HIV-1-Specific CTL Exhaustion at Immune Synapse

 描述(申请人提供)：如果不坚持联合抗逆转录病毒疗法(CART)，大多数艾滋病毒感染者最终会死于慢性感染。然而，一些被称为“精英控制员(EC)”的人展示了卓越的病毒控制能力，即使在没有治疗的情况下也能保持几乎无法检测到的病毒载量。虽然众所周知，HIV特异性细胞毒性淋巴细胞(CTL)对病毒控制至关重要，而且人类白细胞抗原单倍型与病毒进展状态有关，但EC病毒控制的机制仍不清楚。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高质量的免疫突触(IS)，穿孔素的生物发生更快，裂解颗粒负载更有效。我们还发现，抑制性受体程序性死亡-1(PD-1，CTL耗竭的标志)与HIV特异性CTL结合，特异性地诱导适配蛋白鸡肿瘤病毒#10调节蛋白激酶(Crk)的磷酸化。我们假设，慢性HIV导致CTL功能损害是通过PD-1等抑制性受体上调，从而导致Crk下游磷酸化，进而通过破坏CTL靶细胞突触的结构和信号来阻止CTL激活。抑制性受体表达增加是慢性HIV患者CTL耗竭的最早标志之一。EC患者的PD-1表达水平低于慢性进展者(CP)患者。在目标1中，我们将比较衰竭(CP)和非衰竭(EC)HIV特异性CTL的突触结构和功能。HIV特异性CTL将被还原的脂质双层系统或在没有PD-1‘S一级配体PD-L1的情况下被HIV感染的靶细胞所刺激。IS将使用超分辨率受激发射耗尽显微镜和全内反射荧光(TIRF)显微镜可视化。有许多关于IS的重要问题只能通过超分辨率成像来解决(例如，F-肌动蛋白和溶解颗粒在IS的分布)。拟议的高分辨率单细胞分析将使我们能够确定抑制性受体信号对IS结构和功能的具体影响，并将对这些与瓶子控制(EC与CP)和单倍型的关系产生关键的见解。我们假设，耗竭细胞中抑制受体的上调导致pCrk增加，阻断关键的Crk依赖的激活信号，如Vav-1。目的2将剖析耗竭的HIV特异性CTL的信号整合动力学，将CRK磷酸化和关键调节因子的定位以及细胞毒性和病毒抑制与抑制性受体表达水平、单倍型和病毒进展状态联系起来。 信号将在单细胞、突触水平上使用荧光标记的信号分子和超分辨率STED显微镜进行跟踪。虽然其他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次针对下游的中央信号枢纽。我们的长期目标是利用这一知识增强艾滋病毒和其他慢性病毒感染的T细胞反应。

 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.