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.
authors.affiliation: Institute of Infection and Immunity, Cardiff University School of Medicine, University Hospital, Cardiff CF14 4XN, Wales, United Kingdom;
authors.forename: Katie
authors.initials: K
authors.lastname: Tungatt
authors.affiliation: Institute of Infection and Immunity, Cardiff University School of Medicine, University Hospital, Cardiff CF14 4XN, Wales, United Kingdom;
authors.forename: Valentina
authors.initials: V
authors.lastname: Bianchi
authors.affiliation: Institute of Infection and Immunity, Cardiff University School of Medicine, University Hospital, Cardiff CF14 4XN, Wales, United Kingdom;
authors.forename: Michael D
authors.initials: MD
authors.lastname: Crowther
authors.affiliation: Institute of Infection and Immunity, Cardiff University School of Medicine, University Hospital, Cardiff CF14 4XN, Wales, United Kingdom;
authors.forename: Wendy E
authors.initials: WE
authors.lastname: Powell
authors.affiliation: Institute of Infection and Immunity, Cardiff University School of Medicine, University Hospital, Cardiff CF14 4XN, Wales, United Kingdom;
authors.forename: Andrea J
authors.initials: AJ
authors.lastname: Schauenburg
authors.affiliation: Institute of Infection and Immunity, Cardiff University School of Medicine, University Hospital, Cardiff CF14 4XN, Wales, United Kingdom;
authors.forename: Andrew
authors.initials: A
authors.lastname: Trimby
authors.affiliation: Center for Cancer Immune Therapy, Herlev University Hospital, DK-2730 Herlev, Denmark;
authors.forename: Marco
authors.initials: M
authors.lastname: Donia
authors.affiliation: Institute of Infection and Immunity, Cardiff University School of Medicine, University Hospital, Cardiff CF14 4XN, Wales, United Kingdom; QIMR Berghofer Medical Research Institute, Brisbane, Queensland 4029, Australia; and.
authors.forename: John J
authors.initials: JJ
authors.lastname: Miles
authors.affiliation: Institute of Infection and Immunity, Cardiff University School of Medicine, University Hospital, Cardiff CF14 4XN, Wales, United Kingdom;
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Holland
authors.affiliation: Institute of Infection and Immunity, Cardiff University School of Medicine, University Hospital, Cardiff CF14 4XN, Wales, United Kingdom;
authors.forename: David K
authors.initials: DK
authors.lastname: Cole
authors.affiliation: Institute of Infection and Immunity, Cardiff University School of Medicine, University Hospital, Cardiff CF14 4XN, Wales, United Kingdom;
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Godkin
authors.affiliation: Department of Immunobiology, King's College London School of Medicine, Guy's Hospital, London SE1 9RT, United Kingdom.
authors.forename: Mark
authors.initials: M
authors.lastname: Peakman
authors.affiliation: Center for Cancer Immune Therapy, Herlev University Hospital, DK-2730 Herlev, Denmark;
authors.forename: Per Thor
authors.initials: PT
authors.lastname: Straten
authors.affiliation: Center for Cancer Immune Therapy, Herlev University Hospital, DK-2730 Herlev, Denmark;
authors.forename: Inge Marie
authors.initials: IM
authors.lastname: Svane
authors.affiliation: Institute of Infection and Immunity, Cardiff University School of Medicine, University Hospital, Cardiff CF14 4XN, Wales, United Kingdom; sewellak@cardiff.ac.uk.
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Sewell
authors.affiliation: Institute of Infection and Immunity, Cardiff University School of Medicine, University Hospital, Cardiff CF14 4XN, Wales, United Kingdom;
authors.forename: Garry
authors.initials: G
authors.lastname: Dolton
mesh_terms: D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D010798:Phycocyanin / Q000737:chemistry; D011485:Protein Binding / Q000276:immunology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D013194:Staining and Labeling / Q000379:methods*; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*
chemical_list: D000906:Antibodies; D005456:Fluorescent Dyes; D047428:Protein Kinase Inhibitors; D011948:Receptors, Antigen, T-Cell; C002497:allophycocyanin; D010798:Phycocyanin