Figure 2.

Jak/STAT3 pathway inhibitor JSI-124 activates DCs. DCs were generated from mice BM cells in the presence of control 3T3 or CT26 CM. CD11c⁺ DCs were isolated using magnetic beads separation technique. Cells were then treated with 0.5 μM JSI-124 or vc, and cultured again with 3T3 or CT26 CM. On day 4 cells were collected and their phenotype and function was evaluated. The level of surface markers expression was evaluated by flow cytometry. Mean results of three performed experiments (A) and typical example (B) are shown. MFI- mean fluorescence intensity. Dotted line represents CD11c⁺ DCs cultured for 4 days with DMSO, solid line − treated with JSI-124. Expression of MHC class II (IA^d), CD86, and CD40 molecules was evaluated by flow cytometry as described in Materials and Methods. Cells were also used as stimulators of allogeneic T cells in MLR (C) and syngenic T cells in the presence of HA peptide (D). Two experiments with the same results were performed. (E) - DC ability to process antigen was evaluated by uptake of FITC-Dextran. MFI- mean fluorescence intensity. The level of FITC-dextran uptake at 4°C was subtracted from uptake at 37°C. Mean results of three performed experiments are shown. (F). DCs were generated from bone marrow HPC using GM-CSF and IL-4. CD11c⁺ cells were isolated and treated for 4 days with either 0.5 μM JSI-124 or DMSO (vc). After that time DCs were pulsed with 10 μg/ml of H2K^b matched peptide (IYSTVASSL). Naïve BALB/c mice were immunized s.c. with 2×10⁵ DCs. Immunization was repeated once 10 days later. Seven days after the second immunization mice were sacrificed, splenocytes isolated and stimulated with either control (C.P.) or specific peptide (S.P.). The number of IFN-γ producing cells was measured in ELISPOT assay. Each group included 3 mice and each experiment was performed in quadruplicates. Naïve − non-immunized mice. Mean ± St. Dev. are shown. (G, H) Human DCs were generated in vitro from peripheral blood MNC with GM-CSF and IL-4 and purified by Metrizamide gradient centrifugation. Cells were then treated with 0.5 μM JSI-124 or DMSO (vc) for 5 days. (G) After that DCs were collected and labeled with PE-conjugated lineage cocktail antibody (Anti-CD3, CD14, CD19, CD56), PerCP-conjugated anti-HLA-DR antibody, and FITC-conjugted anti-CD86, CD40, CD83 antibodies. Cells were analyzed by flow cytometry (FACSCalibur). Fluorescence intensity was measured within the population of lin^- HLA-DR⁺ DCs. MFI- mean fluorescence intensity. (H) DCs treated with JSI-124 or DMSO (vc) were mixed in triplicates in U-bottom 96-well plates at different ratios with 10⁵ MNC from a different donor. Cells were incubated for 5 days. ³H-Thymidine was added 18 hr before cell harvesting. cpm − count per minute. Mean results of two experiments are shown. Spontaneous proliferation of MNC was less than 3,000 CPM.