Fig. 3

Surface-expressed and soluble immunoregulatory mediators upregulated by hfcMSCs following IFNγ stimulation. a Density plots showing dual staining of cell surface expressed co-inhibitory receptors PD-L1 and PD-L2. b Scatter dot plots showing median fluorescence intensity (MFI; bar indicates mean ± standard error of the mean [SEM]) for surface-expressed PD-L1 and c PD-L2, which were both constitutively expressed in non-stimulated hfcMSCs, and increased significantly in response to IFNγ stimulation, but dropped to background levels or below after removal of the cytokine. d Soluble (s)PD-L1 was detected in the media after 7 days of IFNγ stimulation, whereas e detectable levels of sPD-L2 were secreted from resting hfcMSCs. An increase in sPD-L2 could be measured after IFNγ stimulation. Both sPD-L1 and sPD-L2 dropped back to or below background levels after withdrawal of the pro-inflammatory stimulus. f Upregulation of the IDO1 gene was reflected in an increased consumption of tryptophan shown as reduced levels, in addition to g elevated levels of the metabolite kynurenine, serving as a surrogate marker for IDO expression and activity. This was observed in hfcMSCs from all donors analyzed, as demonstrated by the kynurenine to tryptophan ratios. IDO activity rapidly returned to baseline levels upon withdrawal of IFNγ, shown as retained tryptophan levels and concomitantly reduced kynurenine levels 2 days after media replacement. The experiments were performed on cells from four different donors (n = 4). Graph data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001