - coding joint formation D J V4 7 12 9 23
Fully recombined VDJ regions in same transcriptional orientation
No DNA is deleted 4.

32. Steps of TcR gene recombination
Recombination activating gene products, (RAG1 & RAG 2) and ‘high mobility group proteins’ bind to the RSS V 7 23 9 D 7 12 9 J V 7 23 9
The two RAG1/RAG 2 complexes bind to each other and bring the V region adjacent to the DJ region D 7 12 9 J
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 7 23 9 12 7 23 9 12 V D J

33. Steps of TcR gene recombinationV D J 7 23 9 12
A number of other proteins, (Ku70:Ku80, XRCC4 and DNA dependent protein kinases) bind to the hairpins and the heptamer ends. V D J
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 V D J 7 23 9 12
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.

34. Junctional diversity: P nucleotide additions7 12 9 J V 23 7 V 23 9
TC CACAGTG
AG GTGTCAC
AT GTGACAC
TA CACTGTG 7 D 12 9 J
The recombinase complex makes single stranded nicks at random sites close to the ends of the V and D region DNA. D J V TC AG AT TA U 7 D 12 9 J V 23
CACAGTG
GTGTCAC
GTGACAC
CACTGTG TC AG AT TA
The 2nd strand is cleaved and hairpins form between the complimentary bases at ends of the V and D region.

35. V2 V3 V4 V8 V7 V6 V5 V9 7 23 9
CACAGTG
GTGTCAC 12
GTGACAC
CACTGTG
Heptamers are ligated by DNA ligase IV V TC AG U D J AT TA V TC AG U D J AT TA
V and D regions juxtaposed

36. Generation of the palindromic sequenceV TC AG U D J AT TA
Regions to be joined are juxtaposed
Endonuclease cleaves single strand at random sites in V and D segment V TC AG U D J AT TA
The nicked strand ‘flips’ out V
TC~GA AG D J AT
TA~TA
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.

37. Junctional Diversity – N nucleotide additions
Terminal deoxynucleotidyl transferase (TdT) adds nucleotides randomly to the P nucleotide ends of the single-stranded V and D segment DNA V
TC~GA AG D J AT
TA~TA
CACTCCTTA
TTCTTGCAA V
TC~GA AG D J AT
TA~TA
CACACCTTA
TTCTTGCAA
Complementary bases anneal D J
TA~TA
Exonucleases nibble back free ends V
TC~GA
CACACCTTA
TTCTTGCAA V D J
DNA polymerases fill in the gaps with complementary nucleotides and DNA ligase IV joins the strands
TC~GA AG AT
TA~TA
CACACCTTA
TTCTTGCAA V TC D TA
GTT AT AT
AG C

38. V D J TCGACGTTATAT AGCTGCAATATA TTTTT Junctional Diversity
Germline-encoded nucleotides
Palindromic (P) nucleotides - not in the germline
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.

39. How does somatic recombination work?
How is an infinite diversity of specificity generated from finite amounts of DNA?
Combinatorial diversity and junctional diversity
How do V region find J regions and why don’t they join to C regions? rule
How does the DNA break and rejoin?
Imprecisely, with the random removal and addition of nucleotides to generate sequence diversity.

40. Why do V regions not join to J or C regions?Vb Db Jb C
IF the elements of the TcR did not assemble in the correct order, diversity of specificity would be severely compromised
DIVERSITY 2x 1x
Full potential of the beta chain for diversity needs V-D-J-C joining - in the correct order
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

41. Location of junctional diversity
TcR  chain
TcR  chain
CDR3
CDR1
CDR2
V-J
Join
V-D
Join
D-J
join
Variability
Amino acid No.
of TcR chain
CDR = Complemantarity determining region

42. T-cell Receptor
T cells also express other membrane receptors that do not recognize antigen but participate in responses to antigens: these are collectively called accessory molecules.
2/9/04

43. T cell co-receptor molecules
Lck PTK
CD4 and CD8 can increase the sensitivity of T cells to peptide antigen MHC
complexes by ~100 fold
TcR  
CD8
CD4
MHC Class I
MHC Class II 3 2

44. CD8 and CD4 contact points on MHC
class I and class II
MHC class I
MHC class II
CD8 binding site
CD8 binding site

45. TcR-CD3 complex         TcR 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

46. 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:
The mitogen-activated protein kinase (MAP kinase) pathway
An increase in intracellular calcium ion concentration mediated by IP3
The activation of Protein Kinase C mediated by DAG

48. 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-g, 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

49. Estimate of the number of human TcR and Ig
Excluding somatic hypermutation
Immunoglobulin
 TcR
Element H
  
Variable segments 40 59 52
~70
Diversity segments 27 2
D segments in
all 3 frames - -
Yes
Yes
Joining segments 6 9 13 61
Joints with N & P
nucleotides 2
(1)* 2 1
2360
3640
No. of V gene pairs
Junctional diversity
~1013
~1013
Total diversity
~1016**
~1016
* Only half of human k chains have N & P regions
**No of distinct receptors increased further by somatic hypermutation

50. Why do TcR not undergo somatic mutation?
Antigen presentation Y B
T cell help
Self Antigen
Foreign antigen
APC
Antibody
Anergy or deletion
of anti-self cells Y T Y B No
T cell help
Affinity maturation due to somatic mutation

51. Why do TcR not undergo somatic mutation?B Y B Y B
Occasional B cell
that somatically mutates
to become self reactive
Affinity maturation due to somatic mutation

52. The lack of somatic mutation in TcR helps to prevent autoimmunity
T cell help Y T Y B
Occasional B cell
that somatically mutates
to become self reactive X
No T cell help Y T
T cell that doesn’t mutate
can not help the
self reactive B cell
T cell that mutates can may help the self reactive B cell
Autoantibody production

53. 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

54. 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

55. Why do B cell receptors need to mutate?
Neutralisation of
bacterial toxins Y `
Ab-Ag interaction must be of high affinity to capture and neutralise toxins in extracellular fluids
There is a powerful selective advantage to B cells that can somatically mutate their receptors to increase affinity
SOMATIC MUTATION Y `
Toxin binding
blocked
Prevents
toxicity

56. An alternative TcR: 
Discovered as Ig-homologous, rearranging genes in non  TcR T cells V V J C
3x D
3x J
1x C
Human  locus
3x J
C1
2x J
C2
12x V1
The  locus is located between the V and J regions
V to J rearrangement deletes D, Jand CTcR cells can not express g TcR
Few V regions, but considerable junctional diversity as  chain can use 2 D regions

57.  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

60. 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