共抑制分子VISTA调节T淋巴细胞功能障碍在脓毒症免疫抑制中的作用研究

T cell dysfunction was the main cause of sepsis associated immunosuppression. Co-inhibitory molecules played an important role in the development of immunosuppression during sepsis, and the blockade of these pathways improved T cell functions. VISTA, a new member in B7 family, has been demonstrated to exert both ligand and receptor function. VISTA could function as a ligand to negatively regulate T cell activation. Our preliminary experiments found that VISTA was up-regulated on the splenic T cells in septic mice, and anti-VISTA antibody improved survival during sepsis. This study was designed to further establish the specific role of VISTA in sepsis induced T cells dysfunction. First, VISTA expression on T cells will be examined in septic patients and the relationship between VISTA expression and immune function as well as patients outcome will be analyzed. VISTA Ig and anti-VISTA antibody will be used to investigate the regulatory effects of VISTA on T cells and the underlying mechanisms during sepsis. Finally, we will evaluate the targeted therapeutic effects of anti-VISTA antibody on T cells dysfunction by using septic mice. This study will illuminate the definite role of VISTA in sepsis-induced T cells dysfunction, so as to provide new target in the prevention and therapy of sepsis associated immunosuppression.

T淋巴细胞功能障碍是脓毒症免疫抑制的重要原因。共抑制分子表达上调参与脓毒症免疫抑制的形成，阻断共抑制分子通路可以改善T细胞的功能。VISTA是B7家族的新成员，同时具备配体和受体的信号活性，负性调节T细胞的活化。我们在前期的预实验中发现，脓毒症小鼠脾脏T细胞表达VISTA升高，抗VISTA抗体可以改善小鼠的生存率。为求进一步明确VISTA是否参与调控脓毒症T淋巴细胞功能障碍，本课题拟检测脓毒症患者T细胞VISTA的表达变化，明确其与患者免疫功能和预后的相关性；利用VISTA Ig和抗VISTA抗体明确VISTA对脓毒症T细胞功能障碍的调控作用及机制；最后在体探讨抗VISTA抗体对T细胞功能障碍的靶向治疗价值。力图阐明VISTA在脓毒症T淋巴细胞功能障碍中的调控作用，为脓毒症免疫抑制提供新的防治靶点。

研究背景.B7家族成员及其受体是重要的免疫检查点分子，在脓毒症免疫炎症反应中发挥重要的调节作用。VISTA是B7家族的新成员，同时具备配体和受体的信号活性，活化VISTA信号通路可以负性调节T细胞和骨髓源性免疫细胞的功能。本研究拟应用激动性VISTA抗体（MH5A）在体干预脓毒症小鼠，以探讨其潜在的治疗作用及机制。.研究内容及结果.利用雄性C57BL/6小鼠构建腹腔感染的脓毒症模型，于建模后24h和72h小鼠，分别检测外周血、脾脏及重要脏器中的VISTA表达变化。检测MH5A抗体对脓毒症小鼠7天生存率、重要脏器病理、细菌清除率、外周血细胞因子、胸腺及脾脏淋巴细胞凋亡。体外纯化培养脓毒症或假手术组小鼠来源的CD3+T细胞，并给予TNF-α诱导细胞凋亡，观察MH5A抗体对T细胞凋亡的影响。体外培养腹腔来源的巨噬细胞，观察MH5A抗体对LPS诱导巨噬细胞分泌细胞因子的影响。结果发现，小鼠脾脏、肺、肝脏及外周血的淋巴细胞和单核细胞上均大量表达VISTA分子，建立模型后的小鼠各脏器免疫细胞上VISTA分子表达并无显著变化。通过尾静脉注射MH5A抗体可以改善脓毒症小鼠的生存率，减轻重要脏器的损伤，降低全身的炎症反应和细菌负荷，逆转胸腺和脾脏的淋巴细胞凋亡。体外实验进一步证实抗VISTA抗体可以抑制淋巴细胞的凋亡、降低巨噬细胞对LPS刺激引起的细胞因子释放。.科学意义.本课题通过体内外的实验，初步明确了共抑制分子VISTA参与调控脓毒症T淋巴细胞和巨噬细胞的功能，利用MH5A抗体可以靶向治疗脓毒症相关的免疫功能紊乱。本研究发现VISTA与其他免疫检查点分子有所不同，在重要脏器和未活化的免疫细胞中表达丰富，提示其在维持脓毒症早期免疫反应中即可发挥重要作用，本课题可为脓毒症免疫炎症调控提供了新的机制和治疗方向。