Tungatt, Katie2; Bianchi, Valentina2; Crowther, Michael D2; Powell, Wendy E2; Schauenburg, Andrea J2; Trimby, Andrew2; Donia, Marco2; Miles, John J2; Holland, Christopher J2; Cole, David K2; Godkin, Andrew J2; Peakman, Mark2; Straten, Per Thor2; Svane, Inge Marie3; Sewell, Andrew K2; Dolton, Garry2
1 Department of Clinical Medicine, Department of Clinical Medicine, Faculty of Health and Medical Sciences, Københavns Universitet2 unknown3 Department of Clinical Medicine, Department of Clinical Medicine, Faculty of Health and Medical Sciences, Københavns Universitet
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.
Journal of Immunology (baltimore, Md. : 1950), 2015, Vol 194, Issue 1, p. 463-474