Journal: Frontiers in Immunology

Article Title: The Antigen Presenting Potential of CD21 low B Cells

doi: 10.3389/fimmu.2020.535784

Figure Lengend Snippet: (A) General gating strategy for different CD21 low and CD21 pos B-cell subsets. CD19 pos B cells delineated by CD19 and CD21 were separated into naïve (a), IgM memory (IgM mem) (b), switched memory (sw mem) (c), and CD27 neg IgD neg memory (CD27 neg IgD neg mem) B cells (d) by expression of IgD and CD27. Subsequent gating on CD21 pos CD38 low-intermediate and CD21 low CD38 low B cells allowed the differentiation of CD21 pos and CD21 low B cells, respectively. (B) FACS plots showing CD21 and CD11c, FCRL5, CD95, ICAM-1, CD40 and CXCR5 in naive, IgM mem, sw mem and CD27 neg IgD neg mem B cells in one representative AI patient. (C) Graphs show the MFI of CD80, CD86 and HLA-DR in CD21 pos B-cell subpopulations of healthy donors (HD) and CD21 pos and CD21 low B-cell subsets of patients with autoimmune (AI) diseases in naïve, IgM mem, sw mem and CD27 neg IgD neg mem B cells. Statistical differences were considered significant at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. RA, SLE, sarcoidosis, psoriatic arthritis, and spondyloarthropathy were marked by different symbols as indicated in the legend. ns, not significant.

Article Snippet: Anti-CD19 APC-Cy7, anti-CD38 PerCp-Cy5.5, anti-CD4 PE-Cy7, anti-CD25 PerCP-Cy5.5, anti-CD86 APC, anti-CD80 BV421, anti-CD45RA BV605, anti-ICOS PE, anti-CD21 PE-Cy7, anti-CD80 BV421, anti-CD27 PerCp-Cy5.5, anti-PD-1 APC, anti-CD45RA APC-Cy7, anti-HLA-DR BV605, anti-CD11c AF700, anti-CD86 BV711, anti-CD95 BV650, anti-IgD BV786, anti-FCRL5 APC, anti-CXCR3 BV421, anti-CCR6 BV605, anti-CD19 BV650, anti-CD19 BV605, anti-PD-L1 BV421, anti-PD-L2 PE, anti-ICOS-L PE, anti-OX40L PE, anti-IL-10 PE, anti-IL-10 APC, anti-TNF-α APC-Cy7 (all obtained from BioLegend); anti-IgD FITC and anti-IgD PE (both from Southern Biotech); anti-CD27 PE (Dako); and anti-CD21 PE-Cy7, anti-CD69 FITC, anti-HLA-DR FITC, anti-CD27 BV605, anti-CD40 FITC, anti-CD40L PE, anti-ICAM-1 PE, anti-CXCR5 BUV395 and anti-CD27 BUV395 (all from BD Biosciences).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: The Antigen Presenting Potential of CD21 low B Cells

doi: 10.3389/fimmu.2020.535784

Figure Lengend Snippet: (A) Representative histogram overlays for the expression of CD80 and CD86 on naïve B cells of HD unstimulated, stimulated with SEB, CpG and in T-B co-cultures without or in the presence of SEB. (B) Histogram overlays for CD80 (upper line) and CD86 (bottom line) of naïve, IgM mem, switched mem and CD27 neg IgD neg mem CD21 pos B cells of one representative HD (black solid line) and CD21 pos (blue solid line) and CD21 low B cells (red solid line) of one representative AI patient in Staphylococcus enterotoxin B (SEB-) stimulated co-cultures with allogenic T cells and statistical analysis thereof. Differences were considered significant at *p < 0.05 and **p < 0.01. The different diseases were marked by different symbols as indicated in the legend.

