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  • Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells.

Comparison of peptide-major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells.

Clinical and experimental immunology (2014-03-29)
G Dolton, A Lissina, A Skowera, K Ladell, K Tungatt, E Jones, D Kronenberg-Versteeg, H Akpovwa, J M Pentier, C J Holland, A J Godkin, D K Cole, M A Neller, J J Miles, D A Price, M Peakman, A K Sewell
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Biotin, SAJ special grade, ≥98.0%
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Biotin, ≥99.0% (T)
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Biotin, tested according to Ph. Eur.
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Biotin, meets USP testing specifications
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Biotin, powder, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99%
Supelco
Biotin, certified reference material, TraceCERT®
USP
Biotin, United States Pharmacopeia (USP) Reference Standard
SAFC
BIOTIN
Dextran 250 for calibration, European Pharmacopoeia (EP) Reference Standard
Supelco
Biotin, Pharmaceutical Secondary Standard; Certified Reference Material
Dextran 40 for performance test, European Pharmacopoeia (EP) Reference Standard
Dextran Vo, European Pharmacopoeia (EP) Reference Standard
Biotin, European Pharmacopoeia (EP) Reference Standard
Supelco
Dextran, analytical standard, for GPC, 270,000
Supelco
Dextran, analytical standard, for GPC, 670,000
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Dextran, analytical standard, for GPC, 150,000
Supelco
Dextran, analytical standard, for GPC, 80,000
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Dextran, analytical standard, for GPC, 410,000
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Dextran from Leuconostoc spp., Mr 15,000-25,000
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Dextran, analytical standard, for GPC, 5,000
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Dextran from Leuconostoc spp., Mr ~40,000
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Dextran from Leuconostoc spp., Mr ~2,000,000
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Dextran from Leuconostoc spp., Mr ~6,000
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Dextran from Leuconostoc spp., Mr ~70,000
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Dextran from Leuconostoc spp., Mr 450,000-650,000
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Dextran from Leuconostoc mesenteroides, Mr ~200,000
Supelco
Dextran from Leuconostoc mesenteroides, analytical standard, for GPC, Mw 12,000
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Dextran from Leuconostoc mesenteroides, analytical standard, for GPC, Mw 1,000