HIV-1-Specific CTL Exhaustion at Immune Synapse

免疫突触处 HIV-1 特异性 CTL 耗尽

• 批准号: 9137404
• 负责人: Dongfang Liu
• 金额: $ 25.05万
• 依托单位: METHODIST HOSPITAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-05 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9137404
• 关键词: Actins; Adaptor Signaling Protein; Address; Alleles; Apoptosis; Biogenesis; Biological Assay; CD8-Positive T-Lymphocytes; CD8B1 gene; Cell Differentiation process; Cell physiology; Cells; Cessation of life; Chickens; Chronic; Cytoplasmic Granules; Cytotoxic T-Lymphocyte-Associated Protein 4; Data; Development; F-Actin; Fluorescence Microscopy; Goals; HIV; HIV Infections; HIV-1; HLA A*0201 antigen; HLA-A2 Antigen; HLA-B 2705 antigen; HLA-B27 Antigen; Haplotypes; Host Defense; Human; Image; Imaging Techniques; Immune; Immunologics; Individual; Infection; Killer Cells; Knowledge; Ligands; Lipid Bilayers; Lymphocyte; Lymphocyte Activation; Lytic; Malignant Neoplasms; Mediating; Microscopy; Modeling; Molecular; Natural Killer Cells; Oncogenic Viruses; PDCD1LG1 gene; Patients; Phosphorylation; Phosphotransferases; Play; Protein Family; RNA; Resolution; Role; Signal Transduction; Signaling Molecule; Staging; Structure; Synapses; System; T cell response; T-Lymphocyte; Techniques; Up-Regulation; Vial device; Viral; Viral Load result; Virus; Virus Diseases; Work; ZAP-70 Gene; antiretroviral therapy; cytotoxic; cytotoxicity; exhaust; exhaustion; functional disability; immunological synapse; immunological synapse formation; inhibitor/antagonist; insight; perforin; prevent; programs; public health relevance; receptor; receptor expression; research study; src-Family Kinases

 DESCRIPTION (provided by applicant): Most HIV-infected people ultimately succumb to chronic infection if not adherent to combination antiretroviral therapy (cART). Some, however, known as "elite controllers (EC)", demonstrate superior virus control maintaining virtually undetectable viral loads even in the absence of treatment. While it is well known that HIV- specific cytotoxic lymphocytes (CTL) are critical for viral control and that HLA haplotype is associated with viral progression status, the mechanisms underlying EC virus control remain unknown. Recently, we showed that HIV-specific CTL from EC form better quality immunological synapses (IS) with HIV-infected target cells, with more rapid biogenesis of perforin and more effective lytic granule loading. We also found that engagement of inhibitory receptor programmed death-1 (PD-1, a marker for CTL exhaustion) on HIV-specific CTL specifically induces phosphorylation of the adaptor protein Chicken Tumor Virus #10 Regulator of Kinase (Crk). We hypothesize that chronic HIV leads to functional impairment of the CTL IS via upregulation of inhibitory receptors like PD-1 and that the resulting downstream phosphorylation of Crk, in turn, prevents CTL activation by disrupting the structure and signaling of the CTL-target cell synapse. Increased inhibitory receptor expression is one of the earliest markers of CTL exhaustion in chronic HIV. EC patients express lower levels of PD-1 than do their chronic progressor (CP) counterparts. In Aim 1 we will compare synapse structure and function in exhausted (CP) vs. non-exhausted (EC) HIV-specific CTL. HIV-specific CTL will be stimulated by a reductionist lipid bilayer system or by HIV-infected target cells in the presence o absence of PD-1's primary ligand PD-L1. IS will be visualized using super-resolution stimulated emission depletion microscopy and total internal reflection fluorescence (TIRF) microscopy. There are a number of important questions about IS that can only be addressed through super-resolution imaging (e.g. distribution of F-actin and lytic granules at IS). The proposed high-resolution, single cell assays will allow us to determine the specific impact of inhibitory recepto signaling on IS structure and function and will yield critical insights into how these relate to vial control (EC vs. CP) and haplotype. We hypothesize that upregulation of inhibitory receptor in exhausted cells results in increased pCrk, blocking critical Crk-dependent activation signals like Vav-1. Aim 2 will dissect the dynamics of signal integration at the IS of exhausted HIV-specific CTL, relating Crk phosphorylation and localization of key regulators, as well as cytotoxicity and viral inhibition, to inhibitory receptor expression level, haplotype, and viral progression status. Signaling will be tracked at a single-cell, synapse level using fluorescently tagged signal molecules and super-resolution STED microscopy. While other groups are attempting to boost T cell responses by targeting individual inhibitory receptors (e.g. PD-1 and CTLA-4), the proposed experiments will be the first to address a downstream, central signaling hub. It is our long-term goal to use the knowledge to augment T cell responses in HIV and other chronic viral infections.

 描述（由申请人提供）：如果不坚持联合抗逆转录病毒治疗（cART），大多数HIV感染者最终死于慢性感染。然而，一些被称为“精英控制者（EC）"的病毒控制表现出上级，即使在没有治疗的情况下也维持几乎不可检测的病毒载量。虽然众所周知HIV特异性细胞毒性淋巴细胞（CTL）对于病毒控制是关键的，并且HLA单倍型与病毒进展状态相关，但EC病毒控制的潜在机制仍然未知。最近，我们发现，来自EC的HIV特异性CTL与HIV感染的靶细胞形成更好质量的免疫突触（IS），具有更快的穿孔素生物合成和更有效的裂解颗粒负载。我们还发现抑制性受体程序性死亡-1（PD-1，CTL耗竭的标志物）对HIV特异性CTL的参与特异性诱导接头蛋白鸡肿瘤病毒#10激酶调节因子（Crk）的磷酸化。我们假设慢性HIV通过上调抑制性受体如PD-1导致CTL IS的功能受损，并且由此产生的下游Crk磷酸化反过来通过破坏CTL靶细胞突触的结构和信号传导来阻止CTL活化。抑制性受体表达增加是慢性HIV中CTL耗竭的最早标志之一。EC患者表达的PD-1水平低于其慢性进展者（CP）对应者。在目标1中，我们将比较耗尽（CP）与非耗尽（EC）HIV特异性CTL的突触结构和功能。在存在或不存在PD-1的主要配体PD-L1的情况下，HIV特异性CTL将被还原脂质双层系统或被HIV感染的靶细胞刺激。将使用超分辨率受激发射损耗显微镜和全内反射荧光（TIRF）显微镜观察IS。有一些关于IS的重要问题只能通过超分辨率成像来解决（例如IS处F-肌动蛋白和溶解颗粒的分布）。提出的高分辨率，单细胞测定将使我们能够确定抑制性受体信号传导对IS结构和功能的具体影响，并将产生关键的见解，这些如何与小瓶控制（EC与CP）和单倍型。我们推测，在耗竭细胞中抑制性受体的上调导致pCrk增加，阻断关键的Crk依赖性激活信号，如Vav-1。目的2将剖析信号整合的动力学在耗尽的HIV特异性CTL的IS，相关的Crk磷酸化和本地化的关键监管机构，以及细胞毒性和病毒抑制，抑制受体表达水平，单倍型，和病毒的进展状态。 将使用荧光标记的信号分子和超分辨率STED显微镜在单细胞突触水平上跟踪信号传导。虽然其他研究小组正试图通过靶向单个抑制性受体（例如PD-1和CTLA-4）来增强T细胞反应，但拟议的实验将是第一个解决下游中央信号中枢的实验。我们的长期目标是利用这些知识来增强HIV和其他慢性病毒感染中的T细胞反应。