Effect of ESAT-6 on human T cell Function

描述（由申请人提供）：结核病仍然是一个主要的全球卫生问题，疫苗开发是一个关键的优先事项。t细胞在抵抗结核分枝杆菌方面起着关键作用。然而，已知结核分枝杆菌可抑制人体免疫，但其机制尚不清楚。早期分泌的6kd抗原靶标（ESAT-6）是一种免疫原性蛋白，是一种潜在的候选疫苗，但也有助于哺乳动物细胞的毒力和细胞溶解。我们的初步数据表明，ESAT-6直接抑制人T细胞增殖和IFN-g分泌，这些作用不是由于细胞毒性或细胞凋亡增加。我们的目标是了解ESAT-6抑制人类T细胞IFN-g产生的分子机制。具体目标是：1。确定ESAT-6抑制T细胞IFN-g产生的信号通路。我们将使用启动子下拉试验和染色质免疫沉淀（目的1.1）来确定ESAT-6对结合IFN-g近端启动子的转录因子的影响。我们将评估ESAT-6对通过t细胞受体介导信号传导的分子（aim 1.2）和中间信号传导分子，丝裂原活化蛋白激酶（aim 1.3）的影响，使用免疫沉淀，Western blotting和功能激酶测定2。鉴定ESAT-6在人t细胞上的哺乳动物配体。我们将使用生物素化的ESAT-6从纯化的t细胞提取物中提取蛋白质，洗脱蛋白质，用SDS-PAGE分离它们，并用质谱法鉴定它们（目的2.1）。假设的配体将被克隆和表达，ESAT-6与其配体之间的相互作用将通过表面等离子体共振和蛋白质覆盖测定来确认（目标2.2）。为了确定ESAT-6与其假定的配体之间相互作用的功能意义，我们将确定中和ESAT-6配体的抗体和/或siRNA是否会消除ESAT-6对t细胞的抑制作用（目的2.3）。公共卫生相关性：结核病仍然是世界范围内一个巨大的公共卫生问题，有效的疫苗将大大有助于疾病控制。早期分泌6kd抗原靶点（ESAT-6）是一种结核分枝杆菌蛋白，是一种有希望的候选疫苗，但也有助于毒力和组织损伤。该提案旨在了解ESAT-6抑制人类t细胞免疫功能的机制，这一信息对于开发改进的结核病疫苗和治疗策略至关重要。

DESCRIPTION (provided by applicant): Tuberculosis remains a major global health problem and vaccine development is a critical priority. T-cells play a pivotal role in protection against Mycobacterium tuberculosis. However, M. tuberculosis is known to inhibit human immunity, but the mechanisms are not well understood. Early secreted antigenic target of 6 kD (ESAT-6) is an immunogenic protein that is a potential vaccine candidate, but also contributes to virulence and cytolysis of mammalian cells. Our preliminary data indicate that ESAT-6 directly inhibits human T cell proliferation and IFN-g secretion, and these effects are not due to cytotoxicity or increased apoptosis. Our goal is to understand the molecular mechanisms by which ESAT-6 inhibits human T cell IFN-g production. The specific aims are: 1. Identify the signaling pathways by which ESAT-6 inhibits T cell IFN-g production. We will determine the effect of ESAT-6 on transcription factors that bind to the IFN-g proximal promoter, using promoter pull-down assays and chromatin immunoprecipitation (aim 1.1). We will evaluate the effect of ESAT-6 on molecules that mediate signaling through the T-cell receptor (aim 1.2) and on the intermediary signaling molecules, mitogen-activated protein kinases (aim 1.3), using immunoprecipitation, Western blotting and functional kinase assays 2. Identify a mammalian ligand for ESAT-6 on human T-cells. We will use biotinylated ESAT-6 to pull down proteins from extracts of purified T-cells, elute the proteins, separate them by SDS-PAGE and identify them by mass spectrometry (aim 2.1). The putative ligand will be cloned and expressed, and interactions between ESAT-6 and its ligand will be confirmed by surface plasmon resonance and protein overlay assays (aim 2.2). To determine the functional significance of the interaction between ESAT-6 and its putative ligand, we will determine if neutralizing antibodies and/or siRNA to the ESAT-6 ligand abrogate the inhibitory effects of ESAT-6 on T-cells (aim 2.3). PUBLIC HEALTH RELEVANCE: Tuberculosis remains a tremendous public health problem world-wide, and an effective vaccine would contribute greatly to disease control. Early secreted antigenic target of 6 kD (ESAT-6) is a Mycobacterium tuberculosis protein that is a promising vaccine candidate, but also contributes to virulence and tissue damage. This proposal seeks to understand the mechanisms by which ESAT-6 inhibits human T-cell immune function, information that is critical for development of improved vaccines and treatment strategies for tuberculosis.