Migratory Properties of HIV-infected T cells in vivo

描述（由申请人提供）：HIV-1使用T细胞、树突细胞和巨噬细胞表达的CD 4膜蛋白作为其主要进入受体，引起人体免疫系统的全身感染。虽然细胞外病毒粒子在受感染个体的体液中丰富，但目前尚不清楚游离病毒是否有助于体内感染和未感染细胞之间的传播。体外研究表明，与游离病毒体相比，T细胞之间的直接相互作用，以及T细胞与感染或未感染的树突状细胞和反式呈递HIV的巨噬细胞之间的直接相互作用，大大增加了病毒传播的效率。在这里，我们建议使用多光子活体显微镜（MP-IVM）的时空分辨可视化的HIV感染的T细胞在淋巴结和女性生殖道（FRT）粘膜的人源化BLT小鼠的行为。采用调色板的重组HIV菌株，赋予荧光无论是生产性感染的细胞或病毒粒子，我们的目标是获得洞察感染的T细胞在组织中的迁移和他们的贩运到远程器官通过淋巴和血液在本地和系统的HIV传播的作用。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的HIV靶点中传播HIV。目的1将探索HIV感染的T细胞在淋巴结中的迁移特征（子目的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（子目的1.2和1.3）。我们还将研究在通过次级淋巴器官的T细胞再循环的生理过程中，感染的T细胞是否作为HIV的细胞载体，从而促进其全身传播（子目标1.4）。目的2将检查淋巴结中易感T细胞与HIV+细胞的相互作用。具体而言，我们将测量易感T细胞在HIV感染的淋巴结中感染的效率（子目标2.1），并分析T细胞与HIV感染的T细胞相遇的频率、持续时间、动力学和化学计量（子目标2.2）。我们还将探讨这些相互作用是否以及在何种情况下促进细胞物质（包括病毒）的细胞间转移（子目标2.2）。目标3将 开发显微外科技术（子目标3.1），通过MP-IVM研究传播和早期局部病毒扩增期间FRT中HIV感染的细胞动力学（子目标3.2和3.3）。

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).