Defining the Pathogenesis of Immune Deficiency in Chronic HIV Infection

该项目申请由克利夫兰免疫病理学联盟（CLIC）的成员提交，该联盟是一组代表美国和加拿大10个学术和研究机构的研究人员，他们从事了三年多的协调研究工作，旨在揭示HIV感染导致进行性免疫缺陷的机制。这组经验丰富，优秀的研究人员利用互补的研究技能和资源，并提出了一个跨学科的计划，包括由两个核心协调和支持的4个项目：行政核心，负责项目和标本采集核心的总体协调和管理，Alan Landay，PI，负责确保肠道和淋巴结临床标本的持续供应，以支持项目研究人员。这些项目包括一系列相互作用的研究平台，从基础实验室研究到实验动物模型再到转化项目，每个项目都旨在探索免疫激活驱动慢性HIV感染中CD4 + T细胞耗竭和功能障碍的决定因素和机制。项目#1：旁观者激活驱动慢性HIV感染中的T细胞损失-PI：Michael M.莱德曼，医学博士，斯科特·F Sieg博士- 病例，将检验以下假设：在慢性HIV感染中，次级淋巴组织中微生物TLR配体和常见γ链细胞因子的全身水平增加驱动中枢记忆T细胞活化和周转。项目#2：HIV感染中肠道屏障功能的丧失-Alan Levine博士病例，将检查HIV感染中肠粘膜的完整性，以记录微生物产物通过受损肠道的增强易位的机制细节，我们提出这有助于慢性HIV感染中细胞丢失的发病机制。项目#3：SIV感染的非人灵长类动物的免疫激活和艾滋病发病机制-Guido Silvestri，医学博士，宾夕法尼亚大学，将尝试通过激活先天免疫系统在非致病性SIV感染的白眉猴中诱导疾病，并将测试阻断先天免疫激活是否会减弱受感染恒河猴的疾病发病机制。项目#4：免疫激活促进慢性HIV感染中的PD1表达和免疫功能障碍-Rafick Pierre Sekaly博士- 蒙特利尔大学，将通过HIV RNA和TLR 7/8对PDL-1和2表达的相互作用及其对HIV反应性T细胞功能和存活的影响来研究先天免疫系统激活的作用。 项目1：旁观者激活驱动慢性HIV感染中的T细胞损失（Michael Lederman） 项目1描述（由申请人提供）：来自我们合作小组的最新数据提供了一个新的HIV发病机制模型的背景，将在这里进行测试。在该模型中，中央记忆（CM）CD4 + T细胞的周转是HIV感染的进行性细胞损失的核心。我们认为这是由HIV复制对次级淋巴组织细胞因子环境的间接影响和原位暴露于从受损肠道易位的微生物TLR配体之间的相互作用驱动的。HIV复制是必要的，但不足以促进T细胞周转;微生物TLR配体通过两种不同的机制提供额外的信号。首先，它们通过增加C型凝集素CD69的表达促进效应CD8 + T细胞在次级淋巴组织中的非特异性保留增强，所述C型凝集素CD69干扰允许活化细胞从淋巴结离开所需的鞘氨醇-1磷酸受体的表面表达。效应细胞的隔离增强了这些组织中的细胞因子"风暴"，导致我们在这些部位定量的常见γ链受体细胞因子IL-2和IL-15的爆炸性水平。TLR配体还激活CM T细胞以失去对死亡信号的特征性抗性，该抗性是由于FOXO 3a磷酸化和失活。这导致CM CD4 + T细胞的周转和选择性死亡增加，从而导致HIV感染的免疫缺陷。我们的目标是：1）表征选定的TLR配体和常见γ链受体细胞因子促进中枢记忆CD 4+和CD 8 + T细胞激活的细胞间相互作用和机制。这将通过详细说明T细胞活化的特定ARC要求，通过探索细胞周期进展，凋亡和存活的信号特征的选定基因表达模式，并使用这些结果来识别使HIV感染中的CM T细胞更容易受到死亡信号影响的途径来实现。2)建立次级淋巴组织中TLR配体和常见γ链受体细胞因子暴露后旁观者T细胞活化和隔离的体外模型。这将首先通过使用悬浮的外周血单核细胞定义模型，然后在淋巴结组织培养实验中确认模型来实现。3)确定慢性HIV感染中免疫激活的近因。我们将测定慢性HIV感染者血浆中选定的微生物TLR激动剂、HIV RNA和细菌PGN、LPS和DNA的水平。基于SpAim 1实验的结果，我们将开发一组基于流动的试剂，其将表征将应用于慢性HIV感染中的血液、淋巴结和肠道淋巴细胞的离体分析的旁观者T细胞活化的特征。

DESCRIPTION (provided by applicant): This program project application is submitted by the members of the Cleveland Immunopathogenesis Consortium (CLIC) a group of investigators representing 10 academic and research institutions in the United States and Canada who have engaged for more than three years in a coordinated research effort aimed at unraveling the mechanisms whereby HIV infection results in progressive immune deficiency. This group of experienced, outstanding investigators capitalizes on complementary research skills and resources and proposes an interdisciplinary program comprising 4 projects that are coordinated and supported by two cores: Administrative Core, charged with overall coordination and administration of the program and Specimen Acquisition Core, Alan Landay, PI, charged with assuring a sustained supply of clinical specimens of gut and lymph nodes to support project investigators. The projects comprise a series of interacting research platforms from basic laboratory research to experimental animal models to translational projects, each designed to explore the determinants and mechanisms whereby immune activation drives CD4+ T cell depletion and dysfunction in chronic HIV infection. Project #1: Bystander activation drives T cell losses in chronic HIV infection - PIs: Michael M. Lederman, M.D., Scott F. Sieg Ph.D. - Case, will test the hypothesis that increased systemic levels of microbial TLR ligands and common gamma chain cytokines in secondary lymphoid tissues drive central memory T cell activation and turnover in chronic HIV infection. Project #2: Loss of intestinal barrier function in HIV infection - Alan Levine, Ph.D. Case, will examine the integrity of the intestinal mucosa in HIV infection to document the mechanistic details underlying the enhanced translocation of microbial products through the damaged gut that we propose contributes to the pathogenesis of cell loss in chronic