Abstract：
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.
摘要：
HHLA2是免疫检查点球员B7家族的成员，与各种癌症有关。本研究旨在确定HHLA2在肝细胞癌（HCC）中的表达和生物学功能，以及它与TMIGD2的连接。首先,在Huh-7或HepG2细胞中HHLA2敲低或过表达后，我们将T细胞与HCC细胞共培养转染48h。使用流式细胞术和FlowCytomix检测试剂盒检测T细胞增殖和细胞因子释放。随后，我们使用GSEA数据库筛选了过表达或低表达HHLA2的细胞中的差异表达基因,并分析了它们富集的通路.我们用免疫荧光进一步检测了STAT3和STAT2的核易位。之后,我们通过激光共聚焦显微镜观察了HHLA2和TMIGD2在肝癌细胞中的亚细胞定位，其次是RIP和救援实验。我们发现，与过表达HHLA2的HCC细胞共培养后，T细胞的增殖和细胞因子的释放显着减少，而与HHLA2表达低的细胞共培养则具有相反的结果。HHLA2与TMIGD2结合,从而抑制T细胞增殖和活化。HHLA2过表达显著促进STAT2和STAT3的核转位,从而激活JAK/STAT通路。随后，我们发现，使用JAK/STAT信号通路拮抗剂后，HCC细胞的免疫耐受显着减弱。HHLA2的异常过表达通过与TMIGD2结合激活JAK/STAT信号通路,从而促进HCC细胞的免疫耐受。
