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The JAK-inhibitor ruxolitinib impairs dendritic cell function in vitro and in vivo

Annkristin Heine, Stefanie Andrea Erika Held, Solveig Nora Daecke, Stephanie Wallner, Sowmya Parampalli Yajnanarayana, Christian Kurts, Dominik Wolf and Peter Brossart

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Article Figures & Data

Figures

  • Figure 1

    Ruxolitinib impairs phenotype and function of DCs. (A) Human monocytes cultured under DC-driving conditions with final LPS stimulation were exposed every other day to different concentrations of ruxolitinib (0.2 µM, 0.5 µM, and 1 µM on day 0, 2, 4, 6; ▪) or DMSO (□) throughout the differentiation period and analyzed for expression of DC and activation markers. Results are from 1 experiment representative of at least 3. (B) Expression of DC and activation markers after exposure of moDCs to ruxolitinib (1 µM, 5 µM, and 10 µM) on day 5, followed by subsequent final maturation with LPS on day 6, are shown. Filled black graphs represent negative controls. (C) Monocytes were cultured under DC-driving conditions and treated with ruxolitinib once on day 5, followed by LPS activation on day 6. Supernatants were collected on day 7 and analyzed for IL-12 production using a commercially available enzyme-linked immunosorbent assay. Results of 1 representative donor are shown. The significance was calculated according to the 1-way ANOVA Dunnett multiple comparison test and is related to the vehicle control. *P < .1; **P < .01; ***P < .001.

  • Figure 2

    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.

  • Figure 3

    Ruxolitinib impairs migratory behavior of DCs. (A) Ruxolitinib-treated and LPS-stimulated human moDCs (▪) were assessed for their migratory behavior toward CCL19/MIP-3β in Transwell assays. □, Results of vehicle-exposed, LPS-stimulated DCs. Results are from 1 experiment representative of at least 3. The significance was calculated according to 1-way ANOVA Dunnett’s multiple comparison test and is related to the vehicle control. *P < .1; **P < .01; ***P < .001. (B) Overnight ruxolitinib-exposed, CFSE-labeled ex vivo–generated immature bmDCs (▪) were subcutaneously injected into the hock of TBI-FIA (M tuberculosis in FIA)–injected recipient mice together with vehicle-challenged, eFluor670-labeled immature bmDCs (□) and then quantified in the local draining lymph node by FACS. Both bmDC groups were further injected into solvent-injected recipients (“unprimed”) as control for spontaneous migration. Untreated (no ruxolitinib, no vehicle exposure), but CFSE- or eFluor670-labeled bmDCs were used as additional controls to exclude changes in migratory behavior due to the dying process (“untreated”). Numbers indicate percentage of migrated bmDCs of all lymph node cells. (C) Histograms represent results of pooled independent experiments with a total of n = 14 (10 µM), n = 8 (5 µM), n = 7 (1 µM) mice. The significance was calculated according to the Mann-Whitney test and is related to the vehicle control. *P < .1; **P < .01; ***P < .001.

  • Figure 4

    Ruxolitinib impairs the T-cell stimulatory function of DCs in vivo. (A) Mice were fed twice via oral gavage either ruxolitinib (▪) or vehicle (□) prior to and after injection of OVA/CpG, followed by an analysis of MHC expression and activation markers on CD11c+CD8+ splenic DCs 20 hours after priming. Gray columns represent mice vaccinated with OVA/CpG without receiving vehicle or ruxolitinib feeding, to exclude any effects of the oral gavage per se. (B-G) CFSE-labeled OT-I cells (2 × 106) were adoptively transferred to naive C57/BL6N recipient mice injected with OVA/CpG. Mice were fed 6 hours prior to as well as 6 and 18 hours after priming with OVA/CpG with ruxolitinib (gray shaded) or its vehicle (black line). (B) OT-I cell proliferation shown as histogram and (C) division index as well as (D-E) CD25 expression and (F-G) IFN-γ production (analyzed by intracellular cytokine staining) were assessed 2.5 days after priming with OVA/CpG. (H) Using the entirely endogenous T-cell repertoire of nontransgenic C57/B6N mice primed with OVA/CpG, OVA-specific cytotoxicity on day 5 was assessed. Mice had been treated either with ruxolitinib or its vehicle in the concentrations and schedule indicated above. Results are from 1 experiment (n = 4 per group) representative of at least 3. The significance was calculated according to (A) the 1-way ANOVA Dunnett multiple comparison test or (C,E,G,H) Mann-Whitney test and is always related to the vehicle control. *P < .1; **P < .01.

  • Figure 5

    Ruxolitinib-treated mice show a delay in clearing adenovirus infection. (A) C57/BL6N mice were infected with 5 × 106 plaque-forming units of AdLGO, a recombinant adenovirus expressing OVA, enhanced green fluorescent protein, and click-beetle luciferase, followed by vaccination with OVA/CpG or left unprimed as control. Mice were fed 6 hours before as well as 6 hours and 18 hours after vaccination ruxolitinib or the respective vehicle control. (B) Time course of bioluminescence measurements is shown on a logarithmic scale. Results are from 1 experiment (n = 5 per group) representative of 3. The significance is related to the vehicle control. *P < .1; **P < .01; ***P < .001.