Article Snippet: Anti-CD19 APC-Cy7, anti-CD38 PerCp-Cy5.5, anti-CD4 PE-Cy7, anti-CD25 PerCP-Cy5.5, anti-CD86 APC, anti-CD80 BV421, anti-CD45RA BV605, anti-ICOS PE, anti-CD21 PE-Cy7, anti-CD80 BV421, anti-CD27 PerCp-Cy5.5, anti-PD-1 APC, anti-CD45RA APC-Cy7, anti-HLA-DR BV605, anti-CD11c AF700, anti-CD86 BV711, anti-CD95 BV650, anti-IgD BV786, anti-FCRL5 APC, anti-CXCR3 BV421, anti-CCR6 BV605, anti-CD19 BV650, anti-CD19 BV605, anti-PD-L1 BV421, anti-PD-L2 PE, anti-ICOS-L PE, anti-OX40L PE, anti-IL-10 PE, anti-IL-10 APC, anti-TNF-α APC-Cy7 (all obtained from BioLegend); anti-IgD FITC and anti-IgD PE (both from Southern Biotech); anti-CD27 PE (Dako); and anti-CD21 PE-Cy7, anti-CD69 FITC, anti-HLA-DR FITC, anti-CD27 BV605, anti-CD40 FITC, anti-CD40L PE, anti-ICAM-1 PE, anti-CXCR5 BUV395 and anti-CD27 BUV395 (all from BD Biosciences).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: The Antigen Presenting Potential of CD21 low B Cells

doi: 10.3389/fimmu.2020.535784

Figure Lengend Snippet: (A) Representative histogram overlay displaying CD69 expression in unstimulated T cells (T only) or T-B co-cultures with naïve CD21 pos B cells of a HD or naïve CD21 low B cells of an AI patients in the absence (T + (B) or presence of SEB (T + B + SEB) gated on naïve T cells. (B – D) Histogram overlays for CD69, CD25 and Inducible Co-stimulator (ICOS) on naïve (CD45RA pos ) CD4 T cells co-cultured with naïve, IgM mem, sw mem and CD27 neg IgD neg mem B-cell populations of CD21 pos B cells of one representative HD and CD21 pos and CD21 low populations of one representative patient and statistical analysis thereof. T cells cultured alone and with or without stimulation with SEB were obtained as control. Subsets marked in green (*) were significantly different to the SEB-stimulated T-cell control (p < 0.05). All subsets were statistically significant to unstimulated T cells. Differences were considered significant at *p < 0.05 * and **p < 0.01. The different diseases were marked by different symbols as indicated in the legend.

Article Snippet: Anti-CD19 APC-Cy7, anti-CD38 PerCp-Cy5.5, anti-CD4 PE-Cy7, anti-CD25 PerCP-Cy5.5, anti-CD86 APC, anti-CD80 BV421, anti-CD45RA BV605, anti-ICOS PE, anti-CD21 PE-Cy7, anti-CD80 BV421, anti-CD27 PerCp-Cy5.5, anti-PD-1 APC, anti-CD45RA APC-Cy7, anti-HLA-DR BV605, anti-CD11c AF700, anti-CD86 BV711, anti-CD95 BV650, anti-IgD BV786, anti-FCRL5 APC, anti-CXCR3 BV421, anti-CCR6 BV605, anti-CD19 BV650, anti-CD19 BV605, anti-PD-L1 BV421, anti-PD-L2 PE, anti-ICOS-L PE, anti-OX40L PE, anti-IL-10 PE, anti-IL-10 APC, anti-TNF-α APC-Cy7 (all obtained from BioLegend); anti-IgD FITC and anti-IgD PE (both from Southern Biotech); anti-CD27 PE (Dako); and anti-CD21 PE-Cy7, anti-CD69 FITC, anti-HLA-DR FITC, anti-CD27 BV605, anti-CD40 FITC, anti-CD40L PE, anti-ICAM-1 PE, anti-CXCR5 BUV395 and anti-CD27 BUV395 (all from BD Biosciences).

