title: Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells.
abstract: Fluorochrome-conjugated peptide-major histocompatibility complex (pMHC) multimers are widely used for flow cytometric visualization of antigen-specific T cells. The most common multimers, streptavidin-biotin-based 'tetramers', can be manufactured readily in the laboratory. Unfortunately, there are large differences between the threshold of T cell receptor (TCR) affinity required to capture pMHC tetramers from solution and that which is required for T cell activation. This disparity means that tetramers sometimes fail to stain antigen-specific T cells within a sample, an issue that is particularly problematic when staining tumour-specific, autoimmune or MHC class II-restricted T cells, which often display TCRs of low affinity for pMHC. Here, we compared optimized staining with tetramers and dextramers (dextran-based multimers), with the latter carrying greater numbers of both pMHC and fluorochrome per molecule. Most notably, we find that: (i) dextramers stain more brightly than tetramers; (ii) dextramers outperform tetramers when TCR-pMHC affinity is low; (iii) dextramers outperform tetramers with pMHC class II reagents where there is an absence of co-receptor stabilization; and (iv) dextramer sensitivity is enhanced further by specific protein kinase inhibition. Dextramers are compatible with current state-of-the-art flow cytometry platforms and will probably find particular utility in the fields of autoimmunity and cancer immunology.
authors.affiliation: Institute of Infection and Immunity, Cardiff University School of Medicine, Cardiff, UK.
authors.forename: G
authors.initials: G
authors.lastname: Dolton
authors.forename: A
authors.initials: A
authors.lastname: Lissina
authors.forename: A
authors.initials: A
authors.lastname: Skowera
authors.forename: K
authors.initials: K
authors.lastname: Ladell
authors.forename: K
authors.initials: K
authors.lastname: Tungatt
authors.forename: E
authors.initials: E
authors.lastname: Jones
authors.forename: D
authors.initials: D
authors.lastname: Kronenberg-Versteeg
authors.forename: H
authors.initials: H
authors.lastname: Akpovwa
authors.forename: J M
authors.initials: JM
authors.lastname: Pentier
authors.forename: C J
authors.initials: CJ
authors.lastname: Holland
authors.forename: A J
authors.initials: AJ
authors.lastname: Godkin
authors.forename: D K
authors.initials: DK
authors.lastname: Cole
authors.forename: M A
authors.initials: MA
authors.lastname: Neller
authors.forename: J J
authors.initials: JJ
authors.lastname: Miles
authors.forename: D A
authors.initials: DA
authors.lastname: Price
authors.forename: M
authors.initials: M
authors.lastname: Peakman
authors.forename: A K
authors.initials: AK
authors.lastname: Sewell
mesh_terms: D001710:Biotin / Q000737:chemistry; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D002999:Clone Cells; D003911:Dextrans / Q000737:chemistry; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000737:chemistry; D015789:HLA-A2 Antigen / Q000737:chemistry; D015798:HLA-DR1 Antigen / Q000737:chemistry / Q000378:metabolism; D006389:Hemagglutinins, Viral / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D008285:Major Histocompatibility Complex / Q000276:immunology*; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry; D037182:T-Cell Antigen Receptor Specificity / Q000276:immunology; D019098:Telomerase / Q000378:metabolism
chemical_list: D003911:Dextrans; D005456:Fluorescent Dyes; D015789:HLA-A2 Antigen; D015798:HLA-DR1 Antigen; D006389:Hemagglutinins, Viral; D007328:Insulin; D010446:Peptide Fragments; D011498:Protein Precursors; C013737:preproinsulin; D001710:Biotin; D019809:Streptavidin; C437311:telomerase reverse transcriptase (540-548); D019098:Telomerase
keywords: T cell receptors; T cells; autoimmunity; diabetes; tumour immunology