Recognition of Mtb-Infected Cells by Non-Classically Restritcted CD8+ T Cells

非经典限制性 CD8 T 细胞对 Mtb 感染细胞的识别

• 批准号: 8397519
• 负责人: DAVID M. LEWINSOHN
• 金额: --
• 依托单位: PORTLAND VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8397519
• 关键词: A549; Adult; Afghanistan; Alleles; Alveolar Macrophages; Antibodies; Antigens; Area; Biological Assay; Biomedical Research; Biotin; Blocking Antibodies; CD8B1 gene; Calmette-Guerin Bacillus; Cell Fractionation; Cell Wall; Cell surface; Cells; Characteristics; Chemicals; Clinical; Collaborations; Colorado; Confocal Microscopy; Containment; Cytotoxic T-Lymphocytes; Development; Electron Microscopy; Epithelial Cells; Excision; Flow Cytometry; Frequencies; Funding; Genetic; Grant; HLA Antigens; Human; Immune response; Immune system; Individual; Infection; Interferons; Iraq; Knock-out; Label; Libraries; Location; Lung; Lymphocyte; MHC Class I Genes; Military Personnel; Mutagenesis; Mycobacterium smegmatis; Mycobacterium tuberculosis; Organelles; Pathway interactions; Peptide/MHC Complex; Peripheral Blood Mononuclear Cell; Persons; Phagocytosis; Phagosomes; Play; Population; Post-Translational Protein Processing; Prevalence; Prevention; Process; Property; Protein Biosynthesis; Proteins; Receptor Cell; Recording of previous events; Relative (related person); Research; Research Institute; Research Proposals; Role; Sorting - Cell Movement; Surface; T cell response; T-Cell Receptor; T-Lymphocyte; Testing; Therapeutic; TimeLine; Tuberculosis; Tuberculosis Vaccines; Umbilical Cord Blood; Universities; Vaccines; Western Blotting; Work; antigen processing; cell type; enzyme linked immunospot assay; improved; inhibitor/antagonist; loss of function; macrophage; mortality; multicatalytic endopeptidase complex; mutant; mycobacterial; pathogen; peripheral blood; response; tuberculosis immunity

DESCRIPTION (provided by applicant): Project Summary Objectives Tuberculosis (TB) remains a leading cause of infectious mortality worldwide. At present, the only available vaccine for TB, Bacillus Calmette-Guerin (BCG), has not proven effective in the prevention of adult tuberculosis. Increasingly, military personnel have been deployed to areas of the world where TB is endemic, with recent examples including Iraq and Afghanistan. As a result, an improved vaccine for TB is urgently needed. We have recently identified and characterized human, M. tuberculosis (Mtb)-reactive, non-classically restricted Cluster of differentiation 8+ (CD8+) T cells restricted by the non-classical Human Leukocyte Antigen 1b (HLA-Ib) molecule MR1. This molecule has not previously been shown to present pathogen associated antigens. In this application, we will define the role of MHC class I-related protein 1 (MR1) in the recognition of Mtb infected cells, and will define the antigen presented by MR1. AIM 1: Define the role of MR1 in CD8+ T cell recognition of Mtb-infected cells. a) Determine whether or not MR1-restricted Mtb-reactive T cells are innately acquired. b) Define the relative contribution of MR1, HLA-E, and CD1 to HLA-Ib-restricted CD8+ T cell TB immunity. c) Determine if primary lung epithelial cells can present Mtb antigens in the context of MR1, and if MR1- restricted CD8+ T cells are present in the lung. AIM 2: Define the antigen processing pathway for MR1. a) Identify the sub-cellular location in which TB antigens and MR1 become associated. b) Define the role of the proteasome, do-novo protein synthesis, TAP, and endosomal acidification in the processing of MR1 antigen(s). AIM 3: Determine the Mtb-derived antigen(s) that is (are) presented by MR1. a) Use an M. smegmatis transposon mutagenesis library to identify the MR1 antigen (s). Approach MR1-resctricted T cells from human cord blood, from peripheral blood adults with evidence of infection with Mtb, and in peripheral blood from those without infection will be compared by T-cell Receptor Excision Circles (TREC) for evidence of prior replication. Blocking antibodies will be used to determine the relative contribution of each HLA-Ib allele. Tracheal epithelial cells, type II pneumoncytes, and alveolar macrophages will be prepared, and tested for their ability to process and present Mtb and antigens found within the Mtb cell wall. Flow cytometry will be used to enumerate CD8 T cells expressing the MR-1 associated V17 T-cell Receptor (TCR), and these cells tested for their ability to recognize Mtb infected cells using IFN-3 ELISPOT. A combination of confocal microscopy and flow organellometry will be used to evaluate the presence or absence of MR1 in the Mtb phagosome. Fluorescently tagged MR1 suitable for lentivirial expression will be developed. Using chemical blockers as well as protein blockers of TAP, the requirement for TAP, acidificaton, and de-novo protein synthesis will be determined. A genetic approach will be used to define the mycobacterial MR1 antigen. In collaboration with Dr David Sherman (Seattle Biomedical Research Institute) an M. smegmatis transposon library will be developed, and tested for loss of function against MR1 restricted T cell clones. Confirmation of these results will be performed with Dr Karen Dobos (Colorado State University) and Bill Bishai (Johns Hopkins) using available mutants from a mariner transposon library.

