Journal: Frontiers in Immunology

Article Title: CD38 Deficiency Ameliorates Chronic Graft- Versus -Host Disease Murine Lupus via a B-Cell-Dependent Mechanism

doi: 10.3389/fimmu.2021.713697

Figure Lengend Snippet: Decreased frequencies of GC B cells and plasma cells upon induction of chronic graft- versus -host disease (cGVHD) in Cd38 −/− recipients. WT (closed circles) and Cd38 −/− (open circles). (A) Representative flow cytometry plot showing CD19 lo-int CD138 hi plasma cells and CD19 + CD138 − B cells from the spleen cells gated on singlets. (B) Flow cytometry plot of CD95 + GL7 hi GC B cells from gated CD19 + CD138 − cells. (C) Percentages of GC B cells (of CD19 + B cells). (D) Total number GC B cells. (E) Percentages of plasma cells in spleen cells. (F) Total number of plasma cells in spleen cells. (G) Percentages of CD19 + B cells in the spleen. (H) Total number of CD19 + B cells over time upon the adoptive transfer of bm12 cells. In panels (C–H) WT mice (closed circles) and Cd38 −/− mice (open circles). The symbols are the mean values and the vertical bars represent ± SEM. P -values are shown for Welch’s t -test. The results are cumulative data from two to four different experiments per time point and mouse type, each with three to five mice per experiment. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cells were then separated in different panels and stained with the following anti-mouse antibodies: CD4-PercP (1:250 dilution) (BD Biosciences Cat# 561090, RRID: AB_10562560), TCR-β-FITC (1:500 dilution) (BD Biosciences Cat# 553170, RRID: AB_394682), CXCR5-biotin (1:350 dilution) (BD Biosciences Cat# 551960, RRID: AB_394301), PD1-APC (1:350) (BD Biosciences Cat# 562671, RRID: AB_2737712), CD19-PE-CF594 (1:350 dilution) (BD Biosciences Cat# 562329, RRID: AB_11154580), CD138-PE (1:350 dilution) (BD Biosciences Cat# 561070, RRID: AB_2033998), B220-PE (1:350 dilution) (BD Biosciences Cat# 553089, RRID: AB_394619), CD95-Alexa Fluor 647 (1:350 dilution) (BD Biosciences Cat# 563647, RRID: AB_2738346), GL7-FITC (1:350 dilution) (BD Biosciences Cat# 562080, RRID: AB_10894953), CD38-FITC (1:350 dilution) (BD Biosciences Cat# 558813, RRID: AB_397126), CD19-APC (1:350 dilution) (BD Biosciences Cat# 550992, RRID: AB_398483), CD183-VioBright FITC (1:50 dilution) (Miltenyi Biotec Cat# 130-111-092, RRID: AB_2655744), CD11c-PE (1:50 dilution) (Miltenyi Biotec Cat# 130-110-701, RRID: AB_2654708), and Streptavidin-PE (1:350) (BioLegend Cat# 405203).

Techniques: Flow Cytometry, Adoptive Transfer Assay

Journal: Frontiers in Immunology

Article Title: CD38 Deficiency Ameliorates Chronic Graft- Versus -Host Disease Murine Lupus via a B-Cell-Dependent Mechanism

doi: 10.3389/fimmu.2021.713697

Figure Lengend Snippet: (A–C) Decreased frequencies and numbers of T-bet + CD11c hi B cells in Cd38 −/− cGVHD mice. (D–F) Decreased anti-ssDNA autoantibody serum levels in Cd38 −/− cGVHD mice. Correlation with the frequencies of T-bet + CD11c hi B cells. (A) Representative flow cytometry plot showing T-bet + CD11c hi cells and T-bet −/lo CD11c − on gated CD19 + B cells from spleen. (B) Percentages of CD11c hi T-bet + cells (of CD19 + B cells). Mean ± SEM. P -values are shown for Welch’s t -test. (C) Total number of CD11c hi T-bet + cells. Mean ± SEM. P -values are shown for Welch’s t -test. (D) Anti-ssDNA serum levels. Median values. P -values are shown for Mann–Whitney test. (E) Total IgG serum levels. Median values. P -values are shown for Mann–Whitney test. (F) Positive correlation between anti-ssDNA serum levels from cGVHD mice and the frequencies of CD11c hi T-bet + B cells (simple linear regression). The anti-ssDNA levels and frequencies are shown in log scale. The results are cumulative data from two to four different experiments per time point and mouse type, each with three to five mice per experiment. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ***** P < 0.00001.

Article Snippet: Cells were then separated in different panels and stained with the following anti-mouse antibodies: CD4-PercP (1:250 dilution) (BD Biosciences Cat# 561090, RRID: AB_10562560), TCR-β-FITC (1:500 dilution) (BD Biosciences Cat# 553170, RRID: AB_394682), CXCR5-biotin (1:350 dilution) (BD Biosciences Cat# 551960, RRID: AB_394301), PD1-APC (1:350) (BD Biosciences Cat# 562671, RRID: AB_2737712), CD19-PE-CF594 (1:350 dilution) (BD Biosciences Cat# 562329, RRID: AB_11154580), CD138-PE (1:350 dilution) (BD Biosciences Cat# 561070, RRID: AB_2033998), B220-PE (1:350 dilution) (BD Biosciences Cat# 553089, RRID: AB_394619), CD95-Alexa Fluor 647 (1:350 dilution) (BD Biosciences Cat# 563647, RRID: AB_2738346), GL7-FITC (1:350 dilution) (BD Biosciences Cat# 562080, RRID: AB_10894953), CD38-FITC (1:350 dilution) (BD Biosciences Cat# 558813, RRID: AB_397126), CD19-APC (1:350 dilution) (BD Biosciences Cat# 550992, RRID: AB_398483), CD183-VioBright FITC (1:50 dilution) (Miltenyi Biotec Cat# 130-111-092, RRID: AB_2655744), CD11c-PE (1:50 dilution) (Miltenyi Biotec Cat# 130-110-701, RRID: AB_2654708), and Streptavidin-PE (1:350) (BioLegend Cat# 405203).

