Migratory Properties of HIV-infected T cells in vivo

描述(申请人提供)：HIV-1引起人类免疫系统的系统性感染，使用T细胞、树突状细胞和巨噬细胞表达的CD4膜蛋白作为其主要进入受体。虽然感染者的体液中含有丰富的胞外病毒粒子，但目前尚不清楚游离病毒是否在体内感染和未感染的细胞之间起到了传播作用。体外研究表明，与游离病毒粒子相比，T细胞之间以及T细胞与感染或未感染的树突状细胞和巨噬细胞之间的直接相互作用极大地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小鼠的淋巴和雌性生殖道(FRT)粘膜中HIV感染的T细胞的行为进行时间空间分辨可视化。利用一组重组HIV毒株，这些毒株向感染细胞或病毒粒子发出荧光，我们的目标是深入了解受感染T细胞在组织中的迁移以及它们通过淋巴和血液运输到远程器官在局部和全身艾滋病毒传播中的作用。我们还将研究感染艾滋病毒的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艾滋病毒易感目标中传播艾滋病毒。目的1将探讨HIV感染的T细胞在淋巴结中的迁移特征(子目标1.1)，特别是病毒蛋白Nef和Env在观察到的其运动行为改变中的作用(子目标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，从而促进其系统性传播(子目标1.4)。目的2将研究易感T细胞与淋巴结中HIV+细胞的相互作用。具体来说，我们将测量易感T细胞在HIV感染的淋巴结中的感染效率(SubAim 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、动态和化学计量(SubAim 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(次级目标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中艾滋病毒感染的细胞动力学(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).