Role of clonal expansion in HIV-1 persistence

PROJECT SUMMARY Despite effective antiretroviral therapy (ART), HIV-1 persists in memory CD4+ T cells as the major barrier to cure. ART inhibits viral enzyme function but does not kill infected cells. Around 12 million HIV-1-infected CD4+ T cells establish the HIV-1 reservoir. HIV-1-infected cells survive viral cytopathic effects, evade cytotoxic CD8+ T cell killing, and persist over time. What is even worse, these HIV-1-infected cells proliferate: more than 50% of the HIV-1 reservoir undergo clonal expansion through antigen stimulation, homeostatic cytokine stimulation, or HIV- 1 integration site-driven proliferation. Understanding mechanisms of the clonal expansion of HIV-1-infected cells is a top priority to stop the proliferation of HIV-1-infected cells and guide the design of HIV-1 cure. However, understanding HIV-1 persistence in people living with HIV-1 is extremely challenging. First, CD4+ T cells are highly heterogeneous, having different T cell polarization (such as Th1, Th2, Th17, and T reg), memory differentiation (such as naïve, central memory, effector memory, and effector), activation states, exhaustion states, proliferation capacity, and antigen specificity. Second, HIV-1-infected cells harboring inducible HIV-1 are extremely rare in HIV-1-infected individuals, accounting for ~1–100/million CD4+ T cells (<0.01%) in the peripheral blood. Third, there is no cellular marker that can precisely identify HIV-1-infected cells for single-cell profiling. We recently used a cutting-edge single-cell multi-omic profiling method called ECCITEseq to decipher the heterogeneity of immune cell types and capture the rare HIV-1-infected cells. This single-cell multi-omic method allows us to capture single-cell transcriptome at the genome-wide level, profile more than 100 cellular protein markers, detect HIV-1 RNA+ cells, and capture T cell proliferation dynamics by T cell receptor (TCR) sequencing, all within the same single cell. This allows us to determine immune programs, clonal expansion dynamics, and cell markers for HIV-1 RNA+ cells for high-dimensional, multi-omic, and genome-wide understanding of HIV-1 persistence without ex vivo stimulation. Using advanced single-cell multi-omic approach, we tracked the clonally expanding CD4+ T cells between different anatomical locations in the body. In addition, we identified a pro-survival cellular factor that is upregulated in HIV-1-infected cells and may promote the proliferation of HIV-1-infecte cells. We hypothesize that HIV-1-infected CD4+ T cells traffic to different tissue compartments and persist under cytokine cues in the tissue compartment. We further hypothesize that HIV-1- infected cells can resist viral cytopathic effect by upregulating this pro-survival cellular factor. Our goal is to track the clonal expansion dynamics of HIV-1+ cells over space and time (Aim 1) and decipher how HIV-1-infected cells survive viral cytopathic effect and proliferate (Aim 2). Overall, we will provide an unprecedented understanding of HIV-1 reservoir regarding mechanisms of HIV-1 persistence and clonal expansion dynamics of HIV-1-infected cells at the single cell level. We will identify therapeutic targets and guide HIV-1 eradication strategies.