Techniques: Flow Cytometry, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: CD38 Deficiency Ameliorates Chronic Graft- Versus -Host Disease Murine Lupus via a B-Cell-Dependent Mechanism

doi: 10.3389/fimmu.2021.713697

Figure Lengend Snippet: Decreased frequencies of GC B cells and plasma cells upon induction of chronic graft- versus -host disease (cGVHD) in Cd38 −/− recipients. WT (closed circles) and Cd38 −/− (open circles). (A) Representative flow cytometry plot showing CD19 lo-int CD138 hi plasma cells and CD19 + CD138 − B cells from the spleen cells gated on singlets. (B) Flow cytometry plot of CD95 + GL7 hi GC B cells from gated CD19 + CD138 − cells. (C) Percentages of GC B cells (of CD19 + B cells). (D) Total number GC B cells. (E) Percentages of plasma cells in spleen cells. (F) Total number of plasma cells in spleen cells. (G) Percentages of CD19 + B cells in the spleen. (H) Total number of CD19 + B cells over time upon the adoptive transfer of bm12 cells. In panels (C–H) WT mice (closed circles) and Cd38 −/− mice (open circles). The symbols are the mean values and the vertical bars represent ± SEM. P -values are shown for Welch’s t -test. The results are cumulative data from two to four different experiments per time point and mouse type, each with three to five mice per experiment. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cells were then separated in different panels and stained with the following anti-mouse antibodies: CD4-PercP (1:250 dilution) (BD Biosciences Cat# 561090, RRID: AB_10562560), TCR-β-FITC (1:500 dilution) (BD Biosciences Cat# 553170, RRID: AB_394682), CXCR5-biotin (1:350 dilution) (BD Biosciences Cat# 551960, RRID: AB_394301), PD1-APC (1:350) (BD Biosciences Cat# 562671, RRID: AB_2737712), CD19-PE-CF594 (1:350 dilution) (BD Biosciences Cat# 562329, RRID: AB_11154580), CD138-PE (1:350 dilution) (BD Biosciences Cat# 561070, RRID: AB_2033998), B220-PE (1:350 dilution) (BD Biosciences Cat# 553089, RRID: AB_394619), CD95-Alexa Fluor 647 (1:350 dilution) (BD Biosciences Cat# 563647, RRID: AB_2738346), GL7-FITC (1:350 dilution) (BD Biosciences Cat# 562080, RRID: AB_10894953), CD38-FITC (1:350 dilution) (BD Biosciences Cat# 558813, RRID: AB_397126), CD19-APC (1:350 dilution) (BD Biosciences Cat# 550992, RRID: AB_398483), CD183-VioBright FITC (1:50 dilution) (Miltenyi Biotec Cat# 130-111-092, RRID: AB_2655744), CD11c-PE (1:50 dilution) (Miltenyi Biotec Cat# 130-110-701, RRID: AB_2654708), and Streptavidin-PE (1:350) (BioLegend Cat# 405203).

Techniques: Flow Cytometry, Adoptive Transfer Assay

Journal: Frontiers in Immunology

Article Title: CD38 Deficiency Ameliorates Chronic Graft- Versus -Host Disease Murine Lupus via a B-Cell-Dependent Mechanism

doi: 10.3389/fimmu.2021.713697

Figure Lengend Snippet: Decreased frequencies of GC B cells and plasma cells upon induction of chronic graft- versus -host disease (cGVHD) in Cd38 −/− recipients. WT (closed circles) and Cd38 −/− (open circles). (A) Representative flow cytometry plot showing CD19 lo-int CD138 hi plasma cells and CD19 + CD138 − B cells from the spleen cells gated on singlets. (B) Flow cytometry plot of CD95 + GL7 hi GC B cells from gated CD19 + CD138 − cells. (C) Percentages of GC B cells (of CD19 + B cells). (D) Total number GC B cells. (E) Percentages of plasma cells in spleen cells. (F) Total number of plasma cells in spleen cells. (G) Percentages of CD19 + B cells in the spleen. (H) Total number of CD19 + B cells over time upon the adoptive transfer of bm12 cells. In panels (C–H) WT mice (closed circles) and Cd38 −/− mice (open circles). The symbols are the mean values and the vertical bars represent ± SEM. P -values are shown for Welch’s t -test. The results are cumulative data from two to four different experiments per time point and mouse type, each with three to five mice per experiment. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cells were then separated in different panels and stained with the following anti-mouse antibodies: CD4-PercP (1:250 dilution) (BD Biosciences Cat# 561090, RRID: AB_10562560), TCR-β-FITC (1:500 dilution) (BD Biosciences Cat# 553170, RRID: AB_394682), CXCR5-biotin (1:350 dilution) (BD Biosciences Cat# 551960, RRID: AB_394301), PD1-APC (1:350) (BD Biosciences Cat# 562671, RRID: AB_2737712), CD19-PE-CF594 (1:350 dilution) (BD Biosciences Cat# 562329, RRID: AB_11154580), CD138-PE (1:350 dilution) (BD Biosciences Cat# 561070, RRID: AB_2033998), B220-PE (1:350 dilution) (BD Biosciences Cat# 553089, RRID: AB_394619), CD95-Alexa Fluor 647 (1:350 dilution) (BD Biosciences Cat# 563647, RRID: AB_2738346), GL7-FITC (1:350 dilution) (BD Biosciences Cat# 562080, RRID: AB_10894953), CD38-FITC (1:350 dilution) (BD Biosciences Cat# 558813, RRID: AB_397126), CD19-APC (1:350 dilution) (BD Biosciences Cat# 550992, RRID: AB_398483), CD183-VioBright FITC (1:50 dilution) (Miltenyi Biotec Cat# 130-111-092, RRID: AB_2655744), CD11c-PE (1:50 dilution) (Miltenyi Biotec Cat# 130-110-701, RRID: AB_2654708), and Streptavidin-PE (1:350) (BioLegend Cat# 405203).

