Fig. 2

DRD3-signalling in B cells attenuates disease severity in an EAE model that does not depend on the APC-function of B cells. BM chimeric mice harbouring Drd3-deficient or Drd3-sufficient B cells were generated by the i.v. transfer of a 4:1 mixed BM from μMT and Drd3^−/− mice (grey symbols) or 4:1 mixed BM from μMT and Drd3^+/+ mice (black symbols), respectively, into γ-irradiated μMT recipient mice. A Schematic illustration of chimeric mice generation. B–E EAE was induced in chimeric mice by immunization with pMOG_35-55 in CFA followed by pertussis toxin injection. B Disease severity was evaluated throughout the time-course of the disease development. Values represent the mean ± SEM; n = 8 mice per group. C–E At the peak of disease severity (day 15 post-induction), mononuclear cells were isolated from the CNS (C, D) and the spleen (E) followed by ex vivo stimulation with PMA/ionomycin in the presence of brefeldin A, and intracellular cytokine staining analysis in CD4⁺ T cells was carried out by flow cytometry. C Representative dot-plots in the CD4⁺ gate are shown. Numbers indicate the percentage of cells in the corresponding quadrant. D, E Quantification of the frequency of CD4⁺ T cells producing IFNγ, IL-17, GM-CSF or expressing FoxP3; n = 3 mice per group. Each symbol represents data obtained from an individual mouse. The mean ± SEM are depicted. Data representative from one out of two independent experiments are shown. *, p < 0.05; **, p < 0.01; by Mann–Whitney U test (B) or two-way ANOVA followed by Sidak’s post hoc test (D, E)