Dissecting HIV integration vs production in CD4+ T cells

剖析 HIV 整合与 CD4 T 细胞的产生

• 批准号: 6746810
• 负责人: Una T O'Doherty
• 金额: $ 31.7万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6746810
• 关键词: CD28 molecule; clinical research; cytokine; flow cytometry; fluorescent in situ hybridization; helper T lymphocyte; human immunodeficiency virus 1; human subject; immunologic memory; latent virus infection; leukocyte activation /transformation; polymerase chain reaction; provirus; tissue /cell culture; virus DNA; virus infection mechanism; virus integration

DESCRIPTION (provided by applicant): HIV-1 infected individuals accumulate a reservoir of treatment-resistant, latently infected resting CD4+ T cells, even when successful combination therapy clears their viremia. A newly developed technique, polychromatic flow cytometry (PFC), has revealed that the body's population of resting T cells comprises a complex mixture of cells with a variety of different proteins on their surfaces, presumably all specialized for different tasks and in a range of activation states. Thus, T cell activation is far from an "all or none" process. We have begun to investigate the role(s) that the growing number of resting T cell subtypes and states of partial activation may play in the development of viral reservoirs. Using PFC and a quantitative assay for HIV-1 integration we plan to dissect the activation requirements for establishment of stable HIV-1 latency (versus complete productive infection) and the resting T cell subtypes comprising the latent reservoir in infected individuals. A number of pertinent variables will be examined, including: 1. The relative efficiency of integration vs production in naive, central and effector memory resting CD4+ T cells exposed to virus in vitro without activation. 2. The effect of antigen independent intercellular interactions on integration vs production in resting CD4+ T cells. 3. The effects of distinct stimuli alone and in combinations on the efficiency of integration vs production. Stimuli to be tested include individual cytokines and costimulators (e.g., CTLA-4) immobilized on artificial immunostimulatory beads 4. The relative frequency of integrated HIV-1 proviral DNA in memory and naive CD4+ T cells isolated from the blood of HIV-1 infected individuals, compared to the same populations purified from uninfected mononuclear cells and exposed to HIV-1 in vitro. Long term Objectives: Because latently infected resting T cells are the barrier to curing HIV disease, we hope to better define the mechanisms underlying latency, including the cellular activation requirements for its efficient establishment. We will then apply what we learn from our in vitro studies to define more precisely the phenotype(s) of the CD4+ resting T cells that are latently infected in patients.

描述（由申请人提供）：HIV-1感染的个体积累了耐治疗，潜在感染的静息CD4+ T细胞的储层，即使成功的联合疗法可以清除其病毒性血症。一种新开发的技术，多色流式细胞仪（PFC），揭示了人体的静止T细胞种群包含细胞的复杂混合物和表面上各种不同蛋白质的混合物，大概都是专门用于不同任务和激活状态的不同任务。因此，T细胞激活远非“全部或无”过程。我们已经开始研究静息T细胞亚型的数量越来越多，部分激活可能在病毒储层的发展中发挥作用。使用PFC和用于HIV-1整合的定量测定法，我们计划剖析建立稳定的HIV-1潜伏期（相对于完全生产感染）的激活要求，以及在受感染个体中包含潜在储层的静息T细胞亚型。将检查许多相关变量，包括： 1。整合与生产的相对效率与幼稚，中央和效应子记忆中静止的CD4+ T细胞在体外暴露于病毒的情况下，而无需激活。 2。抗原独立的细胞间相互作用对静息CD4+ T细胞中整合与产生的影响。 3。单独刺激和组合对整合效率与生产的效率的影响。要测试的刺激包括固定在人工免疫刺激珠上 4。与从未感染的单核细胞纯净的同一种群相比，从HIV-1感染个体的血液中分离出的HIV-1前病毒DNA和从HIV-1感染个体的血液中分离出来的NAIVE CD4+ T细胞的相对频率，并在体外暴露于HIV-1。 长期目标：由于潜在感染的静息T细胞是治愈HIV疾病的障碍，因此我们希望更好地定义潜伏期的机制，包括其有效建立的细胞激活要求。然后，我们将应用我们从体外研究中学到的知识来更准确地定义患者潜在感染的CD4+静息T细胞的表型。