共抑制分子Tim3介导CD8+T细胞功能低下在脓毒症继发感染中的作用及机制研究

Sepsis-induced immunosuppression is associated with uncontrolled infection. CD8+ T cell dysfunction is one of the major manifestions.Our previous studies showed that inhibitory molecures, i.e., CTLA-4, PD-1, were involved in immune dysfunction during sepsis. T-cell Ig- and mucin-domain-containing molecule-3 (Tim3) was identified as a co-inhibitoy molecule expressed on T helper (Th)1 and CD8+ T cells. Interaction of Tim3 with its ligand, galectin-9, regulates negative immune responses by promoting the death of Th1 cells and induces peripheral tolerance. Our preliminary study showed that Tim3 expression was upregulated significantly on CD8+ lymphocytes in septic mice. We speculated that Tim3 were involved in the pathogenesis of immunosuppression induced by sepsis. The present study was designed to measure the expression of Tim3 on CD8+ T lymphocytes of septic patients and its correlation with CD8+ T cell function. The reversal effect of Tim3 on CD8+ T cell function will also be observed. The expression of Tim3 on CD8+ T cell and its correlation with CD8+ cell function will be investigated during different stage of CLP-induced sepsis. Blockade of Tim3 on its ablility to control a secondary bacterial or fungi infection and CD8+ T cell immunity will be investigated by a two-hit septic animal model. Tim3 mediated CD8+ T cell dysfuntion and the underlying signal transduction mechanism will be validated by an in vivo experiment in CLP mice. This study aimed to illuminate the role of Tim3 in CD8+ T cell dysfunction and the underlying mechanisms, which might provide experimental evidence for treating sepsis via Tim3 signaling in future.

脓毒症免疫抑制期易合并继发感染，CD8+T细胞功能低下是其重要表现。我们前期研究证实，共抑制分子CTLA-4、PD-1介导脓毒症T细胞无能；新近实验发现，共抑制分子Tim3在脓毒症小鼠脾脏CD8+T细胞表达上调。Tim3主要表达于Th1与CD8+T细胞，产生负性调控信号，导致细胞凋亡。脓毒症时Tim3是否介导CD8+T细胞功能低下，促进继发感染这一问题值得深入探讨。本研究拟测定脓毒症患者CD8+T细胞Tim3表达及其与病情关系；利用基因敲除小鼠研究Tim3在脓毒症CD8+ T细胞功能和继发条件致病菌感染中的作用；研究阻断Tim3对脓毒症小鼠抵抗条件致病菌感染的效果；探索Tim3介导脓毒症CD8+T细胞无能的信号机制。本研究力图通过分子、细胞、动物及临床患者,应用流式细胞术等手段，揭示Tim3介导脓毒症后CD8+T细胞无能，继发条件致病菌感染的机制，为脓毒症防治提供理论和实验依据。

Tim3是免疫调节蛋白Tim(T cell immunoglobulin and mucin domain)家族成员，主要表达于T辅助细胞和CD8+T淋巴细胞表面。Tim3分子和其内源性配体Galectin9作用，能特异性结合凋亡细胞上的磷脂酰丝氨酸，从而产生负性调控信号，引发细胞凋亡。研究已证明，Tim3是一种负性调控分子，通过使T淋巴细胞产生免疫抑制反应，而在多种免疫性疾病中发挥重要作用。脓毒症(sepsis)是指感染合并全身炎症反应综合征，通常合并有较重的脏器损坏，有时还可能伴随有多器官功能障碍综合征。随着疾病的发展，脓毒症呈现更为复杂的免疫应答过程。随着危重病治疗手段日益成熟，绝大多数脓毒症患者可以在促炎反应时期生存下来，步入紧接着的免疫抑制期。本项研究深入研究了Tim3在脓毒症小鼠或患者免疫调节过程中的作用，其是否在其病理生理机制中发挥关键作用。我们在动物研究中发现，脓毒症小鼠脾脏CD8+T细胞Tim3分子表达显著增加，抗Tim3抗体可改善脓毒症小鼠7d生存率。抗Tim3抗体对脓毒症小鼠的保护作用可能与其改善脓毒症小鼠肝脏、肺脏病理损伤程度，抑制脓毒症小鼠胸腺和脾脏细胞凋亡，提高腹腔和血液细菌清除率，以及调节腹腔机体免疫功能中性粒细胞数量相关。我们在临床研究中发现，脓毒症患者外周血CD8+T细胞Tim3表达显著上调，其PD-1表达水平也显著升高。通过对123例脓毒症、重症脓毒症、脓毒性休克患者血清标本的ELISA分析发现，脓毒症时外周血Galectin9水平显著升高，其与患者免疫抑制程度相关，是脓毒症患者预后的分子标志物。综合上述结果和分析，本研究认为Tim3在脓毒症病理生理中具有重要的作用，是有潜力的“免疫调节点”靶标，值得进一步深入研究。