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Engineered T-cell receptor tetramers bind MHC-peptide complexes with high affinity


In this study we extend tetramerization technology to T-cell receptors (TCRs). We identified TCR αβ pairs in the absence of accessory molecules, ensuring isolation of high-affinity TCRs that maintain stable binding characteristics after tetramerization. Subtle changes in cognate peptide levels bound to the class I molecule were accurately reflected by parallel changes in the mean fluorescence intensity of cells that bound TCR tetramers, allowing us to accurately assess the binding affinity of a panel of peptides to major histocompatibility complex (MHC) class I. Using a TCR tetramer specific for the Mamu-A*01 allele, we identified animals expressing this restricting class I allele from a large cohort of outbred rhesus macaques. TCR tetramers should facilitate analysis of the MHC-peptide interface and, more generally, the design of immunotherapeutics and vaccines.

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Figure 1: A schematic representation of the screening of TCR αβ chain pairs using a peptide-specific TL8-Mamu-A*01 tetramer complex.
Figure 2: Tat-TL8 TCR tetramer binds specifically to TL8 peptide–pulsed Mamu-A*01–expressing cells.
Figure 3: Changes in the mean fluorescence intensity of TCR-tetramer staining reflect changes in bound levels of the cognate Tat-TL8 peptide.
Figure 4: TCR tetramer identifies animals that express the Mamu-A*01 allele in a cohort of outbred rhesus macaques.


  1. Davis, M.M. et al. Ligand recognition by alpha beta T cell receptors. Annu. Rev. Immunol. 16, 523–544 (1998).

    Article  CAS  PubMed  Google Scholar 

  2. Valitutti, S., Muller, S., Cella, M., Padovan, E. & Lanzavecchia, A. Serial triggering of many T-cell receptors by a few peptide-MHC complexes. Nature 375, 148–151 (1995).

    Article  CAS  PubMed  Google Scholar 

  3. Altman, J.D. et al. Phenotypic analysis of antigen-specific T lymphocytes. Science 274, 94–96 (1996).

    Article  CAS  PubMed  Google Scholar 

  4. Kuroda, M.J. et al. Analysis of Gag-specific cytotoxic T lymphocytes in simian immunodeficiency virus-infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex. J. Exp. Med. 187, 1373–1381 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kuroda, M.J. et al. Human immunodeficiency virus type 1 envelope epitope-specific CD4(+) T lymphocytes in simian/human immunodeficiency virus-infected and vaccinated rhesus monkeys detected using a peptide-major histocompatibility complex class II tetramer. J. Virol. 74, 8751–8756 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ogg, G.S. & McMichael, A.J. HLA-peptide tetrameric complexes. Curr. Opin. Immunol. 10, 393–396 (1998).

    Article  CAS  PubMed  Google Scholar 

  7. Holmberg, K., Mariathasan, S., Ohteki, T., Ohashi, P.S. & Gascoigne, N.R. TCR binding kinetics measured with MHC class I tetramers reveal a positive selecting peptide with relatively high affinity for TCR. J. Immunol. 171, 2427–2434 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Holler, P.D., Chlewicki, L.K. & Kranz, D.M. TCRs with high affinity for foreign pMHC show self-reactivity. Nat. Immunol. 4, 55–62 (2003).

    Article  CAS  PubMed  Google Scholar 

  9. Starr, T.K., Jameson, S.C. & Hogquist, K.A. Positive and negative selection of T cells. Annu. Rev. Immunol. 21, 139–176 (2003).

    Article  CAS  PubMed  Google Scholar 

  10. Holler, P.D. & Kranz, D.M. Quantitative analysis of the contribution of TCR/pepMHC affinity and CD8 to T cell activation. Immunity 18, 255–264 (2003).

    Article  CAS  PubMed  Google Scholar 

  11. Cho, B.K. et al. Differences in antigen recognition and cytolytic activity of CD8(+) and CD8(−) T cells that express the same antigen-specific receptor. Proc. Natl. Acad. Sci. USA 98, 1723–1727 (2001).

    Article  CAS  PubMed  Google Scholar 

  12. Garcia, K.C. et al. CD8 enhances formation of stable T-cell receptor/MHC class I molecule complexes. Nature 384, 577–581 (1996).

    Article  CAS  PubMed  Google Scholar 

  13. Sykulev, Y. et al. Kinetics and affinity of reactions between an antigen-specific T cell receptor and peptide-MHC complexes. Immunity 1, 15–22 (1994).

    Article  CAS  PubMed  Google Scholar 

  14. Egan, M.A. et al. Use of major histocompatibility complex class I/peptide/beta2M tetramers to quantitate CD8(+) cytotoxic T lymphocytes specific for dominant and nondominant viral epitopes in simian-human immunodeficiency virus-infected rhesus monkeys. J. Virol. 73, 5466–5472 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Allen, T.M. et al. CD8+ Lymphocytes from Simian Immunodeficiency virus-infected rhesus macaques recognize 14 different epitopes bound by the major histocompatibility complex class I molecule Mamu-A*01: Implication for vaccine design and testing. J. Virol. 75, 738–749 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Barouch, D.H. et al. Eventual AIDS vaccine failure in a rhesus monkey by viral escape from cytotoxic T lymphocytes. Nature 415, 335–339 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Adams, E.J. & Parham, P. Species-specific evolution of MHC class I genes in the higher primates. Immunol. Rev. 183, 41–64 (2001).

