Article open access publication

Peptide–MHC Class I Tetramers Can Fail To Detect Relevant Functional T Cell Clonotypes and Underestimate Antigen-Reactive T Cell Populations

The Journal of Immunology, The American Association of Immunologists, ISSN 0022-1767

Volume 200, 7, 2018

DOI:10.4049/jimmunol.1700242, Dimensions: pub.1101235493, PMC: PMC5857646, PMID: 29483360,



  1. (1) Cardiff University, grid.5600.3
  2. (2) University Hospital of Lausanne, grid.8515.9
  3. (3) Herlev Hospital, grid.411900.d, Capital Region
  4. (4) King's College London, grid.13097.3c
  5. (5) University of Warwick, grid.7372.1


United Kingdom






Peptide-MHC (pMHC) multimers, usually used as streptavidin-based tetramers, have transformed the study of Ag-specific T cells by allowing direct detection, phenotyping, and enumeration within polyclonal T cell populations. These reagents are now a standard part of the immunology toolkit and have been used in many thousands of published studies. Unfortunately, the TCR-affinity threshold required for staining with standard pMHC multimer protocols is higher than that required for efficient T cell activation. This discrepancy makes it possible for pMHC multimer staining to miss fully functional T cells, especially where low-affinity TCRs predominate, such as in MHC class II-restricted responses or those directed against self-antigens. Several recent, somewhat alarming, reports indicate that pMHC staining might fail to detect the majority of functional T cells and have prompted suggestions that T cell immunology has become biased toward the type of cells amenable to detection with multimeric pMHC. We use several viral- and tumor-specific pMHC reagents to compare populations of human T cells stained by standard pMHC protocols and optimized protocols that we have developed. Our results confirm that optimized protocols recover greater populations of T cells that include fully functional T cell clonotypes that cannot be stained by regular pMHC-staining protocols. These results highlight the importance of using optimized procedures that include the use of protein kinase inhibitor and Ab cross-linking during staining to maximize the recovery of Ag-specific T cells and serve to further highlight that many previous quantifications of T cell responses with pMHC reagents are likely to have considerably underestimated the size of the relevant populations.


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Times Cited: 27

Field Citation Ratio (FCR): 9.08

Relative Citation ratio (RCR): 2.91

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