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

Tungatt, Katie, Bianchi, Valentina, Crowther, Michael D., Powell, Wendy E., Schauenburg, Andrea J., Trimby, Andrew, Donia, Marco, Miles, John J., Holland, Christopher J., Cole, David K., Godkin, Andrew J., Peakman, Mark, Straten, Per Thor, Svane, Inge Marie, Sewell, Andrew K., and Dolton, Garry (2015) Antibody stabilization of peptide-MHC multimers reveals functional T cells bearing extremely low-affinity TCRs. Journal of Immunology, 194 (1). pp. 463-474.

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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 (K(D) >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.

Item ID: 45053
Item Type: Article (Non-Refereed Research)
ISSN: 1550-6606
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© 2014 The Authors. This is an open-access article distributed under the terms of the CC-BY 3.0 Unported license.

Date Deposited: 09 Aug 2016 06:02
FoR Codes: 11 MEDICAL AND HEALTH SCIENCES > 1107 Immunology > 110702 Applied Immunology (incl Antibody Engineering, Xenotransplantation and T-cell Therapies) @ 100%
SEO Codes: 92 HEALTH > 9201 Clinical Health (Organs, Diseases and Abnormal Conditions) > 920108 Immune System and Allergy @ 100%
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