HHLA2 Activates the JAK/STAT Signaling Pathway by Binding to TMIGD2 in Hepatocellular Carcinoma Cells

HHLA2, a member of the B7 family of immune checkpoint players, has been implicated in various cancers. The study set to determine the expression and biological function of HHLA2 in hepatocellular carcinoma (HCC), and its connection to TMIGD2. First, after HHLA2 knockdown or overexpression in Huh-7 or HepG2 cells, we co-cultured T cells with HCC cells after transfection for 48 h. T cell proliferation and cytokine release were detected using flow cytometry and the FlowCytomix assay kit. Subsequently, we screened differentially expressed genes in cells overexpressing or under-expressing HHLA2 using GSEA database and analyzed the pathways enriched by them. We further detected the nuclear translocation of STAT3 and STAT2 using immunofluorescence. After that, we observed the subcellular localization of HHLA2 and TMIGD2 in HCC cells by laser confocal microscopy, followed by RIP and rescue experiments. We found that the proliferation of T cells and the release of cytokines were significantly reduced after co-culture with HCC cells overexpressing HHLA2, while co-culture with cells low in HHLA2 expression had the opposite results. HHLA2 bound to TMIGD2, thus inhibiting T cell proliferation and activation. Overexpression of HHLA2 significantly promoted the nuclear translocation of STAT2 and STAT3, thereby activating the JAK/STAT pathway. Subsequently, we showed that the immune tolerance of HCC cells was significantly attenuated after using a JAK/STAT signaling pathway antagonist. Aberrant overexpression of HHLA2 activates the JAK/STAT signaling pathway by binding to TMIGD2, thereby promoting immune tolerance in HCC cells.