Fig. 6

The role of MSC secreted soluble factors and cell–cell contact in T-bet expression. A Purified HNT CD4⁺ T cells were activated with anti-CD3 and anti-CD28 mAbs and cultured in the presence of PGE2 at 1 ng/ml (PGE2), MSC-A conditioned media (Sup), MSC-A, MSC-A plus indomethacin (INDO) or left untreated without MSCs (Non-Tr). After 24 h, CD4⁺ T cells were harvested and the expression of intracellular T-bet was analyzed by FACS. Mean fluorescence intensity (MFI) minus that of the isotype control relative to the non-treated control is indicated. Values are represented as mean ± SEM. Data from 3 independent experiments is presented. B Purified HNT CD4⁺ T cells were activated with anti-CD3 and anti-CD28 mAbs and cultured in the presence of MSC-A, Mitotracker Deep Red-labeled MSC-A or Mitotracker Deep Red-labeled MSC-A in a trans-well insert (TW). After 12 h, harvested T cells were analyzed by FACS to assess Mitotracker Deep Red (MTDR) fluorescence. Data from one representative experiment is presented. Values represent percentage of Mitotracker Deep Red⁺ cells ± SEM. C Purified HNT CD4⁺ T cells were activated with anti-CD3 and anti-CD28 mAbs and cultured either alone (Non-Tr), in the presence of MSC-A or MSC-A in a trans-well insert (TW). After 4 days, activated HNT CD4⁺ T cells were restimulated with PMA and ionomycin in the presence of brefeldin A and production of intracellular IFNγ was assessed by FACS. Percentage of cytokine-producing T cells is indicated. Values are represented as mean ± SEM. D Purified HNT CD4⁺ T cells were activated with anti-CD3 and anti-CD28 mAbs and cultured either alone (Non-Tr), in the presence of MSC-A or MSC-A in a trans-well insert (TW). After 24 h, CD4⁺ T cells were harvested and the expression of intracellular T-bet was analyzed by FACS. Mean fluorescence intensity (MFI) minus that of the isotype control relative to the non-treated control is indicated. Values are represented as mean ± SEM. Data from 3 independent experiments is presented in panels A to D