IL-12/IL-12R与CCR5通过JAK/STAT通路相互调控对分离aGVHD与GVL的机制研究

Acute graft-versus-host disease (aGVHD) is one of the main transplant-related-complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT), and the mortality is high for severe cases. Steroids and second-line agents, such as anti-CD25 monoclonal antibodies, can reduce the graft-versus-leukemia (GVL) effect, leading to the recurrence of leukemia, although the morbidity of aGVHD would be decreased. How to control aGVHD while retaining GVL effect is not clear. Our recently studies indicated that IL-12/IL-12R and chemokine CCR5 were increased and positively correlated in patients with allo-HSCT, and JAK-STAT signaling pathway was heavily activated in severe aGVHD patients, and CD4+CD25high-Treg cells were correlated with GVL effect. We speculated that IL-12/IL-12R and CCR5 might have very important role in separating aGVHD and GVL via JAK/STAT signal transduction. In this project, we want to establish the allo-HSCT mice model, in order to investigate whether IL-12/IL-12R and CCR5 activate and regulate donor T-cells through JAK-STAT signaling pathway in the pathogenesis of aGVHD; to study the impact of IL-12/IL-12R and CCR5 on the activation of Treg cells and the residual tumor of the mice, in order to demonstrate their influence on the effect of GVL; to further explore the outcome of JAK inhibitors blocking JAK/ STAT signal transduction on the aGVHD and GVL effect. Our projects not only reveal the new mechanisms of separation of aGVHD and GVL after HSCT, but also investigate the role of JAK inhibitor to control aGVHD but keep GVL effect. This project would provide the experiment foundation for the separation of aGVHD and GVL, and also provide a new treatment strategy for the control of severe or refractory aGVHD but retain the GVL effects in the clinical practice.

急性移植物抗宿主病（aGVHD）是异基因造血干细胞移植主要并发症之一，如何控制aGVHD但保留移植物抗白血病（GVL）效应，尚不明确。课题组最近发现aGVHD患者IL-12/IL-12R水平与趋化因子受体CCR5呈正相关，且重症患者存在JAK-STAT信号通路异常活化。推测IL-12/IL-12R与CCR5通过JAK/STAT信号通路对分离aGVHD与GVL具有重要作用。本课题拟通过移植小鼠模型，首先明确IL-12R与CCR5是否通过JAK/STAT信号通路激活供者T细胞并相互调控导致aGVHD；再研究两者对Treg细胞活化和移植小鼠肿瘤负荷的影响，阐明对GVL潜在作用规律；最后探讨JAK抑制剂阻断JAK/STAT通路对aGVHD和GVL效应的分离。本项目不仅揭示aGVHD和GVL发生与分离的新机制，同时阐明JAK抑制剂控制aGVHD并保留GVL的作用，为临床治疗重症GVHD提供新的策略。

异基因造血干细胞移植（allo-HSCT）是治愈高危或复发难治恶性血液病的唯一有效方法。如何更好的降低造血干细胞移植后重症或难治aGVHD的发生，同时保留移植后GVL效应，对减少移植相关死亡及提高移植后无病生存，具有重要的临床意义。本项目通过建立同种异基因造血干细胞移植小鼠模型，开展了以下研究：（1）首先，通过体外及体内试验研究IL-12/ IL-12R、CCR5在供者T淋巴细胞活化、增殖以及促进T细胞向GVHD靶器官聚集的作用；通过免疫沉淀和Western印迹检测JAK1/JAK-2/JAK-3磷酸化水平，以及STAT1/ STAT3/ STAT4/ STAT5/ STAT6的表达，明确IL-12/IL-12R信号通路与CCR5相互调控对JAK-STAT磷酸化水平的影响，阐明IL-12/IL-12R与CCR5是否通过JAK-STAT信号通路相互调控在aGVHD发生中的重要作用。（2）通过流式细胞仪检测IL-12R与CCR5被激活前后Treg细胞含量的变化，了解IL-12/IL-12R信号通路与CCR5相互协同作用是否增加Treg细胞的含量与功能。对小鼠白血病模型与实体瘤模型进行同种异基因造血干细胞移植（HSCT），通过生物发光成像技术检测小鼠体内残存的肿瘤负荷，明确了IL-12/IL-12R与CCR5通过JAK-STAT信号通路相互调控在GVL效应中发挥的作用。（3）最后，通过注射JAK抑制剂（Ruxolitinib）之后，阻断IL-12/IL-12R及CCR5介导的JAK-STAT信号通路，降低aGVHD的发生；同时明确JAK抑制剂在抑制aGVHD的同时具有保留GVL效应的作用。 . 本项目以信号通路的形式，探讨造血干细胞移植后GVHD和GVL的发生及分离的机制，阐明了IL-12/IL-12R与CCR5通过JAK/STAT信号通路的相互调控对分离aGVHD与GVL效应的作用及机制；同时揭示了JAK抑制剂在控制aGVHD和保留GVL效应中的作用机制，为实现GVHD和GVL的分离奠定了实验基础，为临床治疗重症或难治性GVHD但保留GVL效应提供了全新的治疗策略。本课题共发表论著6篇：其中SCI期刊5篇，中华血液学杂志1篇。培养硕士研究生3名，博士后1名（目前在读）。国际会议发言1次，国内会议发言2次。