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

Cristina Rius; Meriem Attaf; Katie Tungatt; Valentina Bianchi; Mateusz Legut; Amandine Bovay; Marco Donia; Per Thor Straten; Mark Peakman; Inge Marie Svane; Sascha Ott; Tom Connor; Barbara Szomolay; Garry Dolton; Andrew K Sewell

doi:10.4049/jimmunol.1700242

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

Cristina Rius, Meriem Attaf, Katie Tungatt, Valentina Bianchi, Mateusz Legut, Amandine Bovay, Marco Donia, Per Thor Straten, Mark Peakman, Inge Marie Svane, Sascha Ott, Tom Connor, Barbara Szomolay, Garry Dolton, Andrew K Sewell

Institut for Klinisk Medicin

35 Citationer (Scopus)

39 Downloads (Pure)

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.

Originalsprog	Engelsk
Tidsskrift	The Journal of Immunology
Vol/bind	200
Udgave nummer	7
Sider (fra-til)	2263-2279
ISSN	0022-1767
DOI	https://doi.org/10.4049/jimmunol.1700242
Status	Udgivet - 1 apr. 2018

Adgang til dokumentet

10.4049/jimmunol.1700242Licens: CC BY

2263.fullForlagets udgivne version, 4,32 MBLicens: CC BY

Citationsformater

Rius, C., Attaf, M., Tungatt, K., Bianchi, V., Legut, M., Bovay, A., Donia, M., Thor Straten, P., Peakman, M., Svane, I. M., Ott, S., Connor, T., Szomolay, B., Dolton, G., & Sewell, A. K. (2018). 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, 200(7), 2263-2279. https://doi.org/10.4049/jimmunol.1700242

Rius, C, Attaf, M, Tungatt, K, Bianchi, V, Legut, M, Bovay, A, Donia, M, Thor Straten, P, Peakman, M, Svane, IM, Ott, S, Connor, T, Szomolay, B, Dolton, G & Sewell, AK 2018, '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, bind 200, nr. 7, s. 2263-2279. https://doi.org/10.4049/jimmunol.1700242

@article{e2670f58f7934676ad971f4cdf6d46a0,

title = "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.",

keywords = "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",

author = "Cristina Rius and Meriem Attaf and Katie Tungatt and Valentina Bianchi and Mateusz Legut and Amandine Bovay and Marco Donia and {Thor Straten}, Per and Mark Peakman and Svane, {Inge Marie} and Sascha Ott and Tom Connor and Barbara Szomolay and Garry Dolton and Sewell, {Andrew K}",

note = "Copyright {\textcopyright} 2018 The Authors.",

year = "2018",

month = apr,

day = "1",

doi = "10.4049/jimmunol.1700242",

language = "English",

volume = "200",

pages = "2263--2279",

journal = "Journal of Immunology",

issn = "0022-1767",

publisher = "American Association of Immunologists",

number = "7",

}

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

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 - Journal of Immunology

JF - Journal of Immunology

IS - 7

ER -

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

Abstract

Adgang til dokumentet

Fingeraftryk

Citationsformater