ICOS/ICOSL信号通路在调节性T细胞促进急性髓系白血病细胞免疫逃避中的作用及其机制研究

How to promote the immune system to fight against tumor cells is always a key problem in tumor treatment. In our previous paper,we have found that the regulatory T cell (Treg) frequencies were markedly higher in peripheral blood (PB) and bone marrow (BM) from patients with acute myeloid leukemia (AML) than those from healthy volunteers (HVs). Suppression of the proliferation of CD4+CD25- cells mediated by Treg was higher in AML patients compared to HVs. In our preliminary studies, we have shown that the frequencies of ICOS+ Treg were dramatically increased in BM from patients with AML, and the reason remains unclear. The expression of ICOSL was also found in most AML cell lines as well as primary AML cells from patients with AML. We hypothesize that the ICOS/ICOSL costimulatory pathway plays a central role in the immune evasion of AML cells mediated by Treg in patients with AML. We will investigate the role and mechanism of the ICOS/ICOSL costimulatory pathway in the production, differentiation, and expansion of Treg in BM from patients with AML using cell sorting, in vitro co-culture, over-expression, RNA interference, etc. We will elucidate the role and mechanism of the ICOS/ICOSL costimulatory pathway in the inhibition of immune effector cell killing AML cells mediated by Treg, by which we investigate the potential therapeutic targets for immunotherapy. We will also evaluate the antitumor effect of anti-ICOSL mAb in an AML mouse model engrafted with mouse AML cell line C1498. These studies will clarify the main mechanism of immune escape in AML and provide a theoretical basis for AML immunotherapy and contribute to the prevention and treatment of AML.

如何促进机体免疫系统杀伤肿瘤细胞一直是肿瘤治疗的关键问题。我们前期研究发现急性髓系白血病（AML）患者骨髓和外周血中调节性T细胞（Treg）频率增多，抑制免疫效应细胞功能强大，进一步分析发现骨髓Treg中ICOS+Treg增多，其原因目前尚不清楚。结合我们前期研究发现AML细胞表达ICOSL，本项目提出AML细胞通过ICOS/ICOSL信号通路调控Treg增多，并借此逃避免疫效应细胞杀伤的假说。为验证此假说，本项目拟用过表达、RNA干扰、细胞分选、共培养、动物实验等方法，阐明ICOS/ICOSL信号通路在AML患者骨髓Treg产生、分化及扩增中的作用及机制；阐明ICOS/ICOSL信号通路在AML患者骨髓Treg抑制免疫效应细胞杀伤AML细胞中的作用及机制；探讨抗ICOSL抗体调控体内免疫系统杀伤AML细胞的作用。本项目对阐明AML细胞免疫逃避机制有重要意义，为AML免疫治疗提供理论基础。

急性髓系白血病(AML)是起源于造血干细胞的恶性克隆性疾病，常伴随机体免疫系统异常，如何促进机体免疫系统杀伤肿瘤细胞一直是肿瘤治疗的关键问题。调节性T细胞（Treg）是一类免疫抑制性T细胞，通过多种机制抑制免疫效应细胞发挥免疫抑制作用，是肿瘤免疫逃避的关键因素。与健康志愿者相比，AML患者骨髓和外周血中调节性T细胞（Treg）频率增多，抑制免疫效应细胞功能强大，骨髓Treg中ICOS+Treg增多。该项目目的是研究ICOS/ICOSL信号通路在Treg促进AML细胞免疫逃避中的作用及机制，有助于AML的免疫治疗。通过该项目研究，我们发现：1）急性髓系白血病初发患者骨髓Treg、ICOS+Treg频率明显高于健康志愿者，患者骨髓中CD4+ Foxp3+细胞增殖高于CD4+ Foxp3-细胞。部分AML细胞株和原代细胞高表达ICOSL，高表达ICOSL的细胞株可以促进CD4+CD25-细胞转化为CD4+CD25+细胞，以及促进CD4+CD25+细胞增殖。低表达ICOSL的HL-60细胞株通过转染使其过表达ICOSL，与HL-60相比，过表达ICOSL的HL-60更明显促进CD4+CD25-细胞转化为CD4+CD25+细胞以及CD4+Foxp3+频率，anti-ICOSL可以部分阻断此作用。过表达ICOSL的HL-60与CD4+CD25-共培养促进细胞因子IL-10和IL-35分泌。IL-10、IL-35可以促进AML细胞增殖，AML细胞p-Akt、p-ERK、p-STAT3表达增加。2）我们建立了过表达ICOSL的小鼠AML细胞株C1498，尾静脉输注ICOSL+C1498细胞立小鼠急性髓系白血病模型，结果发现模型小鼠脾脏增大，脾脏、外周血、骨髓中Treg、Th17细胞增多，Th1细胞减少。3）AML初发患者骨髓白血病细胞ICOSL表达率、Treg、ICOS+Treg频率与预后呈负相关。