BTLA对DC抗结核免疫作用的影响及其作为抗结核免疫治疗靶点的研究

It has long been noted that patients with tuberculosis (TB) are associated with altered dendritic cell (DC) function manifested by the reduced capacity for innate immune response along with lower capability to initiate adaptive immune response. Despite past extensive studies, the underlying mechanisms, however, largely remain elusive. Given the role of DCs played in against Mtb infection, they are also considered to be a pivotal target for developing effective immunotherapeutic strategies. We now found in patients with active pulmonary tuberculosis that the number for CD11c+BTLA+ cells in the peripheral blood is significantly higher than that in normal controls. Importantly, DCs with enhanced BTLA expression are concordantly with lower expression of co-stimulatory molecules. We further noticed increased BTLA expression in the tuberculosis granuloma tissue of patients with pulmonary tuberculosis. Interestingly, administration of soluble BTLA was found to promote DCs expression of B7-1. Based on these findings, we herein hypothesize that Mtb induce DCs expression of BTLA, and by which it impairs the capability of DCs against Mtb infection. To test the hypothesis, we will employ DCs with either overexpression of BTLA or deficient in BTLA to dissect the impact of BTLA expression on the functionality of DCs such as their capacity for antigen uptake, process and presentation, migration to draining lymph nodes and activation of na?ve T cells. We will next generate a mouse model with depleted DCs, and exogenous DCs with either BTLA overexpression or BTLA deficiency 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 BTLA expression on DCs for the clearance of invaded Mtb and on mice for the development of latent infection. The molecular mechanisms by which BTLA impairs the functionality of DCs will be also investigated. Finally, we will employ BTLA-specific lipid nanoparticle RNAi to silence BTLA expression in DCs and to explore the feasibility of this approach for the development of immunotherapeutic strategies for TB prevention and treatment.

抗结核免疫治疗是控制耐药性结核菌流行的有效措施。DC在抗结核免疫中起重要作用，结核菌感染导致DC功能低下的机制未完全清楚。我们前期研究发现：活动性肺结核患者外周血BTLA阳性DC增多，且其表面共刺激分子表达显著低于BTLA阴性DC；结核性肉芽组织内DC高表达BTLA；可溶性BTLA可上调DC表面B7-1表达。据此推测：DC高表达BTLA可能是结核菌感染中DC功能低下的重要原因。本项目将以高表达BTLA DC或BTLA缺陷DC为细胞模型；建立清除DC的结核菌感染小鼠模型，并输注高表达BTLA DC或BTLA缺陷DC，观察BTLA对小鼠DC吞噬/杀伤结核菌、成熟、迁移、抗原提呈及活化T细胞能力的影响；借助免疫纳米脂质体RNAi技术，研究靶向沉默DC内BTLA基因治疗结核病的效果。探讨DC高表达BTLA降低机体抗结核免疫功能的作用及机制和靶向沉默DC中BTLA表达作为抗结核免疫治疗措施的可行性。

BTLA属于CD28家族成员免疫检测点抑制分子，HVEM为期受体。BTLA主要表达于T、B细胞表面，在巨噬细胞、DC、NK细胞等也有表达。BTLA介导的免疫抑制信号参与正常机体自身耐受的维持，在免疫性疾病发生、发展中起十分重要的作用。但其在结核病发病中的作用未见报道。本研究以结核病患者为研究对象，采用流式细胞术对患者外周血表达CD11c的抗原提呈细胞（CD11cAPC）和树突状细胞（DC）的数量进行了检测，并对BTLA在CD11cAPC和树突状细胞中的表达进行了分析，同时检测BTLA的表达对CD11cAPC及树突状细胞及其亚群生物学性质及功能的影响并探讨其机制。结果发现活动性肺结核患者外周血中CD11cAPC细胞数量高于对照组，但DC及其亚群数量降低，治疗后细胞数量逆转。患者与正常人DC表达表面成熟分子（CCR7、CD83）、HLA II分子和共刺激分子（CD80 、CD86）存在差异。有趣的是BTLA在ATB患者CD11cAPC和DC中表达升高，在正常人和肺结核患者中BTLA+DC细胞与BTLA-DC相比高表达DC成熟分子（CD83、CCR7）、HLA-DR和CD80，但低表达CD86。而且ATB患者与正常人外周血中BTLA+DC细胞表面分子的表达存在差异。同时患者BTLA+DC摄取抗原能力高于正常对照组。BTLA表达与活动性肺结核患者外周血CD11cAPC活化同种T细胞功能下降相关，其机制可能是BTLA的表达下调了患者CD11cAPC表面HLA-DR的表达，并抑制其IL-6的分泌。正常人BTLA+DC刺激同种异型CD3+T细胞和CD8+T的活性显著低于BTLA-DC，而结核病患者中BTLA+DC与BTLA-DC对同种T细胞的刺激作用没有差异，但患者的DC细胞活化同种T细胞的能力显著低于正常对照组。以上结果提示结核菌感染可导致患者抗原提呈细胞数量和功能改变，BTLA在肺结核患者抗原提呈细胞中的表达升高，并与其抗原摄取和处理能力的改变有关，同时BTLA的表达影响抗原提呈细胞活化同种T细胞的能力，其机制与BTLA影响抗原提呈细胞的成熟、抗原提呈分子的表达相关，本研究对阐明结核病患者免疫功能低下的原因，以及结核病的治疗和预防有重要意义。