anti d3 primary antibodies  (Alomone Labs)

Journal: Journal of Neuroinflammation

Article Title: Dopaminergic stimulation leads B-cell infiltration into the central nervous system upon autoimmunity

doi: 10.1186/s12974-021-02338-1

Figure Lengend Snippet: 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 )

Article Snippet: For the immunostaining of DRD2, DRD3, and DRD5, the rabbit anti-DRD2 antibody (ADR-002, Alomone labs), the rabbit anti-DRD3 antibody (ADR-003, Alomone labs), or the rabbit anti-DRD5 antibody (ADR-005, Alomone labs) was directly used or pre-incubated, respectively, with the antigenic peptide DRD2 11-26 (DDLERQNWSRPFNGSE), with the antigenic peptide DRD3 15-29 (CGAENSTGVNRARPH) or with the antigenic peptide DRD5 199-211 (EEGWELEGRTENC) used to develop the antibodies (in a mixture of 0.8 mg/ml antibody and 0.4 mg/ml peptide) for 30 min as a control to abolish the specific immunostaining.

Techniques: Generated, Irradiation, Injection, Isolation, Ex Vivo, Staining, Flow Cytometry, Expressing, MANN-WHITNEY

Journal: Journal of Neuroinflammation

Article Title: Dopaminergic stimulation leads B-cell infiltration into the central nervous system upon autoimmunity

doi: 10.1186/s12974-021-02338-1

Figure Lengend Snippet: Drd3 deficiency in B cell abrogates disease manifestation in an EAE model that depends on the APC-function of B cells. BM chimeric mice harbouring Drd3 -deficient (grey symbols) or Drd3 -sufficient (black symbols) B cells were generated as described in Fig. A. Next, EAE was induced in chimeric mice by immunization with huMOG in CFA followed by pertussis toxin injection. A Disease severity was evaluated throughout the time-course of the disease development. Values represent the mean ± SEM; n = 4 mice per group. B – D At the peak of disease severity (day 15 post-induction), mononuclear cells were isolated from the CNS ( B and C ) and the spleen ( D ) followed by ex vivo stimulation with PMA/ionomycin in the presence of brefeldin A, and intracellular cytokine staining analysis in CD4 + Tcells was carried out by flow cytometry. B Representative dot-plots in the CD4 + gate are shown. Numbers indicate the percentage of cells in the corresponding quadrant. C , D Quantification of the frequency of CD4 + T cells producing IFNγ, IL-17, GM-CSF or expressing FoxP3. n = 3–4 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 Mann–Whitney U test ( A ) or two-way ANOVA followed by Sidak’s post hoc test ( C , D )

Article Snippet: For the immunostaining of DRD2, DRD3, and DRD5, the rabbit anti-DRD2 antibody (ADR-002, Alomone labs), the rabbit anti-DRD3 antibody (ADR-003, Alomone labs), or the rabbit anti-DRD5 antibody (ADR-005, Alomone labs) was directly used or pre-incubated, respectively, with the antigenic peptide DRD2 11-26 (DDLERQNWSRPFNGSE), with the antigenic peptide DRD3 15-29 (CGAENSTGVNRARPH) or with the antigenic peptide DRD5 199-211 (EEGWELEGRTENC) used to develop the antibodies (in a mixture of 0.8 mg/ml antibody and 0.4 mg/ml peptide) for 30 min as a control to abolish the specific immunostaining.

Techniques: Generated, Injection, Isolation, Ex Vivo, Staining, Flow Cytometry, Expressing, MANN-WHITNEY

Journal: Journal of Neuroinflammation

Article Title: Dopaminergic stimulation leads B-cell infiltration into the central nervous system upon autoimmunity

doi: 10.1186/s12974-021-02338-1

Figure Lengend Snippet: 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. A. 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

Article Snippet: For the immunostaining of DRD2, DRD3, and DRD5, the rabbit anti-DRD2 antibody (ADR-002, Alomone labs), the rabbit anti-DRD3 antibody (ADR-003, Alomone labs), or the rabbit anti-DRD5 antibody (ADR-005, Alomone labs) was directly used or pre-incubated, respectively, with the antigenic peptide DRD2 11-26 (DDLERQNWSRPFNGSE), with the antigenic peptide DRD3 15-29 (CGAENSTGVNRARPH) or with the antigenic peptide DRD5 199-211 (EEGWELEGRTENC) used to develop the antibodies (in a mixture of 0.8 mg/ml antibody and 0.4 mg/ml peptide) for 30 min as a control to abolish the specific immunostaining.

