Background
HIV latency is a driver of persistence; however, cells expressing HIV RNA and proteins can still be detected after years of antiretroviral therapy (ART) and appear to drive ongoing stimulation of HIV-specific T-cells – indicating that some infected cells may be recognized by cytotoxic T lymphocytes (CTL) but are resistant to elimination. In this study, we use a primary in vitro infection model and cells from people living with HIV (PLWH) to evaluate mechanisms of resistance.
Methods
CD4+ T-cells were infected with either a WT HIVJRSCF strain (targets) or a variant containing an escape mutation in the Gag-TW10 epitope (bystanders), and co-cultured with TW10-specific CTLs. Infected, HIV-expressing target cells that survived CTL attack (survivors) and bystander infected cells were profiled by ECCITE-seq after sorting based on HIV-Env expression. SCENITH metabolic profiling and DCFH-DA tagging by flow cytometry were used to measure metabolic flux and reactive oxygen species. Killing assays were also performed with pre-treatment of infected cells with the hypoxia-inducing, FDA-approved drug Deferoxamine (DFO) to induce oxidative stress. Memory CD4+ T-cells from PLWH on ART were sorted based on ROS levels and assayed by IPDA.
Results
Survivors exhibited reduced expression of aerobic respiration and glycolysis gene-sets among the most differentially expressed pathways (Figure 1A). Markedly reduced bioenergetic capacity and glucose metabolism were confirmed in survivors by SCENITH profiling (Figure 1B). This reduced metabolic flux led to an enrichment of cells expressing lower levels of ROS (P=0.037). Pre-treatment of infected cells with DFO enhanced susceptibilities of infected cells to elimination by CTL. IPDA analysis of memory CD4+ T-cells from PLWH sorted based on ROS levels revealed a trend towards increased levels of total and intact HIV DNA in ROS-lo populations (n=4, P = 0.071 and P = 0.078), and this is being extended to additional donors.
Conclusions
Oxidative stress contributes to CTL-mediated killing. Our results identify a quiescent metabolic program with low production of ROS as a protective feature of infected, HIV-expressing cells to CTL attack. Treatment with DFO improves CTL-mediated elimination of infected cells. CD4+ T-cells with low oxidative stress are enriched for total and intact proviruses in PLWH on ART and may provide a sanctuary for HIV persistence. Targeting metabolic balance may be a strategy to enhance elimination of persistent HIV-infected cells.