PD-1+Treg化疗后优势存活在急性髓系白血病免疫逃逸中的作用及机制研究

Acute myeloid leukemia (AML) is the most common subtype of acute leukemia in adults, accounts for the largest number of annual deaths from leukemia. Treatment of AML with current chemotherapy regimens achieved overall survival rates at 5 years no more than 40%。Relapse after chemotherapy are responsible for the predominant mortality of AML. However, the mechanism of post-chemotherapy relapse remains unclear. Evasion from immune attack of residual myeloblasts might be one of the prerequisites of AML relapse. Therefore, exploring the mechanism of relapse by focusing on immune evasion of residual myeloblasts after chemotherapy are critical for prevention of AML relapse.. The evidence of PD-L1/PD-1 axis mediating immune evasion in tumor settings including AML were well established. However, the data largely came from high leukemic loads circumstances such as de novo AML settings or complete hematological relapse settings, in which most studies focus on the PD-L1/PD-1 axis and T cell exhaustion. Notably, AML patients in complete remission are exposed in very low leukemic antigen burden, Moreover, recovery of normal hematopoiesis after remission generates new-born T cells. Significantly Lower exhausted T cells proportions in remission than in hematological relapse were reported. These date indicated that T cell exhaustion in remission status might be not as important as in higher leukemic load settings. Other mechanisms might be responsible for the immune evasion in early stage after chemotherapy. Recently, PD-L1/PD-1 axis were demonstrated to play an important role in maintenance of function and amounts of regulatory T cells (Treg). PD-1+Treg displayed more intensive immunosuppressive capacity than PD-1(-)Treg . . Our previous data showed that PD-L1(+)AML patients displayed higher percentages of PD-1+Treg than that PD-L1(-)AML patients after reduction therapy. Moreover, higher frequency of PD-1+Treg were observed in PD-L1(+)AML patients after chemotherapy than that before treatment. To demonstrate whether increased PD-1(+)Treg after chemotherapy were associated with its resistance to chemo-agents, studies in vitro were performed. We found lower apoptosis of PD-1+Treg than that PD-1(-)Treg after doxorubicin treatment. In addition, PD-1+Treg expressed higher ABC transporter: ABCB1 than PD-1(-) Treg. Intriguingly, PD-L1 stimulation could induce upregulation of ABCB1 expression in PD-1+Treg, which were related to PTEN/AKT pathway. It was reported that AKT could promote foxo3a to translocate out of nuclei and induce its degeneration in cell plasma. FOXO3a could bind to the region near the promotor of ABCB1, thereby inducing the upregulation of ABCB1. Take above data into consideration. We hypothesize that PD-L1/PD-1 axis could induce survival advantages of PD-1+Treg after chemotherapy by upregulation of ABCB1 expression through PTEN/AKT/FOXO3a pathway ,subsequently contribute to immune evasion of residual AML blasts. . Experiments in vitro and vivo were designed to verify the hypothesis mentioned above. This project for the first time explore the mechanism of immune evasion of residual myeloblasts after chemotherapy from the insight of chemoresistance induced survival advantages of PD-1+Treg by PD-L1/PD-1 axis. The fulfillment of the project would provide potential immune targets for prevention AML relapse and indication for the time to initiate the immune intervention.

复发是导致急性髓系白血病（AML）患者死亡的重要原因，AML残留免疫逃逸是复发的重要环节，然其机制未明。研究发现PD-L1/PD-1轴与AML复发相关，而PD-1+Treg较PD-1(-)Treg具有更强免疫抑制功能，我们预实验发现PD-L1(+)AML患者化疗后PD-1+Treg比例高于化疗前,体外实验发现PD-1+Treg对阿霉素耐药，PD-L1刺激可上调PD-1+Treg 耐药蛋白ABCB1表达，且与PTEN/AKT通路有关；而研究证实PTEN/AKT通路可上调FOXO3a，而后者促进ABCB1表达。因此，我们推测：PD-L1/PD-1轴可能通过PTEN/AKT/FOXO3a途径上调ABCB1表达而介导PD-1+Treg化疗后优势存活，继而产生更强的免疫抑制效应以参与化疗后AML的免疫逃逸。本课题拟在体外、动物及临床水平证实该假设，其完成将为AML复发的预防时机及干预靶点提供新线索。

AML化疗后复发是导致AML死亡的主要原因，残留白血病的免疫逃逸是复发的前提，然其形成机制迄今未明。本项目通过体内外实验研究取得如下结果：.1、证实AML细胞可通过PD-L1/PD-1轴通过上调ABCB1介导PD-1+Treg化疗后的优势存活。2、证实PD-L1/PD-1轴介导Treg的ABCB1表达上调与PI3K/ AKT/FOXO3a通路相关。3、证实AML可通过PD-L1/PD-1轴可介导PD-1+Treg化疗后的优势存活，4、证实PD-1+Treg化疗后的优势存活抑制CD8+T细胞功能，参与AML残留的免疫逃逸并导致AML的复发。这些研究成果对AML化疗后早期免疫逃逸机制的阐明，对临床中传统化疗免疫治疗联合提供了线索，为化疗终止后早期免疫治疗干预提供了靶点，同时对于免疫治疗时机也提供了理论依据。本项目发表SCI论文2篇，ASH Poster 1篇。