Techniques: Expressing, Cell Culture

Journal: Frontiers in Immunology

Article Title: The Antigen Presenting Potential of CD21 low B Cells

doi: 10.3389/fimmu.2020.535784

Figure Lengend Snippet: Histogram overlays for CD69 (A) , CD25 (B) , and ICOS (C) expression on memory (CD45RA neg ) CD4 T cells co-cultured with naïve, IgM mem, sw mem and CD27 neg IgD neg mem B-cell populations of CD21 pos B cells of one representative HD and CD21 pos and CD21 low populations of one representative patient and statistical analysis thereof. T cells cultured alone and with or without stimulation with SEB were obtained as control. (D) Representative FACS plot for CD4 and PD-1 or the FMO in unstimulated T cells, T cells stimulated with SEB and T cells co-cultured with naïve CD21 pos B cells of HD or naive CD21 low B cells of a patient in the absence or presence of SEB. Graph shows the proportion of PD-1 positive memory T cells in T-cell cultures stimulated as indicated to unstimulated T cells. Subsets marked with green stars were significantly different to the SEB-stimulated T-cell controls. Differences were considered significant at *p < 0.05. The different diseases were marked by different symbols as indicated in the legend. **p < 0.01.

Article Snippet: Anti-CD19 APC-Cy7, anti-CD38 PerCp-Cy5.5, anti-CD4 PE-Cy7, anti-CD25 PerCP-Cy5.5, anti-CD86 APC, anti-CD80 BV421, anti-CD45RA BV605, anti-ICOS PE, anti-CD21 PE-Cy7, anti-CD80 BV421, anti-CD27 PerCp-Cy5.5, anti-PD-1 APC, anti-CD45RA APC-Cy7, anti-HLA-DR BV605, anti-CD11c AF700, anti-CD86 BV711, anti-CD95 BV650, anti-IgD BV786, anti-FCRL5 APC, anti-CXCR3 BV421, anti-CCR6 BV605, anti-CD19 BV650, anti-CD19 BV605, anti-PD-L1 BV421, anti-PD-L2 PE, anti-ICOS-L PE, anti-OX40L PE, anti-IL-10 PE, anti-IL-10 APC, anti-TNF-α APC-Cy7 (all obtained from BioLegend); anti-IgD FITC and anti-IgD PE (both from Southern Biotech); anti-CD27 PE (Dako); and anti-CD21 PE-Cy7, anti-CD69 FITC, anti-HLA-DR FITC, anti-CD27 BV605, anti-CD40 FITC, anti-CD40L PE, anti-ICAM-1 PE, anti-CXCR5 BUV395 and anti-CD27 BUV395 (all from BD Biosciences).

Techniques: Expressing, Cell Culture

Journal: Frontiers in Immunology

Article Title: The Antigen Presenting Potential of CD21 low B Cells

doi: 10.3389/fimmu.2020.535784

Figure Lengend Snippet: Patients’ characteristics.

Article Snippet: Anti-CD19 APC-Cy7, anti-CD38 PerCp-Cy5.5, anti-CD4 PE-Cy7, anti-CD25 PerCP-Cy5.5, anti-CD86 APC, anti-CD80 BV421, anti-CD45RA BV605, anti-ICOS PE, anti-CD21 PE-Cy7, anti-CD80 BV421, anti-CD27 PerCp-Cy5.5, anti-PD-1 APC, anti-CD45RA APC-Cy7, anti-HLA-DR BV605, anti-CD11c AF700, anti-CD86 BV711, anti-CD95 BV650, anti-IgD BV786, anti-FCRL5 APC, anti-CXCR3 BV421, anti-CCR6 BV605, anti-CD19 BV650, anti-CD19 BV605, anti-PD-L1 BV421, anti-PD-L2 PE, anti-ICOS-L PE, anti-OX40L PE, anti-IL-10 PE, anti-IL-10 APC, anti-TNF-α APC-Cy7 (all obtained from BioLegend); anti-IgD FITC and anti-IgD PE (both from Southern Biotech); anti-CD27 PE (Dako); and anti-CD21 PE-Cy7, anti-CD69 FITC, anti-HLA-DR FITC, anti-CD27 BV605, anti-CD40 FITC, anti-CD40L PE, anti-ICAM-1 PE, anti-CXCR5 BUV395 and anti-CD27 BUV395 (all from BD Biosciences).

