FIG. 4.

MSC use multiple immune suppressive pathways to abrogate DC activation. IFNγ-treated MSC and DC were cocultured (1:10 ratio) for 24 hr and activated with LPS (50 ng/mL) for 24 hr. The MSC:DC cocultures were also incubated with the following immune pathway inhibitors: aminoguanidine (0.5 nM), indomethacin (100 μM), CSC-A2 inhibitor (0.2 μM), SB-43154 (10 μM), 1-methyltryptophan (MT) (500 μM), or programmed death ligand-1 (PD-L1) blocking antibody (10 μg/mL). Controls included medium with DMSO or methanol vehicle or with an isotype matched antibody for PD-L1 (data not shown). (i) DC:MSC cocultures or (ii) DC alone were incubated at 37°C for 24 hr in the presence of inhibitors and LPS. DC were subsequently harvested and immunostained for flow cytometric quantitation of expression of: (A) MCHII, (B) CD86, and (C) CD40 on CD11c⁺ events. Horizontal dashed lines denote the average maximal level of suppression of MHCII, CD86, and CD40 expression that is observed in DC cultures treated with IFNγ+MSC+LPS. Data are representative of four separate experiments. P values calculated for statistical variance were determined using a paired two-tailed Mann–Whitney test. *P < 0.05, **P < 0.01, and ***P < 0.005.