结核杆菌脂糖诱导的Be2抑制CD4+TCM形成的作用及机制研究

A de?ning feature of vertebrate immunity is the acquisition of immunological memory. CD4+ T cells play an important role in adaptive immune responses against Mycobacterial Tuberculosis (M. tb), but CD4+ memory T cell generation in anti-M. tb immunity is incompletely understood. Generation of immunological memory is associated with not only the antigens but also immune cell interactions and cytokines. Mannosylated lipoarabinomannan (ManLAM), which has an immunosuppressive effects on immune cells, is a lipoglycan and major virulence factor in Mycobacterium. There have been no reports so far about the effects of ManLAM-stimulated immune cells on generation of CD4+ central memory cells (CD4+ TCM). In recent years, we have been conducting researches on ManLAM-related-M. tb pathogenic mechanism. In our preliminary experiments, we found that ManLAM induced na?ve B cell to differentiate into effector B2 (Be2) cells and Be2 inhibited the CD4+ TCM generation. In our current work, we plan to illustrate how the CD4+ TCM generation is controlled by Be2 induced by ManLAM in human cells and mouse cells (in vitro and in vivo).We propose that IL-10 secreted by Be2 cells and decrease of ICOSL expression on Be2 will be involved in suppressing CD4+ TCM generation. Moreover, we will construct the recombinant BCG (BCGΔ2196) which has mutant LAM lacking the mannose cap. We hope mice will enhance the memory immune response to M. tb by vaccination with BCGΔ2196. Our work will help to understand the memory immune response to M. tb.

在抗结核杆菌（M. tb）感染免疫应答中，CD4+T细胞发挥重要作用，但记忆性CD4+T细胞形成机制的研究严重滞后。免疫记忆的形成与抗原相关，还与免疫细胞相互作用、细胞因子效应关系密切。M. tb脂糖通过诱导免疫细胞功能变化和细胞因子释放影响中央记忆性CD4+T细胞（CD4+TCM）形成的机制尚待研究。申请人一直进行甘露糖修饰脂阿拉伯甘露聚糖（ManLAM）相关M. tb感染机制研究，前期发现ManLAM刺激B细胞向Be2（效应性B细胞2型）分化，Be2减少CD4+TCM形成。本项目拟用人和小鼠细胞，从体内外探讨Be2是否通过分泌IL-10或/和下调ICOSL(inducible costimulator ligand)抑制CD4+TCM形成及其机制；并对BCG疫苗ManLAM进行改造，以增强抗M. tb感染记忆性免疫应答。本项目的完成将帮助明确抗M. tb感染记忆性免疫应答的具体机制。

在结核分枝杆菌（M. tb）感染过程中，M. tb利用自身的毒力因子调控宿主的免疫应答，尤其是抑制Th1 CD4+T细胞的抗M. tb作用，但具体调控机制不清楚。甘露糖修饰脂阿拉伯甘露聚糖（ManLAM）是M. tb胞壁中重要毒力成分，本项目开展了M. tb ManLAM诱导的免疫调控机制研究，发现ManLAM与TLR2受体结合，诱导B细胞向Be2型分化并分泌IL-10，进而负调控Th1 CD4+T细胞的抗M. tb作用；发现ManLAM与巨噬细胞表面甘露糖受体（MR）和CD44分子结合，发挥双向调控。运用自主研发的小分子拮抗剂阻断ManLAM与MR受体的结合，可抑制巨噬细胞产生IL-10。通过动物实验（恒河猴）证实，以上小分子拮抗剂能在动物体内增强BCG（Bacillus Calmette-Guérin）的免疫效能。本项目的完成不仅明确了B细胞分泌的IL-10在M. tb感染中的负性调控作用，研制的小分子BCG ManLAM拮抗剂将有潜力作为疫苗佐剂提高BCG疫苗的免疫效能。项目执行过程中超额完成了各项预期目标。项目负责人获得湖北省杰出青年基金（主持人）。共发表11篇文章（均标注基金），其中项目负责人以第一（共同第一）以及通讯（共同通讯）作者发表文章SCI论文5篇（Cell子刊1篇，IF>10文章1篇，二区文章1篇）。参与著作编写3部，相关研究成果获得教育部高等学校科学研究优秀成果奖自然科学奖二等奖（排名第三），培养硕士研究生10人（获得学位毕业3人）。