OX40表达对双阴性T调节细胞介导的免疫抑制功能的影响及机制研究

Double negative regulatory T cells (DN Treg cells) contribute to antigen specific immune tolerance in organ transplantation. Costimulatory factor OX40 plays a critical role in the regulation of immune T cells. However, immunosuppression function and mechanism of OX40 in DN Treg cells remain poorly defined. Our previous work showed that immunosuppression function of DN Treg cells decreased significantly, when knockout the gene of OX40. Therefore, we hypothesized that OX40 might be involved in the DN Treg cell immunesuppression function. We will first, study the expression of perforin/granzyme and T cells suppression function in OX40 knockout DN T cells. Second, we plan to deliver OX40 signals to DN Treg cells using agonist monoclonal antibody (OX86) and explore the immunoregulatory functions of DN Treg cells when overexpression of OX40. Third, we will intend to study how OX40 recruit the TRAF family members and activate NF-κB pathway by gain/loss-of-function approaches and chromatin immunoprecipitation analysis and investigate the issue of OX40 through which terminal target sites regulate immunesuppression function. Finally, we will improve immunesuppression function of DN Treg cells by regulate this mechanism with IL-2 or OX86 and prolong alloantigen-specific MHC-mismatched skin and islet allograft survival in vivo. Our study is of great significance on reveal the immune regulation function and mechanism of OX40 in double negative regulatory T cells. These findings will provide new ideas for immune tolerance of transplantation strategy.

利用双阴性T调节细胞进行细胞治疗是一种特异性的诱导器官移植免疫耐受的治疗策略。共刺激因子OX40在T细胞的免疫调节过程中起关键作用，但OX40对双阴性T调节细胞的调节机制尚不清楚。我们前期工作发现：相比敲除其他共刺激因子，敲除OX40后双阴性T调节细胞的免疫抑制功能明显下降。据此提出OX40可能参与其功能的调控。本课题拟通过检测颗粒酶/穿孔素的表达和效应T细抑制实验，初步研究敲除OX40对DN Treg细胞免疫抑制功能的影响。其次利用OX86激活OX40通路，结合小鼠皮肤移植模型，深入探索OX40表达对双阴性T调节细胞的调控规律。最后利用功能性获得/缺失实验阐明OX40如何通过下游TRAF家族成员激活NF-κB通路，进而调节免疫抑制功能，并通过染色质免疫共沉淀技术找到OX40的终末靶位点。拟利用信号通路药物增强双阴性T调节细胞的免疫抑制能力，为探索细胞诱导的移植免疫耐受策略提供实验依据。

利用双阴性T调节细胞（DNT）进行细胞治疗是一种特异性的诱导器官移植免疫耐受的治疗策略。共刺激因子OX40在T细胞的免疫调节过程中起关键作用，但OX40对DNT的调节机制尚不清楚。我们前期工作发现：相比敲除其他共刺激因子，敲除OX40后DNT的免疫抑制功能明显下降。据此提出OX40可能参与其功能的调控。.我们利用体内和体外实验研究了OX40对DNT转化率和功能的影响。我们发现OX86（一种OX40激动剂）能提高DNT的转化率，但不能提高DNT的抑制功能。OX40缺失可显著降低DNT的转化率和抑制功能。这种抑制功能的下降是由于OX40对增殖和凋亡双方面的影响，而与穿孔素、颗粒酶和Fas配体无关。除此以外OX40还通过多种信号影响DNT的功能，如Cxcr3、Cd160和Cd30。 .综上所述，我们阐明了OX40促进DNT的转化和维持；Ox40缺乏可通过调节增殖、凋亡和抑制相关基因来降低DNT的体内外调节功能。.这项研究为探索细胞诱导的移植免疫耐受策略提供实验依据。