Fig. 5: Flow cytometry using TAPBPR-exchanged pMHC-I tetramers.
From: High throughput pMHC-I tetramer library production using chaperone-mediated peptide exchange
a Representative flow cytometric analysis of top row; murine cells expressing the B4.3.2 TCR, middle row; DMF5 human T cells expressing the MART-1 TCR, bottom row; NY-ESO-1 human T cells expressing the NY-ESO-1 TCR. Columns depict staining with a fixed excess (1 µg/mL) of P18-I10/H2-Dd, MART-1/HLA-A*02:01 and NY-ESO-1/HLA-A*02:01 tetramers prepared either by conventional refolding, exchange of peptide on stoichiometric MHC-I/TAPBPR complexes, TAPBPR-mediated peptide exchange using a catalytic (1:100) TAPBPR to MHC-I molar ratio or by stoichiometric exchange of a mismatched peptide, not recognized by the respective TCRs. b Histogram plots of tetramer staining. c Tetramer titration of P18-I10/H-2Dd (top), MART-1/HLA-A*02:01(middle) and NY-ESO-1/HLA-A*02:01 (bottom), prepared by exchange of each peptide on the corresponding stoichiometric MHC-I/TAPBPR complexes. Percentage of cells staining positive with tetramer over a serial two-fold dilution series were plotted and EC50 values calculated by curve fitting to a sigmoidal line (with R2 values in the 0.97–0.99 range), using Graph Pad Prism version 8 (GraphPad Software, La Jolla California USA). Data shown are representative of triplicate assays (n = 3) and error-bars are standard deviations from the mean. Gating strategies used for sorting tetramer-positive cells are outlined in Supplementary Fig. 4. Source data are provided as a Source Data file.