Techniques: Flow Cytometry, Adoptive Transfer Assay

Journal: Frontiers in Immunology

Article Title: CD38 Deficiency Ameliorates Chronic Graft- Versus -Host Disease Murine Lupus via a B-Cell-Dependent Mechanism

doi: 10.3389/fimmu.2021.713697

Figure Lengend Snippet: Long-term decreased frequencies and numbers of T follicular helper (Tfh) cells in Cd38 −/− mice after adoptive transfer of bm12 spleen cells. WT mice (closed circles) or Cd38 −/− mice (open circles) were i.p. injected with bm12 cells and euthanized at the indicated time points and spleens were analyzed. (A, B) Representative flow cytometry contour plots showing gating strategy for Tfh cells. (A) First, gating on CD4 + cells, then gating on CXCR5 + PD-1 + . (B) Then, gating on FoxP3 − cells: CD4 + PD-1 hi CXCR5 + FoxP3 − . (C) Percentages of Tfh cells (of CD4 + T cells) in spleen at the indicated time points. (D) Total number of Tfh cells (in spleen) over time. (E) Percentages of CD4 + T cells (CD4 + TCR-β + ) in spleens. (F) Total number of CD4 + T cells in spleen. (G) Total number of spleen cells over time. (H) Spleen length (cm) over time upon the adoptive transfer. In all panels, the symbols are the mean values and the vertical bars represent ± SEM. P -values are shown for Welch’s t -test. The results are cumulative data from two to four different experiments per time point and mouse type, each with three to five mice per experiment. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then separated in different panels and stained with the following anti-mouse antibodies: CD4-PercP (1:250 dilution) (BD Biosciences Cat# 561090, RRID: AB_10562560), TCR-β-FITC (1:500 dilution) (BD Biosciences Cat# 553170, RRID: AB_394682), CXCR5-biotin (1:350 dilution) (BD Biosciences Cat# 551960, RRID: AB_394301), PD1-APC (1:350) (BD Biosciences Cat# 562671, RRID: AB_2737712), CD19-PE-CF594 (1:350 dilution) (BD Biosciences Cat# 562329, RRID: AB_11154580), CD138-PE (1:350 dilution) (BD Biosciences Cat# 561070, RRID: AB_2033998), B220-PE (1:350 dilution) (BD Biosciences Cat# 553089, RRID: AB_394619), CD95-Alexa Fluor 647 (1:350 dilution) (BD Biosciences Cat# 563647, RRID: AB_2738346), GL7-FITC (1:350 dilution) (BD Biosciences Cat# 562080, RRID: AB_10894953), CD38-FITC (1:350 dilution) (BD Biosciences Cat# 558813, RRID: AB_397126), CD19-APC (1:350 dilution) (BD Biosciences Cat# 550992, RRID: AB_398483), CD183-VioBright FITC (1:50 dilution) (Miltenyi Biotec Cat# 130-111-092, RRID: AB_2655744), CD11c-PE (1:50 dilution) (Miltenyi Biotec Cat# 130-110-701, RRID: AB_2654708), and Streptavidin-PE (1:350) (BioLegend Cat# 405203).

Techniques: Adoptive Transfer Assay, Injection, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: CD38 Deficiency Ameliorates Chronic Graft- Versus -Host Disease Murine Lupus via a B-Cell-Dependent Mechanism

doi: 10.3389/fimmu.2021.713697

Figure Lengend Snippet: Decreased frequencies of GC B cells and plasma cells upon induction of chronic graft- versus -host disease (cGVHD) in Cd38 −/− recipients. WT (closed circles) and Cd38 −/− (open circles). (A) Representative flow cytometry plot showing CD19 lo-int CD138 hi plasma cells and CD19 + CD138 − B cells from the spleen cells gated on singlets. (B) Flow cytometry plot of CD95 + GL7 hi GC B cells from gated CD19 + CD138 − cells. (C) Percentages of GC B cells (of CD19 + B cells). (D) Total number GC B cells. (E) Percentages of plasma cells in spleen cells. (F) Total number of plasma cells in spleen cells. (G) Percentages of CD19 + B cells in the spleen. (H) Total number of CD19 + B cells over time upon the adoptive transfer of bm12 cells. In panels (C–H) WT mice (closed circles) and Cd38 −/− mice (open circles). The symbols are the mean values and the vertical bars represent ± SEM. P -values are shown for Welch’s t -test. The results are cumulative data from two to four different experiments per time point and mouse type, each with three to five mice per experiment. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cells were then separated in different panels and stained with the following anti-mouse antibodies: CD4-PercP (1:250 dilution) (BD Biosciences Cat# 561090, RRID: AB_10562560), TCR-β-FITC (1:500 dilution) (BD Biosciences Cat# 553170, RRID: AB_394682), CXCR5-biotin (1:350 dilution) (BD Biosciences Cat# 551960, RRID: AB_394301), PD1-APC (1:350) (BD Biosciences Cat# 562671, RRID: AB_2737712), CD19-PE-CF594 (1:350 dilution) (BD Biosciences Cat# 562329, RRID: AB_11154580), CD138-PE (1:350 dilution) (BD Biosciences Cat# 561070, RRID: AB_2033998), B220-PE (1:350 dilution) (BD Biosciences Cat# 553089, RRID: AB_394619), CD95-Alexa Fluor 647 (1:350 dilution) (BD Biosciences Cat# 563647, RRID: AB_2738346), GL7-FITC (1:350 dilution) (BD Biosciences Cat# 562080, RRID: AB_10894953), CD38-FITC (1:350 dilution) (BD Biosciences Cat# 558813, RRID: AB_397126), CD19-APC (1:350 dilution) (BD Biosciences Cat# 550992, RRID: AB_398483), CD183-VioBright FITC (1:50 dilution) (Miltenyi Biotec Cat# 130-111-092, RRID: AB_2655744), CD11c-PE (1:50 dilution) (Miltenyi Biotec Cat# 130-110-701, RRID: AB_2654708), and Streptavidin-PE (1:350) (BioLegend Cat# 405203).

