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Tracking epitope-specific T cells

Nature Protocols volume 4, pages 565–581 (2009)Cite this article

Abstract

The tracking of antigen-specific T cells in vivo is a useful approach for the study of the adaptive immune response. This protocol describes how populations of T cells specific for a given peptide–major histocompatibility complex (pMHC) epitope can be tracked based solely on T-cell receptor (TCR) specificity as opposed to other indirect methods based on function. The methodology involves the adoptive transfer of TCR transgenic T cells with defined epitope specificity into histocompatible mice and the subsequent detection of these cells through the use of congenic or clonotypic markers. Alternatively, endogenous epitope-specific T cells can be tracked directly through the use of pMHC tetramers. Using magnetic bead-based enrichment and advanced multiparameter flow cytometry, populations as small as five epitope-specific T cells can be detected from the peripheral lymphoid organs of a mouse. The adoptive transfer procedure can be completed within 3 h, whereas analysis of epitope-specific cells from mice can be completed within 6 h.

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Figure 1: Graphical representation of in vivo T-cell-tracking strategies.
Figure 2: Analysis of TCR transgenic T cells before adoptive transfer.
Figure 3: Analysis of donor TCR transgenic T cells from a high-frequency adoptive transfer.
Figure 4: Analysis of donor TCR transgenic T cells from a low-frequency adoptive transfer.
Figure 5: Analysis of endogenous epitope-specific T cells.

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References

  1. Corradin, G., Etlinger, H.M. & Chiller, J.M. Lymphocyte specificity to protein antigens. I. Characterization of the antigen-induced in vitro T cell-dependent proliferative response with lymph node cells from primed mice. J. Immunol. 119, 1048–1053 (1977).

    CAS  PubMed  Google Scholar 

  2. Lamoreaux, L., Roederer, M. & Koup, R. Intracellular cytokine optimization and standard operating procedure. Nat. Protoc. 1, 1507–1516 (2006).

    Article  CAS  PubMed  Google Scholar 

  3. Anthony, D.D. & Lehmann, P.V. T-cell epitope mapping using the ELISPOT approach. Methods 29, 260–269 (2003).

    Article  CAS  PubMed  Google Scholar 

  4. Chattopadhyay, P.K., Yu, J. & Roederer, M. Live-cell assay to detect antigen-specific CD4+ T-cell responses by CD154 expression. Nat. Protoc. 1, 1–6 (2006).

    Article  CAS  PubMed  Google Scholar 

  5. Chattopadhyay, P.K., Yu, J. & Roederer, M. A live-cell assay to detect antigen-specific CD4+ T cells with diverse cytokine profiles. Nat. Med. 11, 1113–1117 (2005).

    Article  CAS  PubMed  Google Scholar 

  6. Jenkins, M.K. et al. In vivo activation of antigen-specific CD4 T cells. Annu. Rev. Immunol. 19, 23–45 (2001).

    Article  CAS  PubMed  Google Scholar 

  7. Kearney, E.R., Pape, K.A., Loh, D.Y. & Jenkins, M.K. Visualization of peptide-specific T cell immunity and peripheral tolerance induction in vivo . Immunity 1, 327–339 (1994).

    Article  CAS  PubMed  Google Scholar 

  8. Pape, K.A. et al. Use of adoptive transfer of T-cell-antigen-receptor-transgenic T cell for the study of T-cell activation in vivo . Immunol. Rev. 156, 67–78 (1997).

    Article  CAS  PubMed  Google Scholar 

  9. Blattman, J.N. et al. Estimating the precursor frequency of naive antigen-specific CD8 T cells. J. Exp. Med. 195, 657–664 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hataye, J., Moon, J.J., Khoruts, A., Reilly, C. & Jenkins, M.K. Naive and memory CD4+ T cell survival controlled by clonal abundance. Science 312, 114–116 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Stemberger, C. et al. A single naive CD8+ T cell precursor can develop into diverse effector and memory subsets. Immunity 27, 985–997 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. Moon, J.J. et al. Naive CD4(+) T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude. Immunity 27, 203–213 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Obar, J.J., Khanna, K.M. & Lefrancois, L. Endogenous naive CD8+ T cell precursor frequency regulates primary and memory responses to infection. Immunity 28, 859–869 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kotturi, M.F. et al. Naive precursor frequencies and MHC binding rather than the degree of epitope diversity shape CD8+ T cell immunodominance. J. Immunol. 181, 2124–2133 (2008).

    Article  CAS  PubMed  Google Scholar 

  15. Ford, M.L. et al. Antigen-specific precursor frequency impacts T cell proliferation, differentiation, and requirement for costimulation. J. Exp. Med. 204, 299–309 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Foulds, K.E. & Shen, H. Clonal competition inhibits the proliferation and differentiation of adoptively transferred TCR transgenic CD4 T cells in response to infection. J. Immunol. 176, 3037–3043 (2006).

