GPR15/GRK5/β-arrestin信号轴调节T细胞活化在急性移植物抗宿主病发生中的作用研究

Intervention on activation of T cells remains a key therapy in counteracting acute graft-versus-host disease (aGVHD). G protein-coupled receptor 15 (GPR15) has been implied to involve in several kinds of inflammatory diseases. Expression of GPR15 was up-regulated on T cells and antigen presenting cells (APCs) after activation of these cells. We previously found that GPR15 plays a role in mediating murine aGVHD. GPR15 mediated suppression on activation of T cells in association with up-regulation of G-protein coupled receptor kinase 5 (GRK5). However, the mechanism, through which GPR15 regulates activation of T cells, remains unknown. Based on all these findings, we postulate that GPR15 might be a promising target in regulating activation of T cells. In this project, we plan to study the role of GPR15/GRK5/β-arrestin signaling pathway in regulating activation of T cells as well as mediating pathogenesis of aGVHD in mice. We also plan to explore the biased ligands, which would selectively stimulate GPR15/GRK5/β-arrestin signaling pathway. Our study should expand our knowledge in T cell-activation. Identification of GPR15/GRK5/β-arrestin biased ligands would provide us promising new strategies for separating aGVHD and GVL not only in allogeneic hematopoietic stem cell transplantation, but also in other adoptive transfer therapeutics such as allogeneic CAR-T treatment.

精准靶向调节T细胞活化是防治急性移植物抗宿主病（aGVHD）的理想追求。G蛋白偶联受体15（GPR15）主要表达于活化T细胞和抗原提呈细胞表面，我们前期研究发现，该受体参与小鼠aGVHD的发生，GPR15抑制T细胞活化与G蛋白偶联受体激酶（GRKs）信号通路密切相关，提示GPR15可能是调控T细胞活化的潜在有效靶标，但其调控T细胞活化的分子机制尚不明确。为此，本项目拟在前期研究的基础上，运用条件性基因敲除小鼠，深入探索GPR15/GRK5/β-arrestin信号通路对T细胞活化的影响，明确该通路在aGVHD发生中的作用，并进一步探索选择性激活此信号通路的配体。如获成功，则可进一步深入解析aGVHD中T细胞活化的分子机制，而探索GPR15/GRK5/β-arrestin通路的选择性配体可为异基因造血干细胞移植和CAR-T细胞治疗后aGVHD和GVL分离策略提供新思路。

精准靶向T细胞活化是防治急性移植物抗宿主病（aGVHD）的理想追求。G蛋白偶联受体15（GPR15）主要表达于效应性T细胞和调节性T细胞表面。本项目研究发现，GPR15受体及其下游的βarrestin2通路可抑制T细胞活化并减轻aGVHD。经过筛选GPCR化合物库，我们鉴定出TRAP-6（thrombin receptor activating peptide-6）可结合GPR15受体并激活下游βarrestin2通路，通过抑制NF-κB活化减少效应性T细胞的含量。得到如下结论：① GPR15L可减轻小鼠aGVHD，增加T细胞中GRK5的表达水平，减少T细胞NF-κB和EKR的磷酸化水平；② 敲除GPR15可加重aGVHD，并取消GPR15L介导的抗aGVHD作用，敲除GPR15取消GPR15L在体外抑制混合淋巴细胞反应的作用，敲除GPR15减少T细胞内βarrestin2通路的活化水平；③ 在GPCR化合物库中筛选得出8种可激活GPR15/βarrestin2通路的小分子化合物，其中TRAP-6（thrombin receptor activating peptide-6）可激活T细胞的GPR15/βarrestin2通路，TRAP-6和结合GPR15并抑制体外T细胞在体外活化；④ TRAP-6可减轻小鼠aGVHD，该作用依赖于T细胞表达GPR15；⑤ TRAP-6抑制T细胞活化的机制为：TRAP-6激活βarrestin2，βarrestin2可通过结合TAB1抑制TAK1和NF-κB磷酸化；⑥ TRAP-6原本为F2R（coagulation factor II thrombin receptor）的配体，通过氨基酸突变的方式获得的TRAP-6突变体（F->A）保留了与GPR15相互作用的效应，但避免了与F2R结合而导致血小板聚集的效应。通过本研究，我们证实了GPR15在aGVHD中的调控作用，也发现了可用于靶向调节GPR15受体的潜在手段。