MSC-DCs inhibit allo-DTH. (A) Treatment of allo-DTH by MSC-DCs. BALB/C mice were immunized on the dorsal flank by subcutaneous inoculation of C57BL/6 spleen cells (10⁸ cells/mouse) on day 0, and challenged on day 7 and day 14 at the hind footpad by injecting the same antigens (10⁸ cells/mouse). H-2K^d–APCs from BALB/C mice or H-2K^b–MSC-DCs from C57BL/6 (−EGFP) mice were injected intraperitoneally (3 × 10⁶ cells/mouse) on days −6, −4, 0, 3, and 6. Footpad thickness was then measured on day 8 and day 15. Data are representative of 3 independent experiments, showing the mean (± SD) of footpad swelling on indicated day. *P < .05. (B) MSC-DCs proliferated and up-regulated their CD11c and Ia expression in vivo. EGFP⁺MSC-DC–treated BALB/C mice were killed on days 8 and 15, and all of the splenocytes isolated from these mice were analyzed for the levels of EGFP and CD11c expression by flow cytometry. Subsequently, CD11c⁺ cells were enriched from splenocytes by magnetic-activated cell sorting. MSC-DC proliferation and Ia expression in vivo were measured by I-A/E and EGFP double-staining FACS analysis and showed with a percentage change. A representative of at least 3 independent experiments is shown. (C) MSC-DCs induced a donor-specific tolerance. Normal or C57BL/6-MSC-DC–treated BALB/C mice were killed 15 days later, and splenocytes were used as responder cells in MLC and mitogen proliferative assay. Lethally irradiated splenocytes from C57BL/6 mice or ConA (5 μg/mL) were used as stimulators. Splenocytes from normal mice served as control. The proliferative responses were assessed by CFSE label and FACS. Dotted line, unstimulated splenocytes.