ATM介导活化信号轴NKG2D/NKG2DLs激活NK细胞抑制重型再生障碍性贫血CTL免疫亢进的作用机制研究

The pathophysiology of severe aplastic anemia (SAA) is immune mediated, with activated CD8+ cytotoxic T lymphocytes (CTLs) implicated in most cases. Previous studies published by the applicant suggested that NK cells could restrain CTL immunity by recognizing NKG2DLs on CTLs via NKG2D. With the imbalance of T cell immunity, the immuno-regulatory functions of NK cells enhanced in SAA. Further study indicated that the expressions of NKG2DLs on CTLs relied on the phosphorylation of ATM, an upstream signal protein. Accordingly, the applicant put forward the hypothesis that after ATM activation, NKG2DLs on CTLs were up-regulated, NK cells exerted cytotoxicity to autologous CTLs through signal axis NKG2D/NKG2DLs. In this study, the applicant will further verify the regulatory roles of ATM on the expressions of NKG2DLs in CTLs and its related signaling pathways. The immuno-regulatory effect of NK cells on autologous CTLs under ATM over-expression or ATM agonists will be explored using in vitro cell co-culture techniques and bone marrow failure animal models. This research may help with proposing a new potential approach to SAA treatment.

CD8+细胞毒T细胞（CTL）的功能亢进被认为是重型再生障碍性贫血（SAA）发病的关键机制。申请人前期已发表研究提示SAA患者的NK细胞可通过NKG2D识别表达在CTL上的NKG2DLs，从而抑制CTL效应功能；随着T细胞免疫平衡的紊乱，NK细胞的免疫调节功能增强。进一步研究发现，SAA患者CTL表达NKG2DLs依赖于上游信号蛋白ATM的磷酸化。据此，申请人提出假说，激动ATM可上调CTL表面NKG2DLs的表达，从而促进NK细胞通过信号轴NKG2D/NKG2DLs杀伤自身免疫性CTL。本课题拟在前期工作基础上，利用体外细胞共培养和骨髓衰竭动物模型，探索过表达ATM或加入ATM激动剂及相关信号分子阻断剂对CTL表达NKG2DLs的调控作用及相关信号通路，进一步验证ATM调控下NK细胞对自体CTL的免疫调节作用，为SAA治疗提供新的思路和实验基础。

重型再生障碍性贫血（SAA）是一种以骨髓造血衰竭为表现的自身免疫性疾病，CD8+细胞毒T细胞（CTLs）的功能亢进为主要发病机制。本人前期研究发现SAA患者NK细胞可以通过NKG2D/NKG2DLs途径杀伤自身CTLs。本项目对NKG2DLs的上游激活途径进行了更加细致的研究。我们探索了ATM激活剂及抑制剂干预下NK细胞对CTLs功能分子及凋亡水平的影响；在蛋白和mRNA水平证实了ATM激动剂（磷酸氯喹，CQ）存在增加CTLs细胞表面NKG2DLs（包括MICA、ULBP1、ULBP3等）的作用，同时ATM抑制剂降低了其表达。SAA患者自体NK细胞与CTLs共培养试验表明CQ具有通过上调CTLs表面NKG2DLs表达从而诱导CTLs接受NK细胞的杀伤作用，继而CTLs功能降低，凋亡增加。信号通路的研究提示CQ通过磷酸化ATM上调NKG2DLs表达，其可能途径与mTOR的磷酸化有关。本研究为进一步完善SAA免疫发病机制提供新的思路及细胞治疗方向。