Techniques: Expressing, Generated, Injection, Isolation, Flow Cytometry, Fluorescence, Irradiation, FACS, Incubation, In Vitro, Marker, Two Tailed Test, Quantitative RT-PCR

Journal: Journal of Neuroinflammation

Article Title: Dopaminergic stimulation leads B-cell infiltration into the central nervous system upon autoimmunity

doi: 10.1186/s12974-021-02338-1

Figure Lengend Snippet: 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. A. 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

Article Snippet: For the immunostaining of DRD2, DRD3, and DRD5, the rabbit anti-DRD2 antibody (ADR-002, Alomone labs), the rabbit anti-DRD3 antibody (ADR-003, Alomone labs), or the rabbit anti-DRD5 antibody (ADR-005, Alomone labs) was directly used or pre-incubated, respectively, with the antigenic peptide DRD2 11-26 (DDLERQNWSRPFNGSE), with the antigenic peptide DRD3 15-29 (CGAENSTGVNRARPH) or with the antigenic peptide DRD5 199-211 (EEGWELEGRTENC) used to develop the antibodies (in a mixture of 0.8 mg/ml antibody and 0.4 mg/ml peptide) for 30 min as a control to abolish the specific immunostaining.

Techniques: Generated, Injection, Isolation, Expressing, Flow Cytometry, Irradiation, FACS, Incubation, In Vitro, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: Dopaminergic stimulation leads B-cell infiltration into the central nervous system upon autoimmunity

doi: 10.1186/s12974-021-02338-1

Figure Lengend Snippet: DRD3-signalling in lymphocytes is required for the development of CNS autoimmunity. A BM chimeric mice harbouring Drd3 -deficient or Drd3 -sufficient lymphocytes were generated by the i.v. transfer of a 4:1 mixed BM from Rag1 −/− and Drd3 −/− mice (grey symbols) or 4:1 mixed BM from Rag1 −/− and Drd3 + / + mice (white symbols), respectively, into γ-irradiated Rag1 −/− recipient mice. Afterwards, EAE was induced in chimeric mice by immunization with pMOG 35-55 in CFA followed by pertussis toxin injection and disease severity was determined throughout the time-course of the disease development. n = 4–5 mice per group. Top panel shows an illustration of the experimental strategy for generation of chimeric mice. Bottom panel shows the quantification of clinical score for different experimental groups. ( B ) Primary progressive EAE was induced in mice bearing Drd3 -deficient (grey symbols) or Drd3 -sufficient (white symbols) CD4 + T cells by the i.v. transfer of transgenic naïve CD4 + T cells (Tn; 7.5 × 10 5 cells per mouse) isolated from Drd3 −/− 2D2 or Drd3 + / + 2D2 mice into Rag1 −/− recipient mice. Disease severity was determined throughout the time-course of the disease development. n = 4 mice per group. Top panel illustrates the experimental design to induce primary-progressive EAE. Bottom panel shows the quantification of clinical score for different experimental groups. C EAE was induced in wild-type C57BL/6 mice by immunization with pMOG 35-55 (black symbols) or huMOG (grey symbols) in CFA followed by pertussis toxin injection. Disease severity was evaluated throughout the time-course of the disease development. n = 6–8 mice per group. D , E At the peak of disease severity (indicated by an arrow in C ), mononuclear cells were isolated from the spleen, draining lymph nodes (dLN) and central nervous system (CNS) and the frequency of CD19 + B cells from the CD45 + gate ( D ) and the percentage of DRD3 expression in CD19 + B cells ( E ) were evaluated. A control group (white symbols) without immunization (-) was included in the analysis. D , E n = 3–9 mice per group. A – E Each symbol represents data obtained from an individual mouse. The mean ± SEM are depicted. *, p < 0.05; **, p < 0.01; ****, p < 0.0001 by Mann–Whitney U test ( A – C ) or one-way ANOVA followed by Tukey’s post hoc t -test ( D , E )

Article Snippet: For the immunostaining of DRD2, DRD3, and DRD5, the rabbit anti-DRD2 antibody (ADR-002, Alomone labs), the rabbit anti-DRD3 antibody (ADR-003, Alomone labs), or the rabbit anti-DRD5 antibody (ADR-005, Alomone labs) was directly used or pre-incubated, respectively, with the antigenic peptide DRD2 11-26 (DDLERQNWSRPFNGSE), with the antigenic peptide DRD3 15-29 (CGAENSTGVNRARPH) or with the antigenic peptide DRD5 199-211 (EEGWELEGRTENC) used to develop the antibodies (in a mixture of 0.8 mg/ml antibody and 0.4 mg/ml peptide) for 30 min as a control to abolish the specific immunostaining.

