IL-28B下调调节性T细胞的分子机制及对免疫微环境的影响

Regulatory T cells (Treg) down-regulated the immune responses in tumor and chronic infection and expressed Foxp3 which was specific marker. We found that the cytokine IL-28B could down-regulate the frequency of Treg and had an anti-tumor activity. Our study also showed that IL-28B reduced the memory response, which suggested that the cytokine had an influence on immune microenvironment. However, the molecular mechanisms of IL-28B down-regulating regulatory T cells and its effects on immune microenvironment were unclear, which limited the application in the field of immunological therapy. In this study, those factors which were associated with the expression of Foxp3 were detected after the production of Treg was treated by IL-28B. And the target of IL-28B was determined and will be verified by a knockout mouse. Meanwhile, the epigenetic changes were detected. Then the findings were tested in mice. Furthermore, the tumor-bearing mice were used to evaluate the effects of IL-28B on immune microenvironment. The work will be a theoretical basis for application of IL-28B in the fields of tumor and chronic infection therapies.

调节性T细胞(Treg)在肿瘤和慢性感染性疾病中发挥重要的免疫抑制作用，表达特征性转录因子Foxp3。我们发现细胞因子IL-28B能够下调Treg，具有明显抗肿瘤作用。我们还发现IL-28B下调Treg后减弱免疫记忆反应，提示IL-28B可能影响局部免疫微环境。然而IL-28B下调Treg的分子机制及对免疫微环境的影响尚不清楚，限制了IL-28B在免疫治疗中的应用。本课题拟在体外诱导Treg生成过程中，IL-28B干预后检测影响Foxp3转录的分子，确定IL-28B作用靶分子，应用靶分子基因敲除小鼠验证IL-28B的作用靶点；同时检测Foxp3基因表观遗传学变化，阐明IL-28B影响Foxp3表达的表观遗传学机制；接着通过小鼠体内试验验证；最后应用小鼠肿瘤模型，IL-28B治疗后检测肿瘤局部免疫细胞和分子，阐明IL-28B对免疫微环境的调节作用，为IL-28B在免疫治疗中的应用提供依据。

调节性T细胞(Treg)在肿瘤和慢性感染性疾病中发挥重要的免疫抑制作用，已发现IL-28B能够下调小鼠Treg细胞比例，但下调Treg的机制及对免疫功能的影响尚不清楚。本课题分离小鼠脾细胞和胸腺细胞，在体外利用TGF-β诱导Treg生成过程中IL-28B干预后发现Treg比例无明显降低，提示IL-28B并不直接下调Treg；IL-28B干预肿瘤小鼠模型后显示出一定的抗肿瘤作用，MTT检测淋巴细胞增殖能力显示淋巴细胞增殖能力升高，蛋白芯片技术检测血清及肿瘤微环境中的可溶性免疫分子发现血清FasL和IL-7水平明显升高，肿瘤微环境中FasL、CD30L、RANTES水平明显高于血清中；IL-28B干预细粒棘球蚴感染小鼠模型后显示出一定的抗包虫作用，FCM检测发现Foxp3+T细胞占脾淋巴细胞的比例明显降低，质谱流式检测发现B细胞有升高趋势、固有免疫细胞（DC、巨噬细胞、粒细胞）无变化、CD4+T细胞和CD8+T细胞无变化、CD8+CD69+T细胞降低趋势、TCM无变化而TEM有升高趋势、CD4+CD25+Treg无变化；蛋白芯片技术检测小鼠血清细胞因子，发现Th17型细胞因子（IL-17、IL-17F、IL-21、IL-22、IL-23）明显升高，Th1（IL-2、IFN-γ）和Th2型细胞因子（IL-4）无明显变化，抑制性细胞因子（IL-10、TGF-β1）明显升高，炎性细胞因子（IL-6、TNF-α）明显升高；IL-28B联合MASP-2干预泡球蚴感染小鼠模型后，蛋白芯片技术检测小鼠血清细胞因子，发现Th1型（IFN-γ、IL-12P70）和Th17型细胞因子（IL-17F、IL-21、IL-22、IL-23）水平升高，Th2型细胞因子（IL-4、IL-5、IL-13）水平无明显变化；IL-28B联合Fluconazole干预白色念珠菌感染小鼠模型后，显示一定的抗真菌作用，FCM检测脾细胞亚群发现CD4+T细胞比例升高。从以上结果得出结论，IL-28B不直接抑制Treg的生成或作用于Treg而下调Treg，而可能通过影响其他免疫细胞而调节Treg。该研究将为IL-28B治疗Treg增多的疾病如肿瘤或慢性感染性疾病的临床应用奠定基础。