Antibody Stabilization of Peptide–MHC Multimers Reveals Functional T Cells Bearing Extremely Low-Affinity TCRs

Katie Tungatt; Valentina Bianchi; Michael D Crowther; Wendy E Powell; Andrea J Schauenburg; Andrew Trimby; Marco Donia; John J Miles; Christopher J Holland; David K Cole; Andrew J Godkin; Mark Peakman; Per Thor Straten; Inge Marie Svane; Andrew K Sewell; Garry Dolton

doi:10.4049/jimmunol.1401785

Antibody Stabilization of Peptide–MHC Multimers Reveals Functional T Cells Bearing Extremely Low-Affinity TCRs

Katie Tungatt, Valentina Bianchi, Michael D Crowther, Wendy E Powell, Andrea J Schauenburg, Andrew Trimby, Marco Donia, John J Miles, Christopher J Holland, David K Cole, Andrew J Godkin, Mark Peakman, Per Thor Straten, Inge Marie Svane, Andrew K Sewell, Garry Dolton

Institut for Klinisk Medicin

40 Citationer (Scopus)

Abstract

Fluorochrome-conjugated peptide-MHC (pMHC) multimers are commonly used in combination with flow cytometry for direct ex vivo visualization and characterization of Ag-specific T cells, but these reagents can fail to stain cells when TCR affinity and/or TCR cell-surface density are low. pMHC multimer staining of tumor-specific, autoimmune, or MHC class II-restricted T cells can be particularly challenging, as these T cells tend to express relatively low-affinity TCRs. In this study, we attempted to improve staining using anti-fluorochrome unconjugated primary Abs followed by secondary staining with anti-Ab fluorochrome-conjugated Abs to amplify fluorescence intensity. Unexpectedly, we found that the simple addition of an anti-fluorochrome unconjugated Ab during staining resulted in considerably improved fluorescence intensity with both pMHC tetramers and dextramers and with PE-, allophycocyanin-, or FITC-based reagents. Importantly, when combined with protein kinase inhibitor treatment, Ab stabilization allowed pMHC tetramer staining of T cells even when the cognate TCR-pMHC affinity was extremely low (KD >1 mM) and produced the best results that we have observed to date. We find that this inexpensive addition to pMHC multimer staining protocols also allows improved recovery of cells that have recently been exposed to Ag, improvements in the recovery of self-specific T cells from PBMCs or whole-blood samples, and the use of less reagent during staining. In summary, Ab stabilization of pMHC multimers during T cell staining extends the range of TCR affinities that can be detected, yields considerably enhanced staining intensities, and is compatible with using reduced amounts of these expensive reagents.

Originalsprog	Engelsk
Tidsskrift	Journal of immunology (Baltimore, Md. : 1950)
Vol/bind	194
Udgave nummer	1
Sider (fra-til)	463-474
Antal sider	12
ISSN	0022-1767
DOI	https://doi.org/10.4049/jimmunol.1401785
Status	Udgivet - 1 jan. 2015

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10.4049/jimmunol.1401785

463.full.pdfForlagets udgivne version, 2,75 MBLicens: CC BY

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Tungatt, K., Bianchi, V., Crowther, M. D., Powell, W. E., Schauenburg, A. J., Trimby, A., Donia, M., Miles, J. J., Holland, C. J., Cole, D. K., Godkin, A. J., Peakman, M., Straten, P. T., Svane, I. M., Sewell, A. K., & Dolton, G. (2015). Antibody Stabilization of Peptide–MHC Multimers Reveals Functional T Cells Bearing Extremely Low-Affinity TCRs. Journal of immunology (Baltimore, Md. : 1950), 194(1), 463-474. https://doi.org/10.4049/jimmunol.1401785

Tungatt, K, Bianchi, V, Crowther, MD, Powell, WE, Schauenburg, AJ, Trimby, A, Donia, M, Miles, JJ, Holland, CJ, Cole, DK, Godkin, AJ, Peakman, M, Straten, PT, Svane, IM, Sewell, AK & Dolton, G 2015, 'Antibody Stabilization of Peptide–MHC Multimers Reveals Functional T Cells Bearing Extremely Low-Affinity TCRs', Journal of immunology (Baltimore, Md. : 1950), bind 194, nr. 1, s. 463-474. https://doi.org/10.4049/jimmunol.1401785

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title = "Antibody Stabilization of Peptide–MHC Multimers Reveals Functional T Cells Bearing Extremely Low-Affinity TCRs",

abstract = "Fluorochrome-conjugated peptide-MHC (pMHC) multimers are commonly used in combination with flow cytometry for direct ex vivo visualization and characterization of Ag-specific T cells, but these reagents can fail to stain cells when TCR affinity and/or TCR cell-surface density are low. pMHC multimer staining of tumor-specific, autoimmune, or MHC class II-restricted T cells can be particularly challenging, as these T cells tend to express relatively low-affinity TCRs. In this study, we attempted to improve staining using anti-fluorochrome unconjugated primary Abs followed by secondary staining with anti-Ab fluorochrome-conjugated Abs to amplify fluorescence intensity. Unexpectedly, we found that the simple addition of an anti-fluorochrome unconjugated Ab during staining resulted in considerably improved fluorescence intensity with both pMHC tetramers and dextramers and with PE-, allophycocyanin-, or FITC-based reagents. Importantly, when combined with protein kinase inhibitor treatment, Ab stabilization allowed pMHC tetramer staining of T cells even when the cognate TCR-pMHC affinity was extremely low (KD >1 mM) and produced the best results that we have observed to date. We find that this inexpensive addition to pMHC multimer staining protocols also allows improved recovery of cells that have recently been exposed to Ag, improvements in the recovery of self-specific T cells from PBMCs or whole-blood samples, and the use of less reagent during staining. In summary, Ab stabilization of pMHC multimers during T cell staining extends the range of TCR affinities that can be detected, yields considerably enhanced staining intensities, and is compatible with using reduced amounts of these expensive reagents.",