Techniques: Generated, Irradiation, Injection, Transgenic Assay, Isolation, Expressing, MANN-WHITNEY

Journal: Journal of Neuroinflammation

Article Title: Dopaminergic stimulation leads B-cell infiltration into the central nervous system upon autoimmunity

doi: 10.1186/s12974-021-02338-1

Figure Lengend Snippet: 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 )

Article Snippet: For the immunostaining of DRD2, DRD3, and DRD5, the rabbit anti-DRD2 antibody (ADR-002, Alomone labs), the rabbit anti-DRD3 antibody (ADR-003, Alomone labs), or the rabbit anti-DRD5 antibody (ADR-005, Alomone labs) was directly used or pre-incubated, respectively, with the antigenic peptide DRD2 11-26 (DDLERQNWSRPFNGSE), with the antigenic peptide DRD3 15-29 (CGAENSTGVNRARPH) or with the antigenic peptide DRD5 199-211 (EEGWELEGRTENC) used to develop the antibodies (in a mixture of 0.8 mg/ml antibody and 0.4 mg/ml peptide) for 30 min as a control to abolish the specific immunostaining.

Techniques: Generated, Irradiation, Injection, Isolation, Ex Vivo, Staining, Flow Cytometry, Expressing, MANN-WHITNEY

Journal: Journal of Neuroinflammation

Article Title: Dopaminergic stimulation leads B-cell infiltration into the central nervous system upon autoimmunity

doi: 10.1186/s12974-021-02338-1

Figure Lengend Snippet: Drd3 deficiency in B cell abrogates disease manifestation in an EAE model that depends on the APC-function of B cells. BM chimeric mice harbouring Drd3 -deficient (grey symbols) or Drd3 -sufficient (black symbols) B cells were generated as described in Fig. A. Next, EAE was induced in chimeric mice by immunization with huMOG in CFA followed by pertussis toxin injection. A Disease severity was evaluated throughout the time-course of the disease development. Values represent the mean ± SEM; n = 4 mice per group. B – D At the peak of disease severity (day 15 post-induction), mononuclear cells were isolated from the CNS ( B and C ) and the spleen ( D ) followed by ex vivo stimulation with PMA/ionomycin in the presence of brefeldin A, and intracellular cytokine staining analysis in CD4 + Tcells was carried out by flow cytometry. B Representative dot-plots in the CD4 + gate are shown. Numbers indicate the percentage of cells in the corresponding quadrant. C , D Quantification of the frequency of CD4 + T cells producing IFNγ, IL-17, GM-CSF or expressing FoxP3. n = 3–4 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 Mann–Whitney U test ( A ) or two-way ANOVA followed by Sidak’s post hoc test ( C , D )

Article Snippet: For the immunostaining of DRD2, DRD3, and DRD5, the rabbit anti-DRD2 antibody (ADR-002, Alomone labs), the rabbit anti-DRD3 antibody (ADR-003, Alomone labs), or the rabbit anti-DRD5 antibody (ADR-005, Alomone labs) was directly used or pre-incubated, respectively, with the antigenic peptide DRD2 11-26 (DDLERQNWSRPFNGSE), with the antigenic peptide DRD3 15-29 (CGAENSTGVNRARPH) or with the antigenic peptide DRD5 199-211 (EEGWELEGRTENC) used to develop the antibodies (in a mixture of 0.8 mg/ml antibody and 0.4 mg/ml peptide) for 30 min as a control to abolish the specific immunostaining.

Techniques: Generated, Injection, Isolation, Ex Vivo, Staining, Flow Cytometry, Expressing, MANN-WHITNEY

Journal: Journal of Neuroinflammation

Article Title: Dopaminergic stimulation leads B-cell infiltration into the central nervous system upon autoimmunity

doi: 10.1186/s12974-021-02338-1

Figure Lengend Snippet: 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. A. 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

Article Snippet: For the immunostaining of DRD2, DRD3, and DRD5, the rabbit anti-DRD2 antibody (ADR-002, Alomone labs), the rabbit anti-DRD3 antibody (ADR-003, Alomone labs), or the rabbit anti-DRD5 antibody (ADR-005, Alomone labs) was directly used or pre-incubated, respectively, with the antigenic peptide DRD2 11-26 (DDLERQNWSRPFNGSE), with the antigenic peptide DRD3 15-29 (CGAENSTGVNRARPH) or with the antigenic peptide DRD5 199-211 (EEGWELEGRTENC) used to develop the antibodies (in a mixture of 0.8 mg/ml antibody and 0.4 mg/ml peptide) for 30 min as a control to abolish the specific immunostaining.

