Figure 8: Bafetinib inhibits TLR-induced cytokine production and signalling in human pDCs.

CAL-1 cells (a) or human PBMCs (b–i) were pre-treated for 1 h with bafetinib (5 μM) or DMSO control and then stimulated with R848. (a,b) Viability was assessed by flow cytometry, while type I IFN and TNF protein levels in the supernatant were assessed by bioassay and ELISA, respectively, at 15 h post-stimulation. (c,d) TNF and IFN-α were determined by flow cytometry at 6 h post-stimulation. Co-expression of TNF and IFN-α in gated pDCs (c). Percentage of pDCs single positive to IFN-α and TNF staining (d). MFI of IFN-α and TNF in gated IFN-α⁺ and TNF⁺ pDCs (e). (f–i) The level of p-NF-κB (f), p-IRF-7 (g), p-p38 (h) and p-MTOR (i) were assessed by flow cytometry in gated pDCs at 15 (f,g,i) or 30 (h) min after stimulation. Lines in graph connect the same donor and histograms depict a representative donor; dotted lines, unstimulated; solid lines, R848 stimulation; black line, DMSO; red line, bafetinib treated. Data are representative of five independent experiments (a) or four (b–f) and five (g–i) donors processed separately. Graphs depict mean±s.d. of replicates within one representative experiment (a–e) or individual donors (f–i). Dunnett’s multiple comparisons test (a,b) and two-ways Student’s t-test (d–i) were used for statistical analyses. *P<0.05, **P<0.01, ***P<0.001.