Antigen processing for MSC-mediated antigen presentation. (A) C57BL/6 MSCs, DC2.4, or MEFs (5 × 104 cells) were cocultured for 20 hours with ovalbumin-specific MHC class II-restricted T-T hybridomas (MF2.2D9; 105 cells) in the presence of 2.5 mg/mL soluble ovalbumin. Where indicated, MSCs were treated with IFNγ (50 ng/mL final). Where indicated, MSCs were first fixed with paraformaldehyde prior to coculture. After 20 hours, supernatant was collected and tested for IL-2 release by ELISA. (B) C57BL/6 MSCs or DC2.4 (5 × 104 cells) were first incubated with soluble ovalbumin (2.5 mg/mL) and IFNγ (50 ng/mL final) for 20 hours, then fixed with paraformaldehyde and cocultured for 20 hours with MF2.2D9 hybridomas (105 cells). (C) MSCs (5 × 104 cells) were cocultured for 20 hours with MF2.2D9 cells (105 cells) in the presence of increasing doses of soluble ovalbumin. Where indicated, MSCs were treated with chloroquine (100 μM) 30 minutes prior to and during antigen exposure. Means of triplicates ± standard deviations of 1 of 2 representative experiments are shown.