Techniques: Expressing, Generated, Injection, Isolation, Flow Cytometry, Fluorescence, Irradiation, FACS, Incubation, In Vitro, Marker, Two Tailed Test, Quantitative RT-PCR

Journal: Journal of Neuroinflammation

Article Title: Dopaminergic stimulation leads B-cell infiltration into the central nervous system upon autoimmunity

doi: 10.1186/s12974-021-02338-1

Figure Lengend Snippet: 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. A. 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

Article Snippet: For the immunostaining of DRD2, DRD3, and DRD5, the rabbit anti-DRD2 antibody (ADR-002, Alomone labs), the rabbit anti-DRD3 antibody (ADR-003, Alomone labs), or the rabbit anti-DRD5 antibody (ADR-005, Alomone labs) was directly used or pre-incubated, respectively, with the antigenic peptide DRD2 11-26 (DDLERQNWSRPFNGSE), with the antigenic peptide DRD3 15-29 (CGAENSTGVNRARPH) or with the antigenic peptide DRD5 199-211 (EEGWELEGRTENC) used to develop the antibodies (in a mixture of 0.8 mg/ml antibody and 0.4 mg/ml peptide) for 30 min as a control to abolish the specific immunostaining.

Techniques: Generated, Injection, Isolation, Expressing, Flow Cytometry, Irradiation, FACS, Incubation, In Vitro, Fluorescence

Journal: Journal of Neuroinflammation

Article Title: Dopaminergic stimulation leads B-cell infiltration into the central nervous system upon autoimmunity

doi: 10.1186/s12974-021-02338-1

Figure Lengend Snippet: DRD3-signalling in lymphocytes is required for the development of CNS autoimmunity. A BM chimeric mice harbouring Drd3 -deficient or Drd3 -sufficient lymphocytes were generated by the i.v. transfer of a 4:1 mixed BM from Rag1 −/− and Drd3 −/− mice (grey symbols) or 4:1 mixed BM from Rag1 −/− and Drd3 + / + mice (white symbols), respectively, into γ-irradiated Rag1 −/− recipient mice. Afterwards, EAE was induced in chimeric mice by immunization with pMOG 35-55 in CFA followed by pertussis toxin injection and disease severity was determined throughout the time-course of the disease development. n = 4–5 mice per group. Top panel shows an illustration of the experimental strategy for generation of chimeric mice. Bottom panel shows the quantification of clinical score for different experimental groups. ( B ) Primary progressive EAE was induced in mice bearing Drd3 -deficient (grey symbols) or Drd3 -sufficient (white symbols) CD4 + T cells by the i.v. transfer of transgenic naïve CD4 + T cells (Tn; 7.5 × 10 5 cells per mouse) isolated from Drd3 −/− 2D2 or Drd3 + / + 2D2 mice into Rag1 −/− recipient mice. Disease severity was determined throughout the time-course of the disease development. n = 4 mice per group. Top panel illustrates the experimental design to induce primary-progressive EAE. Bottom panel shows the quantification of clinical score for different experimental groups. C EAE was induced in wild-type C57BL/6 mice by immunization with pMOG 35-55 (black symbols) or huMOG (grey symbols) in CFA followed by pertussis toxin injection. Disease severity was evaluated throughout the time-course of the disease development. n = 6–8 mice per group. D , E At the peak of disease severity (indicated by an arrow in C ), mononuclear cells were isolated from the spleen, draining lymph nodes (dLN) and central nervous system (CNS) and the frequency of CD19 + B cells from the CD45 + gate ( D ) and the percentage of DRD3 expression in CD19 + B cells ( E ) were evaluated. A control group (white symbols) without immunization (-) was included in the analysis. D , E n = 3–9 mice per group. A – E Each symbol represents data obtained from an individual mouse. The mean ± SEM are depicted. *, p < 0.05; **, p < 0.01; ****, p < 0.0001 by Mann–Whitney U test ( A – C ) or one-way ANOVA followed by Tukey’s post hoc t -test ( D , E )

Article Snippet: For the immunostaining of DRD2, DRD3, and DRD5, the rabbit anti-DRD2 antibody (ADR-002, Alomone labs), the rabbit anti-DRD3 antibody (ADR-003, Alomone labs), or the rabbit anti-DRD5 antibody (ADR-005, Alomone labs) was directly used or pre-incubated, respectively, with the antigenic peptide DRD2 11-26 (DDLERQNWSRPFNGSE), with the antigenic peptide DRD3 15-29 (CGAENSTGVNRARPH) or with the antigenic peptide DRD5 199-211 (EEGWELEGRTENC) used to develop the antibodies (in a mixture of 0.8 mg/ml antibody and 0.4 mg/ml peptide) for 30 min as a control to abolish the specific immunostaining.

Techniques: Generated, Irradiation, Injection, Transgenic Assay, Isolation, Expressing, MANN-WHITNEY