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Broad TCR repertoire and diverse structural solutions for recognition of an immunodominant CD8+ T cell epitope

Abstract

A keystone of antiviral immunity is CD8+ T cell recognition of viral peptides bound to MHC-I proteins. The recognition modes of individual T cell receptors (TCRs) have been studied in some detail, but the role of TCR variation in providing a robust response to viral antigens is unclear. The influenza M1 epitope is an immunodominant target of CD8+ T cells that help to control influenza in HLA-A2+ individuals. Here we show that CD8+ T cells use many distinct TCRs to recognize HLA-A2–M1, which enables the use of different structural solutions to the problem of specifically recognizing a relatively featureless peptide antigen. The vast majority of responding TCRs target a small cleft between HLA-A2 and the bound M1 peptide. These broad repertoires lead to plasticity in antigen recognition and protection against T cell clonal loss and viral escape.

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Figure 1: Diversity of CD8+ T cell repertoire: dominant usage of TRAV38, and CDR3α and CDR3β sequence motifs, in the HLA-A2–M1-specific response.
Figure 2: TCRα-TCRβ pairs bind HLA-A2–M1 and stimulate T cell signaling.
Figure 3: Structural comparison of three TCRs docked onto HLA-A2–M1.
Figure 4: The LS10 TCR uses conserved 15-mer CDR3α and xGxY CDR3β motifs to select an M1 peptide conformation with Phe5-p occupying the notch between peptide and MHC.
Figure 5: The LS01 TCR uses CDR3β Phe98 to occupy the notch between peptide and MHC with additional interactions from CDR1α, CDR3α, and CDR3β.
Figure 6: Different structural solutions to high-avidity binding of a featureless peptide.

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References

  1. La Gruta, N.L. & Turner, S.J. T cell mediated immunity to influenza: mechanisms of viral control. Trends Immunol. 35, 396–402 (2014).

    Article  CAS  PubMed  Google Scholar 

  2. Guo, H., Santiago, F., Lambert, K., Takimoto, T. & Topham, D.J. T cell-mediated protection against lethal 2009 pandemic H1N1 influenza virus infection in a mouse model. J. Virol. 85, 448–455 (2011).

    Article  CAS  PubMed  Google Scholar 

  3. Tan, A.C.L. et al. The design and proof of concept for a CD8+ T cell-based vaccine inducing cross-subtype protection against influenza A virus. Immunol. Cell Biol. 91, 96–104 (2013).

    Article  CAS  PubMed  Google Scholar 

  4. Gotch, F., McMichael, A., Smith, G. & Moss, B. Identification of viral molecules recognized by influenza-specific human cytotoxic T lymphocytes. J. Exp. Med. 165, 408–416 (1987).

    Article  CAS  PubMed  Google Scholar 

  5. Gotch, F., Rothbard, J., Howland, K., Townsend, A. & McMichael, A. Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2. Nature 326, 881–882 (1987).

    Article  CAS  PubMed  Google Scholar 

  6. Assarsson, E. et al. Immunomic analysis of the repertoire of T-cell specificities for influenza A virus in humans. J. Virol. 82, 12241–12251 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Keskin, D.B. et al. Physical detection of influenza A epitopes identifies a stealth subset on human lung epithelium evading natural CD8 immunity. Proc. Natl. Acad. Sci. USA 112, 2151–2156 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Moss, P.A. et al. Extensive conservation of alpha and beta chains of the human T-cell antigen receptor recognizing HLA-A2 and influenza A matrix peptide. Proc. Natl. Acad. Sci. USA 88, 8987–8990 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Lehner, P.J. et al. Human HLA-A0201-restricted cytotoxic T lymphocyte recognition of influenza A is dominated by T cells bearing the Vβ17 gene segment. J. Exp. Med. 181, 79–91 (1995).

