GM-CSF licensing induces IFN-γR1/R2 assembly on the cell surface and nuclear translocation of IRF-1 required for MDSC function. (A) Upregulation of IFN-γR1 and IFN-γR2 in western blots of whole cell extracts after 3 days. (B) Colocalization of IFN-γR1 and IFN-γR2 by confocal microscopy in BM cells cultured for 3 days with the indicated cytokines (n = 4). Original magnification ×400. (C) Exemplified cell surface expression of IFN-γR1 and IFN-γR2 of Ly-6C^high monocytes by FACS analysis after BM cell culture for 3 days with the indicated cytokines; bar graphs for statistical evaluation of MFI values (n = 5). (D) Confocal microscopy analysis of IRF-1 nuclear translocation in fresh or WT or Irf1^−/− L-Mono cells stimulated or not with IFN-γ for 1 hour (n = 3). Note that granulocytic cells do not upregulate IRF-1 (lower cell in each panel). (E) Suppressor capacity of L-Mono from WT, Irf-1^+/−, or Irf-1^−/− mice. Representative of 3 separate experiments. Statistics using 1-way ANOVA, with Tukey posttest. ***P < .001. (F) NO production by L-Mono of the indicated mouse strains after overnight stimulation with LPS/IFN-γ or cytokines (n = 2 from duplicates, unpaired Student t test for each comparison). **P < .01. (G) Clinical autoimmune symptoms in the EAE model with or without 15 daily GM-CSF injections (day 10 to day 5 of EAE) in WT or Irf1^−/− mice. Representative of 3 separate experiments. Two-way ANOVA with Bonferroni posttest. *P < .05, **P < .01.