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

描述（由申请人提供）： 结核病（TB）仍然是全球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），尚未证明对预防成人结核病有效。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，迫切需要改进的结核病疫苗。我们最近发现并鉴定了人类M.结核病（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MR 1限制。该分子以前未显示出呈递病原体相关抗原。在本申请中，我们将定义MHC I类相关蛋白1（MR 1）在识别Mtb感染细胞中的作用，并定义由MR 1呈递的抗原。目的1：明确MR 1在Mtb感染细胞的CD8 + T细胞识别中的作用。a）确定MR1限制性Mtb反应性T细胞是否是先天获得的。B）确定MR 1、HLA-E和CD 1对HLA-Ib限制性CD 8 + T细胞TB免疫的相对贡献。c）确定原代肺上皮细胞是否可以在MR 1的情况下呈递Mtb抗原，以及肺中是否存在MR 1限制性CD8 + T细胞。目的2：确定MR 1的抗原加工途径。a）鉴定TB抗原和MR1结合的亚细胞位置。B）定义蛋白酶体、从头蛋白合成、TAP和内体酸化在MR 1抗原加工中的作用。目的3：确定由MR 1呈递的Mtb衍生抗原。a）使用M。smeglobin转座子诱变文库以鉴定MR 1抗原。方法将通过T细胞受体切除环（TREC）比较来自人脐带血、来自具有Mtb感染证据的外周血成人和来自未感染者的外周血中的MR1抑制的T细胞的先前复制的证据。将使用封闭抗体来确定每个HLA-Ib等位基因的相对贡献。将制备气管上皮细胞、II型肺细胞和肺泡巨噬细胞，并检测其处理和呈递Mtb和Mtb细胞壁内发现的抗原的能力。将使用流式细胞术计数表达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。将使用共聚焦显微镜和流式细胞器测定法的组合来评价Mtb吞噬体中是否存在MR 1。将开发适合于慢病毒表达的标记的MR 1。使用化学阻断剂以及TAP的蛋白质阻断剂，将确定TAP、酸化和从头蛋白质合成的需要。将使用遗传学方法来定义分枝杆菌MR1抗原。与西雅图生物医学研究所的大卫谢尔曼博士合作，M.将开发smeglycoprotein转座子文库，并测试针对MR 1限制性T细胞克隆的功能丧失。这些结果的确认将由Karen Dobos博士（科罗拉多州立大学）和Bill Bishai（约翰霍普金斯）使用来自水手转座子文库的可用突变体进行。 公共卫生相关性： 结核病仍然是全世界感染性死亡的主要原因。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，迫切需要改进的结核病疫苗。该应用程序的重点是了解人类CD8 T细胞可以识别感染结核分枝杆菌（Mtb）的细胞的机制，结核病的病原体。结核分枝杆菌存在于细胞内。因此，CD8 T细胞能够独特地找到那些携带这种细菌的细胞。该应用程序的重点是了解分子MR1如何用于提醒免疫系统Mtb的存在。由于MR1在所有个体之间都是高度保守的，因此了解这种分子的功能对于开发改进的疫苗和诊断方法具有应用价值。

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. PUBLIC HEALTH RELEVANCE: Project Narrative Tuberculosis remains a leading cause of infectious mortality worldwide. 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. This application is focused on understanding the mechanisms by which human CD8 T cells can identify those cells infected with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. Mtb resides within cells. As a result, CD8 T cells are uniquely able to find those cells that harbor this bacteria. This application is focused on understanding how the molecule MR1 is used to alert the immune system to the presence of Mtb. Because MR1 is highly conserved between all individuals, understanding how this molecule functions has application for the development of improved vaccines and diagnostics.