Techniques:

Journal: Frontiers in Immunology

Article Title: The Antigen Presenting Potential of CD21 low B Cells

doi: 10.3389/fimmu.2020.535784

Figure Lengend Snippet: (A) General gating strategy for different CD21 low and CD21 pos B-cell subsets. CD19 pos B cells delineated by CD19 and CD21 were separated into naïve (a), IgM memory (IgM mem) (b), switched memory (sw mem) (c), and CD27 neg IgD neg memory (CD27 neg IgD neg mem) B cells (d) by expression of IgD and CD27. Subsequent gating on CD21 pos CD38 low-intermediate and CD21 low CD38 low B cells allowed the differentiation of CD21 pos and CD21 low B cells, respectively. (B) FACS plots showing CD21 and CD11c, FCRL5, CD95, ICAM-1, CD40 and CXCR5 in naive, IgM mem, sw mem and CD27 neg IgD neg mem B cells in one representative AI patient. (C) Graphs show the MFI of CD80, CD86 and HLA-DR in CD21 pos B-cell subpopulations of healthy donors (HD) and CD21 pos and CD21 low B-cell subsets of patients with autoimmune (AI) diseases in naïve, IgM mem, sw mem and CD27 neg IgD neg mem B cells. Statistical differences were considered significant at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. RA, SLE, sarcoidosis, psoriatic arthritis, and spondyloarthropathy were marked by different symbols as indicated in the legend. ns, not significant.

Article Snippet: Anti-CD19 APC-Cy7, anti-CD38 PerCp-Cy5.5, anti-CD4 PE-Cy7, anti-CD25 PerCP-Cy5.5, anti-CD86 APC, anti-CD80 BV421, anti-CD45RA BV605, anti-ICOS PE, anti-CD21 PE-Cy7, anti-CD80 BV421, anti-CD27 PerCp-Cy5.5, anti-PD-1 APC, anti-CD45RA APC-Cy7, anti-HLA-DR BV605, anti-CD11c AF700, anti-CD86 BV711, anti-CD95 BV650, anti-IgD BV786, anti-FCRL5 APC, anti-CXCR3 BV421, anti-CCR6 BV605, anti-CD19 BV650, anti-CD19 BV605, anti-PD-L1 BV421, anti-PD-L2 PE, anti-ICOS-L PE, anti-OX40L PE, anti-IL-10 PE, anti-IL-10 APC, anti-TNF-α APC-Cy7 (all obtained from BioLegend); anti-IgD FITC and anti-IgD PE (both from Southern Biotech); anti-CD27 PE (Dako); and anti-CD21 PE-Cy7, anti-CD69 FITC, anti-HLA-DR FITC, anti-CD27 BV605, anti-CD40 FITC, anti-CD40L PE, anti-ICAM-1 PE, anti-CXCR5 BUV395 and anti-CD27 BUV395 (all from BD Biosciences).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: The Antigen Presenting Potential of CD21 low B Cells

doi: 10.3389/fimmu.2020.535784

Figure Lengend Snippet: (A) Representative histogram overlays for the expression of CD80 and CD86 on naïve B cells of HD unstimulated, stimulated with SEB, CpG and in T-B co-cultures without or in the presence of SEB. (B) Histogram overlays for CD80 (upper line) and CD86 (bottom line) of naïve, IgM mem, switched mem and CD27 neg IgD neg mem CD21 pos B cells of one representative HD (black solid line) and CD21 pos (blue solid line) and CD21 low B cells (red solid line) of one representative AI patient in Staphylococcus enterotoxin B (SEB-) stimulated co-cultures with allogenic T cells and statistical analysis thereof. Differences were considered significant at *p < 0.05 and **p < 0.01. The different diseases were marked by different symbols as indicated in the legend.

