Fig. 1.
FACS analysis of pMHCI binding using TAP deficient T2 cells. We used TAP deficient T2 cells, which lack the ability to transport peptide fragments to the endoplasmic reticulum to form stable pMHCI, to analyze the stability of the natural and heteroclitic pMHCIs using FACS analysis. No peptide, and the HLA-B*3501-restricted EBNA-1407–415 (HPVGEADYF) peptide were used as negative controls of binding, and the HLA A*0201-restricted influenza M158–66 (GILGFVFTL) peptide was used as a positive control of binding. The mean fluorescence intensity (MFI) of cell surface pMHCI staining with an anti-human HLA A2:RPE antibody revealed that the natural tumor antigen, KIFGSLAFL, was more stable than the heteroclitic variant, KLFGSLAFV, whereas the natural tumor peptides SLLMWITQC, YLEPGPVA and VISNDVCAQV were less stable than their heteroclitic counterparts: SLLMWITQL, YLEPGPVV and VLSNDVCAQV. Data at a peptide concentration of 100 μM are shown. The binding assay was performed at a range of peptide concentrations (100 μM, 50 μM, 10 μM and 1 μM). LogEC50s (M) for this range are shown in Table 1. The mean standard deviation, representing 4 separate experiments, is shown as error bars.