DNAM1-ERK信号转导通路在Vγ9Vδ2T细胞杀伤急性髓系白血病细胞中的作用及机理研究

Vγ9Vδ2（γδ） T cells have showed great anti-tumor activity，while the mechanism remains unclear. DNAM1 is an activating receptor involved in NK- and T cell-mediating tumor cell killing，may be essential for immune suveillance against tumors. We had an oral presentation recently in 53th Annual Meeting of American Society of Hematology （2011 ASH）that theγδT cells can efficiently kill acute myeloid leukemia（AML）several subsets cells including chemotherapy resistant cells. On this basis, we further found DNAM-1 was needed in the process of killing AML by γδT cells.We also found γδT cell binding to AML cells quickly activated ERK1/2, demonstrated by its tyrosine phosphorylation. Blocking DNAM1 inhibited ERK1/2 activation of γδT cells after contacting with AML cells.According to the results above,we now try making a hypothesis: DNAM-1-ERK signal pathway is necessary in the process of killing AML cells by γδT cells. In this stuty we will define the signal transduction process of DNAM-1-ERK pathway between identify and start killing AML cells，and further confirm the effector mechanisms of DNAM-1-ERK pathway.In this study, for the first time we explore the biological behavior of γδT cells in the process of killing AML cells.New molecular mechanisms of γδT cells to kill tumor cells is expected to be raised from the aspect of signal transduction.This project will also improve clinical practice for AML immunotherapy.

Vγ9Vδ2（γδ）T细胞具有强大的肿瘤杀伤活性，其机制尚不清。DNAM-1参与NK细胞及T细胞介导的肿瘤杀伤，是肿瘤发生的重要免疫监视分子。我们在前期报道"γδT细胞可有效杀伤人急性髓系白血病（AML）细胞"（2011 ASH会议 oral）的基础上，进一步发现γδT细胞启动杀伤AML需要DNAM-1与其配体识别。γδT细胞与AML细胞接触早期即发生ERK快速磷酸化，阻断DNAM-1可拮抗ERK此活化。由此提出假说，DNAM1-ERK信号通路参与了γδT细胞杀伤AML细胞的重要机制。本研究拟深入揭示DNAM-1-ERK在γδT细胞识别AML细胞并启动杀伤活性过程中具体信号转导机制，并系统阐明该信号轴调控的下游杀伤效应机制。本研究首次对γδT细胞杀伤AML全程的生物学行为进行探索，有望从信号转导角度提出γδT细胞杀伤肿瘤细胞的全新分子机制，并指导临床γδT细胞治疗AML奠定坚实的实验基础。

免疫治疗已经成为白血病治疗的趋势，Vγ9Vδ2 T细胞是一种具有很强肿瘤杀伤能力的免疫细胞，甚至可以有效杀伤TKIs耐药的CML细胞，是细胞免疫治疗的“明日之星”。本项目研究Vγ9Vδ2 T细胞杀伤TKIs耐药的CML细胞的作用，并通过羟氯喹（HCQ）的联合作用提高Vγ9Vδ2 T细胞的杀伤功能，并进一步揭示其作用机制，为推动HCQ联合Vγ9Vδ2 T细胞免疫治疗在TKIs耐药CML治疗中的应用奠定基础。项目以K562/GO1细胞（人CML伊马替尼耐药株）和K562细胞（人CML伊马替尼敏感株）为实验对象，通过共培养杀伤实验、脱颗粒实验、Western Blot、流式细胞术、shRNA敲降、荧光定量PCR、透射电子显微镜等技术手段，阐明：1. HCQ可以有效阻断K562/GO1细胞和K562细胞自噬，并且增强了K562/GO1和K562细胞对Vγ9Vδ2 T杀伤作用的敏感性。2. 在ATG7敲降的K562/GO1和K562细胞中HCQ依旧可以提高Vγ9Vδ2 T对肿瘤细胞的杀伤作用，提示HCQ的增敏作用并不依赖于抑制肿瘤细胞自噬。3. HCQ诱导了ULBP4在K562/GO1和K562细胞膜表达，阻断NKG2D与NKG2DL的相互识别后HCQ的增敏作用几乎消失，提示诱导ULBP4在CML细胞表面表达是HCQ促进Vγ9Vδ2 T杀伤CML细胞的关键。4. HCQ并没有提高ULBP4的转录及合成，而是诱导细胞内潴留的ULBP4转移到细胞膜。综上，项目研究发现HCQ诱导ULBP4从K562/GO1和K562细胞浆向细胞膜转移，促进了Vγ9Vδ2 T细胞对K562/GO1和K562细胞的识别和杀伤。