    Article  CAS  PubMed  Google Scholar 

  17. Garcia, Z. et al. Competition for antigen determines the stability of T cell-dendritic cell interactions during clonal expansion. Proc. Natl. Acad. Sci. USA 104, 4553–4558 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Marzo, A.L. et al. Initial T cell frequency dictates memory CD8+ T cell lineage commitment. Nat. Immunol. 6, 793–799 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Smith, A.L., Wikstrom, M.E. & Fazekas de St Groth, B . Visualizing T cell competition for peptide/MHC complexes: a specific mechanism to minimize the effect of precursor frequency. Immunity 13, 783–794 (2000).

    Article  CAS  PubMed  Google Scholar 

  20. Reinhardt, R.L., Khoruts, A., Merica, R., Zell, T. & Jenkins, M.K. Visualizing the generation of memory CD4 T cells in the whole body. Nature 410, 101–105 (2001).

    Article  CAS  PubMed  Google Scholar 

  21. Germain, R.N. et al. Making friends in out-of-the-way places: how cells of the immune system get together and how they conduct their business as revealed by intravital imaging. Immunol. Rev. 221, 163–181 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Davis, M.M. The alphabeta T cell repertoire comes into focus. Immunity 27, 179–180 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Luxembourg, A.T. et al. Biomagnetic isolation of antigen-specific CD8+ T cells usable in immunotherapy. Nat. Biotechnol. 16, 281–285 (1998).

    Article  CAS  PubMed  Google Scholar 

  24. Bodinier, M. et al. Efficient detection and immunomagnetic sorting of specific T cells using multimers of MHC class I and peptide with reduced CD8 binding. Nat. Med. 6, 707–710 (2000).

    Article  CAS  PubMed  Google Scholar 

  25. Lemaitre, F. et al. Detection of low-frequency human antigen-specific CD4(+) T cells using MHC class II multimer bead sorting and immunoscope analysis. Eur. J. Immunol. 34, 2941–2949 (2004).

    Article  CAS  PubMed  Google Scholar 

  26. Barnes, E. et al. Ultra-sensitive class I tetramer analysis reveals previously undetectable populations of antiviral CD8+ T cells. Eur. J. Immunol. 34, 1570–1577 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Keenan, R.D. et al. Purification of cytomegalovirus-specific CD8 T cells from peripheral blood using HLA-peptide tetramers. Br. J. Haematol. 115, 428–434 (2001).

    Article  CAS  PubMed  Google Scholar 

  28. Lucas, M. et al. Ex vivo phenotype and frequency of influenza virus-specific CD4 memory T cells. J. Virol. 78, 7284–7287 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Hohn, H. et al. MHC class II tetramer guided detection of Mycobacterium tuberculosis-specific CD4+ T cells in peripheral blood from patients with pulmonary tuberculosis. Scand. J. Immunol. 65, 467–478 (2007).

    Article  CAS  PubMed  Google Scholar 

  30. Jager, E. et al. Peptide-specific CD8+ T-cell evolution in vivo: response to peptide vaccination with Melan-A/MART-1. Int. J. Cancer 98, 376–388 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Jang, M.H., Seth, N.P. & Wucherpfennig, K.W. Ex vivo analysis of thymic CD4 T cells in nonobese diabetic mice with tetramers generated from I-A(g7)/class II-associated invariant chain peptide precursors. J. Immunol. 171, 4175–4186 (2003).

    Article  CAS  PubMed  Google Scholar 

  32. McDermott, A.B., Spiegel, H.M., Irsch, J., Ogg, G.S. & Nixon, D.F. A simple and rapid magnetic bead separation technique for the isolation of tetramer-positive virus-specific CD8 T cells. Aids 15, 810–812 (2001).

    Article  CAS  PubMed  Google Scholar 

  33. Day, C.L. et al. Ex vivo analysis of human memory CD4 T cells specific for hepatitis C virus using MHC class II tetramers. J. Clin. Invest. 112, 831–842 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  35. Vollers, S.S. & Stern, L.J. Class II major histocompatibility complex tetramer staining: progress, problems, and prospects. Immunology 123, 305–313 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Falta, M.T. et al. Class II major histocompatibility complex-peptide tetramer staining in relation to functional avidity and T cell receptor diversity in the mouse CD4(+) T cell response to a rheumatoid arthritis-associated antigen. Arthritis Rheum. 52, 1885–1896 (2005).

    Article  CAS  PubMed  Google Scholar 

  37. Reichstetter, S. et al. Distinct T cell interactions with HLA class II tetramers characterize a spectrum of TCR affinities in the human antigen-specific T cell response. J. Immunol. 165, 6994–6998 (2000).

