共生菌调控肺脏γδT细胞抗肿瘤免疫应答的细胞与分子机制

Commensal microbiota signals play important role in maintaining mucosal immune homeostasis, which is closely related to infectious, metabolic and chronic inflammatory diseases. However, its effect on anti-tumor immune responses is unclear. Our previous study has found that antibiotics-treated (ABX) mice were more susceptible to lung melanoma and Lewis lung cancer, with significant decrease in the number and percentage of γδT cells in the lungs. Given the importance of lung γδT cells for their immune effects, and its unknown relation with commensal microbiota, we proposed the project on the basis of the previous study. In this study, the percentage, number, phenotypes and function (cytokines, transcription factors) of each lymphocyte subset will be analyzed and compared between the ABX mice and the control mice to determine the regulation effects of commensal microbiota on lung γδT cell subsets (γδT1 and γδT17), and their anti-tumor immune responses and relations with tumor progression. By using cell transfer/depletion, antibody blockade/neutralization and gene-deficient mice, the roles and molecular mechanisms of γδT cell subsets will be explored, and further their immune regulation on adaptive immune responses of Th1 and CTL. Further, the critical commensal microbiota and their innate immune signals such as TLR/NOD and cytokine signals will be determined to reveal the cellular and molecular mechanisms in regulating lung γδT cells. This study will provide strong evidence for deeply understanding the relationship between the commensal microbiota and the immune system.

共生菌信号维持机体粘膜免疫稳态，与感染性、代谢性及慢性炎性疾病密切相关，但其对肿瘤免疫应答的影响尚不明确。我们已有的研究发现抗生素水饲喂(ABX)小鼠更易发生肺脏黑色素瘤及Lewis肺癌，并伴随γδT数量与比例显著降低。鉴于肺脏γδT细胞重要的免疫学效应，且其与共生菌的关系尚未有报道。本课题拟深入分析ABX小鼠及其肺脏肿瘤形成过程中各淋巴细胞亚群比例、数量、表型及功能（细胞因子谱、转录因子谱）等特性，明确共生菌对肺脏γδT1/17亚群分布、抗肿瘤应答的影响及其与肿瘤发生发展的关系；利用细胞转输/清除、抗体阻断/中和、基因缺陷小鼠等手段验证γδT亚群的关键作用与机制，及其对Th1、CTL等免疫应答的调控；确定发挥关键作用的共生菌种类及其天然免疫信号(TLR、NOD等)及细胞因子信号，揭示共生菌促进肺脏γδT应答的细胞与分子机制。本研究将为深入理解共生菌与机体免疫系统之间的关系提供有力的证据。

共生菌信号维持机体粘膜免疫稳态，与感染性、代谢性及慢性炎性疾病密切相关，但其对肿瘤免疫应答的影响尚不明确。本研究旨在明确共生菌在机体肺脏组织抗肿瘤免疫应答中的调控作用。通过抗生素水饲喂的方式建立菌群缺失小鼠模型（Abt小鼠），利用黑色素肺脏转移瘤模型和Lewis肺癌模型观察比对小鼠肿瘤的发生发展情况。发现Abt小鼠更易发生肺脏黑色素转移瘤及Lewis肺癌，肺脏肿瘤结节数量及大小显著增加，生存期明显缩短，证实共生菌在维持肺脏抗肿瘤免疫应答中发挥重要作用。进一步研究发现菌群缺失导致肺脏组织IL-6、IL-23等细胞因子水平显著降低，进而影响肺脏γδT17细胞功能，是Abt小鼠易发肺脏肿瘤的关键因素。当给予Abt小鼠转输正常功能的γδT细胞或者外源性IL-17细胞因子均可恢复Abt小鼠的抗肿瘤能力。机体产生有效的抗肺脏肿瘤应答依赖于完整共生菌的存在，任何菌群组分的缺失均会导致其抗肿瘤效应的降低。同时发现Abt小鼠肺脏肺泡巨噬细胞数量明显减少，出现大量差异表达的基因，并表现出M2型巨噬细胞的特征，恢复Abt小鼠的共生菌群可逆转肺泡巨噬细胞的M1和M2型分子的表达及功能。在肺脏肿瘤发生发展过程中，Abt小鼠肺泡巨噬细胞向M2型极化，并分泌高水平CCL24分子。当给予Abt小鼠转输正常小鼠来源的肺泡巨噬细胞或中和其体内CCL24分子，均可逆转肺脏的抗肿瘤免疫应答能力，同时证实肺脏巨噬细胞及其高水平分泌的CCL24分子是调控肺脏γδT17细胞免疫应答的关键细胞与分子。本课题研究阐明了共生菌调控肺脏γδT细胞抗肿瘤免疫应答的细胞与分子机制，为深入理解共生菌与机体免疫系统之间的关系提供了有力的证据。