HIV infection. Project #3: Immune activation and AIDS pathogenesis in SIV-infected non-human primates - Guido Silvestri, M.D., Univ of Pennsylvania, will attempt to induce disease in non-pathogenic SIV infection of sooty mangabeys by activation of the innate immune system and will test whether blocking innate immune activation will attenuate disease pathogenesis in infected rhesus macaques. Project #4: Immune activation promotes PD1 expression and immune dysfunction in chronic HIV infection - Rafick Pierre Sekaly Ph.D. - Univ of Montreal, will examine the role of innate immune system activation through the interaction between HIV RNAs and TLR7/8 on the expression of PDL-1 and 2 and their effects on the function and survival of HIV reactive T cells. PROJECT 1: Bystander activation drives T cell losses in chronic HIV infection (Michael Lederman) PROJECT 1 DESCRIPTION (provided by applicant): Recent data from our collaborative group provide background for a new model of HIV pathogenesis that will be tested here. In this model, turnover of central memory (CM) CD4+ T cells is central to progressive cell losses of HIV infection. We propose that this is driven by interplay between indirect effects of HIV replication on the cytokine environment of secondary lymphoid tissues and in situ exposure to microbial TLR ligands translocated from the damaged gut. HIV replication is necessary but not sufficient to promote T cell turnover; microbial TLR ligands provide additional signals via two distinct mechanisms. First, they promote enhanced non-specific retention of effector CD8+ T cells in secondary lymphoid tissues by increasing expression of the C-type lectin CD69 that interferes with surface expression of sphingosine-1 phosphate receptors needed to permit exit of activated cells from lymph nodes. Sequestration of effector cells intensifies the cytokine "storm" in these tissues that results in explosive levels of common gamma chain receptor cytokines IL-2 and IL-15 that we have quantified at these sites. TLR ligands also activate CM T cells to lose characteristic resistance to death signals a resistance that is due to FOXO3a phosphorylation and inactivation. This results in heightened turnover and selective death of CM CD4+ T cells that drives the immune deficiency of HIV infection. Our aims are: 1) To characterize the intercellular interactions and mechanisms whereby selected TLR ligands and common gamma chain receptor cytokines promote activation of central memory CD4+ and CD8+ T cells. This will be achieved by detailing the specific ARC requirements for T cell activation, by exploring selected gene expression patterns for signals characteristic of cell cycle progression, apoptosis and survival and to use these results to identify the pathways that render CM T cells in HIV infection more susceptible to death signals. 2) To establish in vitro models for bystander T cell activation and sequestration after exposure to TLR ligands and common gamma chain receptor cytokines in secondary lymphoid tissues. This will be accomplished first by defining the model using peripheral blood mononuclear cells in suspension and then confirming the model in lymph node histoculture experiments. 3) To identify the proximate causes of immune activation in chronic HIV infection. We will determine the levels of selected microbial TLR agonists, HIV RNA and bacterial PGN, LPS and DNA in the plasma of chronically HIV infected persons. Based upon the results of SpAim 1 experiments, we will develop a panel of flow based reagents that will characterize the signatures of bystander T cell activation that will be applied to ex vivo analyses of blood, lymph node and gut lymphocytes in chronic HIV infection.