Figure 6. Distinct alterations to the peptide repertoire expressed on MHC class I molecules in IFN-γ treated KBM-7 cells upon TAPBPR depletion or tapasin deficiency.
Cytofluorometric analysis of (A) HLA-A2 expression (detected with BB7.2) and (D) HLA-B expression (detected with 4E) on the cell surface of WT, TAPBPR stably depleted (shTAPBPR) and tapasin deficient (TPN-) KBM-7 cells treated with and without IFN-γ for 48 hr. The data is representative of three independent experiments. (B,E) Peptide-MHC class I complexes were isolated using affinity chromatography using (B) BB7.2 (HLA-A2) and (E) B1.23.2 (HLA-B,-C) from IFN-γ treated WT, TAPBPR stably depleted (shTAPBPR) and tapasin deficient (TPN-) KBM-7 cells. Eluted peptide were analysed using LC-MS/MS. Graphs show the total number of peptides. The data was generated from tandem MS analysis performed five times on one immunoprecipitate. Two independent biological repeats comparing the peptide repertoire expressed on MHC class I under conditions of TAPBPR competency and TAPBPR deficiency in two other cell lines have been performed (HeLa-M cells shown in Figure 5 and HeLa-S cell shown in Figure 5—figure supplement 1) in which a similar pattern of increased peptide diversity upon TAPBPR depletion was observed. (C,F) Venn diagrams show the overlap of peptides on (C) HLA-A2 and (F) HLA-B,-C eluted from IFN-γ treated WT, TAPBPR stably depleted (TAPBPR KD) and tapasin deficient (Tapasin KO) KBM-7 cells.