1. Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells.
- Author
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Dolton G, Lissina A, Skowera A, Ladell K, Tungatt K, Jones E, Kronenberg-Versteeg D, Akpovwa H, Pentier JM, Holland CJ, Godkin AJ, Cole DK, Neller MA, Miles JJ, Price DA, Peakman M, and Sewell AK
- Subjects
- Biotin chemistry, CD4-Positive T-Lymphocytes cytology, CD8-Positive T-Lymphocytes cytology, Cell Line, Clone Cells, Dextrans chemistry, Flow Cytometry, Fluorescent Dyes chemistry, HLA-A2 Antigen chemistry, HLA-DR1 Antigen chemistry, HLA-DR1 Antigen metabolism, Hemagglutinins, Viral metabolism, Humans, Insulin metabolism, Peptide Fragments metabolism, Protein Binding, Protein Precursors metabolism, Streptavidin chemistry, T-Cell Antigen Receptor Specificity immunology, Telomerase metabolism, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, Cell Separation methods, Major Histocompatibility Complex immunology
- 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., (© 2014 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)
- Published
- 2014
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