Interrogating the repertoire: broadening the scope of peptide–MHC multimer analysis

Article metrics


Labelling antigen-specific T cells with peptide–MHC multimers has provided an invaluable way to monitor T cell-mediated immune responses. A number of recent developments in this technology have made these multimers much easier to make and use in large numbers. Furthermore, enrichment techniques have provided a greatly increased sensitivity that allows the analysis of the naive T cell repertoire directly. Thus, we can expect a flood of new information to emerge in the coming years.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: The advantage of peptide–MHC tetramers and other multimers for the detection of antigen-specific T cells.
Figure 2: UV-mediated peptide-exchange technology.
Figure 3: The combinatorial tetramer staining concept.
Figure 4: Peptide–MHC tetramer enrichment using magnetic particles.


  1. 1

    Weitkamp, J. H. et al. Generation of recombinant human monoclonal antibodies to rotavirus from single antigen-specific B cells selected with fluorescent virus-like particles. J. Immunol. Methods 275, 223–237 (2003).

  2. 2

    Doucett, V. P. et al. Enumeration and characterization of virus-specific B cells by multicolor flow cytometry. J. Immunol. Methods 303, 40–52 (2005).

  3. 3

    Hayakawa, K., Ishii, R., Yamasaki, K., Kishimoto, T. & Hardy, R. R. Isolation of high-affinity memory B cells: phycoerythrin as a probe for antigen-binding cells. Proc. Natl Acad. Sci. USA 84, 1379–1383 (1987).

  4. 4

    McHeyzer-Williams, L. J. & McHeyzer-Williams, M. G. Analysis of antigen-specific B-cell memory directly ex vivo. Methods Mol. Biol. 271, 173–188 (2004).

  5. 5

    Matsui, K. et al. Low affinity interaction of peptide–MHC complexes with T cell receptors. Science 254, 1788–1791 (1991).

  6. 6

    Schatz, P. J. Use of peptide libraries to map the substrate specificity of a peptide-modifying enzyme: a 13 residue consensus peptide specifies biotinylation in Escherichia coli. Biotechnology 11, 1138–1143 (1993).

  7. 7

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

  8. 8

    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).

  9. 9

    Murali-Krishna, K. et al. In vivo dynamics of anti-viral CD8 T cell responses to different epitopes. An evaluation of bystander activation in primary and secondary responses to viral infection. Adv. Exp. Med. Biol. 452, 123–142 (1998).

  10. 10

    Savage, P. A. & Davis, M. M. A kinetic window constricts the T cell receptor repertoire in the thymus. Immunity 14, 243–252 (2001).

  11. 11

    Choi, E. M. et al. High avidity antigen-specific CTL identified by CD8-independent tetramer staining. J. Immunol. 171, 5116–5123 (2003).

  12. 12

    Lee, P. P. et al. Characterization of circulating T cells specific for tumor-associated antigens in melanoma patients. Nature Med. 5, 677–685 (1999).

  13. 13

    Toebes, M. et al. Design and use of conditional MHC class I ligands. Nature Med. 12, 246–251 (2006).

  14. 14

    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).

  15. 15

    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).

  16. 16

    Reijonen, H. & Kwok, W. W. Use of HLA class II tetramers in tracking antigen-specific T cells and mapping T-cell epitopes. Methods 29, 282–288 (2003).

  17. 17

    Newell, E. W., Klein, L. O., Yu, W. & Davis, M. M. Simultaneous detection of many T-cell specificities using combinatorial tetramer staining. Nature Methods 6, 497–499 (2009).

  18. 18

    Hadrup, S. R. et al. Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers. Nature Methods 6, 520–526 (2009).

  19. 19

    Stadinski, B. D. et al. Diabetogenic T cells recognize insulin bound to IAg7 in an unexpected, weakly binding register. Proc. Natl Acad. Sci. USA 107, 10978–10983 (2010).

  20. 20

    Landais, E. et al. New design of MHC class II tetramers to accommodate fundamental principles of antigen presentation. J. Immunol. 183, 7949–7957 (2009).

  21. 21

    Grotenbreg, G. M. et al. Discovery of CD8+ T cell epitopes in Chlamydia trachomatis infection through use of caged class I MHC tetramers. Proc. Natl Acad. Sci. USA 105, 3831–3836 (2008).

  22. 22

    Bendelac, A., Savage, P. B. & Teyton, L. The biology of NKT cells. Annu. Rev. Immunol. 25, 297–336 (2007).

  23. 23

    Karadimitris, A. et al. Human CD1d–glycolipid tetramers generated by in vitro oxidative refolding chromatography. Proc. Natl Acad. Sci. USA 98, 3294–3298 (2001).

