Characterization of immune responses of human PBMCs infected with Mycobacterium tuberculosis H37Ra: Impact of donor declared BCG vaccination history on immune responses and M. tuberculosis growth

This study characterized the immune responses in early Mycobacterium tuberculosis (Mtb) H37Ra infection of human peripheral blood mononuclear cell (PBMC)-collagen matrix culture and the impact of Bacille Calmette-Guérin (BCG) vaccination history of donor PBMCs on the immune responses to Mtb infection. Aggregates of PBMCs were initially observed on day 3 and the size of aggregates continued to increase on day 8 post-infection, where macrophages and T cell subsets were identified to be present. Similarly, mycobacterial load progressively increased in infected PBMCs during the 8 days of culture but were significantly lower in infected PBMCs from BCG vaccinated (BCG+) donors compared to unvaccinated (BCG-) donors. The levels of INF-γ, TNF-α, IL-4, IL-6, IL-10 and IL-17 in the supernatants of Mtb-infected PBMCs peaked at day 3 and decreased on days 5 and 8. The levels of these cytokines except IL-10 were significantly lower in Mtb-infected PBMCs from BCG+ donors compared to infected PBMCs from BCG- donors. The percentages of activated naïve Th cells, activated effector memory Th cells and activated central memory Tc cells were significantly higher in Mtb-infected PBMCs compared to uninfected PBMCs at day 8 post-infection. Further, the proportion of activated central memory Tc cells was significantly higher in infected PBMCs from BCG+ donors compared to the BCG- donors. This study highlights the possibility that BCG vaccination may confound results that utilize human PBMCs to study Mtb infection.

本研究对人类外周血单个核细胞（human peripheral blood mononuclear cell, PBMC）-胶原蛋白基质共培养体系中，早期结核分枝杆菌（Mycobacterium tuberculosis, Mtb）H37Ra感染后的免疫应答特征，以及供体PBMC的卡介苗（Bacille Calmette-Guérin, BCG）接种史对Mtb感染免疫应答的影响进行了系统表征。感染后第3天首次观察到PBMC聚集体，且该聚集体的尺寸在感染后第8天持续增大，此时可检测到巨噬细胞与T细胞亚群的存在。同样，在为期8天的培养过程中，感染Mtb的PBMC内分枝杆菌载量逐步升高，但卡介苗接种阳性（BCG+）供体的感染PBMC的分枝杆菌载量显著低于未接种（BCG-）供体的感染PBMC。Mtb感染的PBMC培养上清液中，INF-γ、TNF-α、IL-4、IL-6、IL-10及IL-17的水平于第3天达到峰值，并在第5天和第8天逐渐下降。除IL-10外，其余上述细胞因子在BCG+供体的感染PBMC中的水平均显著低于BCG-供体的感染PBMC。感染后第8天，与未感染的PBMC相比，Mtb感染的PBMC中活化初始辅助性T细胞、活化效应记忆辅助性T细胞及活化中枢记忆细胞毒性T细胞的占比显著升高。进一步分析显示，BCG+供体的感染PBMC中，活化中枢记忆细胞毒性T细胞的比例显著高于BCG-供体。本研究提示，卡介苗接种可能会对以人类PBMC为模型开展结核分枝杆菌感染研究的实验结果产生混杂影响。