淋巴细胞活化基因3介导脓毒症时DC和T细胞功能障碍的研究

Dysfunction of dendritic cells (DC) and T lymphocytes is the major cause of immunosuppression during sepsis. Our previous studies showed that PD-1, one type of co-inhibitory molecules, played an important role in sepsis- induced immunosuppression. The lymphocyte activation gene (LAG3) is a ligand for MHC class Ⅱ molecules. CD3/TCR complex-associated LAG3molecules were involved in immune cell functions. However, there is still a debate on the role of LAG3 in DC and T cell function regulation. Whether LAG3 played a role in sepsis was also unclear. Our preliminary experiments showed that LAG3 expression on DC and T cells was upregulated and linearlly correlated with PD-1 level. Blockade of LAG3 inhibited T cell apoptosis, and improved bacterial clearance and survival rate. Our current study was designed to investigate the role of LAG3 on DC and T cell dysfunction during sepsis. First, LAG3 expression on DC and T cells will be detected in septic patients. The relationship between LAG3 expression and DC as well as T cell dysfunction will also be analyzed. Then the regulatory role of LAG3 on DC and T cell dysfunction during sepsis as well as the underlying signaling pathway will be investigated via LAG3-knock-out mice. In this section, the impact of LAG3 on the interaction between DC and T cells will be focused on. Finally, therapeutic value of anti-LAG3 antibody against sepsis as well as DC and T cell dysfunction will be illuminated by CLP mice. This study will illustrate the role of LAG3 in DC and T cell dysfunction during sepsis, so as to provide new target and experimental evidence for therapy of sepsis-induced immunosuppression in future.

DC和T细胞功能障碍是脓毒症免疫抑制的重要原因，我们前期研究证实共抑制分子PD-1在其中发挥重要作用。LAG3是TCR共受体，可与MHC-Ⅱ类分子结合参与调控DC和T细胞功能，但其具体作用尚存在争议。我们预实验发现脓毒症时DC和T细胞上LAG3表达上调，且与PD-1表达线性相关，阻断LAG3可抑制脓毒症小鼠T细胞凋亡并改善细菌清除率和生存率。为验证LAG3是否介导脓毒症时DC和T细胞功能障碍，本课题拟检测脓毒症患者外周血DC和T细胞LAG3表达及其与疾病严重程度和细胞功能障碍的关系；利用基因敲除小鼠明确LAG3对脓毒症时DC和T细胞功能障碍的调控作用和相关信号通路，并且重点探讨LAG3对DC和T细胞相互作用的影响；最后明确抗LAG3抗体对脓毒症小鼠病情发展和DC及T细胞功能障碍的治疗价值。力图揭示LAG3在脓毒症DC和T细胞功能失常中的作用与机制，为脓毒症免疫抑制的防治提供新靶点。

免疫抑制是目前脓毒症患者死亡的主要原因，随着病程的延长，患者往往能渡过脓毒症过度炎症引起的脏器损伤，但是却难以克服后期的顽固性继发感染。共抑制分子是介导脓毒症免疫抑制的关键分子，如PD-1/PD-L1、CTLA-4等。我们前期研究显示，脓毒症小鼠LAG3表达水平也有所升高，作为共抑制分子之一，LAG3也可能参与了脓毒症免疫抑制的发生。因此，本研究拟通过临床试验分析LAG3与脓毒症患者免疫抑制的关系；然后从离体角度研究LAG3对DC和T细胞功能状态的调控作用；最后，通过在体实验探讨LAG3对脓毒症发生发展的在体干预作用。结果显示，脓毒症患者CD4+和CD8+ T细胞LAG3表达水平显著升高，且CD4+ T细胞LAG3表达水平与患者APACHE II评分线性相关；构建LAG3基因敲除小鼠，分离DC和T细胞后发现，LAG3 KO脓毒症小鼠T细胞凋亡率显著下降，分泌IFNγ的能力显著增强，胸腺T细胞数量显著增多，但是DC功能改善不明显；在体以抗LAG3中和抗体干预或LAG3 KO后脓毒症小鼠7d生存率显著改善，细菌清除率增强。并且，以抗LAG3中和抗体干预后，脓毒症小鼠血清炎症因子水平降低、肺脏干湿重比降低，血和脾脏T细胞计数恢复，胸腺和脾脏细胞凋亡减轻。因此，本项目证实了LAG3是脓毒症中介导免疫抑制的关键分子，利用LAG3作为靶点进行干预可有效改善脓毒症小鼠的预后，值得临床推广。本项目目前已发表SCI论文11篇，其中影响因子大于5分的2篇，包括麻醉学领域的顶级期刊《Anesthesiology》1篇。