TCR/NKG2D共刺激诱导CD4+NKG2D+T细胞表达TGF-β的转录调控机制

There are unconventional CD4+NKG2D+T cells with regulatory function in the tumor-bearing mice and the pCD86-RAE-1 transgenic mice, which were identified by our previous research work. With co-ligations of TCR and NKG2D, CD4+NKG2D+T cells are activated to mediate regulatory activity through expression of membrane-bound TGF-β and FasL. However, the molecular mechanisms of TGF-β transcription induced by co-activations of TCR and NKG2D remain unknown. Whether NF-κB could be activated and how activated NF-κB regulates TGF-β transcription in CD4+NKG2D+T cells will be observed firstly in this study. Whether dephosphorylated STAT3 indirectly regulates TGF-β transcription by binding to NF-κB will be also investigated. Next we will analyze whether and how Egr-3 transactivates TGF-β transcription in CD4+NKG2D+T cells. In addition, how nuclear factor AP-1 which is activated by the MAPK/PI3K signaling pathway functions to regulate TGF-β transcription will be investigated. Therefore the mechanisms of TGF-β transcriptional regulation in CD4+NKG2D+T cells induced by co-stimulations of TCR and NKG2D will be finally elucidated.This study will also provide experimental evidences?for development of new anti-tumor drugs based on inhibition of biological activities of CD4+NKG2D+T cells.

我们前期工作发现，荷瘤小鼠及pCD86-RAE-1转基因小鼠体内存在一群独特的调节性CD4+NKG2D+T细胞，受TCR和NKG2D的共同刺激而活化，表达膜型TGF-β和FasL发挥免疫调节作用。然而关于TCR/NKG2D活化后诱导TGF-β转录的分子机制，目前尚不清楚。本研究拟首先观察受TCR/NKG2D共刺激后，该调节性CD4+NKG2D+T细胞内NF-κB的活化情况及其是否调控TGF-β基因的转录，而去磷酸化STAT3是否通过与NF-κB的结合间接调控TGF-β基因的转录；其次分析Egr3是否及如何调控TGF-β基因的转录；并且观察由MAPK/PI3K途径介导的AP-1活化在TGF-β基因转录调控中的作用。研究结果将阐明CD4+NKG2D+T细胞内由TCR/NKG2D受体介导的TGF-β转录调控机制，为基于抑制CD4+NKG2D+T细胞的生物学活性来开发新型抗肿瘤药物提供实验依据。

本项目拟在明确CD4+NKG2D+细胞生物学特性的基础上，探索CD4+NKG2D+细胞在肿瘤微环境中的诱导条件、促肿瘤活性以及该细胞表达TGF-β1的转录调控机制。研究结果表明，CD4+NKG2D+细胞的诱导主要受MHC II类分子和持续性NKG2D配体的共刺激；肿瘤微环境中持续膜表面和可溶性NKG2D配体促进该细胞诱导。CD4+NKG2D+细胞分泌TGF-β1、IL-10和sFasL抑制DC和效应性T细胞活性而促进肿瘤进展。CD4+NKG2D+细胞受CD3抗体和sRAE-1分子联合刺激后，PI3K-p85亚单位活化，继而通过JNK激活下游的 AP-1、NF-κB、STAT3、Egr-2/3活化调节TGF-β1的转录。MAPK、NF-κB、STAT3调节Egr-2表达，而对Egr-3表达无明显影响。EMSA实验表明Egr-2/3可直接结合TGF-β1启动子促进其表达。在CD4+NKG2D+细胞中，SOCS3主要受Egr-3表达的调节。中药单体落新妇苷被认为具有免疫调节活性。本研究发现落新妇苷具有明显诱导CD4+NKG2D+细胞的活性，促进其分泌TGF-β1和 IL-10，诱导其表达CCR6和CCR9。经落新妇苷刺激的CD4+NKG2D+细胞具有抑制CD8+T细胞和巨噬细胞的活性，且该活性依赖TGF-β1和 IL-10的分泌。落新妇苷对DSS-肠炎的保护作用与其诱导CD4+NKG2D+细胞有关。过继输入经落新妇苷刺激的CD4+NKG2D+细胞可明显抑制DSS-肠炎发病。落新妇苷刺激CD4+NKG2D+细胞主要通过激活PI3K、MAPK和STAT3信号来促进TGF-β1分泌。本研究不仅丰富了对CD4+NKG2D+细胞生物学特性的认识，阐明其TGF-β1表达调控分子机制，还为将落新妇苷应用于炎症性疾病治疗提供重要实验依据。