BATF介导PD-1负性调控活动性结核患者T细胞功能的机制研究

Studies have showed that inhibitory immunological factors play an important role in the immunopathogenesis of active tuberculosis. Our preliminary study found that PD-1 may act as inhibitory factors involved in the pathogenesis of active TB. Recent study on HIV infection confirmed that PD-1 inhibited T cell function by upregulating BATF (Basic leucine zipper transcriptionfactor,; member of AP-1 superfamily). And we also found that BATF expression in patients with active tuberculosis was significantly higher than those of individuals of latent tuberculosis infection and health controls, which was associated with PD-1 expression in active tuberculosis. Thus, we presume that BATF molecule maybe mediate PD-1 negative regulation of T cell functions in active tuberculosis. Therefore, we are going to enroll subjects with different status of tuberculosis infection, including active and latent infection to determine the relationship between expressions of PD-1 and BATF, and tuberculosis specific T cell exhaustion using cell staining and flow cytometry. Then artificial activation/ blockade PD-1 pathway in vitro will be employed to understand the effect of PD-1 pathway on BATF expression on tuberculosis-specific T cells. And BATF expressions will be regulated by small interfering RNA (siRNA) to elucidate the influence of BATF on tuberculosis specific T cell functions. These findings will be expected to provide theoretical basis for the development of new immunotherapy strategy targeting BATF in tuberculosis.

研究已证实，免疫抑制因素在活动性结核的发病中起着重要作用。我们前期研究发现，PD-1可能作为抑制性因素参与活动性结核的发病。最近在HIV感染的研究中证实，PD-1可以特异性上调AP-1转录因子超家族成员BATF的表达，从而降低T细胞活性。我们同样也发现，活动性结核患者BATF表达升高，且与PD-1相关。因此，推测PD-1可能通过BATF介导其负性调控抗结核免疫反应。本课题将入选不同结核感染状态的人群，包括活动性结核患者、潜伏结核感染者及健康对照者，利用细胞染色和流式细胞术，确定PD-1、BATF的表达及其与结核特异性T细胞功能耗竭的关系；然后通过体外实验体系激活或阻断PD-1通路，了解其对结核特异性T细胞表达BATF的影响；最后利用siRNA技术上调或沉默BATF基因表达，明确BATF对结核特异性T细胞功能的影响，从而为开发以BATF分子为靶标的新型结核免疫治疗提供理论基础。

研究目的：研究BATF在活动性结核患者中调控结核免疫的作用机制，为开发抗结核免疫治疗提供理论依据。.研究方法：入选三组不同结核感染状态人群：活动性肺结核患者59例（ATB），潜伏性结核感染者16例（LTBI）和健康对照者22例（HC）。检测三组人群外周血中BATF在CD4+ T细胞和CD8+ T细胞的表达情况，以结核特异性抗原PPD刺激，特异性抗体antiPD-1、antiPD-L1、antiPD-L2阻断PD-1/PDLs通路，PD-L1重组蛋白激活PD-1/PDLs通路不同的处理方法，以及RNA干扰技术下调PBMC的BATF的表达。应用流式细胞术和荧光定量PCR方法检测ATB患者外周血T细胞BATF表达的变化和细胞分泌IL-2和IFN-γ水平的变化以及对CD4+ T细胞体外增殖能力的影响。.研究结果：ATB组外周血表达BATF的T细胞占CD4+ 、CD8+ T细胞的百分比显著高于LTBI组（P=0.0003，P=0.0003）和HC组（P<0.0001，P=0.0003）；ATB患者中，结核特异性抗原PPD刺激组表达BATF的CD4+ T细胞百分比中位数（2.81% vs 0.75%，P=0.048）以及CD8+ T细胞百分比中位数（1.48% vs 0.76%，P=0.039）比未刺激组明显增高。阻断PD-1与其配体结合，表达BATF的CD4+ T细胞百分比无明显变化，但表达BATF的CD8+ T细胞百分比中位数在加入PD-1阻断剂时有明显下降（0.85% vs 1.48%，P=0.014）。进一步分析发现，阻断PD-1的配体PD-L1/L2时表达BATF的CD4+ T细胞百分比明显降低（0.89% vs 2.81%，P=0.023），在CD8+ T细胞得到同样的结果（1.01% vs 1.48%，P=0.026）。通过RNA干扰技术下调BATF的PBMCs中发现，细胞分泌细胞因子IL-2（2.2 vs 1，P=0.0485）和IFN-γ（4.4 vs 1，P=0.0473）的能力（mRNA水平）明显升高。且BATF下调的CD4+ T细胞体外增殖能力明显升高（P=0.0041）。.结论：BATF介导PD-1负性调控活动性结核患者T细胞功能，通过下调BATF的表达可以部分恢复细胞分泌细胞因子IL-2和IFN-γ的能力以及T细胞增殖的能力。