Ding Lin, Yu Qian, Yang Shuo, Yang Wen-Jing, Liu Te, Xian Jing-Rong, Tian Tong-Tong, Li Tong, Chen Wei, Wang Bei-Li, Pan Bai-Shen, Zhou Jian, Fan Jia, Yang Xin-Rong, Guo Wei
Department of Laboratory Medicine, Zhongshan Hospital, Fudan University, Shanghai, China.
Department of Laboratory Medicine, Wusong Branch, Zhongshan Hospital, Fudan University, Shanghai, China.
Front Immunol. 2022 Mar 17;13:831101. doi: 10.3389/fimmu.2022.831101. eCollection 2022.
Inhibitory immune checkpoint proteins promote tumor immune escape and are associated with inferior patient outcome. However, the biological functions and regulatory roles of one of its members, HHLA2, in the tumor immune microenvironment have not been explored.
RandomForest analyses (371 cases), qRT-PCR (15 cases), and immunohistochemical staining (189 cases) were used to validate the prognostic value of HHLA2 in hepatocellular carcinoma (HCC) patients. Bioinformatic analyses were further performed to explore the biological functions and potential signaling pathways affected by . Moreover, ESTIMATE, single sample gene set enrichment analysis, CIBERSORT, TIMER, and other deconvolution methods were used to analyze the composition and infiltration level of immune cells. Multiplex immunofluorescence assays were employed to validate the fractions of suppressive immune cells, and -related molecular alterations were investigated. Finally, the clinical response to chemotherapy and immune checkpoint blockade was predicted by TIDE, Submap, and several other in silico analyses.
RandomForest analysis revealed that HHLA2 was the most important inhibitory immune checkpoint associated with HCC patient prognosis (relative importance = 1). Our HCC cohorts further revealed that high HHLA2 expression was an independent prognostic biomarker of shorter overall survival (P<0.01) and time to recurrence (P<0.001) for HCC patients. Bioinformatics experiments revealed that may accelerate the cell cycle of cancer cells. Additionally, we found that high expression of was associated with immune infiltrates, including some immunosuppressive cells, cytokines, chemokines, and corresponding receptors, resulting in an immunosuppressive environment. Notably, HHLA2 expression was positively correlated with the infiltration of exhausted CD8+ T cells, which was validated by immunofluorescence. Genomic alteration analyses revealed that promoter hypermethylation of may be associated with its low expression. More importantly, patients with high HHLA2 expression may be more sensitive to chemotherapy and have better responses to immunotherapy.
High expression of is an independent prognostic biomarker for HCC patients. It can activate the cell cycle and foster an immunosuppressive tumor microenvironment by enriching exhausted CD8+ T cells. Promoter hypermethylation might lead to low expression of in HCC. Thus, targeting HHLA2 may be a practical therapeutic strategy for HCC patients in the future.
抑制性免疫检查点蛋白促进肿瘤免疫逃逸，并与患者预后较差相关。然而，其成员之一HHLA2在肿瘤免疫微环境中的生物学功能和调节作用尚未得到探索。
采用随机森林分析（371例）、qRT-PCR（15例）和免疫组织化学染色（189例）来验证HHLA2在肝细胞癌（HCC）患者中的预后价值。进一步进行生物信息学分析以探索其影响的生物学功能和潜在信号通路。此外，使用ESTIMATE、单样本基因集富集分析、CIBERSORT、TIMER和其他反卷积方法来分析免疫细胞的组成和浸润水平。采用多重免疫荧光测定法验证抑制性免疫细胞的比例，并研究相关分子改变。最后，通过TIDE、Submap和其他几种计算机分析预测对化疗和免疫检查点阻断的临床反应。
随机森林分析显示，HHLA2是与HCC患者预后相关的最重要的抑制性免疫检查点（相对重要性 = 1）。我们的HCC队列进一步显示，HHLA2高表达是HCC患者总生存期较短（P<0.01）和复发时间较短（P<0.001）的独立预后生物标志物。生物信息学实验表明，其可能加速癌细胞的细胞周期。此外，我们发现其高表达与免疫浸润相关，包括一些免疫抑制细胞、细胞因子、趋化因子和相应受体，从而导致免疫抑制环境。值得注意的是，HHLA2表达与耗竭的CD8+ T细胞浸润呈正相关，这通过免疫荧光得到验证。基因组改变分析显示，其启动子高甲基化可能与其低表达相关。更重要的是，HHLA2高表达的患者可能对化疗更敏感，对免疫治疗有更好的反应。
HHLA2高表达是HCC患者的独立预后生物标志物。它可以激活细胞周期，并通过富集耗竭的CD8+ T细胞来促进免疫抑制性肿瘤微环境。启动子高甲基化可能导致HCC中HHLA2低表达。因此，靶向HHLA2可能是未来HCC患者的一种实用治疗策略。
