Peptide-MHC Class I Tetramers Can Fail To Detect Relevant Functional T Cell Clonotypes and Underestimate Antigen-Reactive T Cell Populations
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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.
Original language | English |
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Journal | The Journal of Immunology |
Volume | 200 |
Issue number | 7 |
Pages (from-to) | 2263-2279 |
ISSN | 0022-1767 |
DOIs | |
Publication status | Published - 2018 |
- CD8-Positive T-Lymphocytes/immunology, Cytomegalovirus/immunology, HLA-A2 Antigen/immunology, Herpesvirus 4, Human/immunology, Humans, Lymphocyte Activation/immunology, Lymphocytes, Tumor-Infiltrating/immunology, Melanoma/immunology, Orthomyxoviridae/immunology, Protein Binding/immunology, Protein Kinase Inhibitors/metabolism, RNA-Binding Proteins/immunology, Receptors, Antigen, T-Cell/immunology, Staining and Labeling/methods, Tumor Cells, Cultured
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