38 kDa抗原特异TCR基因修饰iNKT细胞的抗结核活性研究

Generation of drug-resistant Mycobacterium tuberculosis (MTB) is the root cause of refractory tuberculosis (TB), and recrudescence and reinfection owing to host compromised or deficient immunity can not be prevented by chemotherapy. Development of new therapeutics becomes a top priority! It has been noted that iNKT cells play an important role in the early anti-TB immune response. The research group is the first to carry out the study on the anti-TB activity of iNKT cells genetically modified with antigen-specific TCRs. Our results showed that following genetical modification with TCRs specific for MTB 38 kDa antigen, α-GalCer-activated iNKT cells from healthy volunteers display Th1-type cytokine secretion and have potent activity against MTB. On this basis, it is proposed to: (1) Stimulate CD4+ and CD8+ T cells from 30 cases of TB patients with MTB 38 kDa antigen, and screen out the antigen-specific TCRs; (2) Activate iNKT cells from TB patients with α-GalCer; (3) Silence the TCR Vα24 gene of iNKT cells with siRNA; (4) Genetically modify the iNKT cells with TCRs specific for 38 kDa antigen; (5) Identify the anti-TB activity of TCR gene-modified iNKT cells. The project provides new ideas for TB immunotherapy, and a model and a platform for immunotherapy research on other infectious diseases caused by intracellular bacteria.

耐药菌产生是结核难治的根本原因，且药物无法解决因机体免疫低下或缺陷引起的复燃和再感染，开创新治疗方法已成当务之急！iNKT细胞在早期抗结核免疫反应中发挥重要作用。课题组率先开展抗原特异TCR基因修饰iNKT细胞的抗结核活性研究，前期发现α-GalCer活化的健康志愿者iNKT经38kDa抗原特异TCR基因修饰后，分泌Th1型细胞因子并具有显著的抗结核抗原活性。在此基础上，拟：①用38kDa刺激30例结核患者CD4+、CD8+ T细胞，筛选抗原特异TCR；②α-GalCer活化患者iNKT细胞；③siRNA沉默iNKT细胞TCR Vα24基因；④38kDa抗原特异TCR基因修饰iNKT细胞；⑤实验鉴定TCR基因修饰iNKT细胞的抗结核活性。项目为结核病免疫治疗提供新思路，也为其他胞内菌感染性疾病的免疫治疗研究提供模式和平台。

课题组首次建立了抗原特异TCR基因修饰iNKT细胞抗结核活性研究平台。①利用结核抗原38 kDa刺激32名结核患者外周血CD4+、CD8+ T细胞，检测T细胞TCR α、β双链CDR3谱型，找到抗原特异TCR；②采用α-GalCer联合IL-2刺激PMBC，诱导与活化结核患者iNKT细胞；③利用RNAi技术，沉默iNKT细胞固有TCR Vα24基因的表达；④构建携带沉默TCR Vα24基因shRNA的慢病毒，感染结核患者iNKT细胞，成功沉默其TCR Vα24基因表达；⑤将38 kDa抗原特异TCR基因修饰结核患者iNKTVα24 Low细胞，将之与负载38 kDa抗原的DC混合培养，检测TCR基因修饰iNKT细胞的活性。结果显示，TCR基因修饰组iNKTVα24 Low细胞的增殖、IFN-γ和TNF-α分泌、杀伤水平均显著高于其他各组。项目验证了38 kDa抗原特异TCR基因修饰iNKT细胞的抗结核活性，为结核病免疫治疗提供新思路，也为其他胞内菌感染性疾病的免疫治疗研究提供模式和平台。