利用TIGIT+调节性细胞诱导ITP患者Th亚群平衡重建及机制研究

It has been well established that elevated Th1, Th17 cells and decreased Th2 frequencies were involved in the pathophysiological process of chronic ITP. Restoration of Th subset balance could exert beneficial effects on the correction of immune dysregulation. TIGIT+CD4+CD25+ Foxp3+ Tregs were a newly identified Treg cell subset with distinct phenotype and function. TIGIT+ Tregs could inhibit differentiation of Th1 and Th17 cells, but promote Th2 cell differentiation by secretion of sFGL2. In patients with active ITP, we recently observed that frequencies of peripheral TIGIT+ Tregs were decreased. Based on our previous report about ex vivo induction of CD4+CD25+ Foxp3+ Tregs from CD4+CD25- T cells, we intend to expand and purify TIGIT+ Tregs in vitro, and explore their effect on the differentiation and proliferation of different Th subsets derived from chronic ITP patients. Also, we plan to evaluate the possibility of reestablishing Th subset balance in murine model of ITP by adoptive transfer of in vitro-expanded TIGIT+ Tregs, and investigate the underlying mechanisms by interfering with several crucial signaling molecules using RNA interference or antibody blockade technology. Conduction of the project will help to develop novel strategies for the management of ITP.

Th1、Th17细胞增高及Th2细胞降低在慢性ITP发病中起重要作用。恢复Th亚群平衡有助于纠正ITP免疫紊乱。TIGIT+CD4+CD25+ Foxp3+ Tregs是一群表型和功能特异的Treg亚型，其可显著抑制naive T细胞向Th1、Th17细胞分化，并分泌sFGL2促进Th2细胞分化。课题申请者最新发现，ITP患者外周血TIGIT+ Tregs比例明显降低，结合既往课题组利用CD4+CD25- T细胞成功诱导CD4+CD25+ Foxp3+ Tregs经验，本研究拟体外诱导扩增并纯化TIGIT+ Tregs，观察其离体培养状态对慢性ITP患者Th亚群分化增殖影响，同时探讨纯化回输TIGIT+ Tregs重建ITP模型鼠Th亚群平衡的可能性。进一步利用基因干扰或特异性抗体阻断技术，干预TIGIT+ Tregs关键信号传导分子，研究其对Th亚群平衡重建的影响，为ITP治疗提供新思路

Th1、Th17细胞增高及Th2细胞降低在慢性ITP发病中起重要作用，恢复Th亚群平衡有助于纠正ITP免疫紊乱。TIGIT+CD4+CD25+ Foxp3+ Tregs是一群表型和功能特异的Treg亚型，其可显著抑制naive T细胞向Th1、Th17细胞分化，但其在ITP中的表达及功能尚不明确。本研究进行了体外试验和小鼠模型体内实验，结果发现，ITP患者外周血TIGIT+ Tregs水平及免疫抑制功能显著低于健康对照。转录组测序示：ITP患者TIGIT+ Tregs中有673个基因显著上调，548个基因显著下调，其中涉及体液免疫、细胞黏附分子、细胞因子受体等通路差异，呈现出NF-κB、Jun、β-catenin、SP1、STAT1、STAT3信号通路的明显激活。我们在体外将ITP患者CD4+CD25- T细胞成功诱导扩增为TIGIT+Tregs，诱导后该细胞群体FGL2表达及免疫抑制能力明显高于TIGIT- Tregs。在Th1及Th17极化条件下，体外诱导扩增所得TIGIT+Tregs可通过抑制T-bet、RORγt表达及STAT3磷酸化，分别抑制naive T细胞向Th1及Th17方向极化增殖；sFGL2中和后TIGIT+ Tregs对Th1、Th17的抑制作用减弱。体内实验部分，我们分选小鼠naïve T细胞，体外诱导TIGIT+ Tregs及TIGIT- Tregs，构建ITP主动模型小鼠并进行Tregs回输。结果示：TIGIT+ Tregs及sFGL2注射均可显著提升小鼠模型血小板水平。其中，TIGIT+ Tregs输注组血小板水平回升最为明显，且该组小鼠的Th1、Th17亚群恢复至健康对照水平。因此，TIGIT+ Tregs是一个重要的Tregs亚群，可显著抑制Th1、Th17亚群分化增殖，体外扩增TIGIT+ Tregs并体内回输有助于恢复ITP患者Th亚群平衡，重建ITP患者免疫耐受。