MACROPHAGE ANTIGEN PROCESSING OF HIV SUBTYPES

HIV 亚型的巨噬细胞抗原加工

• 批准号: 6638173
• 负责人: KENNETH S KNOX
• 金额: $ 13.16万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6638173
• 关键词: CD4 molecule; CD8 molecule; HIV infections; MHC class I antigen; antigen presentation; antigenic peptide transporter; cell component structure /function; cell mediated cytotoxicity; cellular immunity; cytokine receptors; cytotoxic T lymphocyte; endocytosis; enzyme linked immunosorbent assay; human immunodeficiency virus; human tissue; intracellular transport; lung lavage; macrophage; major histocompatibility complex; protease inhibitor; protein degradation; radiotracer; vaccinia virus; virus antigen; virus infection mechanism

(Adapted from applicant?s abstract) CD8+, MHC-1 restricted cytotoxic T lymphocytes (CTL) are responsible for controlling viremia associated with human immunodeficiency virus (HIV) infection. CTL activity is directed against epitopes from lymphocytotropic (T-tropic) and monocytotropic (M-tropic) strains. T-tropic HIV strains do not cause a productive infection in human monocytes and macrophages. However, we have shown that macrophages exposed to T-tropic or M-tropic HIV are equally able to induce a primary CTL response, indicating that processing of viral antigens is occurring after exposure to both strains. Thus we hypothesize that macrophages can support both entry and processing of T-tropic HIV and subsequently elicit MHC class I-restricted cytotoxic T lymphocyte responses to specific viral epitopes in the absence of a productive infection. We will test this hypothesis by examining each of the steps involved in antigen processing using an in vitro fixed cell model of HIV infection. The following specific aims will be tested: 1) To determine the significance of various entry mechanisms by T-tropic and M-tropic HIV into alveolar macrophages and monocyte derived macrophages on subsequent CTL responses, 2) To determine whether MHC class I-restricted HIV epitopes are generated in the cytoplasmic compartment by proteasomes or in endosomes, 3) To determine if HIV peptide-MHC class I coupling requires synthesis of new MHC class I molecules or can occur with preexisting molecules via a regurgitant type pathway, 4) To determine specific epitopes recognized by CTL primed in vitro, and 5) To determine if specific HIV epitopes require transporter associated with antigen processing (TAP)-dependent MHC class I processing. Understanding these pathways may provide insight to novel therapies aimed at enhancing HIV antigen presentation and the subsequent cellular immune response in HIV. The candidate is currently a pulmonary fellow in the Department of Medicine at Indiana University. At the proposed start-up time the candidate will be a Lecturer on the faculty in the Pulmonary and Critical Care Division with 75 percent protected time allocated for research. To date the candidate has trained in the laboratory of Dr. Homer Twigg, acquiring basic immunologic knowledge and laboratory skills. This proposal is a logical mechanistic extension of this work designed to allow the candidate to develop a basic understanding of antigen processing pathways. Importantly, in this proposal the candidate will develop new research skills by working in the laboratories of Dr. Homer Twigg (CTL cloning, CTL generation and assays), Dr. Janice Blum (intracellular antigen processing pathways, transfection techniques using TAP-deficient cells), Dr. Randy Brukiewicz (working with vaccinia virus and constructs to study specific CTL epitopes), and Dr. Douglas Perry (liposome biology, phagocytic pathways). Each of these investigators have the expertise and resources (money and laboratory space) necessary to ensure successful completion of the training program. By acquiring the knowledge and skills outlined in this proposal, the candidate hopes to fulfill his career goal of becoming a full-time, funded researcher in an academic pulmonary division.

（改编自申请人的摘要）CD8+，MHC-1受限的细胞毒性T 淋巴细胞（CTL）负责控制与 人免疫缺陷病毒（HIV）感染。 CTL活动是定向的 反对来自淋巴细胞（T-Tropic）和单核（M-Tropic）的表位 菌株。 T型艾滋病毒菌株不会引起生产性感染 人类的单核细胞和巨噬细胞。但是，我们已经证明了巨噬细胞 暴露于T-Tropic或M-Tropic HIV同样能够诱导主要CTL 反应，表明病毒抗原的加工正在发生 暴露于两种菌株。因此，我们假设巨噬细胞可以支持 T-Tropic HIV的进入和处理，随后引起了MHC I类限制的MHC 细胞毒性T淋巴细胞对特定病毒表位的反应 缺乏生产性感染。我们将通过检查这一假设 使用体外固定细胞进行抗原加工涉及的每个步骤 HIV感染的模型。将测试以下具体目标：1） 确定T型和M型 - 热带的各种进入机制的重要性 HIV进入肺泡巨噬细胞和单核细胞衍生的巨噬细胞 随后的CTL响应，2）确定MHC I类限制性HIV是否是否 蛋白酶体或在细胞质室中产生表位 内体，3）确定HIV肽-MHC类I耦合是否需要 新的MHC I类分子的合成或可以与先前分子一起出现 通过反流类型途径，4）确定识别的特定表位 通过在体外启动的CTL，5）确定特定的HIV表位是否需要 与抗原加工（TAP）依赖性MHC类I相关的转运蛋白 加工。了解这些途径可能会为新颖的见解提供见识 旨在增强艾滋病毒抗原表现和随后的疗法 HIV中的细胞免疫反应。 该候选人目前是医学系的肺部研究员 印第安纳大学。在拟议的启动时间，候选人将是 75 百分比保护了用于研究的时间。迄今为止，候选人有 在荷马·特威格（Homer Twigg）博士的实验室接受培训，获得了基本的免疫学 知识和实验室技能。该建议是逻辑机械的 这项工作的扩展旨在允许候选人开发基本 了解抗原加工途径。重要的是，在此提案中 候选人将通过在实验室工作来发展新的研究技能 荷马·特威格（Homer Twigg）博士（CTL克隆，CTL生成和分析），Janice Blum博士 （细胞内抗原加工途径，使用Tap缺陷的转染技术 细胞），兰迪·布鲁基维奇（Randy Brukiewicz）博士（使用离甲酸病毒和 研究特定CTL表位的结构）和Douglas Perry博士（脂质体 生物学，吞噬途径）。这些调查人员中的每一个都有专业知识 以及确保成功的资源（金钱和实验室空间） 完成培训计划。通过获取知识和技能 候选人在此提案中概述了，希望实现他的职业目标 成为学术肺部部门的全职，资助的研究员。