The roles of FRCs in the establishment and maintenance of HIV-1 latently infected cells containing intact proviruses

About This Grant

Project Summary. In people living with HIV (PLWH) under antiretroviral therapy (ART), secondary lymphoid organs including lymph nodes harbor latent viral reservoirs, which are a major obstacle to curing acquired immunodeficiency syndrome (AIDS). While follicular helper T cells and follicular dendritic cells in B cell follicles are well studied as viral reservoirs in lymph nodes, recently it has been reported that lymph node CD4+ tissue-resident memory T (TRM) cells also serve as viral reservoirs in PLWH on ART. Our preliminary data demonstrate: 1) fibroblastic reticular cells (FRCs), a type of secondary lymphoid organ stromal cells, generate lymph node CD4+ TRM-like cells from peripheral blood CD4+ T cells; 2) FRC-induced lymph node CD4+ TRM-like cells support HIV-1 latency; and 3) FRCs maintain the survival of CD4+ T cells. However, the following critical aspects remain to be defined: 1) the extent of similarity between FRC-induced CD4+ TRM-like cells and bona fide lymph node CD4+ TRM cells, 2) the capability of FRC-induced CD4+ TRM-like cells to be HIV-1 reservoirs, and 3) the role of FRCs in maintenance of TRM viral reservoirs. Thus, the long-term goal of this project is to elucidate the roles of secondary lymphoid organ stromal cells in viral latency, which is critical to developing a curative strategy for AIDS. The objective of this proposal is to define how FRCs contribute to the generation and maintenance of viral reservoirs. Our central hypothesis is that FRCs facilitate the formation of lymph node CD4+ TRM cells that are highly permissive to HIV-1 latent infection and maintain the survival of latent viral reservoirs. The rationale of the proposed studies is that the completion of the studies will inform about the cellular and molecular mechanisms involved in both the establishment and maintenance of viral reservoirs, as well as possible means of a curative strategy. We plan to test our central hypothesis by pursuing the following three specific aims: In the first aim, we will use single-cell RNA sequencing, single-cell surface phenotyping and DNA sequencing that allows to determine the provirus sequence and phenotype of infected cells at a single-cell resolution, and an in vivo mouse model to establish that FRCs play a role in the generation of lymph node CD4+ TRM cells. In the second aim, to determine the molecular mechanism by which FRC-induced CD4+ TRM-like cells support latent infection, we will focus on a TRM- associated transcription factor and use chromatin immunoprecipitation and immunoprecipitation to define the interactions of the transcription factor with the HIV-1 LTR and host molecules in FRC-induced CD4+ TRM-like cells. In the third aim, we will employ the single-cell surface phenotyping and DNA sequencing that will be used to assess the ability of FRCs to maintain the survival of latently infected FRC-induced CD4+ TRM-like cells containing intact proviruses. This work is innovative because it combines a range of complementary approaches to test a novel hypothesis regarding the roles of FRCs in HIV-1 latency. The proposed research is significant because of the potential to define how latent viral reservoirs are established and maintained in secondary lymphoid organs.