    Article  CAS  PubMed  Google Scholar 

  38. Gebe, J.A. et al. Low-avidity recognition by CD4+ T cells directed to self-antigens. Eur. J. Immunol. 33, 1409–1417 (2003).

    Article  CAS  PubMed  Google Scholar 

  39. Mallet-Designe, V.I. et al. Detection of low-avidity CD4+ T cells using recombinant artificial APC: following the antiovalbumin immune response. J. Immunol. 170, 123–131 (2003).

    Article  CAS  PubMed  Google Scholar 

  40. Kerry, S.E. et al. Interplay between TCR affinity and necessity of coreceptor ligation: high-affinity peptide-MHC/TCR interaction overcomes lack of CD8 engagement. J. Immunol. 171, 4493–4503 (2003).

    Article  CAS  PubMed  Google Scholar 

  41. Daniels, M.A. & Jameson, S.C. Critical role for CD8 in T cell receptor binding and activation by peptide/major histocompatibility complex multimers. J. Exp. Med. 191, 335–346 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Mallone, R. et al. Functional avidity directs T-cell fate in autoreactive CD4+ T cells. Blood 106, 2798–2805 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Laugel, B. et al. Different T cell receptor affinity thresholds and CD8 coreceptor dependence govern cytotoxic T lymphocyte activation and tetramer binding properties. J. Biol. Chem. 282, 23799–23810 (2007).

    Article  CAS  PubMed  Google Scholar 

  44. Gordon, R.D., Mathieson, B.J., Samelson, L.E., Boyse, E.A. & Simpson, E. The effect of allogeneic presensitization on H-Y graft survival and in vitro cell-mediated responses to H-y antigen. J. Exp. Med. 144, 810–820 (1976).

    Article  CAS  PubMed  Google Scholar 

  45. Steinmetz, M., Bluthmann, H., Ryser, S. & Uematsu, Y. Transgenic mice to study T-cell receptor gene regulation and repertoire formation. Genome 31, 652–655 (1989).

    Article  CAS  PubMed  Google Scholar 

  46. Shinkai, Y. et al. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell 68, 855–867 (1992).

    Article  CAS  PubMed  Google Scholar 

  47. Mombaerts, P. et al. RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68, 869–877 (1992).

    Article  CAS  PubMed  Google Scholar 

  48. Anthony, L.S. et al. Priming of CD8+ CTL effector cells in mice by immunization with a stress protein-influenza virus nucleoprotein fusion molecule. Vaccine 17, 373–383 (1999).

    Article  CAS  PubMed  Google Scholar 

  49. Pope, C. et al. Organ-specific regulation of the CD8 T cell response to Listeria monocytogenes infection. J. Immunol. 166, 3402–3409 (2001).

    Article  CAS  PubMed  Google Scholar 

  50. Fujii, S., Shimizu, K., Smith, C., Bonifaz, L. & Steinman, R.M. Activation of natural killer T cells by alpha-galactosylceramide rapidly induces the full maturation of dendritic cells in vivo and thereby acts as an adjuvant for combined CD4 and CD8 T cell immunity to a coadministered protein. J. Exp. Med. 198, 267–279 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Wall, E.M. et al. Spontaneous mammary tumors differ widely in their inherent sensitivity to adoptively transferred T cells. Cancer Res. 67, 6442–6450 (2007).

    Article  CAS  PubMed  Google Scholar 

  52. Ehst, B.D., Ingulli, E. & Jenkins, M.K. Development of a novel transgenic mouse for the study of interactions between CD4 and CD8 T cells during graft rejection. Am. J. Transplant. 3, 1355–1362 (2003).

    Article  CAS  PubMed  Google Scholar 

  53. Huddleston, S.J., Hays, W.S., Filatenkov, A., Ingulli, E. & Jenkins, M.K. CD154+ graft antigen-specific CD4+ T cells are sufficient for chronic rejection of minor antigen incompatible heart grafts. Am. J. Transplant. 6, 1312–1319 (2006).

    Article  CAS  PubMed  Google Scholar 

  54. Kurts, C. et al. Constitutive class I-restricted exogenous presentation of self antigens in vivo . J. Exp. Med. 184, 923–930 (1996).

    Article  CAS  PubMed  Google Scholar 

  55. Chen, Z.M. & Jenkins, M.K. Clonal expansion of antigen-specific CD4 T cells following infection with Salmonella typhimurium is similar in susceptible (Itys) and resistant (Ityr) BALB/c mice. Infect. Immun. 67, 2025–2029 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Becerra, J.C., Arthur, J.F., Landucci, G.R., Forthal, D.N. & Theuer, C.P. CD8+ T-cell mediated tumor protection by Pseudomonas exotoxin fused to ovalbumin in C57BL/6 mice. Surgery 133, 404–410 (2003).

