title: More tricks with tetramers: a practical guide to staining T cells with peptide-MHC multimers.
abstract: Analysis of antigen-specific T-cell populations by flow cytometry with peptide-MHC (pMHC) multimers is now commonplace. These reagents allow the tracking and phenotyping of T cells during infection, autoimmunity and cancer, and can be particularly revealing when used for monitoring therapeutic interventions. In 2009, we reviewed a number of 'tricks' that could be used to improve this powerful technology. More recent advances have demonstrated the potential benefits of using higher order multimers and of 'boosting' staining by inclusion of an antibody against the pMHC multimer. These developments now allow staining of T cells where the interaction between the pMHC and the T-cell receptor is over 20-fold weaker (K(D) > 1 mm) than could previously be achieved. Such improvements are particularly relevant when using pMHC multimers to stain anti-cancer or autoimmune T-cell populations, which tend to bear lower affinity T-cell receptors. Here, we update our previous work to include discussion of newer tricks that can produce substantially brighter staining even when using log-fold lower concentrations of pMHC multimer. We further provide a practical guide to using pMHC multimers that includes a description of several common pitfalls and how to circumvent them.
authors.affiliation: Division of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK.
authors.forename: Garry
authors.initials: G
authors.lastname: Dolton
authors.affiliation: Division of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK.
authors.forename: Katie
authors.initials: K
authors.lastname: Tungatt
authors.affiliation: Division of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK.
authors.forename: Angharad
authors.initials: A
authors.lastname: Lloyd
authors.affiliation: Division of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK.
authors.forename: Valentina
authors.initials: V
authors.lastname: Bianchi
authors.affiliation: Division of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Theaker
authors.affiliation: Division of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Trimby
authors.affiliation: Division of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Holland
authors.affiliation: Centre for Cancer Immune Therapy, Herlev University Hospital, Herlev, Denmark.
authors.forename: Marco
authors.initials: M
authors.lastname: Donia
authors.affiliation: Division of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Godkin
authors.affiliation: Division of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK.
authors.forename: David K
authors.initials: DK
authors.lastname: Cole
authors.affiliation: Centre for Cancer Immune Therapy, Herlev University Hospital, Herlev, Denmark.
authors.forename: Per Thor
authors.initials: PT
authors.lastname: Straten
authors.affiliation: Peter Gorer Department of Immunobiology, King's College London, Guy's Hospital, London, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Peakman
authors.affiliation: Centre for Cancer Immune Therapy, Herlev University Hospital, Herlev, Denmark.
authors.forename: Inge Marie
authors.initials: IM
authors.lastname: Svane
authors.affiliation: Division of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK.
authors.forename: Andrew K
authors.initials: AK
authors.lastname: Sewell
mesh_terms: D000906:Antibodies / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes; D015395:Histocompatibility Antigens Class I / Q000276:immunology; D000949:Histocompatibility Antigens Class II / Q000276:immunology; D006801:Humans; D008285:Major Histocompatibility Complex / Q000276:immunology*; D010455:Peptides / Q000276:immunology; D055503:Protein Multimerization; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000276:immunology*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D000906:Antibodies; D005456:Fluorescent Dyes; D015395:Histocompatibility Antigens Class I; D000949:Histocompatibility Antigens Class II; D010455:Peptides; D016693:Receptors, Antigen, T-Cell, alpha-beta
keywords: T cell; T-cell receptor; peptide-MHC dextramer; peptide-MHC tetramer