    Article  CAS  PubMed  Google Scholar 

  10. Naumov, Y.N. et al. Complex T cell memory repertoires participate in recall responses at extremes of antigenic load. J. Immunol. 177, 2006–2014 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Naumov, Y.N., Hogan, K.T., Naumova, E.N., Pagel, J.T. & Gorski, J. A class I MHC-restricted recall response to a viral peptide is highly polyclonal despite stringent CDR3 selection: implications for establishing memory T cell repertoires in “real-world” conditions. J. Immunol. 160, 2842–2852 (1998).

    CAS  PubMed  Google Scholar 

  12. Naumov, Y.N. et al. Multiple glycines in TCR α-chains determine clonally diverse nature of human T cell memory to influenza A virus. J. Immunol. 181, 7407–7419 (2008).

    Article  CAS  PubMed  Google Scholar 

  13. Stewart-Jones, G.B.E., McMichael,, A.J., Bell, J.I., Stuart, D.I. & Jones, E.Y. A structural basis for immunodominant human T cell receptor recognition. Nat. Immunol. 4, 657–663 (2003).

    Article  CAS  PubMed  Google Scholar 

  14. Ishizuka, J. et al. The structural dynamics and energetics of an immunodominant T cell receptor are programmed by its Vβ domain. Immunity 28, 171–182 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. Turner, S.J., Doherty, P.C., McCluskey, J. & Rossjohn, J. Structural determinants of T-cell receptor bias in immunity. Nat. Rev. Immunol. 6, 883–894 (2006).

    Article  CAS  PubMed  Google Scholar 

  16. Motozono, C. et al. Molecular basis of a dominant T cell response to an HIV reverse transcriptase 8-mer epitope presented by the protective allele HLA-B*51:01. J. Immunol. 192, 3428–3434 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sun, X. et al. Superimposed epitopes restricted by the same HLA molecule drive distinct HIV-specific CD8+ T cell repertoires. J. Immunol. 193, 77–84 (2014).

    Article  CAS  PubMed  Google Scholar 

  18. Turner, S.J. et al. Lack of prominent peptide-major histocompatibility complex features limits repertoire diversity in virus-specific CD8+ T cell populations. Nat. Immunol. 6, 382–389 (2005).

    Article  CAS  PubMed  Google Scholar 

  19. Wang, G.C., Dash, P., McCullers, J.A., Doherty, P.C. & Thomas, P.G. T cell receptor αβ diversity inversely correlates with pathogen-specific antibody levels in human cytomegalovirus infection. Sci. Transl. Med. 4, 128ra42 (2012).

    PubMed  PubMed Central  Google Scholar 

  20. Messaoudi, I., Guevara Patiño, J.A., Dyall, R., LeMaoult, J. & Nikolich-Zugich, J. Direct link between MHC polymorphism, T cell avidity, and diversity in immune defense. Science 298, 1797–1800 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Yager, E.J. et al. Age-associated decline in T cell repertoire diversity leads to holes in the repertoire and impaired immunity to influenza virus. J. Exp. Med. 205, 711–723 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Price, D.A. et al. T cell receptor recognition motifs govern immune escape patterns in acute SIV infection. Immunity 21, 793–803 (2004).

    Article  CAS  PubMed  Google Scholar 

  23. Vojnov, L. et al. GagCM9-specific CD8+ T cells expressing limited public TCR clonotypes do not suppress SIV replication in vivo. PLoS One 6, e23515 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Woodsworth, D.J., Castellarin, M. & Holt, R.A. Sequence analysis of T-cell repertoires in health and disease. Genome Med. 5, 98 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Klarenbeek, P.L. et al. Deep sequencing of antiviral T-cell responses to HCMV and EBV in humans reveals a stable repertoire that is maintained for many years. PLoS Pathog. 8, e1002889 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Link, C.S. et al. Abundant cytomegalovirus (CMV) reactive clonotypes in the CD8+ T cell receptor alpha repertoire following allogeneic transplantation. Clin. Exp. Immunol. 184, 389–402 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Gras, S. et al. A structural basis for varied αβ TCR usage against an immunodominant EBV antigen restricted to a HLA-B8 molecule. J. Immunol. 188, 311–321 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Liu, Y.C. et al. Highly divergent T-cell receptor binding modes underlie specific recognition of a bulged viral peptide bound to a human leukocyte antigen class I molecule. J. Biol. Chem. 288, 15442–15454 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Yang, X. et al. Structural basis for clonal diversity of the public T cell response to a dominant human cytomegalovirus epitope. J. Biol. Chem. 290, 29106–29119 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Clute, S.C. et al. Cross-reactive influenza virus-specific CD8+ T cells contribute to lymphoproliferation in Epstein-Barr virus-associated infectious mononucleosis. J. Clin. Invest. 115, 3602–3612 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Tan, A.C.L., La Gruta, N.L., Zeng, W. & Jackson, D.C. Precursor frequency and competition dictate the HLA-A2-restricted CD8+ T cell responses to influenza A infection and vaccination in HLA-A2.1 transgenic mice. J. Immunol. 187, 1895–1902 (2011).