Techniques: Flow Cytometry, Adoptive Transfer Assay

Journal: Frontiers in Immunology

Article Title: CD38 Deficiency Ameliorates Chronic Graft- Versus -Host Disease Murine Lupus via a B-Cell-Dependent Mechanism

doi: 10.3389/fimmu.2021.713697

Figure Lengend Snippet: (A–C) Decreased frequencies and numbers of T-bet + CD11c hi B cells in Cd38 −/− cGVHD mice. (D–F) Decreased anti-ssDNA autoantibody serum levels in Cd38 −/− cGVHD mice. Correlation with the frequencies of T-bet + CD11c hi B cells. (A) Representative flow cytometry plot showing T-bet + CD11c hi cells and T-bet −/lo CD11c − on gated CD19 + B cells from spleen. (B) Percentages of CD11c hi T-bet + cells (of CD19 + B cells). Mean ± SEM. P -values are shown for Welch’s t -test. (C) Total number of CD11c hi T-bet + cells. Mean ± SEM. P -values are shown for Welch’s t -test. (D) Anti-ssDNA serum levels. Median values. P -values are shown for Mann–Whitney test. (E) Total IgG serum levels. Median values. P -values are shown for Mann–Whitney test. (F) Positive correlation between anti-ssDNA serum levels from cGVHD mice and the frequencies of CD11c hi T-bet + B cells (simple linear regression). The anti-ssDNA levels and frequencies are shown in log scale. The results are cumulative data from two to four different experiments per time point and mouse type, each with three to five mice per experiment. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ***** P < 0.00001.

Article Snippet: Cells were then separated in different panels and stained with the following anti-mouse antibodies: CD4-PercP (1:250 dilution) (BD Biosciences Cat# 561090, RRID: AB_10562560), TCR-β-FITC (1:500 dilution) (BD Biosciences Cat# 553170, RRID: AB_394682), CXCR5-biotin (1:350 dilution) (BD Biosciences Cat# 551960, RRID: AB_394301), PD1-APC (1:350) (BD Biosciences Cat# 562671, RRID: AB_2737712), CD19-PE-CF594 (1:350 dilution) (BD Biosciences Cat# 562329, RRID: AB_11154580), CD138-PE (1:350 dilution) (BD Biosciences Cat# 561070, RRID: AB_2033998), B220-PE (1:350 dilution) (BD Biosciences Cat# 553089, RRID: AB_394619), CD95-Alexa Fluor 647 (1:350 dilution) (BD Biosciences Cat# 563647, RRID: AB_2738346), GL7-FITC (1:350 dilution) (BD Biosciences Cat# 562080, RRID: AB_10894953), CD38-FITC (1:350 dilution) (BD Biosciences Cat# 558813, RRID: AB_397126), CD19-APC (1:350 dilution) (BD Biosciences Cat# 550992, RRID: AB_398483), CD183-VioBright FITC (1:50 dilution) (Miltenyi Biotec Cat# 130-111-092, RRID: AB_2655744), CD11c-PE (1:50 dilution) (Miltenyi Biotec Cat# 130-110-701, RRID: AB_2654708), and Streptavidin-PE (1:350) (BioLegend Cat# 405203).

Techniques: Flow Cytometry, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: CD38 Deficiency Ameliorates Chronic Graft- Versus -Host Disease Murine Lupus via a B-Cell-Dependent Mechanism

doi: 10.3389/fimmu.2021.713697

Figure Lengend Snippet: Normal expansion of Tfr cells and Treg cells in Cd38 −/− cGVHD mice. (A, C) Gating strategy used to detect total Tfr cells (independently of PD-1 expression) and Treg cells. (A) Representative flow cytometry plot showing FoxP3 + Tfr cells and FoxP3 − Tfh cells on gated CD4 + CXCR5 + T cells. (B) Frequencies of Tfr cells (% of CD4 + ) in non-treated (NT) mice or after 2 weeks of i.p. injection of bm12 cells (cGVHD). (C) Representative flow cytometry plot showing FoxP3 + Treg cells on gated CD4 + CXCR5 − T cells. (D) Frequencies of Treg cells (% of CD4 + ) in NT mice or after 2 weeks of i.p. injection of bm12 cells (cGVHD). In panels (B) and (D) WT mice (closed circles) and Cd38 −/− mice (open circles). Horizontal bars represent mean values and vertical bars ± SEM. The P -values are for two-way ANOVA test for multiple comparisons (Fisher’s LSD test). ns, not significant ( P > 0.05), * P < 0.05, ** P < 0.01.

Article Snippet: Cells were then separated in different panels and stained with the following anti-mouse antibodies: CD4-PercP (1:250 dilution) (BD Biosciences Cat# 561090, RRID: AB_10562560), TCR-β-FITC (1:500 dilution) (BD Biosciences Cat# 553170, RRID: AB_394682), CXCR5-biotin (1:350 dilution) (BD Biosciences Cat# 551960, RRID: AB_394301), PD1-APC (1:350) (BD Biosciences Cat# 562671, RRID: AB_2737712), CD19-PE-CF594 (1:350 dilution) (BD Biosciences Cat# 562329, RRID: AB_11154580), CD138-PE (1:350 dilution) (BD Biosciences Cat# 561070, RRID: AB_2033998), B220-PE (1:350 dilution) (BD Biosciences Cat# 553089, RRID: AB_394619), CD95-Alexa Fluor 647 (1:350 dilution) (BD Biosciences Cat# 563647, RRID: AB_2738346), GL7-FITC (1:350 dilution) (BD Biosciences Cat# 562080, RRID: AB_10894953), CD38-FITC (1:350 dilution) (BD Biosciences Cat# 558813, RRID: AB_397126), CD19-APC (1:350 dilution) (BD Biosciences Cat# 550992, RRID: AB_398483), CD183-VioBright FITC (1:50 dilution) (Miltenyi Biotec Cat# 130-111-092, RRID: AB_2655744), CD11c-PE (1:50 dilution) (Miltenyi Biotec Cat# 130-110-701, RRID: AB_2654708), and Streptavidin-PE (1:350) (BioLegend Cat# 405203).

