Fig. 1

ATF4 impairs CD8⁺ T function by direct contact. a IHC of ATF4 and GZMB in human HCC tissues. (Scale bars, 50 μm.) Quantification is shown on the right (n = 41). b CD8⁺ T cell and HCC cell co-culture model. c Histogram shows the mean levels of GZMB and IFNG expression in CD8⁺ T cell co-cultured with ATF4 knock-down MHCC-97H cells and ATF4 overexpressed SNU-387 cells. (n = 3/group). d Graph shows the concentrations of IFNG and GZMB present in culture supernatant and tested by ELISA. (n = 3/group). e CD8⁺ T cells were plated on chamber for indirect co-culture with HCC cells. f qRT-PCR for IFNG and GZMB expression of CD8⁺ T cell indirectly co-cultured with ATF4 knock-down MHCC-97H and ATF4 overexpressed SNU-387 cells. (n = 3/group). g ELISA for CD8⁺ T cells indirectly co-cultured with ATF4 knock-down MHCC-97H and ATF4 overexpressed SNU-387 cells. (n = 3/group). h The left panel shows immunostaining for CD8 (green) and DAPI (blue) in CD8⁺ T cells co-cultured with ATF4 overexpressed SNU-387 cells. (Scale bars, 100 μm.) The right histogram displays the quantification of bound CD8 + T cells as a percentage of total tumor cells. (n = 5/group). i The left panel shows immunostaining for CD8 (red) and DAPI (blue) in CD8.⁺ T cells co-cultured with ATF4 knock-down MHCC-97H cells. (Scale bars, 100 μm.) The right histogram displays the quantification of bound CD8 + T cells as a percentage of total tumor cells. (n = 5/group). Statistical analysis performed using two-tailed t-test (c–h) and one-way ANOVA (c, d, f, g, i). ns for P>0.05, *for P < 0.05, **for P < 0.01, ***for P < 0.001, ****for P < 0.0001