Antibody stabilization of peptide-MHC multimers reveals functional T cells bearing extremely low-affinity TCRs

Tungatt, Katie, Bianchi, Valentina, Crowther, Michael D., Powell, Wendy ORCID: https://orcid.org/0000-0002-4670-1406, Schauenburg, Andrea J. A., Trimby, Andrew R., Donia, M., Miles, John James, Holland, Christopher J., Cole, David ORCID: https://orcid.org/0000-0003-0028-9396, Godkin, Andrew James ORCID: https://orcid.org/0000-0002-1910-7567, Peakman, M., Straten, P. T., Svane, I. M., Sewell, Andrew K. ORCID: https://orcid.org/0000-0003-3194-3135 and Dolton, Garry Michael 2015. Antibody stabilization of peptide-MHC multimers reveals functional T cells bearing extremely low-affinity TCRs. Journal of Immunology 194 (1) , pp. 463-474. 10.4049/jimmunol.1401785

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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 fluorochromeconjugated 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. The Journal of Immunology, 2015, 194: 000–000.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Medicine Systems Immunity Research Institute (SIURI)
Subjects:	R Medicine > R Medicine (General)
Publisher:	American Association of Immunologists
ISSN:	0022-1767
Funders:	Wellcome Trust
Date of First Compliant Deposit:	30 March 2016
Date of Acceptance:	30 October 2014
Last Modified:	15 May 2024 01:15
URI:	https://orca.cardiff.ac.uk/id/eprint/68887

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