Techniques: Expressing, Flow Cytometry, Injection

Journal: Frontiers in Immunology

Article Title: CD38 Deficiency Ameliorates Chronic Graft- Versus -Host Disease Murine Lupus via a B-Cell-Dependent Mechanism

doi: 10.3389/fimmu.2021.713697

Figure Lengend Snippet: Distinct kinetic profiles of PD-1 + and PD-1 − Tfr cells in cGVHD Cd38 −/− versus cGVHD WT mice. (A) Gating strategy to detect PD-1 hi CXCR5 + FoxP3 + or PD1 − CXCR5 + FoxP3 + Tfrs cells in spleen cells from cGVHD mice. Left panel: FoxP3 versus CD4 plot of gated CD4 + CXCR5 + PD-1 − cells, showing the frequency of the PD-1 − FoxP3 + Tfr cells. Middle panel: PD-1 versus CXCR5 plot on gated CD4 + cells showing the gated CXCR5 + PD-1 hi and CXCR5 − PD-1 − subpopulations. Right panel: FoxP3 versus CD4 plot showing the frequencies of PD-1 + FoxP3 + Tfr cells and PD-1 − FoxP3 − Tfr cells. (B) Kinetics of the frequencies of PD1 + (open red circles) and PD-1 − (open black circles) Tfr cells relative to total Tfr cells in spleen cells from cGVHD Cd38 −/− mice. (C) Kinetics of the frequencies of PD1 + (closed red circles) and PD-1 − (closed black circles) Tfr cells relative to total Tfr cells in spleen cells from cGVHD WT mice. (D) Kinetics of total numbers of PD1 + (open red circles) and PD-1 − (open black circles) Tfr cells relative to total Tfr cells in spleen cells from cGVHD Cd38 −/− mice. (E) Kinetics of total numbers of PD1 + (closed red circles) and PD-1 − (closed black circles) Tfr cells relative to total Tfr cells in spleen cells from cGVHD WT mice. The symbols are the mean values and the vertical bars represent ± SEM. P -values are shown for Welch’s t -test. The results are cumulative data from two to three different experiments per time point and mouse type, each with three to four mice per experiment. * P < 0.05, ** P < 0.01, **** P < 0.0001, ***** P < 0.00001.

Article Snippet: Cells were then separated in different panels and stained with the following anti-mouse antibodies: CD4-PercP (1:250 dilution) (BD Biosciences Cat# 561090, RRID: AB_10562560), TCR-β-FITC (1:500 dilution) (BD Biosciences Cat# 553170, RRID: AB_394682), CXCR5-biotin (1:350 dilution) (BD Biosciences Cat# 551960, RRID: AB_394301), PD1-APC (1:350) (BD Biosciences Cat# 562671, RRID: AB_2737712), CD19-PE-CF594 (1:350 dilution) (BD Biosciences Cat# 562329, RRID: AB_11154580), CD138-PE (1:350 dilution) (BD Biosciences Cat# 561070, RRID: AB_2033998), B220-PE (1:350 dilution) (BD Biosciences Cat# 553089, RRID: AB_394619), CD95-Alexa Fluor 647 (1:350 dilution) (BD Biosciences Cat# 563647, RRID: AB_2738346), GL7-FITC (1:350 dilution) (BD Biosciences Cat# 562080, RRID: AB_10894953), CD38-FITC (1:350 dilution) (BD Biosciences Cat# 558813, RRID: AB_397126), CD19-APC (1:350 dilution) (BD Biosciences Cat# 550992, RRID: AB_398483), CD183-VioBright FITC (1:50 dilution) (Miltenyi Biotec Cat# 130-111-092, RRID: AB_2655744), CD11c-PE (1:50 dilution) (Miltenyi Biotec Cat# 130-110-701, RRID: AB_2654708), and Streptavidin-PE (1:350) (BioLegend Cat# 405203).

Techniques:

Journal: Frontiers in Immunology

Article Title: CD38 Deficiency Ameliorates Chronic Graft- Versus -Host Disease Murine Lupus via a B-Cell-Dependent Mechanism

doi: 10.3389/fimmu.2021.713697

Figure Lengend Snippet: Distinct clustering of Cd38 −/− cGVHD mice. The PCA score plots show the distribution of mice according with two principal components that accumulate the greatest variance (71%–85% range) of the 11 features (variables) tested. Principal component 1 (PC1, x -axis) X% and principal component 2 (PC2, y -axis) Y%. Each panel represents one given time. (A) Non-treated (NT) healthy mice ( n = 4, per mouse). (B) Two-weeks cGVHD ( n = 11, WT; n = 7, KO). (C) Four-weeks cGVHD ( n = 7; WT; n = 10, KO). (D) Eight-weeks cGVHD ( n = 7, WT; n = 8, KO). Each dot represents an individual mouse, and except for NT, they are representative of two or more experiments. WT samples are represented by filled red circles. Cd38 −/− samples with filled green circles.

Article Snippet: Cells were then separated in different panels and stained with the following anti-mouse antibodies: CD4-PercP (1:250 dilution) (BD Biosciences Cat# 561090, RRID: AB_10562560), TCR-β-FITC (1:500 dilution) (BD Biosciences Cat# 553170, RRID: AB_394682), CXCR5-biotin (1:350 dilution) (BD Biosciences Cat# 551960, RRID: AB_394301), PD1-APC (1:350) (BD Biosciences Cat# 562671, RRID: AB_2737712), CD19-PE-CF594 (1:350 dilution) (BD Biosciences Cat# 562329, RRID: AB_11154580), CD138-PE (1:350 dilution) (BD Biosciences Cat# 561070, RRID: AB_2033998), B220-PE (1:350 dilution) (BD Biosciences Cat# 553089, RRID: AB_394619), CD95-Alexa Fluor 647 (1:350 dilution) (BD Biosciences Cat# 563647, RRID: AB_2738346), GL7-FITC (1:350 dilution) (BD Biosciences Cat# 562080, RRID: AB_10894953), CD38-FITC (1:350 dilution) (BD Biosciences Cat# 558813, RRID: AB_397126), CD19-APC (1:350 dilution) (BD Biosciences Cat# 550992, RRID: AB_398483), CD183-VioBright FITC (1:50 dilution) (Miltenyi Biotec Cat# 130-111-092, RRID: AB_2655744), CD11c-PE (1:50 dilution) (Miltenyi Biotec Cat# 130-110-701, RRID: AB_2654708), and Streptavidin-PE (1:350) (BioLegend Cat# 405203).

