Simultaneous inhibition of PPAR-γ 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 PPARγ in monocytes turns GM-CSF into a potent inducer of dendritic cell (Mo-DC) differentiation. Remarkably, simultaneous blockade of PPARγ 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 PPARγ 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 PPARγ 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. 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).