TLR2和TLR4配体激活对新型结核疫苗AdAg85a免疫应答机制影响的研究

We try to invstigate the effect of the activation of TLR2 or TLR4 on the number and function of antigen specific T cells in airway lumen in mice immunized with AdAg85a intranasally and the mechanism behind it. Furthermore, we are interested in investigating the impact of activation of such receptors on the role of the vaccine against tuberculosis. According to our plan, BALB/c mice will be immunized with AdAg85a intranasally. Peptidoglycan(TLR2 ligand) or lipopolysaccharide (TLR4 ligand) will stimulate the bronchoavelor cells of the vaccinated mice in vivo and in vitro. Antigen specific T cells in airway lumen will be detected by tetramer technology at different time points. IFN-γ producing CD4 and CD8 T cells will be detected by intracellular cytokine staining. The levels of cytokines and chemokines in bronchoaveolar fluid will be measured by ELISA. Colonies of mycobacteria in the lungs and spleens in vaccinated mice after challenge with H37Rv or H37Ra will be counted. The mechanism of change of antigen specific T cells in airway lumen will be explored by cell labeling with CFSE，administration of FTY720 and staining with TLR2 and TLR4 antibody for flow cytometry.

新型结核疫苗AdAg85a已进入临床，但在宿主肺部细菌感染病理情况下的免疫保护作用及相关机理仍不清楚。本课题拟探讨TLR2或TLR4的激活对Ad5Ag85a吸入免疫小鼠气道腔内抗原特异性T淋巴细胞数目和功能之影响以及影响背后的机制，同时了解此种激活对疫苗针对结核杆菌保护作用的影响。方法学上拟以TLR2配体PGN或TLR4配体LPS暴露于AdAg85a吸入免疫BALB/c小鼠气道，或体外刺激AdAg85a免疫小鼠的气道腔内细胞，在不同时间点用四聚体技术检测气道腔抗原特异性CD8 T细胞数目，用细胞内细胞因子染色技术探测产生γ干扰素的CD4及CD8 T细胞数目，用ELISA法探讨气道腔内各细胞因子及化学素释放情况，同时通过结核菌培养了解结核杆菌攻击后免疫小鼠的肺脾菌落变化，最后以CFSE示踪、FTY720淋巴细胞供应阻断及TLR2或TLR4流式抗体染色等技术探索气道内T淋巴细胞变化之机理。

结核疫苗诱发的气道腔内T淋巴细胞（ALT）反应新近认为是预防结核的关键因素。然而，维持记忆T淋巴细胞的机制尚不清楚，尤其是气道暴露于外界物质如TLR4配体和TLR2配体是否对疫苗诱发的ALT反应有调节作用有待研究。我们用AdAg85a疫苗免疫小鼠，然后将其气道单次或重复暴露于内毒素(TLR4 配体)，发现ALT明显增加，且针对分枝杆菌攻击也有显著反应。相应地，细胞因子TNFa和化学素IP-10及MIG在肺泡灌洗液中水平也显著增加。CSFE标记的气道腔内ALT的增殖试验发现，总的说来内毒素暴露后小鼠ALTG1~9总的增殖率较对照组并未提高；对外周血淋巴细胞的阻断试验也提示， FTY720明显降低内毒素暴露小鼠气道腔内Tet+和 IFN-γ+ CD8+T细胞数目。因此推断，暴露于外界物质如内毒素对疫苗免疫宿主ALT有调节作用，从而揭示ALT的维持可以不依赖于抗原的刺激，而且ALT的增加主要来源于外周淋巴细胞的募集。用AdAg85a疫苗免疫小鼠气道单次暴露于肽聚糖(TLR2 配体)，则ALT中抗原特异性T淋巴细胞明显增加，功能性T淋巴细胞增加不著，其对ALT的调节作用尚不明确。