Hypoxia and TGF-β1 induce tissue resident memory phenotype cells from human peripheral blood T-cells in vitro

Abstract Tissue resident memory cells (TRM) are a recently identified subset of memory T-cells important in frontline defense against viral diseases and recent reports suggest T-cells with this phenotype are important in anti-tumor immunity. However, relatively little is known regarding TRM differentiation and endogenous TRM are notoriously difficult to isolate, impeding their study in basic research and downstream applications. Thus a means to generate TRM in vitro would be desirable. It is known that TGF-β is critical in establishing TRM populations and attempts to identify other factors that can induce differentiation to resident phenotype have focused on cytokines. Oxygen tension is another factor that distinguishes the circulation from peripheral tissues; thus we hypothesized that hypoxia may contribute to a TGF-β1-induced TRM phenotype in human CD8+ T-cells. We demonstrate that differentiation of human peripheral blood CD8+ T-cells in hypoxia and TGF-β1 in vitro led to the development of a TRM phenotype, characterized by a significant increase in CD69+CD103+ cells expressing CD49a, CD101, and PD-1 – hallmarks of human TRM. At the transcriptional level we observed upregulation of genes associated with TRM differentiation and downregulation of genes associated with memory cell recirculation. Hypoxia and TGF-β1 appear to synergize to induce a resident phenotype as their combination produced a significantly larger population of TRM phenotype cells than the additive effects of either condition alone. Our findings identify a previously unreported cue for TRM differentiation and enable a facile means of generating human TRM phenotype cells in vitro for basic studies and translational applications such as adoptive cellular therapies.