中央记忆T细胞对结核疫苗保护作用的免疫机制研究

Since immune memory induced by BCG vaccination wanes with time and correlates with an increased number of pulmonary cases of TB in adluts, based on the tangilbe expermental data of antigen specific central memory T (TCM) cells mediate immune protection against several chronic pathogens infection, we suppose that the inability to establish a significant TCM population may be a key reason to cause low protection against TB in adult humans. In this hypothesis, we would propose that TEM are generated as the primary memory pool after neonatal BCG vaccination and these cells function perfectly adequatedly over the first decade. However, these cells are gradually lost by attrition or exhausted by exposure to the pathogen or environmental non-tuberculosois mycobacteria (NTM). Once the TEM population had been lost or at least significantly reduced, then a lack of an adequate TCM population as a second line of defense would render the host susceptible to TB infection. In order to verify the hypothesis, our preliminary data proved that TCM frequencies in health adults were lower significantly than those in children, then we intend to use flow cytometry techniques such as surface staining, intracellular cytokine staining etc., to analyaze multifunctional T cell frequencies, dynamic profiles of type I cytokines, TCM/TEM phenotypes and functions, and to further define immunological characteristics induced by vaccination, and immune correlation with anti-TB protection. This may be possible to explain a novel mechanism of immune protetion or prtection loss by vaccination, and be also critical for better evaluation or enhancement of immune protetion and rational development of new vaccines agaisnt TB infection.

鉴于卡介苗(BCG)保护成人肺结核效力不足并在接种10-15年后免疫记忆逐渐丧失的事实以及抗原特异性中央记忆T细胞(TCM)与多种慢性病原体感染的免疫保护直接相关的有力证据，我们推测不能有效诱导机体产生高水平的TCM细胞群可能是导致BCG对成人肺结核保护力低下的主要原因之一。为验证此假说，我们在预实验中首先证实了健康成人的TCM细胞频率要明显低于儿童，接着拟综合利用流式细胞表面染色、胞内因子染色等技术手段，分别在不同年龄段人群(健康人/TB患者)以及接种疫苗的小鼠(不同亚单位疫苗) 两水平多层面地综合分析多功能T细胞频率、I型细胞因子动态变化、TCM与TEM细胞亚型及其功能变化等，以系统研究结核疫苗诱导的免疫学特征特别是TCM频率及其功能与抗结核免疫保护之间的关系，旨在尝试解释结核疫苗免疫保护或失保护可能的新机制，从而有助于有效评价并增强结核疫苗的保护效力，并用以指导结核新疫苗的合理设计。

鉴于卡介苗(BCG)保护成人肺结核效力不足并在接种10-15 年后免疫记忆逐渐丧失的事实以及抗原特异性中央记忆T细胞(TCM)与多种慢性病原体感染的免疫保护直接相关的有力证据，我们推测中央记忆性免疫水平不足可能是导致BCG对成人肺结核保护力低下的主要原因之一。为证，我们构建了表达结核分枝杆菌免疫优势抗原Ag85A或嵌合抗原Ag85A- ESAT-6的DNA疫苗或构建了表达Ag85A/B嵌合抗原的重组仙台病毒载体疫苗，分别用这些不同形式的疫苗或免疫策略接种小鼠，综合利用流式细胞表面染色、胞内因子染色等技术手段，分析不同免疫接种策略所诱导产生的多功能T 细胞频率、I 型细胞因子动态变化、TCM与TEM细胞亚型及其功能变化等免疫学特征；同时将免疫小鼠进行结核分枝杆菌标准毒株H37Rv的气雾攻击，利用CFU计数分析疫苗诱导产生的保护作用，用H&E染色分析感染后的病理损伤情况，并同步分析结核菌感染后的二次免疫应答水平及其记忆表型，重点分析疫苗诱导的中央免疫记忆水平在抗结核保护中的作用，以系统研究结核疫苗诱导的免疫记忆特征及其功能与抗结核免疫保护之间的关系。最终，我们完成了不同疫苗诱导的免疫学特征研究和Mtb 毒株攻击实验，确定了不同结核疫苗诱导的免疫记忆差异及其抗Mtb 感染保护力的关系，同时确定了测定分泌IL-2的中央免疫记忆T细胞频率或IL-2的表达量可作为评估抗Mtb 感染保护力的一种新工具，对于结核病新新疫苗的开发或评价具有一定的指导意义。