多重定向调控特异Vβ21+T细胞克隆抗CML效应及其分子机制研究

Our previous finding showed that clonally expanded T cells predominant in Vβ21 could be identified in patients with chronic myeloid leukemia (CML), although the patients underwent severe T cell immunodeficiency. In the present study, further identification of the T cell receptor (TCR) sequences from Vβ21+ T cell clones will be performed in different CML cases and donor T cells induced by CML associated antigen. The epitopes of bcr-abl antigen recognized by Vβ21+T cells will be deduced according the CDR3 motifs of Vβ21+T cells by bioinformatics analysis. By combination with bcr-abl peptide and RAG or BCL11B, upstream regulation in oriented differentiation of naive T cell from CML patients and donors will be attempted. By combination with cytokines such as IL-7, the specific proliferation of Vβ21+T cells will be induced. By gene transfer of TCR signalling factors such as TCRζ, the downstream regulation of Vβ21+T-cell activation will be performed. Therefore, the regulation effect in specific differentiation, proliferation and activation of Vβ21+T cells will be overall discussed on multiple steps, and the immune effect of modified Vβ21+T cells will be proved by analysis of the activation of TCR signalling pathway and the specific cytotoxicity. Finally, its relative molecular mechanism will be characterized by global gene expression profile analysis, and the further validation from different gene and protein expression and molecular blocker. The study will farther explore the mechanism of TCR rearrangement. The potential regulation pattern on anti-CML cytotoxicity T cells and their differentiation and proliferation will be characterized, which is expected to provide the theoretical and experimental evidence for reconstitution of specific anti-CML T cell immunity and prevention of disease relapse.

我们已首次发现慢性粒细胞白血病(CML)病人在细胞免疫缺陷状态下，仍存在一定抗CML的克隆增殖T细胞，主要为表达Vβ21的T细胞。本课题拟获得更多CML和抗原诱导供者的Vβ21克隆的T细胞受体(TCR)基因序列，经生物信息学分析确定Vβ21识别的bcr-abl抗原表位，以此抗原肽联合RAG和BCL11B等上游调控CML病人和供者初始T细胞的定向分化，联合辅助因子如IL-7等中游调控Vβ21+T细胞增殖，通过信号传导分子如TCRζ基因修饰而下游调控细胞活化，从多层面系统地探讨特异Vβ21+T细胞克隆分化、增殖和活化的调控作用，并从TCR信号通路活化和细胞毒活性等证明其免疫效应，最后，通过基因表达谱分析，差异基因和蛋白表达及分子阻断验证而了解其分子机制。从而深化对TCR基因重排机制的认识，明确抗CML的特异T细胞分化增殖的可调控规律，为重建病人抗CML特异细胞免疫，预防疾病复发提供理和实验依据

慢性粒细胞白血病（CML）病人在免疫缺陷状态下，仍可存在抗CML的T细胞克隆，鉴定和扩增这些T细胞克隆，是建立特异T细胞免疫治疗的关键。在前期发现CML中存在克隆性增殖Vβ21+T细胞的基础上，本研究首先构建了TCRVα13-IRES-Vβ21、TCRVα18-IRES-Vβ21、TCRζ和嵌合型TCR Vα13:ζ-IRES- Vβ21: ζ表达载体和转导T细胞，证明这些TCR基因修饰T细胞活化功能上调和具有特异抗CML细胞毒活性效应；并研究了IL-7和IL-12对T细胞克隆增殖和活化的调控机制；还发现了不同疾病状态和伴abl基因突变的伊马替尼耐药CML病人外周血中优势增殖的T细胞克隆（Vβ6和Vβ9），将用于后续的抗CML研究。通过对TCR信号通路活化调控因子分析，发现TCRζ 3′-UTR剪接体改变可影响TCR信号通路分子表达，据此可将TCRζ 缺陷的CML分为WT+AS- CML和WT+AS+ CML两个亚组，根据其基因表达模式差异，开展相应的靶向免疫调控研究。通过基因表达谱分析，发现CML病人T细胞中TCR 信号通路中NFATC1（钙神经素依赖/活化的T细胞途径核因子）表达下调，其上游磷酸激酶GSK3β、及协同效应转录因子JUN基因均上调，这种表达失衡模式可导致其下游多个T细胞活化相关的细胞因子包括IL-2、IFN-γ和TNF-α下调，可能是病人T细胞功能缺陷的重要原因之一。此外，我们还发现CML病人等外周血中CD4+和CD8+干细胞样记忆性T细胞（TSCM）、中心记忆性T细胞（TCM）、效应记忆（TEM）及效应性（TEF）T细胞亚群在不同病例中个体差异程度很大，提示病人T细胞免疫抑制程度及对相关肿瘤抗原免疫反应的异质性。总之，该研究结果深化对CML病人T细胞免疫缺陷和TCR信号通路异常的分子机制的认识，为重建病人抗CML特异细胞免疫提供理和实验依据。