    Article  CAS  PubMed  Google Scholar 

  32. Gil, A., Yassai, M.B., Naumov, Y.N. & Selin, L.K. Narrowing of human influenza A virus-specific T cell receptor α and β repertoires with increasing age. J. Virol. 89, 4102–4116 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Davis, M.M. The problem of plain vanilla peptides. Nat. Immunol. 4, 649–650 (2003).

    Article  CAS  PubMed  Google Scholar 

  34. Bridgeman, J.S., Sewell, A.K., Miles, J.J., Price, D.A. & Cole, D.K. Structural and biophysical determinants of αβ T-cell antigen recognition. Immunology 135, 9–18 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Gras, S. et al. A structural voyage toward an understanding of the MHC-I-restricted immune response: lessons learned and much to be learned. Immunol. Rev. 250, 61–81 (2012).

    Article  PubMed  Google Scholar 

  36. Buslepp, J., Wang, H., Biddison, W.E., Appella, E. & Collins, E.J. A correlation between TCR Vα docking on MHC and CD8 dependence: implications for T cell selection. Immunity 19, 595–606 (2003).

    Article  CAS  PubMed  Google Scholar 

  37. Tynan, F.E. et al. T cell receptor recognition of a 'super-bulged' major histocompatibility complex class I-bound peptide. Nat. Immunol. 6, 1114–1122 (2005).

    Article  CAS  PubMed  Google Scholar 

  38. Jenkins, M.K. & Moon, J.J. The role of naive T cell precursor frequency and recruitment in dictating immune response magnitude. J. Immunol. 188, 4135–4140 (2012).

    Article  CAS  PubMed  Google Scholar 

  39. Neller, M.A. et al. Naive CD8+ T-cell precursors display structured TCR repertoires and composite antigen-driven selection dynamics. Immunol. Cell Biol. 93, 625–633 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Tscharke, D.C., Croft, N.P., Doherty, P.C. & La Gruta, N.L. Sizing up the key determinants of the CD8+ T cell response. Nat. Rev. Immunol. 15, 705–716 (2015).

    Article  CAS  PubMed  Google Scholar 

  41. Costa, A.I. et al. Complex T-cell receptor repertoire dynamics underlie the CD8+ T-cell response to HIV-1. J. Virol. 89, 110–119 (2015).

    Article  PubMed  Google Scholar 

  42. Koning, D. et al. CD8+ TCR repertoire formation is guided primarily by the peptide component of the antigenic complex. J. Immunol. 190, 931–939 (2013).

    Article  CAS  PubMed  Google Scholar 

  43. Choo, J.A.L., Liu, J., Toh, X., Grotenbreg, G.M. & Ren, E.C. The immunodominant influenza A virus M158-66 cytotoxic T lymphocyte epitope exhibits degenerate class I major histocompatibility complex restriction in humans. J. Virol. 88, 10613–10623 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  44. Madura, F. et al. T-cell receptor specificity maintained by altered thermodynamics. J. Biol. Chem. 288, 18766–18775 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Yassai, M. et al. Naive T cell repertoire skewing in HLA-A2 individuals by a specialized rearrangement mechanism results in public memory clonotypes. J. Immunol. 186, 2970–2977 (2011).

