抗原特异性IL-35+调节性B细胞对T细胞抗结核免疫作用的影响

It has long been noted that patient with tuberculosis (TB) are associated with reduced cellular immunity against to Mycobacterium tumerculosis (Mtb). Despite past extensive studies, the underlying mechanisms, however, largely remain elusive. Recently, a regulatory B cell (Breg) subset, producing immunosupressive cytokine IL-35, was reported, which played important roles in autoimmune disease and in Salmonella infection. We now found that the level of IL-35 is elevated in serum from patients with active pulmonary tuberculosis. Moreover, BCG stimulation could increase the number of IL-35-producing Breg (B35) in PBMC from TB patients and this Breg exist in bronchoalveolar lavage fluid from TB patients. Based on these findings, we herein hypothesize that a IL-35-producing Breg subset exert negative immune regulation to T cell immunity against Mtb infection in human being. To test the hypothesis, we will test the immune characterization of tuberculous antigent specific B35 from TB patients and analyze its correlation to cellular immunity. Then we will study the impact of B35 from TB patients on autonogous T cell function against Mtb infection and its underlying mechanisms by means of direct mixed cell culture test and with trenswell mixed cell culture test, respectively. Finally, We will generate a mouse model with B cell deficiency, and exogenous tuberculous antigen specific B35 will be adoptively transferred into these mice, followed by Mtb infection. The capacity of these mice against Mtb infection will be evaluated to assess the impact of B35 on T cell immunity against Mtb. This research will elucidate the mechanisms of reduced cellular immunity in TB patients from a new perspective and will provide experimental evidence for immune therapy to TB and for tuberculous vaccine design targeting IL-35-producing Breg.

结核病患者细胞免疫功能低下，其机制未完全阐明。近期报道，小鼠体内存在一群分泌IL-35的调节性B细胞（Breg，B35），在自身免疫病和沙门菌感染中起重要作用。我们前期研究发现：活动性肺结核患者血清IL-35水平升高；BCG刺激可致患者B35数量升高；患者支气管灌洗液存在B35。申请者认为人体内存在B35亚群，可能负调节机体抗结核免疫反应，是导致结核病患者细胞免疫功能低下的重要原因。本项目拟开展如下研究：①分析肺结核患者抗原特异性B35的免疫学特征，阐明其与患者细胞免疫功能的相关性；②通过直接混合细胞培养并建立Transwell混合细胞培养体系，探讨抗原特异性B35对肺结核患者T细胞应答的影响及机制；③建立B细胞缺陷小鼠结核病模型，通过过继输注抗原特异性B35，观察对抗结核T细胞应答的影响。本项目可望为阐明结核病患者细胞免疫低下的机制和基于B35抗结核免疫治疗与结核疫苗设计提供实验依据。

IL-35属于IL-12家族成员，是一种抑炎因子，主要由T细胞产生。近期发现B细胞也可以分泌IL-35。与IL-10相似，IL-35在可抑制T细胞增殖和分化并诱导Treg极化，并可减轻自身免疫反应并促进感染性疾病的发展。本课题主要以活动性肺结核患者（ATB）和结核菌感染的小鼠模型为研究对象，探讨产IL-35 B细胞在结核病发病中的作用。首先，发现ATB患者血清IL-35水平升高，白细胞和外周血单个核细胞中IL-35两个亚基(p35和EBI3)的mRNA表达增加。在CD4中，结核菌感染与p35或EBI3蛋白的表达有关，且大多数p35+CD4+T细胞和EBI3+CD4+T细胞表达Treg相关标记CD25。进一步采用qRT-PCR和流式细胞术检测，结果显示IL-35两个亚基的mRNA和蛋白质在纯化B细胞中的表达均增加。结核分枝杆菌裂解物体外刺激试验也显示纯化的ATB患者B细胞表达IL-35的水平显著高于正常人对照组。同时发现ATB患者产生IL-35的B细胞具有更强的产生IL-10的能力。此外，免疫组织化学和免疫荧光染色结果显示ATB患者的结核性肉芽肿中浸润B细胞高表达IL-35。在BCG感染的小鼠模型中，发现外周血、脾脏、骨髓和肺组织的单个核细胞中IL-35两个亚基的mRNA表达升高，流式细胞术分析表明BCG感染小鼠外周血、脾脏、骨髓和肺组织中产IL-35 B细胞计数升高，而抗结核药物治疗后产IL-35 B细胞下降。同时，BCG感染可致产IL-35 B细胞浸润到肺组织中，且与肺中的细菌负载量呈正相关；外源性注射IL-35可刺激产IL-35的B细胞数量升高，并与Th1/Th17的下调和Foxp3+Treg的上调有关。我们的研究结果说明结核菌感染导致产IL-35的B细胞升高，而且与效应性T细胞的下调和调节性T细胞升高相关，其机制与该群细胞能产生IL-35和IL-10相关。