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

TY - JOUR

T1 - Peptide-MHC Class I Tetramers Can Fail To Detect Relevant Functional T Cell Clonotypes and Underestimate Antigen-Reactive T Cell Populations

AU - Rius, Cristina

AU - Attaf, Meriem

AU - Tungatt, Katie

AU - Bianchi, Valentina

AU - Legut, Mateusz

AU - Bovay, Amandine

AU - Donia, Marco

AU - Thor Straten, Per

AU - Peakman, Mark

AU - Svane, Inge Marie

AU - Ott, Sascha

AU - Connor, Tom

AU - Szomolay, Barbara

AU - Dolton, Garry

AU - Sewell, Andrew K

N1 - Copyright © 2018 The Authors.

PY - 2018/4/1

Y1 - 2018/4/1

N2 - 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.

AB - 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.

KW - CD8-Positive T-Lymphocytes/immunology

KW - Cytomegalovirus/immunology

KW - HLA-A2 Antigen/immunology

KW - Herpesvirus 4, Human/immunology

KW - Humans

KW - Lymphocyte Activation/immunology

KW - Lymphocytes, Tumor-Infiltrating/immunology

KW - Melanoma/immunology

KW - Orthomyxoviridae/immunology

KW - Protein Binding/immunology

KW - Protein Kinase Inhibitors/metabolism

KW - RNA-Binding Proteins/immunology

KW - Receptors, Antigen, T-Cell/immunology

KW - Staining and Labeling/methods

KW - Tumor Cells, Cultured

U2 - 10.4049/jimmunol.1700242

DO - 10.4049/jimmunol.1700242

M3 - Journal article

C2 - 29483360

SN - 0022-1767

VL - 200

SP - 2263

EP - 2279

JO - The Journal of Immunology

JF - The Journal of Immunology

IS - 7

ER -