Figure 2
Reduced HLA class-I expression in MCC is associated with an impaired antigen processing machinery (APM). (A) RMA normalized expression values of gene expression array GSE22396, were obtained from the GEO database. RMA values were log2 transformed and are depicted as heat map with expression values ranging from 7 (blue = low expression) to 14 (red = high expression). HLA-A, B2M, TAP1, TAP2, LMP2 and LMP7 mRNA expression is shown in comparison to RPLP0. (B) mRNA expression of HLA-A (black), B2M (grey), TAP1 (light blue), TAP2 (dark blue), LMP2 (light green) and LMP7 (dark green) in 4 MCC cell lines was determined by RT-qPCR in triplicates using specific primers; CT values were normalized to RPLP0 and calibrated to a set of ΔCTs of MKL-2; relative mRNA expression is depicted as mean + SEM. (C) Protein expression in 4 MCC cell lines was determined by immunoblot of whole cell lysates using antibodies specific for HLA-A, β2m, TAP1, TAP2, LMP2 and LMP7; β-tubulin served as loading control. (D,E) MCC cell lines with low (BroLi, MKL-1) and intermediate (WaGa) HLA class-I surface expression were incubated with saturating amounts (10 µM) of a flu peptide mix or MCPyV encoded large and small T antigen and VP1 derived epitopes binding with high affinity to the respective HLA-A molecules or an irrelevant peptide cocktail for at least 24 h (WaGa and MKL-1) or 48 h (BroLi). HLA class-I surface expression was determined by flow cytometry using an HLA-ABC detecting antibody. Induction of HLA class-I surface expression after stabilization with a flu peptide mix (green line), exemplarily shown for MKL-1, or the specific MCPyV peptide mix, exemplarily depicted for BroLi (blue line). An irrelevant peptide cocktail (grey filled) was used as control (D). Comparison of HLA class-I surface expression is depicted as geometric mean fluorescence intensity (gMFI) after incubation with MCPyV derived high affinity (blue) or an irrelevant peptide control (grey) for all analyzed MCC cell lines (E).
