Status of activated T cells in the presence of MSCs. (A) Expression of the T-cell activation markers CD25 and CD69 on CD4 or CD8 cells 24 hours after stimulation of splenocytes (1 × 10⁶ cells) in a 12-well dish with anti-CD3/CD28 beads (10 μL) in the presence or absence of 1 × 10⁵ MSCs. The numbers in the top right quadrants indicate the percentage ± the standard deviation (SD). (B) Top panel, cytokine production in the same condition as in panel A. Concentrations of IL-2 and IFN-γ were determined at 48 hours by ELISA. The values are the means ± SD from 3 independent experiments. Bottom panel, intracellular staining of IFN-γ at 24 hours. GolgiStop (monensin) was used for the last 8 hours. The values are the mean percentages of CD8/IFN-γ–positive cells ± SD from 3 independent experiments. (C) MSCs suppress the induction of CD4⁺ and CD8⁺ T-cell proliferation by PMA and ionomycin. Splenocytes (1 × 10⁵), CD4⁺ cells (1 × 10⁵), or CD8⁺ cells (1 × 10⁵) were stimulated in the presence or absence of irradiated MSCs (1 × 10⁴) in the wells of a 96-well plate. The incorporation of [³H]-thymidine is shown relative to that in the absence of MSCs. The values are the means ± SD from 3 independent experiments. *P < .05. NS indicates P > .05.