KLF2对T细胞迁移的双向调控在GVHD与GVL分离中的作用及机制

How to effectively separate graft-versus-host disease (GVHD) and graft-versus-Leukemia (GVL) effects after allogeneic haematopoietic stem cell transplantation is a major challenge. The traditional ways to inhibit functions of T cells are easy to damage GVL effect. Based on the different target organs of GVHD and GVL, increase of donor T cells in lymphatic or hematologic systems as well as decrease of T cells in GVHD target organs might be a effective strategy to separate them. CXCR3 as the earliest expressed chemokine receptor induces T cell trafficking to several GVHD target organs, while CCR7 contributed to GVL effect leads to T cell homing. Although studies have shown the role of CXCR3 and CCR7 in allogeneic haematopoietic stem cell transplantation, there is no report about combined regulation of them to make T cell physiological distribution. In our previous study, we found that KLF2 could up-regulate CCR7 level whereas down-regulate the expression of CXCR3, and GVHD severity were reduced. In the present study, we plan to modify KLF2 levels of donor T cells by genetic and drug strategies, and clear the role of KLF2 in the separation of GVHD and GVL in transplanted mouse model. Furthermore, the regulatory effect of KLF2 on T cell migration, the interaction region and mechanism of KLF2 and CXCR3 or CCR7 will be explored. If succeed, it will provide precious information and new strategies to separate GVHD and GVL.

如何有效分离移植物抗宿主病（GVHD）与移植物抗白血病（GVL）效应是异基因造血干细胞移植亟需解决的难题。传统上抑制T细胞功能的方法极易损伤GVL效应。根据GVHD和GVL靶器官不同，促使供者T细胞在淋巴血液循环，减少其向GVHD靶器官浸润，可能是两者分离的有效策略。CXCR3是促T细胞迁移到GVHD多个靶器官最早表达的趋化因子受体，CCR7是T细胞归巢实现GVL的关键受体。虽然异基因造血干细胞移植中已有CXCR3和CCR7的相关研究，但靶向两者调控T细胞的合理分布却未见报道。我们前期发现KLF2可上调CCR7而下调CXCR3，减轻GVHD严重程度。本项目拟利用基因或药物干预手段调控供者T细胞KLF2表达，明确KLF2对小鼠移植后GVHD和GVL分离的作用及对T细胞迁移的影响，探索KLF2与CXCR3、CCR7作用位点及机制。如期完成将为实现GVHD和GVL的分离提供新的理论依据。

Allo-HSCT后效应T细胞是介导GVHD和GVL的主要成分，促使供者T细胞在循环系统发挥GVL作用，减少其向GVHD靶器官浸润，是减弱GVHD增强GVL的有效策略。本项目利用他汀药物维持活化T细胞中KLF2表达，促使归巢受体CCR7和CD62L在T细胞表达，而CXCR3和CD44表达降低，从而减少GVHD靶器官中浸润效应T细胞数量，减弱GVHD的病理损伤；且发现他汀药物对T-ALL的杀伤效果，并证明其通过Akt通路发挥抗白血病作用。在该项目资助下完成项目任务书中的内容，并新证实直接抑制效应T细胞的迁移相关酶cdc42，亦可纠正效应T细胞在GVHD受鼠中的病理性分布，从而减轻GVHD。具体研究成果概述如下（1）allo-HSCT后效应T细胞浸润到GVHD靶组织中数量急剧升高，第28天左右趋于平稳，且多个靶组织中浸润的T细胞数量动态变化与病理损伤程度及临床评分一致，表明T细胞的靶器官浸润与其病理损伤及aGVHD的严重程度密切关系。（2）他汀药物不仅可在体外有效维持供鼠骨髓和T细胞的KLF2表达，体内应用亦可阻断KLF2 的降解，且供鼠停药后长时间内KLF2表达不降低，重要的是高表达KLF2的供鼠细胞移植后第28天仍检测到KLF2 在受鼠体内的高表达。（3）氟伐他汀可通过减少效应CD3+ T细胞浸润GVHD靶器官数量从而有效减轻受鼠GVHD临床症状和靶器官病理损伤。（4）氟伐他汀对效应T细胞的迁移调控是通过KLF2介导的CCR7和CD62L归巢受体升高，而下调T细胞靶器官迁移的趋化因子受体CXCR3和活化受体CD44完成。（5）氟伐他汀的应用降低T细胞活化后细胞因子分泌水平，但对血清趋化因子水平无影响。（6）他汀类药物抑制T-ALL细胞增殖，诱导凋亡，使其周期阻滞于G1期，且发现这一作用通过抑制Akt的磷酸化实现。（7）抑制Cdc42蛋白活性可直接抑制T细胞F-actin的聚合，进而影响效应T细胞向SLO的迁移，减少GVHD靶组织浸润，减轻aGVHD严重程度。综上，本项目证实他汀药物通过维持KLF2水平影响效应T细胞受鼠分布，调控GVHD和GVL效应，并揭示了其作用机制。此结果将为实现他汀药物在allo-HSCT病人的临床转化应用提供实际参考价值，为将来彻底实现GVHD和GVL的分离提供新的理论依据。