    Article  CAS  PubMed  Google Scholar 

  18. Boyson, J.E. et al. The MHC class I genes of the rhesus monkey. Different evolutionary histories of MHC class I and II genes in primates. J. Immunol. 156, 4656–4665 (1996).

    PubMed  CAS  Google Scholar 

  19. Lin, A.Y. et al. Expression of T cell antigen receptor heterodimers in a lipid-linked form. Science 249, 677–679 (1990).

    Article  CAS  PubMed  Google Scholar 

  20. Lebowitz, M.S. et al. Soluble, high-affinity dimers of T-cell receptors and class II major histocompatibility complexes: biochemical probes for analysis and modulation of immune responses. Cell. Immunol. 192, 175–184 (1999).

    Article  CAS  PubMed  Google Scholar 

  21. O'Herrin, S.M. et al. Analysis of the expression of peptide-major histocompatibility complexes using high affinity soluble divalent T cell receptors. J. Exp. Med. 186, 1333–1345 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Plaksin, D., Polakova, K., McPhie, P. & Margulies, D.H. A three-domain T cell receptor is biologically active and specifically stains cell surface MHC/peptide complexes. J. Immunol. 158, 2218–2227 (1997).

    PubMed  CAS  Google Scholar 

  23. Shusta, E.V., Holler, P.D., Kieke, M.C., Kranz, D.M. & Wittrup, K.D. Directed evolution of a stable scaffold for T-cell receptor engineering. Nat. Biotechnol. 18, 754–759 (2000).

    Article  CAS  PubMed  Google Scholar 

  24. Holler, P.D., Lim, A.R., Cho, B.K., Rund, L.A. & Kranz, D.M. CD8(−) T cell transfectants that express a high affinity T cell receptor exhibit enhanced peptide-dependent activation. J. Exp. Med. 194, 1043–1052 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Kageyama, S., Tsomides, T.J., Sykulev, Y. & Eisen, N.M. Variation in the number of peptide-MHC class I complexes required to activate cytotoxic T cell responses. J. Immunol. 154, 567–576 (1995).

    PubMed  CAS  Google Scholar 

  26. Tsomides, T.J. et al. Naturally processed viral peptides recognized by cytotoxic T lymphocytes on cells chronically infected by human immunodeficiency virus type 1. J. Exp. Med. 180, 1283–1293 (1994).

    Article  CAS  PubMed  Google Scholar 

  27. Howard, M. & Kaplan, D. Applications of enzymatic amplification staining in immunophenotyping hematopoietic cells. Front. Biosci. 7, c33–c43 (2002).

    Article  PubMed  Google Scholar 

  28. De Rosa, S.C., Brenchley, J.M. & Roederer, M. Beyond six colors: a new era in flow cytometry. Nat. Med. 9, 112–117 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Manning, T.C. et al. Alanine scanning mutagenesis of an alphabeta T cell receptor: mapping the energy of antigen recognition. Immunity 8, 413–425 (1998).

    Article  CAS  PubMed  Google Scholar 

  30. Chang, H.C. et al. A general method for facilitating heterodimeric pairing between two proteins: application to expression of alpha and beta T-cell receptor extracellular segments. Proc. Natl. Acad. Sci. USA 91, 11408–11412 (1994).

    Article  CAS  PubMed  Google Scholar 

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We thank Rudolf P. Bohm, Jr. and Andrew A. Lackner from the Tulane National Primate Research Center (TNPRC) for providing monkey blood samples; and David I. Watkins from the Wisconsin Regional Primate Research Center for the Mamu-A*01 expressing 721.221 cell line used in this study. We thank Carol Lord, Karen Hershberger and William Charini for helpful discussions. This work was supported by National Institutes of Health grants RO1 AI48400 (M.J.K.), AI 48394 (J.E.S.), AI20279 (N.L.L.), AI85343 (N.L.L.), the Dana-Farber Cancer Institute/Beth Israel Deaconess Medical Center/Children's Hospital Center for AIDS Research Grant P30 AI28691 (M.J.K.) and the base grant to the TNPRC RR00168.

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Correspondence to Marcelo J Kuroda.

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Supplementary information

Supplementary Fig. 1

Binding of soluble peptide/MCH complexes to the TCR on S2 cells. (PDF 37 kb)

Supplementary Table 1

Peptide binding to Mamu-A*01 (PDF 17 kb)

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Subbramanian, R., Moriya, C., Martin, K. et al. Engineered T-cell receptor tetramers bind MHC-peptide complexes with high affinity. Nat Biotechnol 22, 1429–1434 (2004).

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