Techniques:

Journal: Frontiers in Immunology

Article Title: CD38 Deficiency Ameliorates Chronic Graft- Versus -Host Disease Murine Lupus via a B-Cell-Dependent Mechanism

doi: 10.3389/fimmu.2021.713697

Figure Lengend Snippet: Abnormal cytokine serum levels in Cd38 −/− cGVHD mice. (A) IL-23. (B) IL-22. (C) IL-27. (D) sCD40L. In panels (A–D) WT mice (closed circles) and Cd38 −/− mice (open circles). The symbols are the mean values and the vertical bars represent ± SEM. P -values are shown for Welch’s t -test. The results are cumulative data from two to three different experiments per time point and mouse type, each with three to five mice per experiment. (E) Matrix showing Spearman’s r correlation values between cytokines serum levels (horizontal) and the frequencies of the T- and B-cell subsets analyzed + anti-ssDNA and total IgG. Asterisks below r numbers denote statistical significance, which could be positive correlation (no sign) or negative correlation (−). * P < 0.05.

Article Snippet: Cells were then separated in different panels and stained with the following anti-mouse antibodies: CD4-PercP (1:250 dilution) (BD Biosciences Cat# 561090, RRID: AB_10562560), TCR-β-FITC (1:500 dilution) (BD Biosciences Cat# 553170, RRID: AB_394682), CXCR5-biotin (1:350 dilution) (BD Biosciences Cat# 551960, RRID: AB_394301), PD1-APC (1:350) (BD Biosciences Cat# 562671, RRID: AB_2737712), CD19-PE-CF594 (1:350 dilution) (BD Biosciences Cat# 562329, RRID: AB_11154580), CD138-PE (1:350 dilution) (BD Biosciences Cat# 561070, RRID: AB_2033998), B220-PE (1:350 dilution) (BD Biosciences Cat# 553089, RRID: AB_394619), CD95-Alexa Fluor 647 (1:350 dilution) (BD Biosciences Cat# 563647, RRID: AB_2738346), GL7-FITC (1:350 dilution) (BD Biosciences Cat# 562080, RRID: AB_10894953), CD38-FITC (1:350 dilution) (BD Biosciences Cat# 558813, RRID: AB_397126), CD19-APC (1:350 dilution) (BD Biosciences Cat# 550992, RRID: AB_398483), CD183-VioBright FITC (1:50 dilution) (Miltenyi Biotec Cat# 130-111-092, RRID: AB_2655744), CD11c-PE (1:50 dilution) (Miltenyi Biotec Cat# 130-110-701, RRID: AB_2654708), and Streptavidin-PE (1:350) (BioLegend Cat# 405203).

Techniques:

Journal: Frontiers in Immunology

Article Title: CD38 Deficiency Ameliorates Chronic Graft- Versus -Host Disease Murine Lupus via a B-Cell-Dependent Mechanism

doi: 10.3389/fimmu.2021.713697

Figure Lengend Snippet: (A) Volcano plots showing the differences in protein abundance in spleens of bm12> Cd38 −/− mice (open blue circles, upper left quadrant) relative to bm12>WT mice (closed red circles, upper right quadrant), 2 weeks after the adoptive transfer. Each circle denotes a different protein, and those above the horizontal dotted line showed statistical significant differences in abundance between mice. P < 0.05. (B) Histogram graph showing STAT1 protein abundance in spleens from bm12> Cd38 −/− versus bm12>WT mice at 2 weeks. Histograms represent the mean ± SEM, and the P -values are for unpaired t -test. Proteomic data shown in panels (A, B) are from two biological replicas per mouse type and three technical replicas per sample. (C) Functionally grouped network of GO terms/pathways and genes are visualized using the ClueGO/CluePedia application. Note that in this application the protein accession numbers of the identified proteins are transformed to their gene names. Terms (large purple circles) are linked based on κ score (≥0.3). Edges show the known expression. The edge thickness is scaled between the minimum and maximum scores shown. The size of the terms is related with their statistical significance. (D) “Positive regulation of type I interferon production’” pathway, which showed the highest statistical significance, was investigated in a subnetwork. Gene names of the identified proteins are highlighted in red. Genes not included in the initial selection are highlighted in black. Known activation [activation symbols in green (arrows)] and inhibition (inhibitory symbols in red) effects are shown. Stat1 and some of the genes of the identified proteins (highlighted in red) are acting as activators in this pathway. * P < 0.05.

Article Snippet: Cells were then separated in different panels and stained with the following anti-mouse antibodies: CD4-PercP (1:250 dilution) (BD Biosciences Cat# 561090, RRID: AB_10562560), TCR-β-FITC (1:500 dilution) (BD Biosciences Cat# 553170, RRID: AB_394682), CXCR5-biotin (1:350 dilution) (BD Biosciences Cat# 551960, RRID: AB_394301), PD1-APC (1:350) (BD Biosciences Cat# 562671, RRID: AB_2737712), CD19-PE-CF594 (1:350 dilution) (BD Biosciences Cat# 562329, RRID: AB_11154580), CD138-PE (1:350 dilution) (BD Biosciences Cat# 561070, RRID: AB_2033998), B220-PE (1:350 dilution) (BD Biosciences Cat# 553089, RRID: AB_394619), CD95-Alexa Fluor 647 (1:350 dilution) (BD Biosciences Cat# 563647, RRID: AB_2738346), GL7-FITC (1:350 dilution) (BD Biosciences Cat# 562080, RRID: AB_10894953), CD38-FITC (1:350 dilution) (BD Biosciences Cat# 558813, RRID: AB_397126), CD19-APC (1:350 dilution) (BD Biosciences Cat# 550992, RRID: AB_398483), CD183-VioBright FITC (1:50 dilution) (Miltenyi Biotec Cat# 130-111-092, RRID: AB_2655744), CD11c-PE (1:50 dilution) (Miltenyi Biotec Cat# 130-110-701, RRID: AB_2654708), and Streptavidin-PE (1:350) (BioLegend Cat# 405203).