    Article  CAS  PubMed  Google Scholar 

  46. Venturi, V., Price, D.A., Douek, D.C. & Davenport, M.P. The molecular basis for public T-cell responses? Nat. Rev. Immunol. 8, 231–238 (2008).

    Article  CAS  PubMed  Google Scholar 

  47. Dai, S. et al. Crossreactive T cells spotlight the germline rules for αβ T cell-receptor interactions with MHC molecules. Immunity 28, 324–334 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Selin, L.K. et al. Heterologous immunity: immunopathology, autoimmunity and protection during viral infections. Autoimmunity 44, 328–347 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Selin, L.K. et al. Memory of mice and men: CD8+ T-cell cross-reactivity and heterologous immunity. Immunol. Rev. 211, 164–181 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Che, J.W., Kraft, A.R.M., Selin, L.K. & Welsh, R.M. Regulatory T cells resist virus infection-induced apoptosis. J. Virol. 89, 2112–2120 (2015).

    Article  PubMed  Google Scholar 

  51. Benn, C.S., Netea, M.G., Selin, L.K. & Aaby, P. A small jab—a big effect: nonspecific immunomodulation by vaccines. Trends Immunol. 34, 431–439 (2013).

    Article  CAS  PubMed  Google Scholar 

  52. Valkenburg, S.A. et al. Molecular basis for universal HLA-A*0201-restricted CD8+ T-cell immunity against influenza viruses. Proc. Natl. Acad. Sci. USA 113, 4440–4445 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Han, A., Glanville, J., Hansmann, L. & Davis, M.M. Linking T-cell receptor sequence to functional phenotype at the single-cell level. Nat. Biotechnol. 32, 684–692 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Jia, Q. et al. Diversity index of mucosal resident T lymphocyte repertoire predicts clinical prognosis in gastric cancer. OncoImmunology 4, e1001230 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  55. Clute, S.C. et al. Broad cross-reactive TCR repertoires recognizing dissimilar Epstein-Barr and influenza A virus epitopes. J. Immunol. 185, 6753–6764 (2010).

    Article  CAS  PubMed  Google Scholar 

  56. Robins, H.S. et al. Comprehensive assessment of T-cell receptor β-chain diversity in αβ T cells. Blood 114, 4099–4107 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Carlson, C.S. et al. Using synthetic templates to design an unbiased multiplex PCR assay. Nat. Commun. 4, 2680 (2013).

    Article  PubMed  Google Scholar 

  58. Bolotin, D.A. et al. Next generation sequencing for TCR repertoire profiling: platform-specific features and correction algorithms. Eur. J. Immunol. 42, 3073–3083 (2012).

    Article  CAS  PubMed  Google Scholar 

  59. Best, K., Oakes, T., Heather, J.M., Shawe-Taylor, J. & Chain, B. Computational analysis of stochastic heterogeneity in PCR amplification efficiency revealed by single molecule barcoding. Sci. Rep. 5, 14629 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Yousfi Monod, M., Giudicelli, V., Chaume, D. & Lefranc, M.P. IMGT/JunctionAnalysis: the first tool for the analysis of the immunoglobulin and T cell receptor complex V-J and V-D-J JUNCTIONs. Bioinformatics 20, i379–i385 (2004).

    Article  PubMed  Google Scholar 

  61. Brochet, X., Lefranc, M.P. & Giudicelli, V. IMGT/V-QUEST: the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis. Nucleic Acids Res. 36, W503–W508 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Szymczak, A.L. et al. Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. Nat. Biotechnol. 22, 589–594 (2004).

    Article  CAS  PubMed  Google Scholar 

  63. Liang, X. et al. A single TCR α-chain with dominant peptide recognition in the allorestricted HER2/neu-specific T cell repertoire. J. Immunol. 184, 1617–1629 (2010).

