靶向miR-17~92/NFATC1通路逆转抗CML T细胞免疫缺陷及其分子机制

T-cell immunodeficiency is a common feature in chronic myelogenous leukemia (CML) patients, which is one of the critical reason to limit the effect of gene redirected T cell immunotherapy, little is known how to revise the status. Our previous studies found that the alterations of T cell immunity in CML involved in skewed T cell repertoire, and lower activation etc, based on the gene expression profile analysis, we suppose that nuclear factor of activated T cells C1 (NFATC1) of T cell receptor signal pathway and its upstream regulator miR-17~92 may be the key regulators. Therefore, we aim to investigate (1) the expressed alteration of miR-17~92/NFATC1 in different T cell subsets from CML patients and T cells within different immune status from healthy individuals, (2) the influence on T cell immune function by target regulation of NFATC1 and miR-17~92 in vitro and in vivo by using humanized CML mice model analysis, and (3) to confirm whether miR-17~92 regulate GSK3β or E2F1- p38MAPK phosphorylation, subsequently regulate the activity of NFATC1 and finally inhibit the expression of cytokine such as interleukin-2 (IL-2) and T cell activation in CML. Finally, the role of mediation of miR-17~92/NFATC1 in T cell immunodeficiency will be confirmed and the possibility of inversed regulation and the mechanism will be determined in CML, the precise molecular mechanism of T cell immunodeficiency in different stage will be definite. Overall, the study will provide the novel target and strategy for inversed regulation of T cell immunity in CML patients.

慢性粒细胞白血病（CML）病人常伴T细胞功能不全，是制约基因修饰T细胞免疫疗效的重要因素，如何逆转该状态研究甚少。我们既往研究发现CML中存在T细胞谱系不全、活化缺陷等，基因表达谱分析推测TCR信号通路中NFATC1（活化的T细胞核因子C1）及其上游miR-17~92可能是关键调控分子。故本研究拟（1）分析miR-17~92/NFATC1在CML病人不同T细胞群和不同免疫状态下T细胞中的变化；（2）通过体外和CML人源化小鼠模型分析靶向调控NFATC1和miR-17~92表达对T细胞功能的影响；（3）分析miR-17~92是否通过调控GSK3β或E2F1-p38MAPK磷酸化，而调控NFATC1活性、抑制IL-2和T细胞活化；最终明确靶向调控miR-17~92/NFATC1能否逆转抗CML T细胞免疫缺陷，深化对CML病人T细胞免疫缺陷的精确分子机制的认识，为其逆转调控提供新靶点和策略。

白血病病人T细胞功能异常及其如何恢复T细胞免疫稳态的机制仍不甚明确。本研究主要结果包括1）首先建立了年龄相关健康人T细胞谱系、T细胞免疫衰老和耗竭特点的基础数据，并进一步比较AML和CML病人T细胞亚群表型和分布、T细胞耗竭的异质性特点，发现AML病人外周记忆T细胞趋向终末分化，初发CML病人T细胞存在活化分子表达降低和抑制性免疫检查点表达增加，且对酪氨酸激酶抑制剂耐药病人T细胞免疫缺陷更为明显。2）通过CML病人T细胞基因表达谱分析，发现20个差异基因涉及参与调控TCR信号通路，其中NFATC1和miR-17~92可能参与CML病人T细胞功能失调，验证结果显示IRF4，BACH2和NFATC3与NFATC1之间呈正相关分析，蛋白间相互作用进一步提示IRF4与NFATC1之间存在互作。3）激活NFAT2诱导人脐血CD8+ 初始T细胞分化，增加CD69+T细胞比例，提高IL-2，IFN-γ和TNF-α等细胞因子的表达是miR-20a-5p依赖的；下调miR-20a可显著抑制NFAT2诱导的CD8+初始T细胞活化，分化及其相关细胞因子表达。结合hTFtarget和TargetScan数据库分析发现：调控PD-1，Tim-3，CTLA-4和TIGIT的转录因子RUNX1是miR-20a的潜在靶基因。4）利用NrasG12D突变自发CMML小鼠模型，同样发现CD8+ T细胞亚群发生明显偏倚，不同CD8+ T细胞亚群间基因表达谱的差异和NFATC1的改变。综上，我们推测NFATC1，NFATC3，IRF4和BACH2可能形成转录调控网络共同参与病人CD3+T细胞功能障碍的调控过程；miR-20a/RUNX1/ICs信号通路的异常可能是引起白血病病人T细胞耗竭的机制。该研究深化对CML病人T细胞免疫缺陷的精确分子机制的认识，为其逆转调控提供新靶点和策略。