T-CELLS IN HUMAN TUBERCULOSIS

结核病(TB)是世界上导致死亡的主要原因。 来自一种单一的传染病。努力培养新一代人 有效的结核病疫苗或免疫疗法受到严重限制 对涉及到的机制缺乏了解 人类疾病的免疫发病机制。虽然T淋巴细胞(T 细胞)介导的免疫在保护性和 结核分枝杆菌(Mtb)的致病宿主反应是 不知道它们是由相同还是不同的种群介导的 T细胞。这项研究的长期目标是开发新的疫苗 结核病的治疗方法。这项提案中描述的研究目标 目的是确定T细胞亚群参与发病的机制。 人类的肺结核病。我们提出了一种免疫细胞模型 由巨噬细胞以及α-β和γ-亚群组成的“回路” 负责启动、动态平衡的Delta T细胞 结核分枝杆菌炎性免疫反应的调节和消解。 尽管此电路通常会提供保护，但它的缺失或 功能障碍可能是促进疾病重新激活的原因。这 模型为进一步表征提供了概念性框架 与结核病发病有关的T细胞群。为了测试 在这个模型中，我们建议识别存在的T细胞亚群，并且 它们在不同时间在肺中产生的可溶性因子(细胞因子) 在疾病的自然发展过程中。外周血(PB)和 将分离出支气管肺泡灌洗(BAL)淋巴细胞群 来自正常、健康(PPD)的个人和来自不同群体的 处于结核分枝杆菌感染不同阶段的患者。T细胞亚群将 初步通过流式细胞术和α-β和α-β鉴定 T细胞受体(TCR)的使用将通过敏感的 聚合酶链式反应/Southern杂交技术 确定TCR可变区基因表达谱。TCR 基因表达与TCR蛋白表达通过 分析细胞表面TCR+细胞的分离群体。脱氧核糖核酸 对TCR转录本的序列分析将有助于区分TCR 由感染部位的细胞表达，以及提供 一种估计T细胞谱系和抗原多样性的方法 他们对此做出回应。将使用原位杂交来鉴定和鉴定 列举表达TCR的α-βT细胞和γ-βT细胞亚群 在感染部位，由不同的V区基因原位编码。 结核分枝杆菌感染部位T细胞亚群分泌细胞因子的研究 肺部感染将使用聚合酶链式反应分析来确定 并对分离的T细胞群体中细胞因子基因的表达进行了定量。 将使用原位杂交来确定相对频率 以及表达不同细胞因子基因的T细胞的种群动态。 T细胞亚群的特征 对结核分枝杆菌的炎性免疫反应是理解 宿主如何响应以产生保护或疾病。

Tuberculosis (TB) is the leading cause of death in the world resulting from a single infectious disease. Efforts to develop a new generation of effective TB vaccines or immunotherapy ar severely restricted by the lack of understanding of the mechanisms involved in the immunopathogenesis of the disease in man. Although T lymphocyte (T cells)-mediated immunity is important in both the protective as well as the pathogenic host responses to mycobacterium tuberculosis (Mtb), it is not known if they are mediated by the same or different populations of T cells. The long term goal of this research is to develop new vaccine therapies for TB. The goal of the research described in this proposal is to identify the T cell subsets involved in the pathogenesis of pulmonary TB in humans. We have proposed a model of an immune cell "circuit" consisting of macrophages and subsets of alpha beta and gamma delta T cells that is responsible for the initiation, homeostatic regulation and resolution of the inflammatory immune reaction to Mtb. Although this circuit would normally provide protection, its absence or dysfunction may be responsible for promoting disease reactivation. This model provides the conceptual framework for the further characterization of the T cell populations involved in the pathogenesis of TB. To test this model, we propose to identify the T cell subsets present, and the soluble factors (cytokines) they produce, in the lung at different times during the natural development of the disease. Peripheral blood (PB) and the bronchoalveolar lavage (BAL) lymphocyte populations will be isolated from normal, healthy (PPD) individuals and from different groups of patients at distinct stages of Mtb-infection. T cell subsets will initially be identified by flow cytometry and alpha beta and alpha beta T cell receptor (TCR) usage will be determined using a sensitive polymerase chain reaction (PCR)/Southern hybridization technique thereby defining the profile of TCR variable (V) region gene expression. TCR gene expression will be correlated with TCR protein expression by analyzing fractionated populations of cell surface TCR+ cells. DNA sequence analysis of the TCR-transcripts will serve to distinguish TCRs expressed by cells present in the site of infection as well as providing a means of estimating the diversity of the T cell repertoire and antigens they respond to. In situ hybridization will be used to identify and enumerate subsets of alpha beta and gamma delta T cells expressing TCRs encoded by different V-region genes in situ, in the site of infection. The profile of cytokines produced by T cell subsets at the site of Mtb- infection in the lung will be determined using a PCR assay to identify and quantitate cytokine gene expression in isolated T cell populations. Hybridization in situ will be used to determine the relative frequencies and population dynamics of T cells expressing different cytokine genes. The characterization of the T cell subset that are involved in the inflammatory immune response to Mtb is essential in order to understand how the hosts response is directed to generating protection or disease.