    Article  CAS  PubMed  Google Scholar 

  64. Garboczi, D.N., Hung, D.T. & Wiley, D.C. HLA-A2-peptide complexes: refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc. Natl. Acad. Sci. USA 89, 3429–3433 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Salter, R.D. & Cresswell, P. Impaired assembly and transport of HLA-A and -B antigens in a mutant TxB cell hybrid. EMBO J. 5, 943–949 (1986).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Boulter, J.M. et al. Stable, soluble T-cell receptor molecules for crystallization and therapeutics. Protein Eng. 16, 707–711 (2003).

    Article  CAS  PubMed  Google Scholar 

  67. Willcox, B.E. et al. Production of soluble αβ T-cell receptor heterodimers suitable for biophysical analysis of ligand binding. Protein Sci. 8, 2418–2423 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006).

    Article  PubMed  Google Scholar 

  70. Battye, T.G.G., Kontogiannis, L., Johnson, O., Powell, H.R. & Leslie, A.G.W. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. D Biol. Crystallogr. 67, 271–281 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Morin, A. et al. Collaboration gets the most out of software. eLife 2, e01456 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  73. Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    Article  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the NIH (grants AI038996 (to L.J.S.), AI49320 (to L.K.S.), and AI109858 (to L.J.S. and L.K.S.)) and the Nebraska Research Initiative (grant to D.G.). This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Use of the Lilly Research Laboratories Collaborative Access Team (LRL-CAT) beamline at Sector 31 of the Advanced Photon Source was provided by Eli Lilly Company, which operates the facility. We thank J. Birtley and Z. Maben for assistance with crystallization, freezing, and shipping of crystals; P. Trehn for advice on model analysis; W. Uckert (Max Delbruck Center, Berlin, Germany) for TCRα/β Jurkat J76 cells transfected with human CD8α; and P. Thomas for technical advice on single-cell PCR.

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Authors and Affiliations

Authors

Contributions

I.Y.S. conceived the project, designed the experimental approach, performed single-cell sequencing experiments, characterized TCR transfectants, determined crystal structures, and wrote the manuscript. A.G. performed NGS analyses, performed single-cell sequencing experiments, and edited the manuscript. R.M. performed NGS analyses and edited the manuscript. D.G. performed NGS analyses and edited the manuscript. L.K.S. conceived the project, designed the experimental approach, supervised TCR sequencing analyses, and wrote the manuscript. L.J.S. conceived the project, designed the experimental approach, supervised cellular and molecular studies, and wrote the manuscript.

Corresponding authors

Correspondence to Liisa K Selin or Lawrence J Stern.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Diversity of CD8+ T cell repertoire, dominant usage of TRAV38, and CDR3α and CDR3β sequence motifs in the HLA-A2–M1-specific response: unique clonotypes.

(a) HLA-A1/M1-specific TRBV repertoires assayed for donors 185, 215, 250, 264 directly ex vivo or after culture are similar (Pearson’s correlation coefficient). CD8 T cells isolated from PBMC were stained with M1 tetramer and Vb-specific mAb or were expanded and analyzed by NGS as described in methods. (b-h) As Figure 1 except frequencies are expressed in terms of numbers of unique sequences. (i) Frequencies of xGxY or xRS/Ax motif-harboring TCRβ in the total M1-specific TRBV19 repertoires. Frequencies of xRSx motif are shown in white in each bar and accumulated frequencies of xRSx and xGxY motifs are shown above each bar. (j) Shannon-Weaver and Simpson Diversity index of the TCRα and TCRβ of the six donors.

Supplementary Figure 2 Diverse TRAV pairing with xRSx TRBV19, and restricted TRAV38-TRAJ52 pairing with xGxY TRBV19.

(a) Schematic diagram. HLA-A2/M1-dextramer-specific CD8 T cells from PBMC were sorted directly ex vivo into single wells for TCRα and TCRβ sequencing. (b) TRAV usage of amplified productive TCRα (n=34) and TCRβ (n=82) sequences. TR gene usage and CDR3 sequences of 13 selected TCRα/TCRβ pairs, categorized into four groups, are shown in Supplementary Table I.

Supplementary Figure 3 Inconspicuous HLA-A2-restricted M1 peptide.

