Fig. 5

HSPA4/ALKBH5 inhibits cytotoxicity of CD8⁺ T cells by downregulating CD58 in GC cells. A The correlation between CD58 expression and immune score of CD8⁺ T cells was analyzed within GC patients (spearman’s correlation). B UMAP plot of cells from 3 cases of GC tumor tissues showing the formation of 17 main clusters from 15729 cells (GSE163558). C Violin plots showing the expression distribution of marker genes in tumor epithelial cell clusters (CD24,KRT19, KRT18 and EPCAM), ALKBH5 expression cell clusters (ALKBH5) and CD8⁺ T cell clusters (CD8A and GZMK). D Cell–cell contact interactions analysis showing strength of interactions between ALKBH5 high-expression or low-expression tumor cells and CD8⁺ T cells. E HGC27 cells with HSPA4 overexpression and ALKBH5 knockdown were co-cultured with CD8⁺ T cells for 16 h. Intracellular staining of IFN-ɤ, TNF-α and GZMB were determined by flow cytometry. Representative images and summary of IFN-ɤ, TNF-α and GZMB positive T cells were listed.F,G The GC cells with HSPA4 overexpression and ALKBH5 knockdown were co-cultured with CD8⁺ T cells for 72 h. The expression levels of PDL1 on the surface of tumor cells (F) and PD1 on the surface of CD8⁺ T cells (G) were analyzed by flow cytometry. Representative images and summaries of PDL1 MFI and PD1⁺ T cells ratio were listed.H ALKBH5-KD AGS and control cells were incubated with anti-CD58 or isotype control IgG for 12 h, followed by co-culture with CD8⁺ T cells for 16 h. Intracellular staining of IFN-ɤ, TNF-α and GZMB were determined by flow cytometry. Representative images and ratios of IFN-ɤ, TNF-α and GZMB positive CD8⁺ T cells were summarized. I,J ALKBH5-KD AGS and control cells were incubated with anti-CD58 or IgG antibodies for 12 h, followed by co-culture with CD8⁺ T cells for 72 h. Expression of PDL1 on tumor cells (I) and PD1 on CD8⁺ T cells (J) were analyzed by flow cytometry. Representative images and results of PDL1 MFI (I) and PD1⁺CD8⁺ T cells ratio (J) were summarized. (*, P<0.05; **, P<0.01; ***, P<0.001)