Fig. 5

Drd3 deficiency in B cells impairs the acquisition of CXCR3 and their infiltration into the CNS in an EAE model that depends on the APC-function of B cells. A–C BM chimeric mice harbouring Drd3-deficient (grey symbols) or Drd3-sufficient (white symbols) B cells were generated as described in Fig. 2A. Afterwards, EAE was induced in chimeric mice by immunization with huMOG in CFA followed by pertussis toxin injection. n = 3–6 mice per group. At the peak of disease severity (day 15 post-induction), mononuclear cells were isolated from the spleen and the CNS and the surface expression of CXCR3 and α4 integrin (CD49d) were analysed in the CD19⁺ population by flow cytometry. A Representative histograms for the expression of CXCR3 and CD49d in the CD19⁺ cells are shown. Quantification of the MFI (B) and frequency (C) associated to the surface expression of CXCR3 and CD49d in living (ZAq⁻) CD19⁺ cells isolated from the spleen (top panel) and CNS (bottom panel). B, C Each symbol represents data obtained from an individual mouse. The mean ± SEM are depicted. **, p < 0.01 by two-way ANOVA followed by Sidak’s post hoc test. D, E BM chimeric mice harbouring Drd3-deficient and Drd3-sufficient B cells were generated by the i.v. transfer of a 3:7 mixed BM from Cd45.1^+/+/Cd45.2^−/−/Drd3^+/+ mice (white bars) and Cd45.1^−/−/Cd45.2^+/+/Drd3^−/− mice (grey bars) into γ-irradiated μMT recipient mice. Next, EAE was induced in chimeric mice by immunization with huMOG in CFA followed by pertussis toxin injection. D Schematic illustration of the experimental design. E At the peak of disease severity (day 15 post-induction), mononuclear cells were isolated from peripheral blood (left panels), the spleen (middle panels) and the CNS (right panels) and the frequency of total CD19⁺ B cells was analysed by flow cytometry. Top panels show representative dot-plots of CD45.1⁺ versus CD45.2⁺ cells in the CD19⁺ gate. Numbers indicate the percentage of cells in the corresponding region. Bottom panels show the percentage quantification. Each symbol represents data obtained from an individual mouse; n = 4–8 mice per group. The mean ± SEM are depicted. **, p < 0.01, ***, p < 0.001, ****, p < 0.0001 by two-way ANOVA followed by Sidak’s post hoc test. F, G Naïve B cells (CD19⁺ IgD^hi IgM^int CD11c⁻ TCRβ⁻) were isolated from the spleen of Drd3-deficient (grey histograms/symbols) or Drd3-sufficient (white histograms/symbols) mice by cell-sorting and incubated in vitro in the presence of anti-CD40, anti-IgM, IFNγ and the TLR9-ligand CpG. After 5 days, CXCR3 and Tbet expression were evaluated by flow cytometry. F Representative histograms of CXCR3 and Tbet expression in the CD19⁺ population are shown in top panels. Quantification of the mean fluorescence intensity (MFI) associated to CXCR3 (bottom left panel) and Tbet (bottom right panel) are shown. G Quantification of the percentage of CD19⁺ B cells positive for CXCR3, Tbet or both are shown. F, G Each symbol represents data obtained from an individual mouse; n = 3 mice per group. The mean ± SEM are depicted. **, p < 0.01; by two-way ANOVA followed by Sidak’s post hoc test