Miller-Jensen, K. | bioRxiv Science

bioRxiv2018

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

Efforts to cure HIV are hindered by viral persistence in latently infected memory CD4+ T cells. Targeting T cell death pathways dysregulated by HIV infection offers a novel approach for eradication of the latent reservoir. To identify potential therapeutic targets, we compared signaling and apoptosis in uninfected and latently infected primary cultured CD4+ central memory T cells by mass cytometry following T cell receptor stimulation. We found that HIV-infected cells were sensitized to activation of pro-apoptotic p38 kinase signaling via p53, and to inhibition of anti-apoptotic mTOR kinase signaling, even without HIV protein expression. Simultaneous targeting of p38 and mTOR kinases in resting CD4+ T cells from virally-suppressed HIV+ patients ex vivo reduced cell-associated HIV RNA and DNA. Our results demonstrate how systems biology approaches are useful for identifying novel therapeutic approaches to treat HIV latency, and further suggest that it may be possible to deplete latent HIV-infected T cells without viral reactivation.

systems biology↗

bioRxiv2018

Fold-change detection of NF-κB at target genes with different transcript outputs

The transcription factor NF-{kappa}B promotes inflammatory and stress-responsive gene transcription across a range of cell types in response to the cytokine tumor necrosis factor- (TNF). Although NF-{kappa}B signaling exhibits significant variability across single cells, some target genes exhibit fold-change detection of NF-{kappa}B, which may buffer against stochastic variation in signaling molecules. However, this observation was made at target genes supporting high levels of TNF-inducible transcription. It is unknown if fold-change detection is maintained at NF-{kappa}B target genes with low levels of TNF-inducible transcription, for which stochastic promoter events may be more pronounced. Here we used a microfluidic cell-trapping device to measure how TNF-induced activation of NF-{kappa}B controls transcription in single Jurkat T cells at the promoters of integrated HIV and the endogenous cytokine gene IL6, which produce only a few transcripts per cell. We tracked TNF-stimulated NF-{kappa}B RelA nuclear translocation by live-cell imaging and then quantified transcript number by RNA FISH in the same cell. We found that TNF-induced transcription correlates with fold change in nuclear NF-{kappa}B with similar strength at low versus high abundance target genes. A computational model of TNF-NF-{kappa}B signaling, which implements fold-change detection from competition for binding to {kappa}B motifs, was sufficient to reproduce fold-change detection across the experimentally measured range of transcript outputs. Nevertheless, we found that gene-specific trends in transcriptional noise and levels of promoter-bound NF-{kappa}B predicted by the model were inconsistent with our experimental observations at low abundance gene targets. Our results reveal a gap in our understanding of RelA-mediated transcription for low abundance transcripts and suggest that cells use additional biological mechanisms to maintain robustness of NF-{kappa}B fold-change detection while tuning transcriptional output.

systems biology↗