Figure 4
The effect of JNJ-54271074 on mouse splenic γδ T and NKT cells or human peripheral γδ T and NKT cells.
(A) Dose-dependent inhibition of production of IL-17A, IL-17F and IL-22 in mouse γδ T cells. γδ T cells were isolated separately from four mouse spleens and cultured under stimulation of IL-1β/IL-23 plus anti-IFNγ for 65 hours, in the absence or presence of titrated JNJ-54271074, and accumulated cytokines were measured by ELISA. Data are presented as mean ± SEM of duplicate assays. (B) Flow cytometry analysis for mouse IL-17A- and IL-22-producing γδ T cells, is shown as representative FACS plots from one individual mouse and mean ± SD of four mice is shown in the graph. Intracellular staining was performed on unstimulated, DMSO or 0.1 μM JNJ-54271074-treated cells. (C) Flow cytometry analysis for IL-17A- producing mouse iNKT cells. Splenocytes from 4 individual mice were stimulated with IL-1β/IL-23 plus anti-IFNγ in the absence or presence of 1 μM JNJ-54271074 for 4 days. IL-17A-producing NKT cells were analyzed through intracellular stain on NKT cells that were identified using anti-CD3 and mouse conjugated CD1d-tetramer pre-loaded with alpha-GalCer. Results are shown as representative FACS plots from one individual mouse and graphed as mean ± SD of four mice, and are representative of two independent experiments (D) Flow cytometry analysis for IL-17A- producing γδ+ T cells and IL-17A- producing iNKT cells in human PBMCs. PBMCs isolated from healthy donors were stimulated with either IL-1β/IL-23/IPP for 7 days (γδ+ T cells) or IL-1β/IL-23 for 6 days (iNKT cells), in the absence or presence of 1 μM JNJ-54271074. Results are shown in a histogram as the percent inhibition relative to vehicle and presented as mean ± SD of three to four different donors. The statistical analysis was performed using one way ANOVA Dunnett’s test for Figure B and unpaired t test with Welch’s correction for Figure (C and D), *P < 0.05; **P < 0.01; ***P < 0.005.
