Ruxolitinib impairs the T-cell stimulatory function of DCs in vitro. (A) The ability of human moDCs, treated once with ruxolitinib (0.2-10 µM; ▪) on day 5 and activated with LPS on day 6, to prime allogeneic T-cell responses in vitro, was assessed using a MLR assay. Irradiated stimulator DCs were cultured with responding allogeneic peripheral blood mononuclear cells. Tritium-labeled thymidine incorporation was measured 5 days later. □, vehicle control. (B-I) CFSE-labeled OT-I cells were cocultured with murine OVA-loaded bmDCs. Ruxolitinib was added once on the first day of culture and resulted in (B,D) reduced OT-I proliferation, (C,F) decreased expression of CD25 as well as (E) a reduced division index. Pretreatment of bmDCs with ruxolitinib followed by repeated washout of the compound still resulted in (G) reduced OT-I proliferation, (H) division index, and (I) CD25 expression. (J-K) CFSE-labeled OT-II cells were cocultured with murine OVA-loaded bmDCs, followed by a single addition of ruxolitinib on the first day of culture. OT-II proliferation and (L) CD25 expression are shown. Results are from 1 experiment representative of 3. The significance was calculated according to the 1-way ANOVA Dunnett multiple comparison test and is always related to the vehicle control. *P < .1; **P < .01; ***P < .001.