Fig. 3.

Compound 10 inhibits production of the cytokines IL-2 and TNF by total CD4⁺ murine T cells under conditions where it is not toxic to the cells. Primary murine CD4⁺ T cells were incubated in the absence (DMSO control) or presence of the indicated concentrations of Compound 10 for 1 h, then either left unstimulated or stimulated with 10 nM PMA and 500 nM ionomycin for an additional 4 h. Where indicated, 1 μM of CsA was added 15 min before stimulation. (A–C) After stimulation, the cells were fixed, washed, permeabilized, and assessed for cytokine production by intracellular staining and flow cytometry. (B) Percentage of cells in A expressing IL-2, TNF, or both cytokines. (C) Mean fluorescent intensities (MFI) for the individual cytokines in cells scored as positive in A. (D) Toxicity was assessed by propidium iodide staining and flow cytometry. Cells were treated with Compound 10 as above, then stimulated with PMA and ionomycin for 4 h. The sub-G₀ population is indicated in each panel. Under these conditions, cells treated with 30 or 40 μM of Compound 10 showed no increase in the sub-G₀ population, whereas cells treated with 50 μM Compound 10 showed a twofold increase compared with untreated cells. The data are representative of at least two independent experiments.