  24. 24

    Sidobre, S. & Kronenberg, M. CD1 tetramers: a powerful tool for the analysis of glycolipid-reactive T cells. J. Immunol. Methods 268, 107–121 (2002).

  25. 25

    Li, D., Chen, N., McMichael, A. J., Screaton, G. R. & Xu, X. N. Generation and characterisation of CD1d tetramer produced by a lentiviral expression system. J. Immunol. Methods 330, 57–63 (2008).

  26. 26

    Crowley, M. P. et al. A population of murine γδ T cells that recognize an inducible MHC class Ib molecule. Science 287, 314–316 (2000).

  27. 27

    Wu, J., Groh, V. & Spies, T. T cell antigen receptor engagement and specificity in the recognition of stress-inducible MHC class I-related chains by human epithelial γδ T cells. J. Immunol. 169, 1236–1240 (2002).

  28. 28

    Subbramanian, R. A. et al. Engineered T-cell receptor tetramers bind MHC-peptide complexes with high affinity. Nature Biotech. 22, 1429–1434 (2004).

  29. 29

    Crawford, F. et al. Use of baculovirus MHC/peptide display libraries to characterize T-cell receptor ligands. Immunol. Rev. 210, 156–170 (2006).

  30. 30

    Tessmer, M. S. et al. KLRG1 binds cadherins and preferentially associates with SHIP-1. Int. Immunol. 19, 391–400 (2007).

  31. 31

    Diefenbach, A., Jamieson, A. M., Liu, S. D., Shastri, N. & Raulet, D. H. Ligands for the murine NKG2D receptor: expression by tumor cells and activation of NK cells and macrophages. Nature Immunol. 1, 119–126 (2000).

  32. 32

    Kriegeskorte, A. K. et al. NKG2D-independent suppression of T cell proliferation by H60 and MICA. Proc. Natl Acad. Sci. USA 102, 11805–11810 (2005).

  33. 33

    Ravkov, E. V., Myrick, C. M. & Altman, J. D. Immediate early effector functions of virus-specific CD8+CCR7+ memory cells in humans defined by HLA and CC chemokine ligand 19 tetramers. J. Immunol. 170, 2461–2468 (2003).

  34. 34

    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).

  35. 35

    Chattopadhyay, P. K. et al. Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry. Nature Med. 12, 972–977 (2006).

  36. 36

    Scholler, J. et al. A recombinant human HLA-class I antigen linked to dextran elicits innate and adaptive immune responses. J. Immunol. Methods 360, 1–9 (2010).

  37. 37

    Chen, D. S. et al. Marked differences in human melanoma antigen-specific T cell responsiveness after vaccination using a functional microarray. PLoS Med. 2, e265 (2005).

  38. 38

    Kwong, G. A. et al. Modular nucleic acid assembled p/MHC microarrays for multiplexed sorting of antigen-specific T cells. J. Am. Chem. Soc. 131, 9695–9703 (2009).

  39. 39

    Ramachandiran, V. et al. A robust method for production of MHC tetramers with small molecule fluorophores. J. Immunol. Methods 319, 13–20 (2007).

  40. 40

    Jenkins, M. K., Chu, H. H., McLachlan, J. B. & Moon, J. J. On the composition of the preimmune repertoire of T cells specific for peptide–major histocompatibility complex ligands. Annu. Rev. Immunol. 28, 275–294 (2010).

  41. 41

    Ornatsky, O., Baranov, V. I., Bandura, D. R., Tanner, S. D. & Dick, J. Multiple cellular antigen detection by ICP-MS. J. Immunol. Methods 308, 68–76 (2006).

  42. 42

    Bandura, D. R. et al. Mass cytometry: technique for real time single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass spectrometry. Anal. Chem. 81, 6813–6822 (2009).

  43. 43

    Bendall, S. C. et al. Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332, 687–696 (2011).

  44. 44

    Skinner, P. J. & Haase, A. T. In situ tetramer staining. J. Immunol. Methods 268, 29–34 (2002).

  45. 45

    Skinner, P. J. & Haase, A. T. In situ staining using MHC class I tetramers. Curr. Protoc. Immunol. 64, 17.4 (2005).

  46. 46

    Haanen, J. B. et al. In situ detection of virus- and tumor-specific T-cell immunity. Nature Med. 6, 1056–1060 (2000).

  47. 47

    Khanna, K. M., McNamara, J. T. & Lefrancois, L. In situ imaging of the endogenous CD8 T cell response to infection. Science 318, 116–120 (2007).

  48. 48

    Li, Q. et al. Visualizing antigen-specific and infected cells in situ predicts outcomes in early viral infection. Science 323, 1726–1729 (2009).

  49. 49

    McGavern, D. B., Christen, U. & Oldstone, M. B. Molecular anatomy of antigen-specific CD8+ T cell engagement and synapse formation in vivo. Nature Immunol. 3, 918–925 (2002).

