Migratory Properties of HIV-infected T cells in vivo

描述（由申请人提供）：HIV-1引起人体免疫系统的全身性感染，使用T细胞、树突状细胞和巨噬细胞表达的CD4膜蛋白作为其主要的进入受体。虽然细胞外病毒粒子大量存在于感染者的体液中，但目前尚不清楚游离病毒是否有助于体内感染细胞和未感染细胞之间的传播。体外研究表明，与游离病毒粒子相比，T细胞之间以及T细胞与转染HIV的感染或未感染树突状细胞和巨噬细胞之间的直接相互作用大大提高了病毒传播的效率。在这里，我们建议使用多光子活体显微镜（MP-IVM）对人源化BLT小鼠淋巴结和雌性生殖道（FRT）粘膜中hiv感染T细胞的行为进行时空分辨可视化。利用重组HIV毒株的调色板，将荧光赋予有生产力的感染细胞或病毒粒子，我们的目标是深入了解感染T细胞在组织中的迁移及其通过淋巴和血液运输到远处器官在局部和全身HIV传播中的作用。我们还将研究HIV感染的T细胞与体内其他免疫细胞的相互作用，以及这些相互作用是否有助于在易感HIV靶点之间传播HIV。目的1将探讨hiv感染的T细胞在淋巴结中的迁移特征（参见目的1.1），特别是病毒蛋白Nef和Env在观察到的其运动行为的任何改变中的作用（参见目的1.2和1.3）。我们还将研究在T细胞通过次级淋巴器官再循环的生理过程中，受感染的T细胞是否充当HIV的细胞载体，从而有助于其全身传播（见第1.4条）。目的2将检查淋巴结中易感T细胞与HIV+细胞的相互作用。具体来说，我们将测量在hiv感染的淋巴结中易感T细胞被感染的效率（参见2.1），并分析T细胞与hiv感染的T细胞接触的频率、持续时间、动力学和化学计量学（参见2.2）。我们还将探讨这些相互作用是否以及在什么情况下促进包括病毒在内的细胞物质的细胞间转移（见第2.2条）。目标3将

DESCRIPTION (provided by applicant): HIV-1 causes a systemic infection of the human immune system, using the CD4 membrane protein expressed by T cells, dendritic cells, and macrophages as its primary entry receptor. While extracellular virions are abundant in body fluids of infected individuals, it is currently not known whether free virus contributes to transmission between infected and uninfected cells in vivo. In vitro studies suggest that direct interactions between T cells, but also between T cells and infected or non-infected dendritic cells and macrophages that trans-present HIV, greatly increase the efficiency of viral dissemination compared to free virions. Here we propose to use multiphoton intravital microscopy (MP-IVM) for the temporospatially resolved visualization of the behavior of HIV-infected T cells in lymph nodes and in the female reproductive tract (FRT) mucosa of humanized BLT mice. Employing a palette of recombinant HIV strains that confer fluorescence either to productively infected cells or to virions, we aim to obtain insight into the role of migration of infected T cells in tissues and their trafficking to remote organs via the lymph and bloodstream in local and systemic HIV dissemination. We will also examine the interactions of HIV-infected T cells with other immune cells in vivo and whether these interactions serve to spread HIV among susceptible HIV targets. Aim 1 will explore the migratory characteristics of HIV-infected T cells in lymph nodes (subaim 1.1), and specifically the role of the viral proteins Nef and Env in any alterations of their motile behavior that are observed (subaims 1.2 and 1.3). We will also investigate if the infected T cells serve as cellular vehicles for HIV during the physiological process of T cell recirculation through secondary lymphoid organs, and thus contribute to its systemic dissemination (subaim 1.4). Aim 2 will examine the interactions of susceptible T cells with HIV+ cells in lymph nodes. Specifically we will measure the efficiency at which susceptible T cells are infected in HIV-infected lymph nodes (subaim 2.1) and analyze the frequency, duration, dynamics, and stoichiometry of T cell encounters with HIV-infected T cells (subaim 2.2). We will also explore whether and under what circumstances these interactions facilitate the intercellular transfer of cellular material including virus (subaim 2.2). Aim 3 will develop microsurgical techniques (subaim 3.1) to investigate by MP-IVM the cellular dynamics of HIV infection in the FRT during transmission and early local viral amplification (subaim 3.2 and 3.3).