调节性T细胞通过IL10相关信号通路对急性髓系白血病细胞干性的调控作用

Regulatory T cells (Tregs) can protect normal hematopoietic stem/ progenitor cells (HSPCs) from immune destruction through immunosuppression, and promote the survival and self-renew of HSPCs. But little was known about the role and mechanism that Tregs might directly promote the stemness of leukemia cells. It has been reported that leukemia stem cells (LSCs) are the basis of leukemia relapse and chemoresistance. So, investigating Tregs regulating the stemness of leukemia cells will help us to know the mechanism of leukemia relapse. Our recent results showed a positive correlation between the proportions of Tregs and LSCs in the bone marrow of acute myeloid leukemia patients; Tregs can increase the proportion of side population cells and promote the ability of colony formation in vitro. In this study, we propose the hypothesis that Tregs can promote the stemness of leukemia cells, and validate it by following experiments: 1) to make sure that Tregs increase the population of LSCs in vitro; 2) to find the related signal pathway on stemness regulation of leukemia cells ; 3) to verify the function and related mechanism of Tregs in promoting the stemness of leukemia cells in animal models. From this study, we want to provide a scientific basis for new treatment strategies of targeting leukemia stem cells.

调节性T细胞(Tregs)可通过免疫抑制作用阻止免疫系统对造血干/祖细胞(HSPCs)的杀伤，从而促进HSPCs的存活及自我更新。但迄今对Tregs直接促进白血病细胞干性的作用和机制所知甚少。而且白血病干细胞(LSCs)是白血病复发和化疗耐药的根源。因此，研究Tregs对白血病细胞干性的调控作用可了解白血病复发的关键机制。在我们前期研究中，检测了急性髓系白血病患者骨髓中Tregs与LSCs的比例，发现两者呈正相关，而且Tregs可增加白血病细胞侧群细胞比例及集落形成能力。在此基础上，本项目以Tregs促进白血病细胞干性为假设，将通过Tregs与白血病细胞共培养体外明确Tregs对白血病细胞干性的影响，使用AML1-ETO小鼠发病模型及PDX模型体内验证Tregs对起始白血病的影响，并将深入探索Tregs对白血病细胞干性的调控机制。旨在为靶向LSCs的治疗提供科学依据。

调节性T细胞（Tregs）可通过免疫抑制作用阻止免疫系统对造血干/祖细胞的杀伤，从而促进造血干/祖细胞存活及自我更新。迄今对Tregs直接促进白血病细胞干性的作用和机制所知甚少，而白血病干细胞(LSC)是白血病复发和化疗耐药的根源。因此，研究Tregs对白血病细胞干性的调控可了解白血病复发的关键机制。.Tregs与白血病细胞共培养可增加AML侧群细胞比例、G0期细胞比例及集落形成能力，且增加Oct4和Nanog的表达水平；GSEA分析TCGA数据表明Foxp3高表达可富集到GENTLES_LEUKEMIC_STEM_CELL_UP基因集。且Foxp3高表达患者生存期明显短于低表达患者。细胞因子芯片分析发现Tregs可分泌大量IL10，且TCGA数据表明IL10高表达患者预后较差。Kasumi-1细胞经IL10处理后，进行RNA-seq分析，Enrichr分析表明AKT被显著激活。通过shRNA下调IL10RA、IL10RA中和抗体以及LY290004小分子抑制PI3K/AKT信号通路表明，由Tregs引起的侧群细胞比例、G0期细胞比例及集落形成能力增加的作用均被下调，且OCT4和NANOG（可由AKT调控）的表达也被抑制。当Tregs与A/E小鼠白血病细胞同时经尾静脉注射到小鼠体内，可促进小鼠白血病的发生，小鼠生存期缩短。当A/E发病小鼠外周血GFP阳性率达≈5%，再经尾静脉注射Tregs，3天后小鼠脾脏和骨髓中GFP+细胞G0期的比例增加。使用1mg/kg抑制剂LY294002处理A/E小鼠后，脾脏中GFP+细胞G0期的比例降低。AML患者骨髓BMMNCs中CD4+CD25+CD127-和CD34+CD38-的细胞比例呈正相关。分别分析Tregs比例低与比例高患者BMMNCs中p-PI3K、p-AKT、OCT4和NANOG的表达，发现Tregs比例低的患者p-AKT、OCT4和NANOG的表达水平明显低于Tregs比例高的患者；且进一步使用rhIL10处理原始AML细胞发现，rhIL10可促进p-PI3K、p-AKT、OCT4和NANOG的表达。因此Tregs可通过IL10/IL10RA/PI3K/AKT信号通路直接促进AML细胞的干性，通过干预Tregs与AML细胞之间的作用“桥梁”，可为AML的治疗提供新思路。