Fig 1: IL-1ß induced HLA-G augmentation of the cell membrane of FaDu cells, which facilitated the cell killing ability of anti-HLA-G CAR-T cells(A) HLA-G expression in FaDu cells overexpressing EGFR variants was determined by immunofluorescence.(B) Total HLA-G protein levels were evaluated by western blotting, or (C) membrane HLA-G was analyzed by flow cytometry after incubated with 0, 5, 10, or 20 ng/mL rhIL-1ß for 0, 12, 24, or 48 h in FaDu cells. Flow cytometric data were shown as histogram overlays.(D) Analysis of transduction efficiency of the anti-HLA-G CAR plasmid on primary T cells. Transduction efficiency was evaluated by flow cytometry using fluorescent-tagged protein L to detect variable light chain Ig, together with human HLA-G protein.(E) Anti-HLA-G CAR-T cell population analyzed using CD3 with CD4 or CD8 by flow cytometry.(F) Cell killing ability of mock T cells and anti-HLA-G CAR-T cells in FaDu cells overexpressing EGFR variants.(G) Cell killing ability of anti-HLA-G CAR-T cells on EGFRL858R-overexpressing FaDu cells; E:T ratios of 1:1, 3:1, and 6:1. ß-actin was used as an internal control. Data are expressed as the mean ± SEM of six separate experiments. Scale bars, 20 µm. White arrows indicated positive staining of HLA-G on the cell membrane. *p < 0.05, **p < 0.01, ***p < 0.001 compared to their mock T cells-treated group. #p < 0.05, ###p < 0.001 compared to mock FaDu cells treated with CAR-T cells.