Fig. 1
IFNγ licencing safeguards MSC from negative effects of CsA co-treatment. MSCs were seeded (1 × 104 per well) into a 96 well round bottom plate followed by the addition of anti-CD3/CD28 bead (1 × 104 per well) activated CFSE labelled PBMCs (5 × 104 per well). MSC IFNγ and MSC IFNγ CsA groups were pre-stimulated with IFNγ (50 ng/ml) for 24 h. Some groups were cultured in the presence of CsA (1000 ng/ml). On day four, cells were harvested and stained with anti-CD3 and 7AAD viability dye to analyse CD3+ proliferation by flow cytometry. CsA significantly antagonises the immunosuppressive ability of MSC, n = 7 (a) and representative histogram plots (b). MSC alone or stimulated with IFNγ (50 ng/ml) for 24 h were labelled with eFluor® 670 and co-cultured with anti-CD3/28 driven PBMCs. Labelled MSC were harvested (on day 3), fixed, permeabilised and stained intracellularly with fluorescent antibody for IDO and analysed by flow cytometry Data presented as mean +/− SEM of the % of MSCs producing IDO (n = 5) (c) and representative histograms (d). Statistical analysis was carried out using one way ANOVA Multiple Tukey comparison test and unpaired Student’s t test where *< 0.05, **< 0.01 and *** < 0.001. Stars with no bar are in comparison to the activated PBMC group. ns not significant