Transcriptomic analysis of the impact of a short-term leucine deprivation and evaluation of GCN2 involvement in induced gene differential expression in human primary T cells

We previously demonstrated the proof of concept for the functionality of the NUTRIREG system in T cells. This technology allows for the induction of transgene expression following the consumption of a diet deficient in an essential amino acid, by activating the GCN2-ATF4 signaling pathway. To utilize the NUTRIREG technology for therapeutic purposes in cell therapy with short-term EAA deprivation, we explored the general impact of 6-hour leucine deprivation on primary activated human T cells using RNA-seq technology. We identified 3,431 differentially expressed genes (adjusted p-value < 0.05) between T cells cultured in control media and those cultured in leucine-deprived media for 6 hours. Gene Set Enrichment Analysis revealed that "TNFα signaling via NFκB", "interferon-γ response", and "unfolded protein response" gene sets were positively enriched, while "mTORC1 signaling", "Myc targets", and "oxidative phosphorylation" gene sets were negatively enriched. T cells were cultured with or without a GCN2 inhibitor, which allowed us to assess the involvement of GCN2 kinase in differential gene expression during the 6-hour leucine deprivation. We found that 59% of the differentially expressed genes (DEGs) in our dataset were dependent on GCN2 kinase (n=2028), with 1,140 up-regulated and 888 down-regulated GCN2-dependent genes. Activated human T cells were incubated in 10% FBS-RPMI medium supplemented with IL-2 at 37°C and 5% CO2 for 7 days of expansion. At day 8 post activation, Dulbecco’s Modified Eagle’s Medium (DMEM) (with 10% of dialyzed calf serum) lacking leucine was used (GENAXXON bioscience). A control medium was simultaneously reconstituted by adding all nine EAAs. RPMI-cultured human T cells were collected and washed in PBS with centrifugation at room temperature at 300xg for 5 min. Cells were then resuspended either in reconstituted DMEM control or in DMEM lacking leucine, at a density of 1x106 cells/mL. DMEM was freshly supplemented with IL-2 (50 IU/mL). GCN2 inhibitor (TAP20) was generously provided by Merck KGaA. The compound was resuspended in DMSO and used at a final concentration of 2.5 µM. Cells were cultured in DMEM Control or DMEM lacking leucine +/- GCN2 inhibitor for 6 hours (quadruplicate) and were then collected for mRNA extraction and subsequent RNAseq analysis.