间充质干细胞通过mTOR通路调控滤泡辅助性T细胞治疗抗体介导排斥反应的机制研究

Antibody-mediated rejection (AMR), induced by donor specific antibody (DSA) derived from B lymphocytes, is an important risk factor for renal allograft dysfunction. AMR in kidney transplantation is refractory under current available therapeutic regimens. Follicular helper T cells (Tfh) is the key to B cells differentiation and maturation in germinal centers thus is critical for DSA production. We have completed and reported the first study which demonstrated that mesenchymal stem cells (MSCs) significantly decreased DSA level in renal allograft recipients with AMR (Am J Transplant 2014). Further study showed that the proportion of Tfh reduced in the peripheral blood in AMR recipients after MSCs treatment. Decrease of Tfh proportion in lymph nodes and spleen was seen in murine cardiac transplant acute rejection models after MSCs treatment. Further ex vivo study showed that MSCs suppressed Tfh differentiation and downregulated the level of phosphorylated mTOR, which is a regulatory protein critical for Tfh differentiation. We therefore propose a hypothesis: MSCs inhibit the differentiation and maturation of B cells in germinal centers by suppression of Tfh differentiation via regulation of mTOR pathway, thus reduce DSA production and exert therapeutic effect on AMR. Based on our previous studies, we intend to confirm the effect of MSCs on Tfh differentiation and identify the critical role of mTOR and downstream signaling pathway in it by performing both ex vivo and in vivo studies. This project will provide new theoretical explanations and experimental evidence for an in-depth understanding of the underlying mechanism of the immunomodulatory effect of MSCs on B lymphocytes in transplantation settings.

B细胞产生供体特异性抗体（DSA）介导的排斥反应（AMR）是导致移植肾远期失功的重要危险因素，治疗棘手且疗效不佳。滤泡辅助性T细胞（Tfh）是促进生发中心B细胞分化成熟分泌DSA的关键。我们首次明确了间质干细胞（MSCs）能明显降低AMR患者的DSA水平（Am J Transplant. 2014）；MSCs治疗后AMR患者外周血的Tfh比例降低，且小鼠心脏移植受体的淋巴结和脾脏Tfh减少；体外共培养MSCs能抑制T细胞向Tfh分化，并降低其分化调控蛋白mTOR磷酸化水平。据此提出科学假设：MSCs通过调控mTOR通路抑制Tfh分化，阻碍B细胞分化成熟，减少DSA产生。在前期基础上，拟通过体内外实验进一步明确MSCs对Tfh分化的影响，重点探明mTOR及其下游信号通路在其中的关键作用，将为深入认识MSCs对B细胞的移植免疫调节机制提供新的理论解释和实验依据。

由供体特异性抗体（DSA）攻击移植肾所引起的抗体介导排斥反应（ABMR）是导致肾移植远期失功的重要原因，目前仍缺乏有效的治疗手段。课题组前期探索发现间充质干细胞（MSC）可明显提高肾移植ABMR患者的治疗效果，在此基础上，课题组开展前瞻性临床试验（NCT02563340），结果显示，在肾移植术后慢性ABMR患者中使用MSC能有效降低DSA抗体水平，同时维持肾功能避免其恶化，该方案不良反应极少，患者耐受性佳。课题组进一步开展体内外实验，探明了MSC可通过抑制滤泡辅助T细胞的增殖分化，从而影响生发中心B细胞分化成熟和DSA产生，发挥治疗ABMR、延长移植物存活时间的作用。另外，课题组积极探寻ABMR特异性生物标记物并明确其临床应用价值。课题组明确了B细胞激活因子（BAFF）升高能提示肾移植术后发生ABMR风险增加（RR 1.90），即BAFF是ABMR的预测性标志物，但诊断性试验却证实了BAFF不是ABMR的有效诊断性标志物。课题组还通过临床研究证实了供体特异性游离DNA具有识别肾移植ABMR损伤的能力（AUC-ROC 0.90），是具有良好应用前景的ABMR早筛标志物。