PD-L1抑制性CAR-T细胞治疗人抗鼠异种急性移植物抗宿主病的机制研究

Acute graft-versus-host disease was a major complication of allogeneic hematopoietic stem cell transplantation. Especially, it is that the non-recurrent mortality rate of refractory and severe GVHD was increased. It was a major mechanism that T cells proliferation and activation lead to tissue damage in the development of aGVHD. PD1 / PD-L1 pathway can balance T cell activation and inhibition which maintaining peripheral immune tolerance. Previous studies found that CD4 + and CD8 + T cells in aGVHD patients showed PD1hiPD-L1lo and in human anti-mouse aGVHD model was re-verified. To this end, we propose a scientific hypothesis: T cells with PD1hiPD-L1lo immunophenotype may be promote the progress of aGVHD. To test this hypothesis, we constructed PD1loPD-L1hi CAR-T that can be removed the killing function, retained its proliferative activity, and achieved the dynamic expression of PD-L1 triggered with PD1 of T cells. MLR, western blot, live imaging and human anti-mouse aGVHD model were used to explore the mechanism of PD1loPD-L1hi CAR-T in the treatment of aGVHD. It was that we may explore the role of T cell PD-L1 in aGVHD immune response and provide a new strategy for the treatment of acute graft-versus-host disease with PD1loPD-L1hi CAR-T.

aGVHD是allo-HSCT的主要并发症之一，尤其难治、重度aGVHD增加了非复发死亡。T细胞增殖和激活促进组织损伤导致aGVHD；PD1/PD-L1通过平衡T细胞激活与抑制而维持外周免疫耐受。前期研究发现：aGVHD患者CD4+和CD8+T细胞呈现PD1hiPD-L1lo；并在人抗鼠aGVHD模型中得到再次验证。为此，我们提出科学假说：allo-HSCT中PD1hiPD-L1loT细胞促进了aGVHD的进展，增加T细胞PD-L1的表达能改善aGVHD；为了验证该假说:我们构建了PD1loPD-L1hiCAR-T, 由PD1触发其动态表达PD-L1，并去除CAR-T的杀伤性，增强其增殖活性。采用MLR、WB、活体成像、人抗鼠aGVHD模型等探索PD1loPD-L1hiCAR-T治疗异种aGVHD的作用机制；探索T细胞PD-L1在aGVHD免疫应答中的作用，为aGVHD的治疗提供新的策略。

aGVHD是allo-HSCT的主要并发症之一，尤其难治、重度aGVHD增加了非复发死亡。T细胞增殖和激活促进组织损伤导致aGVHD；PD1/PD-L1通过平衡T细胞激活与抑制而维持外周免疫耐受。我们课题组通过对aGVHD模型中的T细胞单细胞转录组测序，临床aGVHD患者、aGVHD动物模型的数据分析证实：在异基因造血干细胞移植中aGVHD的组织中的T细胞（比如肝脏、肺部），即使T细胞表面PD1，TIM3，CTLA-4，LAG3四个耗竭标志同时表达，仍然不能确定其是否进入终末耗竭。T细胞拟时序分析提示这其中一部分T细胞仍然表现出很强的杀伤功能，而另一部分T细胞杀伤功能逐渐丧失，进入终末耗竭。对这两个T细胞亚群的鉴定，我们课题组正在开展相关研究。 因此，研究者认为allo-HSCT中PD1hiPD-L1loT细胞的意义完全不能等同于肿瘤微环境中的T细胞，其参与并促进了aGVHD的组织损伤。课题组以PD1为靶点，设计全新的PD-L1抑制性CAR-T，祛除CAR-T的杀伤性，增强其增殖活性。同时采用PD-L1膜外域取代传统的单链抗体，成为新型CAR-T的胞外域，通过PD1/PD-L1向T细胞传递抑制信号，并能够实现aGVHD组织中局部的动态扩增，从而实现改善aGVHD的组织损伤。动物实验证实：构建aGVHD模型，小鼠发病状态（治疗，而非预防）时，在没有免疫抑制剂应用的情况下，仅给予PD-L1抑制CAR-T细胞能够延长人抗鼠aGVHD小鼠的生存时间约2周时间。 综上，研究者认为PD-L1抑制性CAR-T有望成为一种安全、有效的aGVHD治疗新策略。