Techniques: Adoptive Transfer Assay, Transformation Assay, Expressing, Selection, Activation Assay, Inhibition

Journal: Frontiers in Immunology

Article Title: CD38 Deficiency Ameliorates Chronic Graft- Versus -Host Disease Murine Lupus via a B-Cell-Dependent Mechanism

doi: 10.3389/fimmu.2021.713697

Figure Lengend Snippet: Representative microphotographs of the histological studies performed on the spleen, liver, and kidney 3-μm sections at 2 weeks of the adoptive transfer of spleen cells from female bm12 to female WT (left panels) or to Cd38 −/− (right panels) mice. (A, B) Spleen, (C, D) liver, and (E–J) kidney. (A–D) Spleen and liver tissue sections stained with H&E. (E–H) Kidney tissue sections stained with PAS. (I, J) Kidney sections stained with Masson’s trichrome. Images were taken with a BH2 Olympus microscope. (A, B) ×20 magnification; (C–F) ×10 magnification; (G–J) 40× magnification. (H, J) Scale bar: 50 μm. Note in WT cGVHD mice and Cd38 −/− cGVHD mice the presence of mild perivascular chronic inflammatory infiltrate in liver [black head arrow in (C) and (D) ]. Note in WT cGVHD mice hyaline casts in kidney [black arrow in (E) ], periglomerular chronic inflammatory infiltrate [black and white arrow in (I) ], and increased glomerular size [asterisks in (G) and (I) ].

Article Snippet: Cells were then separated in different panels and stained with the following anti-mouse antibodies: CD4-PercP (1:250 dilution) (BD Biosciences Cat# 561090, RRID: AB_10562560), TCR-β-FITC (1:500 dilution) (BD Biosciences Cat# 553170, RRID: AB_394682), CXCR5-biotin (1:350 dilution) (BD Biosciences Cat# 551960, RRID: AB_394301), PD1-APC (1:350) (BD Biosciences Cat# 562671, RRID: AB_2737712), CD19-PE-CF594 (1:350 dilution) (BD Biosciences Cat# 562329, RRID: AB_11154580), CD138-PE (1:350 dilution) (BD Biosciences Cat# 561070, RRID: AB_2033998), B220-PE (1:350 dilution) (BD Biosciences Cat# 553089, RRID: AB_394619), CD95-Alexa Fluor 647 (1:350 dilution) (BD Biosciences Cat# 563647, RRID: AB_2738346), GL7-FITC (1:350 dilution) (BD Biosciences Cat# 562080, RRID: AB_10894953), CD38-FITC (1:350 dilution) (BD Biosciences Cat# 558813, RRID: AB_397126), CD19-APC (1:350 dilution) (BD Biosciences Cat# 550992, RRID: AB_398483), CD183-VioBright FITC (1:50 dilution) (Miltenyi Biotec Cat# 130-111-092, RRID: AB_2655744), CD11c-PE (1:50 dilution) (Miltenyi Biotec Cat# 130-110-701, RRID: AB_2654708), and Streptavidin-PE (1:350) (BioLegend Cat# 405203).

Techniques: Adoptive Transfer Assay, Staining, Microscopy

Journal: Frontiers in Immunology

Article Title: CD38 Deficiency Ameliorates Chronic Graft- Versus -Host Disease Murine Lupus via a B-Cell-Dependent Mechanism

doi: 10.3389/fimmu.2021.713697

Figure Lengend Snippet: Representative microphotographs of the histological studies performed on the spleen, liver, and kidney 3-μm sections at 8 weeks of the adoptive transfer of spleen cells from female bm12 to female WT (left panels) or Cd38 −/− (right panels) mice. (A, B) Spleen, (C, D) liver, and (E–J) kidney. (A–D) Spleen and liver tissue sections were stained with H&E. (E–H) Kidney tissue sections were stained with PAS. (I, J) Kidney sections were stained with Masson’s trichrome. (A, B) ×20 magnification; (C–F) ×10 magnification; (G–J) ×40 magnification. (G) Scale bar: 50 μm. Note in the liver from WT cGVHD mice the presence of mild perivascular chronic inflammatory infiltrate [black head arrow in (C) ] and macrovesicular steatosis [black and white head arrow in (C) ]. Note in the kidney of WT cGVHD the periglomerular chronic inflammatory infiltrate [small black arrow in (E) ] and the increased glomerular size [asterisks in (G) and (I) ].

Article Snippet: Cells were then separated in different panels and stained with the following anti-mouse antibodies: CD4-PercP (1:250 dilution) (BD Biosciences Cat# 561090, RRID: AB_10562560), TCR-β-FITC (1:500 dilution) (BD Biosciences Cat# 553170, RRID: AB_394682), CXCR5-biotin (1:350 dilution) (BD Biosciences Cat# 551960, RRID: AB_394301), PD1-APC (1:350) (BD Biosciences Cat# 562671, RRID: AB_2737712), CD19-PE-CF594 (1:350 dilution) (BD Biosciences Cat# 562329, RRID: AB_11154580), CD138-PE (1:350 dilution) (BD Biosciences Cat# 561070, RRID: AB_2033998), B220-PE (1:350 dilution) (BD Biosciences Cat# 553089, RRID: AB_394619), CD95-Alexa Fluor 647 (1:350 dilution) (BD Biosciences Cat# 563647, RRID: AB_2738346), GL7-FITC (1:350 dilution) (BD Biosciences Cat# 562080, RRID: AB_10894953), CD38-FITC (1:350 dilution) (BD Biosciences Cat# 558813, RRID: AB_397126), CD19-APC (1:350 dilution) (BD Biosciences Cat# 550992, RRID: AB_398483), CD183-VioBright FITC (1:50 dilution) (Miltenyi Biotec Cat# 130-111-092, RRID: AB_2655744), CD11c-PE (1:50 dilution) (Miltenyi Biotec Cat# 130-110-701, RRID: AB_2654708), and Streptavidin-PE (1:350) (BioLegend Cat# 405203).

