MACROPHAGE ANTIGEN PROCESSING OF HIV SUBTYPES

(改编自申请者S摘要)CD8+、MHC-1限制性细胞毒T细胞 淋巴细胞(CTL)负责控制与 人类免疫缺陷病毒(HIV)感染。CTL活动是定向的 针对嗜淋巴细胞(T嗜性)和单核细胞嗜性(M嗜性)的表位 菌株。T嗜性HIV毒株不会在 人单核细胞和巨噬细胞。然而，我们已经证明巨噬细胞 暴露于T嗜性或M嗜性艾滋病毒的人同样能够诱导原发CTL 应答，表明病毒抗原的处理发生在 暴露在两种菌株中。因此，我们假设巨噬细胞可以支持 T嗜性HIV的进入和加工，随后引发MHC I类限制性 细胞毒性T淋巴细胞对特定病毒表位的反应 没有产生性感染。我们将通过检验这一假设来检验这一假设 使用体外固定细胞处理抗原的每一步 HIV感染的模型。将测试以下具体目标：1) 用T向和M向确定各种进入机制的意义 HIV进入肺泡巨噬细胞和单核细胞来源的巨噬细胞 随后的CTL反应，2)确定MHC I类限制性HIV 表位是在细胞质中由蛋白酶体或在 内体，3)确定HIV多肽-MHC I类偶联是否需要 合成新的MHC I类分子或可以与先前存在的分子发生 通过返流型途径，4)确定识别的特定表位 通过体外启动的CTL，以及5)确定特定的HIV表位是否需要 与抗原处理相关的转运蛋白(TAP)依赖的MHC-I类 正在处理。了解这些路径可能会为小说提供洞察力 旨在加强艾滋病毒抗原呈递和随后的 HIV中的细胞免疫反应。 候选人目前是医学系的肺部研究员， 印第安纳大学。在建议的启动时间，候选人将成为 肺和重症监护科教职员工讲师 分配给研究的受保护时间百分比。到目前为止，候选人已经 在霍默·特威格博士的实验室接受培训，获得基础免疫学 知识和实验室技能。这个建议是一种合乎逻辑的机械论 这项工作的延伸，旨在使候选人能够制定基本的 对抗原处理途径的理解。重要的是，在这项提案中 应聘者将通过在实验室工作来培养新的研究技能 Hmer Twigg博士(CTL克隆、CTL生成和分析)，Janice Blum博士 (细胞内抗原处理途径，使用TAP缺陷的转染技术 细胞)，Randy Brukiewicz博士(与牛痘病毒和 构建来研究特定的CTL表位)，以及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 和必要的资源(资金和实验室空间)，以确保成功 完成培训计划。通过获取知识和技能 在这份提案中，候选人希望实现他的职业目标 成为学术肺科的全职、有资金支持的研究员。

(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.