γδ T cells from Itch^−/− mice produce IL-4 ex vivo. (A) Naive CD4⁺ T cells were stimulated under Th1- or Th2-polarizing conditions in vitro for 1 week before incubation for 4 hours with PDBu and ionomycin in presence of brefeldin A. IL-4 and IFN-γ production was evaluated by FACS after intracellular staining with specific fluorescent Abs. (B) WT (□) and Itch^−/− (■) lymph node CD4⁺ T cells were stimulated for 1, 2, or 3 days with anti-CD3/anti-CD28 Abs in presence of CD11c⁺ spleen APCs. Cytokine levels were detected by a multiplex fluorescent bead immunoassay. Representative profiles of selected cytokines in 1 of 5 experiments are shown (*P < .05). (C) Splenic WT and Itch^−/− γδ T cells were left unstimulated or activated with PDBu and ionomycin in presence of brefeldin A for 4 hours. IL-4 and IFN-γ production was evaluated by FACS after intracellular staining with fluorescent specific Abs. Representative profile in 1 of 3 experiments are shown. (D) IL-4 production in peritoneal γδ T cells and CD4⁺ αβ T cells was monitored directly ex vivo using 4get WT or 4get Itch^−/− IL-4 reporter mice. Gated γδ T cells (GL3⁺ CD3⁺) or CD4⁺ αβ T cells (H57⁺) are shown, after gating on lymphocytes using side and forward scatter profile. Percentage of CD44⁺ GFP⁺ is indicated.