Figure 4 Increased susceptibility of MIF−/− CD4+ T cells to immunosuppression by Dex in EAE (A) MOG35-55 peptide-activated donor cells from wild-type (Wt)

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Figure 4 Increased susceptibility of MIF−/− CD4+ T cells to immunosuppression by Dex in EAE (A) MOG35-55 peptide-activated donor cells from wild-type (Wt) or macrophage migration inhibitory factor (MIF)−/− mice pretreated with phosphate-buffered saline (PBS) or dexamethasone (Dex) were adoptively transferred into Wt recipient mice as described in the Methods. Increased susceptibility of MIF−/− CD4+ T cells to immunosuppression by Dex in EAE (A) MOG35-55 peptide-activated donor cells from wild-type (Wt) or macrophage migration inhibitory factor (MIF)−/− mice pretreated with phosphate-buffered saline (PBS) or dexamethasone (Dex) were adoptively transferred into Wt recipient mice as described in the Methods. (B) Transfer of Wt donor cells into Wt or MIF−/− recipient mice pretreated with PBS or Dex. Shown is 1 representative experiment of 2–3 independent experiments (n = 5 mice per group; mean ± SEM). *indicates significant difference between recipient groups that received MIF−/− Dex donor cells vs Wt PBS donor cells; †indicates significant difference between recipient groups that received MIF−/− Dex donor cells vs Wt Dex or MIF−/− PBS donor cells (analysis of variance [ANOVA], p < 0.05; NS = not significant). Spleen (C) and brain mononuclear cells (MNCs) (D) of recipient mice on day 23 (A) were harvested and restimulated with MOG35-55 peptide. The frequencies of Th1 and Th17 cytokine-producing T cells from recipients were measured by ELISPOT assay as described in the Methods (n = 5 mice per group; mean ± SEM; ANOVA, *p < 0.05). (E) Preadoptive transfer donor cells were stained after 3-day Ag recall and analyzed by flow cytometry for MOG35-55–specific CD4+ T cells using IAb-MOG38-49 tetramer as described in the Methods. Shown are absolute numbers of Ag-specific CD4+ T cells of different donor groups from pooled data of 5 independent experiments (n = 9–16 mice per group; mean ± SEM; ANOVA, NS). (F, G) Cytokine ELISPOT assays were performed using preadoptive transfer donor cells after 3-day Ag recall as described in the Methods. Shown are pooled data from 3 to 5 independent experiments (F: interleukin (IL)-17, n = 11–15 mice per group; G: interferon (IFN)-γ, n = 9–12 mice per group; mean ± SEM; ANOVA, *p < 0.05, NS). In parallel, preadoptive transfer donor cells were stained and analyzed by flow cytometry for (H) VLA-4 and (I) T-bet expression gated on MOG35-55–specific CD4+ T cells, as described in the Methods. Shown are pooled data from 3 to 5 independent experiments (n = 10–17 mice per group; mean ± SEM; ANOVA, *p < 0.05, **p < 0.01, NS). SFC = spot-forming cells. Niannian Ji et al. Neurol Neuroimmunol Neuroinflamm 2015;2:e139 © 2015 American Academy of Neurology