Fig. 4: ROCK1/2-inhibition reduces stimulatory activity, co-stimulatory molecule expression, Il-6, iNOS, and migratory activity of DCs.

a–d Coculture of T cells and allogeneic BM-derived DCs was performed for 72 hours. DCs were activated with LPS for 24 h and when indicated thereafter treated with 30 µg/ml ROCK1/2-inhibitor for two hours prior to the coculture. a The scatter plot shows the proliferation of CD8⁺ T cells in response to coculture with allogeneic DCs. The experiment was performed three times, each point represents one mouse. The plot shows mean +/− SD and the P-value was calculated using a two-way ANOVA (Dunnett’s multiple comparison). b Representative histogram showing the proliferation of CD8⁺ T cells in response to coculture with allogeneic DCs. c, d The scatter plots show the fold change in the mean fluorescence intensity (MFI) of the surface marker CD86 (c) and CD80 (d) on CD11c⁺ cells incubated in a coculture with T cells. The experiment was performed three times, each point represents one mouse. The plots show mean +/− SD and the P-value was calculated using a two-tailed paired student t-test. e–g The scatter plots show the fold change of iNOS mRNA (e), Il-6 mRNA (f) and Il-10 (g) mRNA isolated from CD11c⁺ cells. BM-derived DCs were treated with LPS for two hours and exposed to ROCK1/2-Inhibitor or vehicle for two hours. The experiment was performed three times, each point represents a mouse, values of the experimental groups were normalized to vehicle control. The plots show mean +/− SD and the P-value was calculated using a two-tailed paired student t-test. h The scatter plot shows the migration of CD11c⁺ cells. BM-derived DCs were treated with LPS for two hours and exposed to ROCK1/2-inhibitor for two hours. Thereafter, they were incubated in a transwell chamber for five hours with a CXCL12 gradient and migration was assessed using FACS. The experiment was performed three times, each point represents one mouse. The plot shows mean +/− SD and the P-value was calculated using a two-tailed paired student t-test. i The scatter plot shows the fold change of MFI for phalloidin in CD11c⁺ cells. BM-derived DCs were treated with LPS for two hours and exposed to ROCK1/2-inhibitor for two hours. The experiment was performed three times, each point represents one mouse, values of the experimental group were normalized to vehicle control. The plot shows mean +/− SD and the P-value was calculated using a two-tailed paired student t-test. j The representative images show staining for phalloidin (green) in CD11c⁺ cells isolated from mice that had undergone allo-HCT. Mice were treated intraperitoneal with vehicle or ROCK1/2- inhibitor (8 mg/kg in 100 µl PBS) from day 4 to day 13. Images (z-stacks) were acquired by structured illumination microscopy (SIM). Superresolution images were processed with Zen Black software and visualized in 3D with Imaris software (maximum intensity projection). All scale bars are 2 µm in overviews and 1 µm in magnifications. k The scatter plot shows the Volume [µm³] of F-actin in CD11c⁺ cells isolated from mice that had undergone allo-HCT. Mice were treated intraperitoneal with vehicle or 8 mg/Kg ROCK1/2-inhibitor from day 4 to day 13. Cells were isolated from mice of two independent experiments and analyzed. Individual symbols represent measurements of cells isolated from the same animal. The plot shows mean +/− SD and P-value was calculated using an two-tailed unpaired student t-test. l Representative histogram showing phalloidin staining of F-actin in CD11c⁺ cells isolated from mice that had undergone allo-HCT as described in (k). m The scatter plot shows the MFI for phalloidin staining of F-actin in CD11c⁺ cells isolated from mice that had undergone allo-HCT. Mice were treated intraperitoneal with vehicle or ROCK1/2-inhibitor (8 mg/kg in 100 µl PBS) from day 4 to day 13. The experiment was performed twice, each point represents one mouse. The plot shows mean +/− SD and the P-value was calculated using an two-tailed unpaired student t-test. n Survival of mice undergoing allo-HCT to induce aGVHD. On the same day, BM-derived DCs were incubated with ROCK1/2-inhibitor or PBS for four hours, thereafter 1 × 10⁶ donor DCs were injected together with the graft. Depicted are 10 mice in each group from two individual experiments. Survival of mice was plotted by using the Kaplan-Meier method and compared by using a log-rank (Mantel Cox) test. o–q Histological aGVHD scoring of liver (o), colon (p) and small intestine (q) of mice on day 14 after allo-HCT as described in (n). The plots show mean +/− SD and the P-value was calculated using a two-way ANOVA (Dunnett’s multiple comparison).