Simultaneous inhibition of PPAR-? and mTORC1 enables GM-CSF to induce the differentiation of monocytes into highly immunogenic dendritic cells

Monocytes can differentiate into macrophages or dendritic cells. When treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) monocytes differentiate into macrophage-like cells. Here, we report that pharmacological blockade of the nuclear receptor PPAR? in monocytes turns GM-CSF into a potent inducer of dendritic cell (Mo-DC) differentiation. Remarkably, simultaneous blockade of PPAR? and mTORC1 in the presence of GM-CSF promoted the differentiation of Mo-DCs with a stronger phenotypic stability and immunogenic profile when compared with canonical Mo-DCs differentiated by treatment with GM-CSF and IL-4. Moreover, and in contrast with the observations made with GM-CSF and IL-4, blockade of PPAR? and mTORC1 was shown to be able to induce the differentiation of monocyte-derived macrophages (Mo-Macs) into Mo-DCs. Transcriptional profiling performed at either early time points, as well as at the end of the differentiation process, revealed marked differences in the gene expression signature between Mo-DCs induced by GM-CSF and IL-4 and Mo-DCs induced by GM-CSF in the presence of PPAR? and/or mTORC1 inhibitors, thus suggesting diverging differentiation pathways. Our observations might contribute, not only to a better understanding of the mechanisms involved in Mo-DCs differentiation but also to improving the efficacy of both, DC vaccines and therapies focusing on the modulation of myeloid cell functions. Overall design: Human monocytes were isolated from buffy coats obtained from healthy individuals using negative selection (RosetteSep™ Human Monocyte Enrichment Cocktail , StemCell) and cultured (1M/ml in RPMI1640 + 10% FBS) for either 8 hours or 5 days in the presence of a) GM-CSF (50ng/ml) alone, to induce MoMac differentiation, or in combination with b) IL4(30ng/ml) , c) Temsirolimus 50nM , d) GW9662 (10uM) or e) Temsirolimus 50nM + GW9662 (10uM), to induce MoDC differentiation. RNAseq was performed after 8h of culture to analyze the early transcriptional profile of monocytes treated with the different experimental conditions (4 donors). Also , RNA was extracted from FACS sorted CD1a+ CD16- MoDCs obtained after 5 days of culture in the presence of GM-CSF + IL4 (GMIL4-MoDC) , GM-CSF + Temsirolimus (GMT-MoDC) , GM-CSF + GW9662 (GMGW-MoDC) and GM-CSF + GW9662 + Temsirolimus (GMTGW-MoDC), in order to compare their transcriptional profile (3 donors).