  50. 50

    Moore, A., Grimm, J., Han, B. & Santamaria, P. Tracking the recruitment of diabetogenic CD8+ T-cells to the pancreas in real time. Diabetes 53, 1459–1466 (2004).

  51. 51

    Delong, J. H. et al. Ara h 1-reactive T cells in individuals with peanut allergy. J. Allergy Clin. Immunol. 127, 1211–1218 (2011).

  52. 52

    Kinnunen, T. et al. Allergen-specific naive and memory CD4+ T cells exhibit functional and phenotypic differences between individuals with or without allergy. Eur. J. Immunol. 40, 2460–2469 (2010).

  53. 53

    Gredmark-Russ, S., Cheung, E. J., Isaacson, M. K., Ploegh, H. L. & Grotenbreg, G. M. The CD8 T-cell response against murine gammaherpesvirus 68 is directed toward a broad repertoire of epitopes from both early and late antigens. J. Virol. 82, 12205–12212 (2008).

  54. 54

    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).

  55. 55

    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).

  56. 56

    Alanio, C., Lemaitre, F., Law, H. K., Hasan, M. & Albert, M. L. Enumeration of human antigen-specific naive CD8+ T cells reveals conserved precursor frequencies. Blood 115, 3718–3725 (2010).

  57. 57

    Kwok, W. W. et al. Direct ex vivo analysis of allergen-specific CD4+ T cells. J. Allergy Clin. Immunol. 125, 1407–1409 (2010).

  58. 58

    Schmidt, J. et al. Immunodominance of HLA-A2-restricted hepatitis C virus-specific CD8+ T cell responses is linked to naive-precursor frequency. J. Virol. 85, 5232–5236 (2011).

  59. 59

    Rizzuto, G. A. et al. Self-antigen-specific CD8+ T cell precursor frequency determines the quality of the antitumor immune response. J. Exp. Med. 206, 849–866 (2009).

  60. 60

    Cobbold, M. et al. Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA–peptide tetramers. J. Exp. Med. 202, 379–386 (2005).

  61. 61

    Knabel, M. et al. Reversible MHC multimer staining for functional isolation of T-cell populations and effective adoptive transfer. Nature Med. 8, 631–637 (2002).

  62. 62

    Maile, R. et al. Antigen-specific modulation of an immune response by in vivo administration of soluble MHC class I tetramers. J. Immunol. 167, 3708–3714 (2001).

  63. 63

    Yuan, R. R. et al. Targeted deletion of T-cell clones using alpha-emitting suicide MHC tetramers. Blood 104, 2397–2402 (2004).

  64. 64

    Kappel, B. J. et al. Remodeling specific immunity by use of MHC tetramers: demonstration in a graft-versus-host disease model. Blood 107, 2045–2051 (2006).

  65. 65

    Penaloza-MacMaster, P., Masopust, D. & Ahmed, R. T-cell reconstitution without T-cell immunopathology in two models of T-cell-mediated tissue destruction. Immunology 128, 164–171 (2009).

  66. 66

    Hess, P. R. et al. Selective deletion of antigen-specific CD8+ T cells by MHC class I tetramers coupled to the type I ribosome-inactivating protein saporin. Blood 109, 3300–3307 (2007).

  67. 67

    Vincent, B. G. et al. Toxin-coupled MHC class I tetramers can specifically ablate autoreactive CD8+ T cells and delay diabetes in nonobese diabetic mice. J. Immunol. 184, 4196–4204 (2010).

  68. 68

    Harmon, M. W., Rota, P. A., Walls, H. H. & Kendal, A. P. Antibody response in humans to influenza virus type B host-cell-derived variants after vaccination with standard (egg-derived) vaccine or natural infection. J. Clin. Microbiol. 26, 333–337 (1988).

  69. 69

    Appay, V., Douek, D. C. & Price, D. A. CD8+ T cell efficacy in vaccination and disease. Nature Med. 14, 623–628 (2008).

  70. 70

    He, X. S. et al. Phenotypic changes in influenza-specific CD8+ T cells after immunization of children and adults with influenza vaccines. J. Infect. Dis. 197, 803–811 (2008).

  71. 71

    Co, M. D., Kilpatrick, E. D. & Rothman, A. L. Dynamics of the CD8 T-cell response following yellow fever virus 17D immunization. Immunology 128, e718–e727 (2009).

  72. 72

    Wei, H. et al. DR*W201/P65 tetramer visualization of epitope-specific CD4 T-cell during M. tuberculosis infection and its resting memory pool after BCG vaccination. PLoS ONE 4, e6905 (2009).