author = "Katie Tungatt and Valentina Bianchi and Crowther, {Michael D} and Powell, {Wendy E} and Schauenburg, {Andrea J} and Andrew Trimby and Marco Donia and Miles, {John J} and Holland, {Christopher J} and Cole, {David K} and Godkin, {Andrew J} and Mark Peakman and Straten, {Per Thor} and Svane, {Inge Marie} and Sewell, {Andrew K} and Garry Dolton",

note = "Copyright {\textcopyright} 2014 The Authors.",

year = "2015",

month = jan,

day = "1",

doi = "10.4049/jimmunol.1401785",

language = "English",

volume = "194",

pages = "463--474",

journal = "Journal of Immunology",

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T1 - Antibody Stabilization of Peptide–MHC Multimers Reveals Functional T Cells Bearing Extremely Low-Affinity TCRs

AU - Tungatt, Katie

AU - Bianchi, Valentina

AU - Crowther, Michael D

AU - Powell, Wendy E

AU - Schauenburg, Andrea J

AU - Trimby, Andrew

AU - Donia, Marco

AU - Miles, John J

AU - Holland, Christopher J

AU - Cole, David K

AU - Godkin, Andrew J

AU - Peakman, Mark

AU - Straten, Per Thor

AU - Svane, Inge Marie

AU - Sewell, Andrew K

AU - Dolton, Garry

PY - 2015/1/1

Y1 - 2015/1/1

N2 - Fluorochrome-conjugated peptide-MHC (pMHC) multimers are commonly used in combination with flow cytometry for direct ex vivo visualization and characterization of Ag-specific T cells, but these reagents can fail to stain cells when TCR affinity and/or TCR cell-surface density are low. pMHC multimer staining of tumor-specific, autoimmune, or MHC class II-restricted T cells can be particularly challenging, as these T cells tend to express relatively low-affinity TCRs. In this study, we attempted to improve staining using anti-fluorochrome unconjugated primary Abs followed by secondary staining with anti-Ab fluorochrome-conjugated Abs to amplify fluorescence intensity. Unexpectedly, we found that the simple addition of an anti-fluorochrome unconjugated Ab during staining resulted in considerably improved fluorescence intensity with both pMHC tetramers and dextramers and with PE-, allophycocyanin-, or FITC-based reagents. Importantly, when combined with protein kinase inhibitor treatment, Ab stabilization allowed pMHC tetramer staining of T cells even when the cognate TCR-pMHC affinity was extremely low (KD >1 mM) and produced the best results that we have observed to date. We find that this inexpensive addition to pMHC multimer staining protocols also allows improved recovery of cells that have recently been exposed to Ag, improvements in the recovery of self-specific T cells from PBMCs or whole-blood samples, and the use of less reagent during staining. In summary, Ab stabilization of pMHC multimers during T cell staining extends the range of TCR affinities that can be detected, yields considerably enhanced staining intensities, and is compatible with using reduced amounts of these expensive reagents.

AB - Fluorochrome-conjugated peptide-MHC (pMHC) multimers are commonly used in combination with flow cytometry for direct ex vivo visualization and characterization of Ag-specific T cells, but these reagents can fail to stain cells when TCR affinity and/or TCR cell-surface density are low. pMHC multimer staining of tumor-specific, autoimmune, or MHC class II-restricted T cells can be particularly challenging, as these T cells tend to express relatively low-affinity TCRs. In this study, we attempted to improve staining using anti-fluorochrome unconjugated primary Abs followed by secondary staining with anti-Ab fluorochrome-conjugated Abs to amplify fluorescence intensity. Unexpectedly, we found that the simple addition of an anti-fluorochrome unconjugated Ab during staining resulted in considerably improved fluorescence intensity with both pMHC tetramers and dextramers and with PE-, allophycocyanin-, or FITC-based reagents. Importantly, when combined with protein kinase inhibitor treatment, Ab stabilization allowed pMHC tetramer staining of T cells even when the cognate TCR-pMHC affinity was extremely low (KD >1 mM) and produced the best results that we have observed to date. We find that this inexpensive addition to pMHC multimer staining protocols also allows improved recovery of cells that have recently been exposed to Ag, improvements in the recovery of self-specific T cells from PBMCs or whole-blood samples, and the use of less reagent during staining. In summary, Ab stabilization of pMHC multimers during T cell staining extends the range of TCR affinities that can be detected, yields considerably enhanced staining intensities, and is compatible with using reduced amounts of these expensive reagents.

U2 - 10.4049/jimmunol.1401785

DO - 10.4049/jimmunol.1401785

M3 - Journal article

C2 - 25452566

SN - 0022-1767

VL - 194

SP - 463

EP - 474

JO - Journal of Immunology

JF - Journal of Immunology

IS - 1

ER -

Antibody Stabilization of Peptide–MHC Multimers Reveals Functional T Cells Bearing Extremely Low-Affinity TCRs

Abstract

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