The document summarizes key aspects of T cell antigen receptor (TcR) structure and generation of diversity. It describes how TcR were discovered using monoclonal antibodies that recognize unique structures on T cell clones. TcR are heterodimers composed of α and β chains that are similar to antibody structures but do not undergo somatic hypermutation. TcR diversity is generated through combinatorial rearrangement of variable (V), diversity (D), and joining (J) gene segments, as well as junctional diversity from imprecise joining and addition of untemplated nucleotides.

1. Topic 6 The T Cell Antigen Receptor Complex © Dr. Colin R.A. Hewitt [email_address]

2. • 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 What you should know by the end of this lecture

3. Discovery of the T cell antigen receptor (TcR) Polyclonal T cells from an immunised strain A mouse 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 Grow and clone a single antigen-specific T cell in-vitro with antigen, IL-2 and antigen presenting cells

4. Making anti- clonotypic TcR antibodies 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 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

5. Anti-TcR Abs that recognise only one clone of T cells are CLONOTYPIC Hypothesise that anti-clonotype Abs recognise the antigen receptor 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) Making anti- clonotypic TcR antibodies Y Y Y Y Y Y Y Y Y Y Y Y Monoclonal antibodies T cell clones Clone used for immunisation

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

7. Structure of the TcR polypeptides Cyanogen bromide digestion of the  and  proteins Biochemical analysis of digestion products Polypeptides contain a variable, clone-dependent pattern of digestion fragments and a fragment common to all TcR Intact TcR chain polypeptides T cell clone A T cell clone B T cell clone C C C C V V V

8. Cloning of the TcR genes 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 B T

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

10. 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 Find two restriction sites that flank the TcR region Ig gene probes can be used as TcR genes will be homologous to Ig genes Cut the T cell cDNA and placental (i.e. germline) DNA and Southern blot the fragments GERMLINE DNA V D J C 32 P V D J C REARRANGED DNA Restriction enzyme sites 32 P

11. Placenta B T Size of digested genomic DNA Gel electrophoresis followed by Southern blot using a TcR probe The TcR genes rearrange, but are not immunoglobulin genes Rearranged allele

12. The T cell antigen receptor V  V  C  C  Carbohydrates Hinge Monovalent Resembles an Ig Fab fragment No alternative constant regions Never secreted Domain structure: Ig gene superfamily Heterodimeric, chains are disuphide-bonded Very short intracytoplasmic tail + + + Positively charged amino acids in the TM region Antigen combining site Antigen combining site made of juxtaposed V  and V  regions 30,000 identical specificity TcR per cell Fab V H V L Fc C L C H V L V H C H C L C H C H C H C H Transmembrane region Cytoplasmic tail

13. C H C L V H V L of Ig C  C  V  V  of the TcR

14. View structures

15. 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 T cell antigen receptor diversity

16. 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

17. Organisation of TcR genes 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 TcR  L & V x70-80 C TcR  D  1 J  1 x 6 C  1 D  2 J  2 x 7 C  2 J x 61 L & V x52

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

19. TcR  gene rearrangement RESCUE PATHWAY There is only a 1:3 chance of the join between the V and J region being in frame  chain tries for a second time to make a productive join using new V and J elements V  n J C V  2 V  1 V  n+1 Productively rearranged TcR  1° transcript

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

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

22. 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 J  7 12 9 D  7 12 9 7 12 9 V  7 23 9 J  7 23 9

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

24. Molecular explanation of the 12-23 rule 23-mer = two turns 12-mer = one turn Intervening DNA of any length 23 V  9 7 12 D  J  7 9

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

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

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

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

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

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

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

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

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

34. Endonuclease cleaves single strand at random sites in V and D segment Generation of the palindromic sequence 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. The nicked strand ‘flips’ out V TC AG U D J AT TA U V TC~ GA AG D J AT TA ~TA The nucleotides that flip out, become part of the complementary DNA strand V TC AG U D J AT TA U Regions to be joined are juxtaposed

35. 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 CACTCCTTA TTCTTGCAA V TC ~ GA AG D J AT TA ~ TA V TC ~ GA AG D J AT TA ~ TA CACACCTTA TTCT T GCAA Complementary bases anneal 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 TTCT T GCAA D J TA ~ TA Exonucleases nibble back free ends V TC ~ GA CACACCTTA TTCT T GCAA V TC D TA GTT AT AT AG C

36. Junctional Diversity TTTTT TTTTT TTTTT 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. V D J TC GA CGTT AT AT AG CT GCAA TA TA

37. 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? 12-23 rule How does the DNA break and rejoin? Imprecisely, with the random removal and addition of nucleotides to generate sequence diversity.

