MACROPHAGE ANTIGEN PROCESSING OF HIV SUBTYPES

（根据申请人？CD 8+，MHC-1限制性细胞毒性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活性是定向的 针对来自嗜淋巴细胞（嗜T细胞）和嗜单核细胞（嗜M细胞）的表位 菌株T嗜性HIV病毒株不会引起生产性感染， 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HIV的人同样能够诱导初级CTL 反应，表明病毒抗原的加工发生在 暴露于两种菌株。因此，我们假设巨噬细胞可以支持 T嗜性HIV的进入和加工，随后引起MHC I类限制性 细胞毒性T淋巴细胞对特异性病毒表位的应答 没有生产性感染。我们将通过检验 使用体外固定细胞进行抗原处理所涉及的每个步骤 艾滋病毒感染模型。将测试以下具体目标：1） 通过T-嗜性和M-嗜性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衍生的巨噬细胞 随后的CTL应答，2）为了确定MHC I类限制性HIV 表位通过蛋白酶体在细胞质区室中产生， 3）为了确定HIV肽-MHC I类偶联是否需要 新的MHC I类分子的合成或可与预先存在的分子一起发生 4）确定识别的特异性表位 通过体外诱导的CTL，以及5）为了确定特定的HIV表位是否需要 抗原加工相关转运蛋白依赖性MHC I类 处理.了解这些途径可能会提供新的见解， 旨在增强HIV抗原呈递和随后的 HIV的细胞免疫反应。 候选人目前是一个肺研究员在医学系， 印第安纳州大学。在拟议的启动时间，候选人将是一名 肺部和重症监护科讲师，75岁 %的时间用于研究。到目前为止，候选人 在霍默·特威格博士的实验室接受过培训，获得了基本的免疫学知识。 知识和实验室技能。这一建议是一个合乎逻辑的机制 这项工作的延伸，旨在让候选人制定一个基本的 了解抗原加工途径。重要的是，在这项提案中， 候选人将通过在实验室工作开发新的研究技能 Homer Twigg博士（CTL克隆，CTL生成和测定），Janice Blum博士 （细胞内抗原加工途径，使用TAP缺陷的转染技术） Randy Brukiewicz博士（研究牛痘病毒， 研究特异性CTL表位的构建体）和道格拉斯佩里博士（脂质体 生物学、吞噬途径）。每个调查员都有专业知识 和资源（资金和实验室空间），以确保成功 完成培训计划。通过获得知识和技能 在这份建议书中，候选人希望实现他的职业目标， 成为一名全职的，有资助的研究人员在一个学术肺部部门。

(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.