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.