T-CELLS IN HUMAN TUBERCULOSIS

结核病（TB）是世界上导致死亡的主要原因， 从一种传染病。 努力培养新一代 有效的结核病疫苗或免疫疗法受到严重限制， 缺乏对有关机制的了解， 虽然T淋巴细胞（T 细胞）介导的免疫在保护性以及 病原宿主对结核分枝杆菌（Mtb）的反应， 不知道它们是否由相同或不同的群体介导。 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 本提案中所述的研究目标 是为了确定T细胞亚群参与的发病机制， 人类的肺结核 我们提出了一个免疫细胞的模型 由巨噬细胞和α、β和γ亚群组成的“回路” δ T细胞负责启动，稳态 调节和消除针对Mtb的炎性免疫反应。 虽然这种电路通常会提供保护，但它的缺乏或 功能障碍可能是促进疾病再激活的原因。 这 模型提供了进一步表征的概念框架 参与结核病发病机制的T细胞群。 测试 在这个模型中，我们建议识别存在的T细胞亚群， 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 将分离支气管肺泡灌洗（BAL）淋巴细胞群 来自正常健康（PPD）个体和来自不同的 结核病感染不同阶段的患者。 T细胞亚群将 最初通过流式细胞术和α β和α β T细胞受体（TCR）的使用将使用敏感的 聚合酶链反应（PCR）/Southern杂交技术， 定义TCR可变（V）区基因表达的谱。 TCR 基因表达与TCR蛋白表达相关， 分析细胞表面TCR+细胞的分级群体。 DNA TCR转录物的序列分析将用于区分TCR 由存在于感染部位的细胞表达， 一种评估T细胞库和抗原多样性的方法 他们回应。 原位杂交将用于鉴定和 计数表达TCR的α β和γ δ T细胞的亚群 在感染部位由不同的V区基因原位编码。 Mtb-位点T细胞亚群产生的细胞因子谱 将使用PCR测定来确定肺中的感染， 并定量分离的T细胞群中的细胞因子基因表达。 原位杂交将用于确定相对频率 和表达不同细胞因子基因的T细胞的群体动力学。 参与免疫应答的T细胞亚群的特征是： 对结核分枝杆菌的炎性免疫反应是理解结核分枝杆菌的重要因素。 宿主的反应是如何产生保护或疾病的。

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.