    Article  PubMed  Google Scholar 

  57. Itano, A.A. et al. Distinct dendritic cell populations sequentially present antigen to CD4 T cells and stimulate different aspects of cell-mediated immunity. Immunity 19, 47–57 (2003).

    Article  CAS  PubMed  Google Scholar 

  58. Lefrancois, L. et al. The role of beta7 integrins in CD8 T cell trafficking during an antiviral immune response. J. Exp. Med. 189, 1631–1638 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Marrack, P., Shimonkevitz, R., Hannum, C., Haskins, K. & Kappler, J. The major histocompatibility complex-restricted antigen receptor on T cells. IV. An antiidiotypic antibody predicts both antigen and I-specificity. J. Exp. Med. 158, 1635–1646 (1983).

    Article  CAS  PubMed  Google Scholar 

  60. Murphy, K.M., Heimberger, A.B. & Loh, D.Y. Induction by antigen of intrathymic apoptosis of CD4+CD8+TCRlo thymocytes in vivo . Science 250, 1720–1723 (1990).

    Article  CAS  PubMed  Google Scholar 

  61. Parish, C.R. Fluorescent dyes for lymphocyte migration and proliferation studies. Immunol. Cell Biol. 77, 499–508 (1999).

    Article  CAS  PubMed  Google Scholar 

  62. Quah, B.J., Warren, H.S. & Parish, C.R. Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Nat. Protoc. 2, 2049–2056 (2007).

    Article  CAS  PubMed  Google Scholar 

  63. Bousso, P. Generation of MHC-peptide tetramers: a new opportunity for dissecting T-cell immune responses. Microbes Infect. 2, 425–429 (2000).

    Article  CAS  PubMed  Google Scholar 

  64. Mallone, R. & Nepom, G.T. MHC class II tetramers and the pursuit of antigen-specific T cells: define, deviate, delete. Clin. Immunol. 110, 232–242 (2004).

    Article  CAS  PubMed  Google Scholar 

  65. Yang, J. et al. Multiplex mapping of CD4 T cell epitopes using class II tetramers. Clin. Immunol. 120, 21–32 (2006).

    Article  CAS  PubMed  Google Scholar 

  66. Scriba, T.J. et al. Ultrasensitive detection and phenotyping of CD4+ T cells with optimized HLA class II tetramer staining. J. Immunol. 175, 6334–6343 (2005).

    Article  CAS  PubMed  Google Scholar 

  67. Cameron, T.O., Cochran, J.R., Yassine-Diab, B., Sekaly, R.P. & Stern, L.J. Cutting edge: detection of antigen-specific CD4+ T cells by HLA-DR1 oligomers is dependent on the T cell activation state. J. Immunol. 166, 741–745 (2001).

    Article  CAS  PubMed  Google Scholar 

  68. Burchill, M.A. et al. Linked T cell receptor and cytokine signaling govern the development of the regulatory T cell repertoire. Immunity 28, 112–121 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Murali-Krishna, K. et al. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8, 177–187 (1998).

    Article  CAS  PubMed  Google Scholar 

  70. Busch, D.H., Pilip, I.M., Vijh, S. & Pamer, E.G. Coordinate regulation of complex T cell populations responding to bacterial infection. Immunity 8, 353–362 (1998).

    Article  CAS  PubMed  Google Scholar 

  71. Dutton, R.W., Bradley, L.M. & Swain, S.L. T cell memory. Annu. Rev. Immunol. 16, 201–223 (1998).

    Article  CAS  PubMed  Google Scholar 

  72. Crawford, F., Kozono, H., White, J., Marrack, P. & Kappler, J. Detection of antigen-specific T cells with multivalent soluble class II MHC covalent peptide complexes. Immunity 8, 675–682 (1998).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the National Institutes of Health (J.J.M., J.H., A.J.P., M.P., J.B.M. and M.K.J.) and the American Heart Association (H.H.C.).

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  1. Jason Hataye

    Present address: Present address: Department of Medicine, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, California 90095, USA.,

  2. Traci Zell

    Present address: Present address: eBioscience, 10255 Science Center Drive, San Diego, California 92121, USA.,

Authors and Affiliations

  1. Department of Microbiology and Center for Immunology, University of Minnesota Medical School, Minneapolis, 55455, Minnesota, USA

    James J Moon, H Hamlet Chu, Jason Hataye, Antonio J Pagán, Marion Pepper, James B McLachlan & Marc K Jenkins

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Moon, J., Chu, H., Hataye, J. et al. Tracking epitope-specific T cells. Nat Protoc 4, 565–581 (2009). https://doi.org/10.1038/nprot.2009.9

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