Systems analysis of latent HIV reversal reveals altered stress kinase signaling and increased cell death in infected T cells

Abstract

Viral latency remains the most significant obstacle to HIV eradication. Clinical strategies aim to purge the latent CD4+ T cell reservoir by activating viral expression to induce death, but are undercut by the inability to target latently infected cells. Here we explored the acute signaling response of latent HIV-infected CD4+ T cells to identify dynamic phosphorylation signatures that could be targeted for therapy. Stimulation with CD3/CD28, PMA/ionomycin, or latency reversing agents prostratin and SAHA, yielded increased phosphorylation of IκBα, ERK, p38, and JNK in HIV-infected cells across two in vitro latency models. Both latent infection and viral protein expression contributed to changes in perturbation-induced signaling. Data-driven statistical models calculated from the phosphorylation signatures successfully classified infected and uninfected cells and further identified signals that were functionally important for regulating cell death. Specifically, the stress kinase pathways p38 and JNK were modified in latently infected cells, and activation of p38 and JNK signaling by anisomycin resulted in increased cell death independent of HIV reactivation. Our findings suggest that altered phosphorylation signatures in infected T cells provide a novel strategy to more selectively target the latent reservoir to enhance eradication efforts.

Induction of selective cell death in HIV-1-infected cells by DDX3 inhibitors leads to depletion of the inducible reservoir

“…We also observed an upward trend in the mRNA expression of the innate antiviral signalling pathway components RIG-I and MAVS in the vRNA high fraction, consistent with previous work demonstrating HIV-1 vRNA's ability to activate these pathways ( Supplementary Fig. 1e and f) 17,18 . Our data therefore indicates that in a preliminary screening model of HIV-1 latency, DDX3 inhibition with RK-33 results in latency reversal and that the RK-33-treated HIV-1 expressing cells harbour markers for apoptosis and are more susceptible to cell death by RK-33 than the vRNA-/GFP-J-Lat 11.1 cells or uninfected parental Jurkat cells.…”

Systems analysis by mass cytometry identifies susceptibility of latent HIV-infected T cells to targeting of p38 and mTOR pathways

“…We recently reported that the antibiotic anisomycin, a potent activator of the p38 and JNK MAPK pathways, increased both p-p38 and p-JNK activation within 30 minutes and cell death at 24 hours in virus-exposed TCM cells relative to uninfected cells (29). We found that anisomycin treatment increased levels of p-p53 and CC3 to a greater extent in virus-exposed cells relative to uninfected cells, and that this could be reversed with p38 inhibition ( Fig.…”

“…We previously showed that TCR stimulation via CD3/CD28 leads to more cell death in latent HIV-infected CD4+ central memory-like T cells infected in vitro relative to uninfected cells (29).…”

“…Cells were infected with a DHIV-GFP/X4 virus and GFP-cells were sorted to isolate uninfected and latently infected populations. Cells were then stimulated with TNFα (Peprotech), and GFP+ cells were sorted into a 96-well plate to establish a clonal line of latently infected cells (29). This parental Jurkat and J-DHIV clonal cell line model provides a fully infected population with minimal divergence between uninfected and infected counterparts.…”

Advancing systems immunology through data-driven statistical analysis

“…To classify HIV+, T-cell depleted, and healthy PBMC samples, partial least squares discriminant analysis (PLS-DA)– which identifies linear combinations of variables to maximally separate known biological states –identified classification signatures based on measurements of 16 cytokines and chemokines, and then the underlying molecular changes could be associated with each group [39]. In a study from our lab, we measured phospho-signaling differences in uninfected and latent HIV-infected T cells and used PLS-DA to define signatures that predicted infection status and then identified changes in stress kinase signaling as a novel target for preferential infected-cell killing [40]. Comparing total immune cell composition in healthy and infected donors, SPADE and t-SNE analyses performed on 26-parameter mass cytometry data demonstrated that infected patients had a higher fraction of highly differentiated effector memory cells following HIV infection and ART treatment in comparison to healthy donors [41].…”