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Published March 2017 | Published
Journal Article Open

Preparation of peptide–MHC and T-cell receptor dextramers by biotinylated dextran doping

Abstract

Peptide–major histocompatibility complex (pMHC) multimers enable the detection, characterization, and isolation of antigen-specific T-cell subsets at the single-cell level via flow cytometry and fluorescence microscopy. These labeling reagents exploit a multivalent scaffold to increase the avidity of individually weak T-cell receptor (TCR)-pMHC interactions. Dextramers are an improvement over the original streptavidin-based tetramer technology because they are more multivalent, improving sensitivity for rare, low-avidity T cells, including self/tumor-reactive clones. However, commercial pMHC dextramers are expensive, and in-house production is very involved for a typical biology research laboratory. Here, we present a simple, inexpensive protocol for preparing pMHC dextramers by doping in biotinylated dextran during conventional tetramer preparation. We use these pMHC dextramers to identify patient-derived, tumor-reactive T cells. We apply the same dextran doping technique to prepare TCR dextramers and use these novel reagents to yield new insight into MHC I–mediated antigen presentation.

Additional Information

© 2017 Eaton Publishing. Received 25 October 2016; accepted 25 January 2017. We thank Shu-ou Shan (Caltech) for the use of her TIRF microscope and Jennifer Keeffe (Caltech) for assistance with HPSEC-MALS. We thank Eugene Barsov and Richard Morgan (NCI) for providing the MSGV vector and Mireille Toebes and Ton Schumacher (NKI) for providing UV-cleavable ligand for HLA-A2 and vectors for HLA-A2, b2m, and BirA ligase expression. We also thank Jost Vielmetter, Director of the Caltech Protein Expression Center for guidance with using the Biacore T200 for SPR analysis. Biotinylated H-2Kb/ovalbumin257-264 monomers were provided by the NIH Tetramer Core Facility (Atlanta, GA). This work was supported by National Institutes of Health grant 5P01CA132681-5 to D.B. and the Prostate Cancer Foundation Challenge Award 15CHAL02 to D.B. and M.T.B. M.T.B is the recipient of a Jane Coffin Childs Postdoctoral Fellowship. A.R. and B.C.-A. were supported by NIH grant R35 CA197633 and the Ressler Family Foundation. This paper is subject to the NIH Public Access Policy. Author contributions: M.T.B. designed the study; M.T.B., B.C.-A., and Y.-H.H.F. performed the experiments; M.T.B. wrote the manuscript with input from B.C.-A., Y.-H.H.F., A.R., and D.B.; B.C.-A. and A.R. provided blood cells from a patient with metastatic melanoma; D.B. supervised the study. The authors declare no competing interests.

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August 19, 2023
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October 25, 2023