Figure 3 Downregulation of T-bet expression in brain-infiltrating MIF−/− CD4+ T cells Macrophage migration inhibitory factor (MIF)−/− and wild-type (Wt)

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Figure 3 Downregulation of T-bet expression in brain-infiltrating MIF−/− CD4+ T cells Macrophage migration inhibitory factor (MIF)−/− and wild-type (Wt) mice were immunized to induce experimental autoimmune encephalomyelitis (EAE) and injected intraperitoneally with 10 mg/kg dexamethasone (Dex) vs phosphate-buffered saline (PBS) control for 3 continuous days upon onset of disease, as shown in figure 1B. Downregulation of T-bet expression in brain-infiltrating MIF−/− CD4+ T cells Macrophage migration inhibitory factor (MIF)−/− and wild-type (Wt) mice were immunized to induce experimental autoimmune encephalomyelitis (EAE) and injected intraperitoneally with 10 mg/kg dexamethasone (Dex) vs phosphate-buffered saline (PBS) control for 3 continuous days upon onset of disease, as shown in figure 1B. Brain mononuclear cells (MNCs) of EAE mice from acute and chronic stages were harvested and cultured for 24 hours with MOG35-55 peptide as described in the Methods. Ag-recalled brain MNCs were stained for CD4 and T-bet for flow cytometry analysis. Shown are representative intracellular staining results of T-bet expression gated on CD4+ T cells (A), pooled results from 2 independent experiments for T-bet expression (B, C), and absolute number of brain-infiltrating T-bet+CD4+ T cells (D, E) from acute stage and chronic stage of EAE (mean ± SEM; acute stage, n = 6–8 mice per group from day 16–24 postimmunization when PBS groups reached peak score of 2 or higher; chronic stage, n = 5–9 mice per group from day 26–36, usually 10 days after PBS groups remitted from peak of disease; analysis of variance; *p < 0.05). (F) T-bet expression in activated CD4+ T cells under non-EAE conditions. Splenocytes from naive Wt and MIF−/− mice were cultured with plate-bound anti-CD3 monoclonal antibody for 48 hours prior to staining for CD4 and T-bet for flow cytometry analysis (Wt, n = 11; MIF−/−, n = 12; shown are pooled results from 4 independent experiments; mean ± SEM; t test). NS = not significant. Niannian Ji et al. Neurol Neuroimmunol Neuroinflamm 2015;2:e139 © 2015 American Academy of Neurology