Figure 5. Role of IFN-γ in the protective properties of CpG-proBs.

(a) Cytokine level determination by multiplex ELISA for Th1/Th2/Th17 cytokines and GM-CSF (expressed in pg ml⁻¹) in supernatants of CpG-proB cells (50,000 cells per 200 μl) stimulated for 5 h with PMA+ionomycin. (b) Constitutive cytokine production by WT CpG-proB cells. Immediately after FACS sorting, WT CpG-proB cells were stained for intracytoplasmic production of cytokines IL-23p19, IL-12p40, IFN-γ, IL-10 and IL-35 using specific antibodies (open histograms) or isotype controls (filled histograms). (c) IFN-γ mRNA levels relative to HPRT1, as determined by qRT–PCR in WT CpG-proBs, their CD45.1⁺ progeny recovered from reactive LN or from spinal cord. Values are expressed as mean±s.e.m. of three experiments (d) EAE clinical score (mean±s.e.m.) in control mice (n=30) and mice injected at day 12 of the disease with 60,000 WT (n=30) or IFN-γ^−/− CpG-proB cells (n=20). Data are pooled from two independent experiments. ***P<0.001 when comparing disease scores for control mice with those obtained after treatment with WT CpG-proB cells by two-way repeated measures ANOVA test. (e) Frequency of CD4⁺ T cells found in reactive LN and spinal cord of control mice versus mice transferred WT or IFN-γ^−/− CpG-proB cells, n=6 mice per group, *P<0.05 (Students't-test). (f–g) Expression of CCR7 analysed by flow cytometry in CD4⁺ and CD8⁺ cells from reactive LN of control mice with EAE versus mice transferred either WT or IFN-γ^−/− CpG-proB cells. Shown are a representative experiment (f) and the mean±s.e.m. of the percentages of CCR7-positive CD4⁺ and CD8⁺ cells out of four experiments (g). (h,i) Expression of CCR7 analysed by flow cytometry either in untreated cells or after an acid wash and shown in a representative experiment (h) and as the mean±s.e.m. of the percentages of CCR7-positive CD4⁺ and CD8⁺ cells out of three experiments (i). *P<0.05, **P<0.005 (Students' t-test).