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).
This project will increase our understanding of the pathogenesis of HIV infection. Specifically, it will create knowledge on how the virus spreads between immune cells in the body, which will inform the development of prophylactic and therapeutic strategies that build on the neutralization or containment of HIV-1 through immunological mechanisms, such as vaccination.
|Fackler, Oliver T; Murooka, Thomas T; Imle, Andrea et al. (2014) Adding new dimensions: towards an integrative understanding of HIV-1 spread. Nat Rev Microbiol 12:563-74|
|Angin, Mathieu; Sharma, Siddhartha; King, Melanie et al. (2014) HIV-1 infection impairs regulatory T-cell suppressive capacity on a per-cell basis. J Infect Dis 210:899-903|
|Murooka, Thomas T; Mempel, Thorsten R (2013) Intravital microscopy in BLT-humanized mice to study cellular dynamics in HIV infection. J Infect Dis 208 Suppl 2:S137-44|