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

治疗HIV-1的主要挑战是静息CD 4+（rCD 4）中潜伏病毒储库（LVR）的持续存在 携带稳定整合的潜伏HIV的T细胞。检查LVR组成的变化是非常困难的 由于半衰期长。最近的数据表明，通过免疫调节，潜伏感染的rCD 4 T细胞的克隆扩增是一个重要的因素。 抗原刺激、稳态增殖和整合位点启动子破坏的组合， 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，以及这三个潜在的机制中的每一个在多大程度上推动了扩散 可能会在这个过程中发挥作用。HIV感染者的实体器官移植及相关的不同T细胞 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独特的机会， 检查在大部分T细胞库被破坏后LVR如何反弹。当局的美好愿望 行动HIV+肾器官移植试验提供了>120个匹配的快速冷冻淋巴结（LN）， 肾同种异体移植物组织和纵向收集的来自PLWH的外周血单核细胞（PBMC）。我们 假设LVR主要通过微环境中潜伏感染细胞的抗原刺激来维持， 淋巴结内的病灶，随后迁移到体内的循环和其他组织中，从而 重建T细胞耗竭疗法后的LVR。 目的1：研究肾移植术后LVR的长期动态变化及其与临床结局的关系。 我们将使用完整前病毒DNA检测（IPDA）每年测量HIV LVR长达10年， 区分完全完整的HIV与有缺陷的、缺失的和高度突变的前病毒DNA， 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：开发组织 移植前LN、血液和器官组织（肾脏）中LVR的特异性图谱，并检查 T细胞诱导后的循环、LN和肾移植物LVR。我们将组装一个HIV+的多模态图谱 通过将CODEX多重免疫荧光（mIF）平台整合到表型淋巴样细胞， 激光捕获显微切割（LCM）和细胞中原病毒的定点下一代测序 分离自不同的LN区域。HIV SMRTcap是一种新型HIV特异性单分子测序检测方法， 提供前病毒序列和匹配整合位点的同时解析，以评估克隆性， LN、PBMC和肾脏内潜伏前病毒的完整性。目标3：确定以下方面的相对贡献： 稳态增殖、抗原刺激和整合位点启动子破坏对LVR维持的影响 以及移植后的重建 这些拟议的研究将使我们能够表征纵向LVR的空间，遗传， 表型上在多个隔室中。该项目将提供关于可行性的重要信息， 通过模拟肾移植物中病毒种群的重新接种来探讨潜在的HIV治愈策略的机制， 淋巴组织和决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.