描述（由申请人提供）： 项目摘要目标 结核病 (TB) 仍然是全世界感染性死亡的主要原因。目前，唯一可用的结核病疫苗卡介苗（BCG）尚未被证明能有效预防成人结核病。越来越多的军事人员被部署到世界上结核病流行的地区，最近的例子包括伊拉克和阿富汗。因此，迫切需要一种改进的结核病疫苗。我们最近鉴定并表征了人类结核分枝杆菌 (Mtb) 反应性、非经典限制性分化簇 8+ (CD8+) T 细胞，其受非经典人类白细胞抗原 1b (HLA-Ib) 分子 MR1 限制。此前尚未证明该分子可呈递病原体相关抗原。在此应用中，我们将定义 MHC I 类相关蛋白 1 (MR1) 在识别 Mtb 感染细胞中的作用，并将定义 MR1 呈递的抗原。目标 1：定义 MR1 在 CD8+ T 细胞识别 Mtb 感染细胞中的作用。 a) 确定 MR1 限制性 Mtb 反应性 T 细胞是否是先天获得的。 b) 定义 MR1、HLA-E 和 CD1 对 HLA-Ib 限制性 CD8+ T 细胞结核免疫的相对贡献。 c) 确定原代肺上皮细胞是否可以在 MR1 背景下呈递 Mtb 抗原，以及肺中是否存在 MR1 限制性 CD8+ T 细胞。目标 2：定义 MR1 的抗原加工途径。 a) 确定 TB 抗原和 MR1 相关的亚细胞位置。 b) 定义蛋白酶体、从头蛋白质合成、TAP 和内体酸化在 MR1 抗原加工中的作用。目标 3：确定 MR1 呈递的 Mtb 衍生抗原。 a) 使用耻垢分枝杆菌转座子诱变文库来鉴定 MR1 抗原。通过 T 细胞受体切除环 (TREC) 比较来自人脐带血、有 Mtb 感染证据的成人外周血以及未感染者外周血中的 MR1 限制性 T 细胞，以获取先前复制的证据。封闭抗体将用于确定每个 HLA-Ib 等位基因的相对贡献。将制备气管上皮细胞、II 型肺炎细胞和肺泡巨噬细胞，并测试它们处理和呈递 Mtb 以及 Mtb 细胞壁内发现的抗原的能力。流式细胞术将用于计数表达 MR-1 相关 V17 T 细胞受体 (TCR) 的 CD8 T 细胞，并使用 IFN-3 ELISPOT 测试这些细胞识别 Mtb 感染细胞的能力。共聚焦显微镜和流式细胞器测量相结合将用于评估 Mtb 吞噬体中是否存在 MR1。将开发适合慢病毒表达的荧光标记 MR1。使用 TAP 的化学阻断剂和蛋白质阻断剂，将确定 TAP、酸化和从头蛋白质合成的要求。将使用遗传学方法来定义分枝杆菌 MR1 抗原。将与 David Sherman 博士（西雅图生物医学研究所）合作开发耻垢分枝杆菌转座子文库，并测试针对 MR1 限制性 T 细胞克隆的功能丧失。 Karen Dobos 博士（科罗拉多州立大学）和 Bill Bishai（约翰霍普金斯大学）将使用水手转座子库中的可用突变体对这些结果进行确认。