sFGL2/FcγRIIb/Btk信号通路在ITP患者GP特异性Treg诱导树突状细胞致耐受性中的作用研究

Functional abnormalities of dendritic cells (DCs) and T cells play important roles in the pathophysiology of ITP. Our previous study demonstrated that CD4+CD25- T cells could be successfully induced into GP-specific CD4+CD25+ regulatory T cells (Tregs) in vitro, which could further drive DCs to differentiate into tolerogenic DCs, and the precise mechanisms remain to be elucidated. Soluble fibrinogen-like protein 2 (sFGL2) is one of the most important inhibitory effector cytokines secreted by Tregs. It has been demonstrated that sFGL2 exerted the inhibitory effect on DCs via acting on the inhibitory FcγRIIb. In addition, our recently published article on Blood revealed that ITP had decreased expression of inhibitory FcγRIIb, which in parallel with the decreased ability of phagocytosing and presenting autologous oposinized platelets by APCs. Based on our above-mentioned study, we speculate that sFGL2/FcγRIIb/Btk signaling pathway played pivotal roles in the induction of tolerogenic DCs by GP-specific regulatory T cells. To test the hypothesis, we intend to use a variety of molecular biological methods, such as blocking antibodies, engineering technologies, to neutralize, downregulate or upregulate the expression of sFGL2, FcγRIIb, Btk, or inhibit the phosphorylation of Btk, and next evaluate the effect of these interventions on the GP-specific tolerogenicity of DCs. Moreover, the feasibility of exogenous sFGL2 or FcγRIIb upregulation for disrupting the pathophysilolgy process of ITP will be evaluated, thus providing new clues for the management of ITP.

DCs、T细胞异常在ITP发病中起关键作用。课题组前期发现：ITP患者CD4+CD25- T细胞可成功诱导为GP特异性CD4+CD25+ Tregs，该Tregs可进一步诱导致耐受性DCs，但其分子机制尚不明确。sFGL2是Tregs的重要效应因子，通过作用于DCs FcγRIIb发挥抑制效应。课题申请者最新发现：ITP患者存在FcγRIIb表达下调，伴单核/巨噬细胞吞噬提呈血小板能力增强。本研究首先提出sFGL2/FcγRIIb/Btk信号通路在Tregs诱导DCs致耐受性过程中发挥关键作用这一假说，为验证该假说，本研究拟应用基因工程等技术中和/阻断/上调sFGL2、FcγRIIb、Btk基因表达，或阻断Btk磷酸化，观察其对DCs免疫致耐受效应的影响，双向验证该通路调节DCs分化的作用；同时探讨外源性sFGL2或上调FcγIIb对临床干预ITP发生发展的可能性，为ITP治疗提供新思路。

研究背景 纠正Tregs数量及功能缺陷对于重建ITP患者免疫耐受有重要意义。前期我们成功体外诱导GP特异性CD4+CD25+ Tregs (iTregs)，并进一步诱导免疫耐受，但其机制尚待明确。 研究内容 本课题在前期研究基础上，鉴定了iTregs相关表型及功能；测定iTregs作用后DCs sFGL2/FcγRIIb/Btk 通路相关信号分子表达情况；利用sFGL2抗体或rhsFGL2阻断或上调培养体系sFGL2作用，检测iTregs诱导DCs表型功能及相关信号分子改变。检测静脉sFGL2 注射对ITP模型鼠影响。 重要结果 1、（1）TIGIT+ iTregs为发挥免疫抑制效应的最主要的iTregs亚群，其高表达效应细胞因子sFGL2；（2）TIGIT+ iTregs作用后DCs其由成熟DCs向非成熟DCs（imDCs）转化，该过程DCs Btk及mTOR活性上调。2、sFGL2阻断抗体、rhsFGL2可分别阻断及促进mDCs向致耐受性DCs分化，Btk、mTOR活性起关键作用。3、ITP模型鼠注射sFGL2后，其血小板水平回升、抗体水平下降，伴脾脏DCs CD83、CD86表达下调及抑制性FcγRII上调。 关键数据 1、（1）T细胞体外诱导后Foxp3+ Tregs比例显著升高且TIGIT、sFGL2表达明显升高，并显著抑制效应性T细胞增殖；（2）TIGIT+ iTregs作用后DCs CD83、CD86、HLA-DR表达明显下调，伴Btk及mTOR底物p70S6K、4E-BP1磷酸化水平明显增加。2、sFGL2阻断抗体可削弱TIGIT+ iTregs-DCs抑制效应性T细胞增殖能力，伴DCs BTK、p70S6K、4E-BP1磷酸化水平下调。 3、rhsFGL2可抑制DCs诱导的效应性T细胞增殖，伴DCs Btk及mTOR底物p70S6K、4E-BP1磷酸化水平上调。4、rhsFGL2注射可提升ITP模型鼠血小板水平，上调脾脏DCs抑制性FcγRII/CD32表达并下调CD83、CD86表达水平，并降低血小板GPIIb/IIIa抗体水平。科学意义 本研究提示：TIGIT+ iTregs可通过sFGL2诱导致耐受性DCs，FcγRIIb、Btk、mTOR为该程关键信号分子。该发现有助于进一步揭示ITP免疫失耐受发病机理。