Fig 1: Oct-1 induces Th2 cytokine expression.(A) Naïve CD4 T cells were activated, transduced with Oct1-MIEG3, Oct2-MIEG3, or control (empty) MIEG3 retroviral vector, and differentiated into Th2 cells. Cytokine expression was measured by intracellular cytokine staining. The plots show the percentages of differentiated cells expressing a particular cytokine. Data are representative of three independent experiments with similar results. (B) The results in (A) were shown as bar graphs. Statistical differences between groups were analyzed by Student’s t-test (*: P < 0.05). (C) Expression levels of Th2 cytokine mRNA. Activated T cells were transduced with Oct-1-MIEG3 or control MIEG3 vector as in (A). GFP+ cells were sorted from samples used in (A) and re-stimulated with anti-CD3 antibody for 24 h. The mRNA expression levels for selected Th2 cytokines were measured by quantitative PCR. Statistical differences between groups were analyzed by Student’s t-test (*: P < 0.05). (D) The effect of Oct1 siRNA on the expression of the Il4 gene. EL4 cells were transfected with control or Oct1 siRNA. The mRNA expression levels for the Il4 gene was measured by quantitative RT-PCR. Bars indicate mean ± SD (n = 3). Statistical difference between groups was analyzed by Student’s t-test (*: P < 0.05). (E) Effect of Oct-1 overexpression on cytokine expression in Th1 cells. Naïve CD4 T cells were transduced with Oct1-MIEG3 or control MIEG3 retroviral vector as in (A) and differentiated into Th1 cells. Cytokine expression was measured by intracellular cytokine staining. The plots show the percentages of cells expressing a particular cytokine. Data are representative of three independent experiments with similar results. (F) IFN-γ-positive cells in (D) were shown as a bar graph.
Fig 2: Central nervous system delivery of anti-CD52 mAb impacts peripheral immune cells in experimental autoimmune encephalomyelitis (EAE) mice. Flow cytometric analysis of immune cell subsets in the spleen of EAE mice treated intrathecally at 10 days post-immunization (dpi) (a–c) or 20 dpi (d–f) with vehicle or anti-CD52 mAb (n = 4). Data are depicted as absolute number of immune cell subsets (a, d), relative number of immune cell subsets (b, e), and percentage of IFNG+, IL17+, IL4+, and FOXP3+ cells in the CD4+ T cell pool (c, f). All replicates were biologically independent.Data are represented as mean ± SEM.*p < 0.05.
Fig 3: Oct-1 stimulates transactivation activity of the Il4 promoter through RHS5.(A) EL4 cells were transfected with expression vectors (Oct1-CMV, Oct2-CMV, GATA-3-CMV, or control CMV) and the RHS5-IL4 reporter construct by electroporation. Cells were allowed to rest for 18 h, followed by treatment with PMA (50 ng/ml) and ionomycin (1 μM) for 4 h. Luciferase activity was then measured. Data are representative of three independent experiments with similar results (NT = no treatment). (B) Mutation sites of Oct-1-binding sites within Il4 promoter and RHS5. Oct-1-binding sites, close to GATA-3-binding site, within Il4 promoter were deleted as indicated (upper), or Oct-1 binding site adjacent to GATA3 binding site (Fig 2E) within RHS5 was mutated as indicated (lower). (C) RHS5(WT)-IL4P(WT) reporter construct or RHS5(mtOct1)-IL4P(WT) construct (RHS5-IL4P reporter construct bearing mutation at Oct-1 binding site within RHS5) were transfected into EL4 cells with expression vectors (Oct1-CMV, GATA-3-CMV, or control CMV) by electroporation. Cells were allowed to rest for 18 h, followed by treatment with PMA (50 ng/ml) and ionomycin (1μM) for 4 h. Luciferase activity was then measured. (D) RHS5(WT)-IL4P(ΔOct1) (RHS5-IL4P construct in which Oct1 binding sites were deleted within the Il4 promoter) or RHS5(mtOct1)-IL4P(ΔOct1) (RHS5-IL4 reporter construct bearing both mutation at Oct-1 binding site within RHS5 and deletion of Oct-1 binding sites within the Il4 promoter) were transfected into EL4 cells with expression vectors (Oct1-CMV, GATA-3-CMV, or control CMV) by electroporation. Luciferase activity was measured as (C). Data are representative of three independent experiments with similar results (NT = no treatment). Bars are shown to indicate mean ± SD (n = 3). Statistical difference between groups was analyzed by Student’s t-test (*: P < 0.05).
Fig 4: CNS delivery of anti-CD52 mAb does not impact peripheral immune cells in healthy mice. Flow cytometric analysis of immune cell subsets in the spleen of healthy mice treated subcutaneously (a–d) or intrathecally (e–h) with vehicle (PBS, n = 5) or anti-CD52 mAb (n = 5). Data are depicted as absolute number of immune cell subsets (a, b, e, f), relative number of immune cell subsets (c, g), and percentage of IFNG+, IL17+, IL4+, and FOXP3+ cells in the CD4+ T cell pool (d, h). All replicates were biologically independent.Data are shown as mean ± SEM.*p < 0.05.**p < 0.01.
Fig 5: Oct-1/2 bind to the Il4 promoter and DHSs within the Th2 LCR.Binding of Oct-1/2 to the Il4 promoter (IL4P) and RHS5, RHS6, and RHS7 within the Th2 LCR was examined by a chromatin immunoprecipitation assay in Th1 and Th2 cells. Data are representative of three independent experiments with similar results. Statistical differences between groups were analyzed by Student’s t-test (*: P < 0.05).
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