Fig. 4

DRD3-signalling favours the expression of α4-integrin and attenuates the immunosuppressive profile in B cells infiltrating the CNS in an EAE model that does not depend 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 pMOG_35-55 in CFA followed by pertussis toxin injection. n = 6–9 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. B Quantification of the mean fluorescence intensity (MFI) associated to the surface expression of CXCR3 and CD49d in living (ZAq⁻) CD19⁺ cells isolated from the spleen (top panel) and CNS (bottom panel). C Quantification of the percentage of 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 < 0.05; ***, p < 0.001 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 1:1 mixed BM from Cd45.1^+/+/Cd45.2^−/−/Drd3^+/+ mice (white symbols) and Cd45.1^−/−/Cd45.2^+/+//Drd3^−/− mice (grey symbols) into γ-irradiated μMT recipient mice. Next, EAE was induced in chimeric mice by immunization with pMOG_35-55 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 panel), the spleen (middle panel) and the CNS (right panel) 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. n = 3–5 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, ****, 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 symbols/histograms) or Drd3-sufficient (white symbols/histograms) mice by cell-sorting, loaded with cell-trace violet (CTV) and incubated in vitro in the presence of anti-CD40, anti-IgM, IFNγ and the TLR9-ligand CpG for 5 days. F The extent of proliferation was evaluated as the dilution of the fluorescence associated to CTV in living (ZAq⁻) CD19⁺ cells by flow cytometry. Representative histograms are shown in the left panel. The marker indicates cells displaying dilution of CTV-associated fluorescence. Quantification of the percentage of cells displaying diluted CTV-associated fluorescence (top right panel) and the MFI of CTV-associated fluorescence (bottom right panel) are shown. G The extent of cell dead was determined as the percentage of ZAq⁺ cells in the CD19⁺ gate. F, G Each symbol represents data obtained from an individual mouse; n = 3 mice per group. The mean ± SEM are depicted. *, p < 0.05 by unpaired two-tailed Student’s t-test. n.s. non-significant. (H) Chimeric mice were treated as shown in D and at the peak of disease severity (day 15 post-induction), CD19⁺ B cells were isolated from the spleen and the levels of cytokine transcripts was analysed by qRT-PCR. The levels of gapdh transcripts were used as a housekeeping. Data were obtained from 4–6 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 two-way ANOVA followed by Sidak’s post hoc test