LAG-3/LSECtin轴在软组织肉瘤逃逸T细胞免疫中的作用及机制

The mechanism of immune escape is the cutting-edge of current oncology research, in which the immune co-inhibitory molecules play an important role. LAG-3 is one of the new immune checkpoint molecules following PD-1. Our previous studies have shown that LAG-3 was highly expressed on the surface of T cells in soft tissue sarcoma tissues and the patients' peripheral blood, and was significantly correlated with disease progression and poor prognosis. Further, LSECtin, a potential ligand of LAG-3, was found to be up-regulated in soft tissue sarcoma tissues and inhibit the function of CD8+T cells in vitro. Protein docking calculation by ZDOCK software revealed that there was a stable binding and interaction between LAG-3 and LSECtin, suggesting that LAG-3 may be involved in the development of soft tissue sarcoma and the process of T-cell immune escape by binding to LSECtin, but the underlying specific effects and mechanisms were not clear. Based on this, the present study will confirm the interaction between LSECtin and LAG-3 by protein interactions technology. The regulative effect and mechanism of the LAG-3/LSECtin axis that mediate T-cell exhaustion/dysfunction leading to tumor immune escape will be explored through in vivo and in vitro experiments. Meanwhile, we will further evaluate the safety and efficacy of treatment with LAG-3 inhibitor alone against that of combined PD-1 inhibitor for B-hPD-L1/hLAG-3 mouse models with soft tissue sarcomas. As such, we intend to provide a new therapeutic target and theoretical basis for the therapy of soft tissue sarcomas.

免疫逃逸机制研究是当前肿瘤学研究的前沿，而共抑制分子在其中发挥重要作用。LAG-3是继PD-1之后发现的新型共抑制分子之一。我们前期发现LAG-3在软组织肉瘤组织及患者外周血中高表达，且与疾病进展和不良预后显著相关；蛋白对接计算发现LAG-3与其潜在配体LSECtin存在稳定结合及残基间的相互作用；LSECtin在软组织肉瘤中表达上调并且在体外可抑制CD8+T细胞功能。提示LAG-3可能通过与LSECtin结合，参与了软组织肉瘤的发生发展及免疫逃逸过程，但具体作用及机制未明。基于此，本课题将利用蛋白互作技术确证LAG-3与LSECtin之间的相互作用。并通过体内及体外实验，探讨LAG-3/LSECtin轴介导T细胞耗竭/功能障碍继而参与免疫逃逸的作用及机制。最后，以人源化小鼠为模型评价LAG-3抗体联合PD-1抗体治疗软组织肉瘤的安全性和有效性，为这一罕见癌肿的治疗提供新的靶点和理论依据。

众所周知，免疫逃逸机制研究是当前肿瘤学研究的前沿，而共抑制分子在其中发挥重要作用。LAG-3是继PD-1之后发现的新型共抑制分子之一。本研究较系统的阐明了LAG-3及其甲基化的功能，并初步探索其在肉瘤免疫微环境中的作用机制及应用价值：（1）发现LAG-3在大部分癌肿中均存在上调表达，其与多癌肿分期、预后密切相关，LAG-3 mRNA可以作为部分肿瘤疾病严重程度的标记物。（2）发现LAG-3的高表达与基因扩增突变有关，首次证实其表达与TMB、MSI和免疫治疗疗效的关系，LAG-3或成为评估免疫治疗反应的指标之一。（3）揭示了泛癌中LAG-3甲基化水平与LAG-3 mRNA及免疫细胞的相关性，阐明了LAG-3不同位点DNA甲基化对软组织肉瘤预后的影响，发现LAG-3 DNA甲基化异常修饰可能参与肉瘤免疫抑制微环境。这为理解LAG-3的免疫抑制功能提供了分子理论基础。（4）发现小剂量的甲基化抑制剂地西他滨可以有效控制肉瘤细胞恶性增殖、促进其凋亡，并可能改善T细胞免疫信号传递，这为理解“去甲基化治疗”理论提供重要的基础，也为肉瘤这一少见癌肿的临床治疗提供新的思路和实验支撑。（5）进一步通过体内动物实验，发现去甲基化并阻断 LAG-3信号通路有助于增强肉瘤免疫应答，二者联合显著地抑制NK细胞和CD4+Foxp3+Tregs，促进IFN-γ分泌，并募集更多的CD8+T细胞，较单药治疗有更好的疗效，为这一热点治疗方案的临床应用提供有应用价值的物质基础。