期刊
CLINICAL AND EXPERIMENTAL IMMUNOLOGY
卷 177, 期 1, 页码 47-63出版社
WILEY
DOI: 10.1111/cei.12339
关键词
autoimmunity; diabetes; T cell receptors; T cells; tumour immunology
类别
资金
- Juvenile Diabetes Research Foundation [17-2012-352]
- UK Department of Health via the National Institute for Health Research (NIHR) Biomedical Research Centre
- King's College London
- Australian National Health and Medical Research (NHMRC)
- BBSRC [BB/H001085/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/H001085/1] Funding Source: researchfish
- Medical Research Council [MR/J006742/1] Funding Source: researchfish
- Wellcome Trust [100326/Z/12/Z] Funding Source: researchfish
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.
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