Techniques: Adoptive Transfer Assay, Staining

Journal: Frontiers in Immunology

Article Title: CD38 Deficiency Ameliorates Chronic Graft- Versus -Host Disease Murine Lupus via a B-Cell-Dependent Mechanism

doi: 10.3389/fimmu.2021.713697

Figure Lengend Snippet: Representative microphotographs of the histological studies performed on the spleen, liver, and kidney 3-μm sections at 4 weeks of the adoptive transfer of spleen cells from female bm12 to female WT (left panels) or Cd38 −/− (right panels) mice. (A, B) Spleen, (C, D) liver, and (E–J) kidney. (A–D) Spleen and liver tissue sections were stained with H&E. ( E–H ) Kidney tissue sections were stained with PAS. (I, J) Kidney sections were stained with Masson’s trichrome. Images were taken with a BH2 Olympus microscope. (A, B) ×20 magnification; (C–F) ×10 magnification; (G–J) ×40 magnification. (F) Scale bar: 50 μm. Note in the liver from WT cGVHD mice the presence of moderate/severe perivascular chronic inflammatory infiltrate [black head arrow in (C) ]. Note in the kidney of WT cGVHD mice the presence of inflammatory infiltrate [small black arrow in (E) ], periglomerular chronic inflammatory infiltrate [black and white arrow in (I) ], and increased glomerular size with increased mesangial matrix [asterisks in (G) and (I) ].

Article Snippet: Cells were then separated in different panels and stained with the following anti-mouse antibodies: CD4-PercP (1:250 dilution) (BD Biosciences Cat# 561090, RRID: AB_10562560), TCR-β-FITC (1:500 dilution) (BD Biosciences Cat# 553170, RRID: AB_394682), CXCR5-biotin (1:350 dilution) (BD Biosciences Cat# 551960, RRID: AB_394301), PD1-APC (1:350) (BD Biosciences Cat# 562671, RRID: AB_2737712), CD19-PE-CF594 (1:350 dilution) (BD Biosciences Cat# 562329, RRID: AB_11154580), CD138-PE (1:350 dilution) (BD Biosciences Cat# 561070, RRID: AB_2033998), B220-PE (1:350 dilution) (BD Biosciences Cat# 553089, RRID: AB_394619), CD95-Alexa Fluor 647 (1:350 dilution) (BD Biosciences Cat# 563647, RRID: AB_2738346), GL7-FITC (1:350 dilution) (BD Biosciences Cat# 562080, RRID: AB_10894953), CD38-FITC (1:350 dilution) (BD Biosciences Cat# 558813, RRID: AB_397126), CD19-APC (1:350 dilution) (BD Biosciences Cat# 550992, RRID: AB_398483), CD183-VioBright FITC (1:50 dilution) (Miltenyi Biotec Cat# 130-111-092, RRID: AB_2655744), CD11c-PE (1:50 dilution) (Miltenyi Biotec Cat# 130-110-701, RRID: AB_2654708), and Streptavidin-PE (1:350) (BioLegend Cat# 405203).

Techniques: Adoptive Transfer Assay, Staining, Microscopy

Journal: Frontiers in Immunology

Article Title: CD38 Deficiency Ameliorates Chronic Graft- Versus -Host Disease Murine Lupus via a B-Cell-Dependent Mechanism

doi: 10.3389/fimmu.2021.713697

Figure Lengend Snippet: Normal cGVHD response in bm12 mice after the adoptive transfer of Cd38 −/− spleen cells. (A) Frequencies of the T- and B-cell subsets analyzed in WT>bm12 mice (closed circles and open bars) and Cd38 −/− >bm12 mice (open circles and gray bars), 2 weeks after the adoptive transfer of cells. (B) Total numbers of the same subsets and mice of panel (A) . (C) Anti-ssDNA serum levels. (D) Total IgG serum levels. Data in (A–D) are from one out of two independent experiments, each symbol representing one mouse. In (A, B) , P -values are for multiple t -tests corrected for multiple comparisons by the Holm–Šidák method. In (C, D) , the P -values are for unpaired t -test. ns, not significant ( P > 0.05), * P < 0.05, **P < 0.01.

Article Snippet: Cells were then separated in different panels and stained with the following anti-mouse antibodies: CD4-PercP (1:250 dilution) (BD Biosciences Cat# 561090, RRID: AB_10562560), TCR-β-FITC (1:500 dilution) (BD Biosciences Cat# 553170, RRID: AB_394682), CXCR5-biotin (1:350 dilution) (BD Biosciences Cat# 551960, RRID: AB_394301), PD1-APC (1:350) (BD Biosciences Cat# 562671, RRID: AB_2737712), CD19-PE-CF594 (1:350 dilution) (BD Biosciences Cat# 562329, RRID: AB_11154580), CD138-PE (1:350 dilution) (BD Biosciences Cat# 561070, RRID: AB_2033998), B220-PE (1:350 dilution) (BD Biosciences Cat# 553089, RRID: AB_394619), CD95-Alexa Fluor 647 (1:350 dilution) (BD Biosciences Cat# 563647, RRID: AB_2738346), GL7-FITC (1:350 dilution) (BD Biosciences Cat# 562080, RRID: AB_10894953), CD38-FITC (1:350 dilution) (BD Biosciences Cat# 558813, RRID: AB_397126), CD19-APC (1:350 dilution) (BD Biosciences Cat# 550992, RRID: AB_398483), CD183-VioBright FITC (1:50 dilution) (Miltenyi Biotec Cat# 130-111-092, RRID: AB_2655744), CD11c-PE (1:50 dilution) (Miltenyi Biotec Cat# 130-110-701, RRID: AB_2654708), and Streptavidin-PE (1:350) (BioLegend Cat# 405203).

Techniques: Adoptive Transfer Assay