Activation of Dendritic Cells via Inhibition of Jak2/STAT3 Signaling

The Jak/STAT3 pathway inhibitor JSI-124 activates DCs. DCs were generated from mouse BM cells in the presence of control 3T3 or CT26 CM. CD11c⁺ DCs were isolated using the 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 were evaluated. The level of surface marker expression was evaluated by flow cytometry. The mean results of three experiments performed (A) and a typical example (B) are shown. MFI, mean fluorescence intensity. The dotted line represents CD11c⁺ DCs cultured for 4 days with DMSO; the solid line represents CD11c⁺ DCs treated with JSI-124. The expressions of MHC class II (IA^d), CD86, and CD40 molecules were evaluated by flow cytometry as described in Materials and Methods. Cells were also used as stimulators of allogeneic T cells in MLR (C) and of syngenic T cells in the presence of HA peptide (D). Two experiments with the same results were performed. E, The ability of DC to process Ag was evaluated by uptake of FITC-DX. The level of FITC-DX uptake at 4°C was subtracted from uptake at 37°C. The mean results of three experiments performed are shown. F, DCs were generated from BM HPC using GM-CSF and IL-4. CD11c⁺ cells were isolated and treated for 4 days with either 0.5 μM JSI-124 or VC. After that time DCs were pulsed with 10 μg/ml H2K^d matched HA-derived peptide. Naive 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 killed, and splenocytes were isolated and stimulated with either control (C.P.) or specific (S.P.) peptide. The number of IFN-γ-producing cells was measured in an ELISPOT assay. Each group included three mice, and each experiment was performed in quadruplicate. Naive, nonimmunized mice. The mean ± SD are shown. G and 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 VC for 5 days. G, Then DCs were collected and labeled with PE-conjugated lineage mixture Ab (anti-CD3, CD14, CD19, and CD56), PerCP-conjugated anti-HLA-DR Ab, and FITC-conjugated anti-CD86, CD40, and CD83 Abs. Cells were analyzed by flow cytometry (FACSCalibur). Fluorescence intensity was measured within the population of lin⁻ HLA-DR⁺ DCs. H, DCs treated with JSI-124 or VC were mixed in triplicate 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 h before cell harvest. The mean results of two experiments are shown. Spontaneous proliferation of MNC was <3000 cpm.

When activated by JSI-124, DCs exert their effect via direct cell-cell contact. A, The RANTES concentration was measured in triplicate in supernatants obtained after 48-h treatment of CD11c⁺ DC with JSI-124 or VC as described in Materials and Methods. B, Splenocytes were isolated from syngeneic BALB/c mice, and 2 × 10⁵ cells were placed in the bottom chamber of U-bottom, 96-well Transwell plates. Cells were stimulated with 0.5 μg/ml anti-CD3 Ab. DCs treated with VC or JSI-124 (JSI) were added to either the bottom chamber (2 × 10³ cells/well; mix) or the upper chamber (4 × 10³ cells/well; Transwell). [³H]Thymidine was added 18 h before cell harvest, and its incorporation was measured on a scintillation counter as described in Materials and Methods. All experiments were performed in triplicate and repeated at least once to assure reproducibility of the results. C, LN cells were isolated from HA-TCR transgenic mice and were placed into the bottom chamber of Transwell wells (10⁵ cells/well). Cells were stimulated with 12.5 μg/ml specific HA-derived peptide. DCs were placed into the bottom chamber (10³/well) or the upper chamber (2 × 10³) of the Transwell. After 4-day culture, the upper chamber was removed, and cells were pulsed for 1 h with BrdU. The proportion of CD3⁺ T cells in the S phase of the cell cycle was evaluated by flow cytometry. Each experiment was performed in triplicate. Two experiments with the same results were performed.

JSI-124 induces translocation of MHC class II to the surface of DCs. Murine BM HPCs were cultured with GM-CSF and IL-4 to generate DCs. After 5-day culture, CD11c⁺ cells were isolated using the magnetic beads separation technique and were then treated with 0.5 μM JSI-124 or VC. A, Total RNA was isolated from cells cultured for 24 and 48 h, followed by RT-PCR as described in Materials and Methods. B, Cells were collected after 48 h in culture, and Western blotting was performed using I-A/I-E Ab as described in Materials and Methods. C, After 48 h in culture, cells were collected onto cytospin slides. Slides were fixed and stained with either isotype control IgG (control) or biotinylated anti-I-A/I-E Ab, followed by streptavidin-FITC, then analyzed by confocal microscopy (×1000).

Inhibition of Jak2/STAT3 signaling activates NF-κB in DCs. CD11c⁺ DCs were generated as described in Fig. 4 and treated with either JSI-124 or VC. A, DCs were collected at the time points indicated, and EMSA with NF-κB-specific probe was performed. B, CD11c⁺ cells were cotransfected with NF-κB or control plasmid and plasmid carrying the Renilla luciferase gene, then treated with JSI-124 or VC for 36 h. Luciferase activity (LA) values for specific and control plasmid were normalized to Renilla LA. The data presented are the fold increase in LA in cells transfected with plasmid containing an NF-κB-responsive element over the LA in cells transfected with the control plasmid. The mean results of three experiments performed are shown. C, DCs were treated with 5 μg/ml LPS for 15–30 min. The level of IκBα was evaluated by Western blotting as described in Materials and Methods. D, Level of IκBα in DCs after 30 and 60 min of treatment with JSI-124. E, DCs were treated with JSI-124 or VC for 18 and 36 h, whole cell lysates were prepared, and the levels of various NF-κB subunits and IκBα were determined by Western blotting.