免疫干预PD-1/PD-L1负性信号通路重塑髓性白血病γδ T细胞功能的研究

Gamma delta (γδ) T cells have good application prospect in the treatment of refractory and relapsed myeloid leukemia. But γδ T cells have different subsets with different functions like anti-tumor immune responses and immunomodulatory effects and so on. In our previous study, we found that some cases of acute myeloid leukemia had high proportion of the γδ T regulatory subsets, which had poor clinical outcome. And we also found a novel type of γδ regulatory cells, PD-1 high γδ regulatory cells, associated with poor prognosis. Related regulation mechanisms of this new type of γδ regulatory cells remain unknown. We proposed a new concept that γδ cells might participate in the mechanism of refractory / relapse in myeloid leukemia by PD-1/PD-L1 negative signaling pathway. So in this research project, immune intervention of PD-1/PD-L1 signal pathway intend to remodel the function of γδ cells. That could be a new approach for remodeling γδ T cell function, reversing the T cells immune tolerance of leukemia and improving cellular immune status, and that might help to treat the refractory and relapsed myeloid leukemia. Meanwhile, that could provide the optimization strategy of adoptive immunotherapy of γδ T cells, and help to select or induce more efficient γδ T functional subsets for the more optimal immunomodulatory therapy strategy of refractory and relapsed myeloid leukemia.

γδ T细胞能特异性识别抗原且不受MHC限制，具有抗肿瘤作用，但其存在不同功能亚群，且疾病状态下有着不同程度的功能缺陷。课题前期研究发现髓性白血病患者γδ T细胞的T细胞受体谱系发生偏移且杀伤功能低下，并发现一类新型的与不良预后相关的PD-1 high γδ调节性亚群，但其调控机制不明。因此我们提出假说：PD-1/PD-L1负性信号通路可能参与γδ T细胞功能亚群的调控机制，免疫干预该信号通路可能重塑γδ T细胞的功能。本课题拟通过miRNA差异表达谱分析明确γδ调节性细胞的表观遗传调控特点和潜在干预靶点，深入分析PD-1/PD-L1负性信号对γδ 调节性亚群的调控作用；通过直接阻断PD-1/PD-L1负性信号传递或转换为CD28/41-BB正性信号来恢复或增强γδ T细胞的杀伤活性，并阻断调节亚群的诱导或转化，从而为难治复发髓性白血病的过继性免疫治疗提供更理想的新策略。

γδ T细胞能特异性识别抗原且不受MHC限制，具有抗肿瘤作用，但其存在不同功能亚群，且疾病状态下有着不同程度的功能缺陷。我们发现，AML患者PD1high γδ T、Foxp3+ γδ T和PD1high Foxp3+ γδ T亚群的表达比例显著增加，且复发组>初发组>缓解组。Foxp3+ γδ T细胞亚群高表达比例是AML患者获得完全缓解的危险因素。TIGIT+ Foxp3+ γδ 调节性T细胞表达比例升高与AML患者总体生存率低相关；且TIGIT+ Foxp3+ γδ 调节性T细胞高表达是预后的独立危险因素。CML患者调节性γδ T细胞亚群表达比例存在一定增高，且Foxp3+ γδ T细胞、PD-1high Foxp3+ Vδ1+ T细胞和TIGIT+ Foxp3+ Vδ1+ T细胞高表达为CML慢性期患者经一线TKI类药物治疗后发生警告治疗反应或治疗失败的危险因素。AML患者的γδ T细胞尤其是γδ调节性T细胞，对白血病细胞株的杀伤效应明显减弱。采用PD-1-siRNA干预γδ T细胞中PD-1的表达，发现AML患者的si-PD1转染组对白血病细胞株（K562、HL-60和NB4）及其耐药细胞株（K562R、HL-60/ADR和NB4/R1）的杀伤能力均有一定程度的提高。通过RNA芯片进行γδ 调节性T细胞的miRNA差异表达谱分析，并进一步预测可能结合PD1的miRNA。将miRNA-155抑制剂分别转染难治复发AML患者的γδ T细胞和γδ 调节性T细胞，发现转染后难治复发AML患者的γδ T细胞和γδ 调节性T细胞对白血病细胞株及白血病耐药细胞株的杀伤效应较转染前有所增强。因此，PD-1负性信号通路可能参与γδ T细胞功能亚群的调控机制，免疫干预该信号通路可能重塑γδ T细胞的功能，为特异性抗难治复发髓性白血病的过继性免疫治疗提供更理想的新策略。