Interferon-g and Tumor Necrosis Factor-a Polarize Bone Marrow Stromal Cells Uniformly to a Th1 Phenotype

Activated T cells polarize mesenchymal stromal cells (MSCs) to a proinflammatory Th1 phenotype which likely has an important role in amplifying the immune response in the tumor microenvironment. We investigated the role of interferon gamma (IFN-g) and tumor necrosis factor alpha (TNF-a), two factors produced by activated T cells, in MSC polarization. Gene expression and culture supernatant analysis showed that TNF-a and IFN-g stimulated MSCs expressed distinct sets of proinflammatory factors. The combination of IFN-g and TNF-a was synergistic and induced a transcriptome most similar to that found in MSCs stimulation with activated T cells and similar to that found in the inflamed tumor microenvironment; a Th1 phenotype with the expression of the immunosuppressive factors IL-4, IL-10, CD274/PD-L1 and indoleamine 2,3 dioxygenase (IDO). Single cell qRT-PCR analysis showed that the combination of IFN-g and TNF-a polarized uniformly to this phenotype. The combination of IFN-g and TNF-a results in the synergist uniform polarization of MSCs toward a primarily Th1 phenotype. The stimulation of MSCs by IFN-g and TNF-a released from activated tumor infiltrating T cells is likely responsible for the production of many factors that characterize the tumor microenvironment. BMSCs (passage 4) were stimulated with 6.5 or 65 ng/ml of IFN-g (R&D System Minneapolis, MN, USA), and with 1.5 or 15 ng/ml of TNF-a (R&D System Minneapolis, MN, USA), or the combination of IFN-g and TNF-a (1.5 ng/ml of TNF-a + 6.5 ng/ml of IFN-g or 15 ng/ml of TNF-a + 65 ng/ml of IFN-g). After the adherent BMSCs were incubated with the cytokines for 48 hours, the BMSCs were harvested using trypsin and they were then evaluated by gene expression profiling.