T-cell depletion and maintenance of the HIV-1 latent reservoir in distinct tissue compartments

治愈HIV-1的主要挑战是潜伏的病毒库(LVR)在静止的CD4(RCD4)中的持久性。 携带稳定整合的潜伏HIV的T细胞。检查LVR成分的变化是极其困难的 由于存在较长的半衰期。最近的数据显示，潜伏感染的rCD4T细胞通过 抗原刺激、内稳态增殖和整合位点启动子破坏的组合是 LVR维护的主要贡献者。目前尚不清楚哪种组织来源是LVR的主要驱动因素 维持，以及这三种潜在机制中每一种对扩散的贡献程度 可能会在这个过程中发挥作用。HIV感染者及相关异种T细胞的实体器官移植 用于预防性同种异体移植排斥治疗的细胞诱导策略提供了一个独特的机会 检查在很大一部分T细胞库被破坏后，LVR是如何反弹的。寄希望于 行动艾滋病毒肾器官移植试验提供了&gt；120匹配的闪存冷冻淋巴(LN)， 移植肾组织，从PLWH纵向采集外周血单个核细胞(PBMC)。我们 假定LVR主要是通过抗原刺激微小潜伏感染细胞来维持的 淋巴结内的病灶，随后迁移到循环和体内其他组织，从而 重建左心室后T细胞耗竭治疗。 目的1：研究肾移植术后长期左心室阻力的动态变化及其与临床预后的关系。 我们将使用完整的前病毒DNA分析(IPDA)在长达10年的时间内每年测量HIV LVR，这是 在接受治疗的个体中，区分完全完整的艾滋病毒与缺陷、缺失和高度突变的前病毒DNA 与移植相关的免疫抑制药物，对艾滋病毒治疗策略感兴趣。目标2：开发一种纸巾 移植前LN、血液和器官组织(肾脏)中LVR的特异性图谱，并检查LN、血液和器官组织(肾脏)的重新种植 T细胞诱导后循环、LN和移植肾的LVR。我们将汇编一份多模式的艾滋病毒地图集 LN通过将Codex多重免疫荧光(MIF)平台整合到表型淋巴细胞和 激光捕获显微切割(LCM)和细胞内前病毒的下一代定点测序 从不同的LN带分离出来的。HIV SMRTCap，一种新的HIV特异性单分子测序方法将 提供前病毒序列和匹配整合位点的同时解析，以评估克隆和 LN、PBMC和肾脏内潜伏前病毒的完整性。目标3：确定以下项目的相对贡献 动态平衡增殖、抗原刺激和整合位点启动子对左心室维持的影响 并在移植后重建。 这些拟议的研究将使我们能够在空间上、遗传上和 表型为多个隔间。该项目将提供有关可行性和 通过模拟病毒群体在移植肾和移植肾中的重新播种来研究潜在的HIV治疗策略的机制 淋巴组织，并确定克隆增殖的驱动机制。

The primary challenge in curing HIV-1 is the persistence of a latent viral reservoir (LVR) in resting CD4+ (rCD4) T cells that harbor stably integrated latent HIV. Examining changes in the LVR composition is incredibly difficult due to the long half-life. Recent data show that clonal expansion of latently infected rCD4 T cells through a combination of antigenic stimulation, homeostatic proliferation, and integration site promotor disruption are major contributors to LVR maintenance. It is unclear which tissue source is the primary driver of LVR maintenance, as well as what level of contribution each of these three potential mechanisms driving proliferation may play in that process. Solid organ transplantation in people living with HIV and the associated different T cell induction strategies prescribed for prophylactic allograft rejection treatment provide a unique opportunity to examine how the LVR rebounds after a large proportion of the T cell repertoire is destroyed. The HOPE in Action HIV+ kidney organ transplantation trial provides access to >120 matched flash frozen lymph nodes (LN), renal allograft tissue, and longitudinally collected peripheral blood mononuclear cells (PBMC) from PLWH. We hypothesize that the LVR is primarily maintained through antigen stimulation of latently infected cells in micro foci within lymph nodes, which subsequently migrate into the circulation and other tissues in the body, thereby reestablishing the LVR post-T cell depletion therapy. Aim 1: Examine long-term LVR dynamics post-renal transplantation and its association with clinical outcomes. We will measure the HIV LVR annually for up to 10 years using the intact proviral DNA assay (IPDA), which distinguishes fully intact HIV from defective, deleted, and hypermutated proviral DNA, in individuals receiving transplant-related immunosuppressive drugs that are of interest to HIV cure strategies. Aim 2: Develop a tissue specific atlas of the LVR in LN, blood, and organ tissue (kidney) pre-transplantation, and examine reseeding of the circulating, LN, and kidney allograft LVR post T cell induction. We will assemble a multi-modal atlas of HIV+ LN by integrating the CODEX multiplexed immunofluorescence (mIF) platform to phenotype lymphoid cells and laser capture microdissection (LCM) and site-directed next-generation sequencing of the proviruses in cells isolated from distinct LN zones. HIV SMRTcap, a novel HIV-specific single molecule sequencing assay will provide simultaneous resolution of proviral sequences and matched integration sites, to evaluate clonality and intactness of latent provirus within the LN, PBMC and kidney. Aim 3: Determine the relative contribution of homeostatic proliferation, antigenic stimulation, and integration site promoter disruption on LVR maintenance and re-establishment post-transplant. These proposed studies will enable us to characterize the longitudinal LVR spatially, genetically, and phenotypically in multiple compartments. This project will provide critical information on feasibility and mechanisms of potential HIV cure strategies by modeling re-seeding of viral populations in kidney allograft and lymphoid tissues and determining driving mechanisms of clonal proliferation.