MSCs can cross-present soluble antigen in vitro. (A) Untreated or IFN-γ–stimulated MSCs were primed with the indicated concentration of OVA for 18 hours and cocultured at various density with primary OT-I CD8⁺ T cells for 48 additional hours. Levels IL-2 produced by activated CD8⁺ T cells were quantified by ELISA. (B) Flow cytometry analysis of surface expression of MHC class I (H2-K^b) on MSC treated with different doses of IFN-γ for 48 hours. (C) Western blot analysis of Tap1 and LMP2 protein in MSCs treated or untreated with 10 ng/mL of IFN-γ for 48 hours. Loading control was performed on α-tubulin. (D) Untreated or IFN-γ–stimulated MSC WT and MSC Tap1^−/− were primed with 1 mg/mL of OVA for 18 hours and cocultured at 10000 cells/well with primary OT-I CD8⁺ T cells for 48 additional hours. Analysis of the assay was performed as described previously in panel A. (E) IFN-γ–stimulated MSCs were treated with the indicated concentration of lactacystin, primed with OVA (1 mg/mL) for 18 hours, and cocultured at 10000 cells/well with OT-I CD8⁺ T cells for 48 hours. Analysis of the assay was performed as described previously in panel A (top). Cell viability of the lactacystin-treated MSCs was assayed by a MTT cell viability assay measured by tetrazolium reduction to a 490-nm absorbing formazan compound (bottom panel). The results of the APC assay and MTT assay show means of triplicates ± SD of 1 of 3 representative experiments.