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

Abstract

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.

Antigen-Specific TCR Signatures of Cytomegalovirus Infection

“…Moreover, the numerical increase of Ag-responsive T cells due to proliferation, as well as the increased absolute amount of TCRb mRNA per cell several days after activation (68), will, in itself, increase resolution and thus the likelihood that rare clonotypes can be detected in samples of limited size. In contrast, HLA/peptide multimer staining can capture T cells that express a specific TCR irrespective of their functional properties; however, despite recent progress (69,70), multimers are still challenging to produce for certain HLA allotypes and epitopes, and they may not always stain the entire Ag-specific T cell population (71).…”

Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8⁺ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector

“…Tetramers can also be stabilized by including anti-fluorochrome unconjugated primary Abs after tetramer staining. Additionally, fluorescence intensity can be increased by addition of a second anti-Ab fluorochrome-conjugated Ab 29,34,35,36 . We optimized the conditions selectively for the tetramers specified in this protocol and did not include PKI or additional Abs.…”

Peptide‐MHC class I and class II tetramers: From flow to mass cytometry

“…These higher order multimers can improve valency and thus, the number of detected cells (Figure ). Notably, the frequency of antigen‐specific T cells may in some cases be underestimated when relying solely on pMHC tetramer staining, and therefore, other techniques can still be beneficial. In such cases, however, higher order pMHC multimers and other staining techniques discussed below can be useful, especially when detecting autoreactive T cells.…”