(a) Structures of representative HLA-A2/peptide complexes shown in surface representation with peptides colored and N-termini at left. (b) Exposed surface areas of individual residues of 131 peptides (see Methods for PDB IDs) bound in unligated HLA-A2/peptide complex structures. (c) Total exposed peptide surface area for 131 HLA-A2/peptide complexes. Average of all peptides is shown as horizontal line. Peptides from panels a and b are shown as colored dots. (d) Total buried peptide surface area for HLA-A2/peptide complexes as in (c).

Supplementary Figure 4 Surface expression, MHC tetramer binding, and T cell activation by paired TCRα/β chains.

(a) TCR surface expression of transiently- transfected TCR-J76-CD8+ cells. (b) Concentration-dependent binding of HLA-A2/M1 tetramer to stably-transfected T cell lines. (c) TCR surface expression of stably-transfected TCR-J76-CD8+ cells with selected paired TCRα/β. (d) Paired TCRα/β can initiate T cell signaling after M1-peptide stimulation. J76-CD8 stably expressing paired TCRα/β were stimulated with antigen presenting cells loaded with M1 (black bars), irrelevant HLA-A2-restricted peptides (hatched bar: BMLF1, tyrosinase) or no peptide (open bar). CD69 expression after peptide stimulation was compared to corresponding unstimulated controls. Representative FACS plots for one TCR (LS12) are shown at right. Error bars represent standard deviations from three independent experiments.

Supplementary Figure 5 Electron density and peptide conformation.

(a) Electron density for LS01-M1-HLA-A*02 and LS10-M1-HLA-A*02 complexes in the region of TCR-peptide-MHC interaction. Composite omit maps were built with final models of each TCR/pMHC complexes and reflection data. Top views from TCR around M1-peptides of final models are shown in the composite omit maps. Yellow: M1-peptide (stick), red: HLA-A*02 (line), orange: TCRa (line), green: TCRb (line). Sigma levels of contour of maps were set as 1.5. (b) Overlaid M1 peptides from LS10-HLA-A2/M1 complex (deep blue) and HLA-C*08/M1 complex, from PDB 4NT6 (green).

Supplementary Figure 6 Functional characterization of residues in TCR-pMHC contact region, and TCR-pMHC contact maps for LS01, LS10, and JM22 TCRs.

(a-c) Effects of alanine mutation of CDR3 residues on HLA-A2/M1- LS10 TCR binding are illustrated in bar graphs with positions of mutated residues marked as red dots above sequence logos. (d) TRAV38-specific residues (Asp31α of CDR1α, Glu52α and Tyr54α of CDR2α) from the LS10 TCR interact with a rarely-contacted MHC residue (Arg157MHC). (e) Mutation of LS10 interface residues Phe98β, Gln100β and Arg101β in CDR3β to alanines abolished M1-tetramer binding. (f) Comparison of TRAJ52-containing CDR3α from different TCR structures. Alanine and tyrosine are underlined in the sequences and depicted as sticks in line representation. (g) Gln155MHC is stabilized by Tyr31α from CDR1α of LS10 TCR. (h) Mutation of LS10 Tyr31α and Asn95α abolished M1-tetramer binding (i) Effect of mutation of CDR1β and CDR2β residues (Asp32β, Gln52β, Ile53β) on HLA-A2/M1 tetramer binding for three TCR. For JM22 all three residues are essential (Ishizuka, J. et al., Immunity 28, 171–182, 2008). (j) Contact maps for three TCR-HLA-A2/M1 complexes. Contact residues from TCR are listed vertically and from pMHC horizontally, with residue numbers shown. Grid boxes are filled with corresponding CDR color if residues from pMHC and TCR contact. Error bars in (a-c,e,h,i) represent standard deviation of duplicate measurements from each of two independent samples.

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Song, I., Gil, A., Mishra, R. et al. Broad TCR repertoire and diverse structural solutions for recognition of an immunodominant CD8+ T cell epitope. Nat Struct Mol Biol 24, 395–406 (2017). https://doi.org/10.1038/nsmb.3383

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