38. Why do V regions not join to J or C regions? IF the elements of the TcR did not assemble in the correct order, diversity of specificity would be severely compromised 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 DIVERSITY 2x DIVERSITY 1x V  D  J  C

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

40. Location of junctional diversity in TcR TcRV  monomer TcR  chain 2 1 3 2 1 3 CDR’s

41. MHC class I and TcR V  /V  MHC class II TcR  /  The trimolecular complex

42. V  and V  of TcR recognising a peptide from MHC class I ribbon plot  TcR recognising a peptide from MHC class II ribbon plot

43. 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 Turn through 90 º

44. TcR contact and anchor residue side chains interact with side chains of TcR

45. Hypervariable loops - CDRs  /  3  /  3  /  2  /  2  /  2  /  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

46. View structures

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

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

49. TcR-CD3 complex The intracytoplasmic region of the TcR chain is too short to transduce a signal Signalling is initiated by aggregation of TcR by MHC-peptide complexes on APC The CD3  or  (zeta)  chains are required for cell surface expression of the TcR-CD3 complex and signalling through the TcR        TcR         CD3 CD3

50. Transduction of signals by the TcR The cytoplasmic domains of the CD3 complex contain 10 Immunoreceptor Tyrosine -based Activation Motifs (ITAMS) - 2 tyrosine residues separated by 9-12 amino acids - Y XX[L/V]X 6-9 Y XX[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       CD3 ITAMs

51. 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 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 by Src kinases Kinase domain Unique region SH3 domain SH2 domain Enzyme domain that phosphorylates tyrosine residues (to give phosphotyrosine) Phosphotyrosine receptor domain Adaptor protein recruitment domain ITAM binding domain

52. Regulation of Src kinases SH2 domain Phosphorylation of ‘Activating Tyrosine’ stimulates kinase activity Phosphorylation of ‘Inhibitory Tyrosine’ inhibits kinase activity by blocking access to the Activating Tyrosine Residue Kinase domain Unique region SH3 domain Activating tyrosine residue Inhibitory tyrosine residue Kinase domain Unique region SH3 domain SH2 domain

53. Early T cell activation Lck Fyn Zap-70 Receptor associated kinases accumulate under the membrane in close proximity to the cytoplasmic domains of the TcR -CD3 complex CD4 MHC II MHC II CD45 As the T cell antigen receptor binds the MHC-peptide antigen, the phosphatase CD45 activates kinases such as Fyn This mechanism of activation is similar to the used to activate Syk in B cells P

54. Fyn phosphorylates the ITAMs of CD3  ,  ,  and  ITAMS T cell activation The tyrosine kinase ZAP-70 binds to the phosphorylated ITAMs of CD3  - further activation requires ligation of the co-receptor, CD4 Zap-70 Lck Fyn CD4 CD45 P MHC II

55. T cell activation Binding of CD4 co-receptor to MHC class II brings Lck into the complex, which then phosphorylates and activates ZAP-70 Lck Fyn P MHC II Zap-70 Tyrosine rich cell membrane associated Linker of Activation in T cells (LAT) and SLP-76 associate with cholesterol-rich lipid rafts LAT SLP-76 Activated ZAP-70 phosphorylates LAT & SLP-76 P P P P ZAP-70 phosphorylates LAT and SLP-76

56. T cell activation PLC-  cleaves phosphotidylinositol bisphosphate (PIP 2 ) to yield diacylglycerol ( DAG ) and inositol trisphosphate ( IP 3 ) 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 Lck Fyn P MHC II Zap-70 LAT SLP-76 P P P P SLP76 binds Tec kinases and activates phospholipase C-  (PLC-  ) Tec Tec

57. Transmission of signals from the cell surface to the nucleus 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 IP 3 The activation of Protein Kinase C mediated by DAG Almost identical to transmission in B cells

58. 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 IP 3 IP 3 , 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 NF  B The activated transcription factors AP-1, NFAT and NF  B induce B cell proliferation, differentiation and effector mechanisms Simplified scheme linking antigen recognition with transcription of T cell-specific genes

59. Element Immunoglobulin  TcR Variable segments Diversity segments D segments in all 3 frames Joining segments Joints with N & P nucleotides No. of V gene pairs Junctional diversity Total diversity H    40 27 Yes 6 2 2360 3640 ~10 13 ~10 13 ~10 16** ~10 16 59 0 - 9 (1)* 52 ~70 2 0 Yes - 13 61 2 1 * Only half of human  chains have N & P regions **No of distinct receptors increased further by somatic hypermutation Estimate of the number of human TcR and Ig Excluding somatic hypermutation

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

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

62. 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 The lack of somatic mutation in TcR helps to prevent autoimmunity Y B Occasional B cell that somatically mutates to become self reactive Y T X No T cell help Y T T cell help Autoantibody production

63. 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

64. 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

65. 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 There is a powerful selective advantage to B cells that can somatically mutate their receptors to increase affinity SOMATIC MUTATION Y ` ` Y ` ` Toxin binding blocked Prevents toxicity

66. An alternative TcR:  Discovered as Ig-homologous, rearranging genes in non  TcR T cells 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 Human  locus 3x D  3x J  1x C  3x J  C  1 2x J  C  2 12x V  1 V  V  V  V  V  J  C 

67.  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

68. • 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 Summary