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 Class I Tetramers Can Fail To Detect Relevant Functional T Cell Clonotypes and Underestimate Antigen-Reactive T Cell Populations. / Rius, Cristina; Attaf, Meriem; Tungatt, Katie; Bianchi, Valentina; Legut, Mateusz; Bovay, Amandine; Donia, Marco; Thor Straten, Per; Peakman, Mark; Svane, Inge Marie; Ott, Sascha; Connor, Tom; Szomolay, Barbara; Dolton, Garry; Sewell, Andrew K.
I: The Journal of Immunology, Bind 200, Nr. 7, 2018, s. 2263-2279.Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
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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
Y1 - 2018
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
VL - 200
SP - 2263
EP - 2279
JO - Journal of Immunology
JF - Journal of Immunology
SN - 0022-1767
IS - 7
ER -
ID: 216458050