  73. 73

    Betts, M. R. et al. Characterization of functional and phenotypic changes in anti-Gag vaccine-induced T cell responses and their role in protection after HIV-1 infection. Proc. Natl Acad. Sci. USA 102, 4512–4517 (2005).

  74. 74

    Pittet, M. J. et al. Ex vivo analysis of tumor antigen specific CD8+ T cell responses using MHC/peptide tetramers in cancer patients. Int. Immunopharmacol. 1, 1235–1247 (2001).

  75. 75

    Rolland, M. et al. Broad and Gag-biased HIV-1 epitope repertoires are associated with lower viral loads. PLoS ONE 3, e1424 (2008).

  76. 76

    Molldrem, J. J. et al. Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia. Nature Med. 6, 1018–1023 (2000).

  77. 77

    Callahan, M. K., Wolchok, J. D. & Allison, J. P. Anti-CTLA-4 antibody therapy: immune monitoring during clinical development of a novel immunotherapy. Semin. Oncol. 37, 473–484 (2010).

  78. 78

    Molldrem, J. J. Vaccination for leukemia. Biol. Blood Marrow Transplant. 12, 13–18 (2006).

  79. 79

    Rosenberg, S. A. & Dudley, M. E. Adoptive cell therapy for the treatment of patients with metastatic melanoma. Curr. Opin. Immunol. 21, 233–240 (2009).

  80. 80

    Britten, C. M. et al. Harmonization guidelines for HLA–peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium. Cancer Immunol. Immunother. 58, 1701–1713 (2009).

  81. 81

    Janetzki, S. et al. “MIATA”-minimal information about T cell assays. Immunity 31, 527–528 (2009).

  82. 82

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

  83. 83

    Warren, L., Bryder, D., Weissman, I. L. & Quake, S. R. Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR. Proc. Natl Acad. Sci. USA 103, 17807–17812 (2006).

  84. 84

    Rodenko, B. et al. Generation of peptide–MHC class I complexes through UV-mediated ligand exchange. Nature Protoc. 1, 1120–1132 (2006).

  85. 85

    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).

  86. 86

    Altman, J. D., Reay, P. A. & Davis, M. M. Formation of functional peptide complexes of class II major histocompatibility complex proteins from subunits produced in Escherichia coli. Proc. Natl Acad. Sci. USA 90, 10330–10334 (1993).

  87. 87

    Cameron, T. O. et al. Labeling antigen-specific CD4+ T cells with class II MHC oligomers. J. Immunol. Methods 268, 51–69 (2002).

  88. 88

    Scott, C. A., Garcia, K. C., Carbone, F. R., Wilson, I. A. & Teyton, L. Role of chain pairing for the production of functional soluble IA major histocompatibility complex class II molecules. J. Exp. Med. 183, 2087–2095 (1996).

  89. 89

    Novak, E. J., Liu, A. W., Nepom, G. T. & Kwok, W. W. MHC class II tetramers identify peptide-specific human CD4+ T cells proliferating in response to influenza A antigen. J. Clin. Invest. 104, R63–R67 (1999).

  90. 90

    Belz, G. T., Altman, J. D. & Doherty, P. C. Characteristics of virus-specific CD8+ T cells in the liver during the control and resolution phases of influenza pneumonia. Proc. Natl Acad. Sci. USA 95, 13812–13817 (1998).

  91. 91

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

  92. 92

    Zajac, A. J. et al. Viral immune evasion due to persistence of activated T cells without effector function. J. Exp. Med. 188, 2205–2213 (1998).

  93. 93

    Gütgemann, I. et al. Induction of rapid T cell activation and tolerance by systemic presentation of an orally administered antigen. Immunity 8, 667–673 (1998).

  94. 94

    Benlagha, K., Weiss, A., Beavis, A., Teyton, L. & Bendelac, A. In vivo identification of glycolipid antigen-specific T cells using fluorescent CD1d tetramers. J. Exp. Med. 191, 1895–1903 (2000).

Download references


We would like to thank our sources of funding for this work. M.M.D. is supported by the Howard Hughes Medical Institute, the Bill and Melinda Gates Foundation (51731) and grants from the US National Institutes of Health (NIH; U19AI057229). J.D.A. is supported by NIH grants to the NIH Tetramer Core Facility (N01A125456M0D13) and the Emory Center for AIDS research (P30 AI050409). E.W.N. is supported by a Steven and Edward Bielfelt Postdoctoral Fellowship from the American Cancer Society.

Author information

Correspondence to Mark M. Davis or John D. Altman.

Ethics declarations

Competing interests

Mark M. Davis and John D. Altman hold a patent for use of peptide–MHC tetramers and receive royalties for their commercial use.

Related links

Related links


Mark M. Davis's homepage

Immune Epitope Database

Rights and permissions

Reprints and Permissions

About this article

Further reading