Fig. 3.

Soluble TAPBPR enhances exogenous peptide association onto surface MHC I. (A) IFN-γ–treated HeLaM cells were incubated ±100 nM soluble TAPBPR^WT or TAPBPR^TN5 for 15 min at 37 °C, followed by detection of surface-bound TAPBPR using PeTe-4. Soluble TAPBPR^WT binding to HeLaM-HLA-ABC^KO cells (MHC−) is included as a control. (B) TAPBPR pulldowns on lysates from IFN-γ–treated HeLaM-TAPBPR^KO cells incubated ±soluble TAPBPR^TN5 or TAPBPR^WT demonstrate that TAPBPR^WT binds to MHC I. Data are representative of three independent experiments. (C) Schematic representation of experimental workflow used to measure peptide exchange by soluble TAPBPR. (D and E) IFN-γ–treated HeLaM cells were incubated ±100 nM soluble TAPBPR^WT or TAPBPR^TN5 for 15 min at 37 °C, followed by incubation ±10 nM ETVSK*QSNV (ETV*), YVVPFVAK*V (YVV*), or EGVSK*QSNG (ETVΔ2/9) for 15 min at 37 °C. In D, histograms show the typical binding observed for ETVSK*QSNV and YVVPFVAK*V and E shows the MFI of fluorescent peptide binding ±SD from three independent experiments. (F) Dose–response curves ±SD from three independent experiments of ETVSK*QSNV binding to IFN-γ–treated HeLaM and HeLaM-HLA-ABC^KO cells treated ±100 nM TAPBPR with increasing concentrations of peptide for 15 min at 37 °C. (G) Bar graph show the MFI of fluorescent peptide binding to IFN-γ–treated HeLaM-HLA-ABC^KO cells reconstituted with HLA-A*02:01 ± SD from two independent experiments with duplicates. Cells were incubated in the absence or presence of 1 µM soluble TAPBPR^WT or TAPBPR^TN5 for 15 min, followed by incubation with 10 nM NLVPK*VATV (NLV*), YVVPFVAK*V (YVV*), YLLEK*LWRL (YLL*), CLGGK*LTMV (CLG*), ETVSK*QSNV (ETV*), or SRYWK*IRTR (SRY*) for 60 min. ***P ≤ 0.001, ****P ≤ 0.0001, n/s not significant, using unpaired two-tailed t test.