Article Snippet: Anti-CD19 APC-Cy7, anti-CD38 PerCp-Cy5.5, anti-CD4 PE-Cy7, anti-CD25 PerCP-Cy5.5, anti-CD86 APC, anti-CD80 BV421, anti-CD45RA BV605, anti-ICOS PE, anti-CD21 PE-Cy7, anti-CD80 BV421, anti-CD27 PerCp-Cy5.5, anti-PD-1 APC, anti-CD45RA APC-Cy7, anti-HLA-DR BV605, anti-CD11c AF700, anti-CD86 BV711, anti-CD95 BV650, anti-IgD BV786, anti-FCRL5 APC, anti-CXCR3 BV421, anti-CCR6 BV605, anti-CD19 BV650, anti-CD19 BV605, anti-PD-L1 BV421, anti-PD-L2 PE, anti-ICOS-L PE, anti-OX40L PE, anti-IL-10 PE, anti-IL-10 APC, anti-TNF-α APC-Cy7 (all obtained from BioLegend); anti-IgD FITC and anti-IgD PE (both from Southern Biotech); anti-CD27 PE (Dako); and anti-CD21 PE-Cy7, anti-CD69 FITC, anti-HLA-DR FITC, anti-CD27 BV605, anti-CD40 FITC, anti-CD40L PE, anti-ICAM-1 PE, anti-CXCR5 BUV395 and anti-CD27 BUV395 (all from BD Biosciences).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: The Antigen Presenting Potential of CD21 low B Cells

doi: 10.3389/fimmu.2020.535784

Figure Lengend Snippet: (A) Representative histogram overlay displaying CD69 expression in unstimulated T cells (T only) or T-B co-cultures with naïve CD21 pos B cells of a HD or naïve CD21 low B cells of an AI patients in the absence (T + (B) or presence of SEB (T + B + SEB) gated on naïve T cells. (B – D) Histogram overlays for CD69, CD25 and Inducible Co-stimulator (ICOS) on naïve (CD45RA pos ) CD4 T cells co-cultured with naïve, IgM mem, sw mem and CD27 neg IgD neg mem B-cell populations of CD21 pos B cells of one representative HD and CD21 pos and CD21 low populations of one representative patient and statistical analysis thereof. T cells cultured alone and with or without stimulation with SEB were obtained as control. Subsets marked in green (*) were significantly different to the SEB-stimulated T-cell control (p < 0.05). All subsets were statistically significant to unstimulated T cells. Differences were considered significant at *p < 0.05 * and **p < 0.01. The different diseases were marked by different symbols as indicated in the legend.

Article Snippet: Anti-CD19 APC-Cy7, anti-CD38 PerCp-Cy5.5, anti-CD4 PE-Cy7, anti-CD25 PerCP-Cy5.5, anti-CD86 APC, anti-CD80 BV421, anti-CD45RA BV605, anti-ICOS PE, anti-CD21 PE-Cy7, anti-CD80 BV421, anti-CD27 PerCp-Cy5.5, anti-PD-1 APC, anti-CD45RA APC-Cy7, anti-HLA-DR BV605, anti-CD11c AF700, anti-CD86 BV711, anti-CD95 BV650, anti-IgD BV786, anti-FCRL5 APC, anti-CXCR3 BV421, anti-CCR6 BV605, anti-CD19 BV650, anti-CD19 BV605, anti-PD-L1 BV421, anti-PD-L2 PE, anti-ICOS-L PE, anti-OX40L PE, anti-IL-10 PE, anti-IL-10 APC, anti-TNF-α APC-Cy7 (all obtained from BioLegend); anti-IgD FITC and anti-IgD PE (both from Southern Biotech); anti-CD27 PE (Dako); and anti-CD21 PE-Cy7, anti-CD69 FITC, anti-HLA-DR FITC, anti-CD27 BV605, anti-CD40 FITC, anti-CD40L PE, anti-ICAM-1 PE, anti-CXCR5 BUV395 and anti-CD27 BUV395 (all from BD Biosciences).

Techniques: Expressing, Cell Culture

Journal: Frontiers in Immunology

Article Title: The Antigen Presenting Potential of CD21 low B Cells

doi: 10.3389/fimmu.2020.535784

Figure Lengend Snippet: Histogram overlays for CD69 (A) , CD25 (B) , and ICOS (C) expression on memory (CD45RA neg ) CD4 T cells co-cultured with naïve, IgM mem, sw mem and CD27 neg IgD neg mem B-cell populations of CD21 pos B cells of one representative HD and CD21 pos and CD21 low populations of one representative patient and statistical analysis thereof. T cells cultured alone and with or without stimulation with SEB were obtained as control. (D) Representative FACS plot for CD4 and PD-1 or the FMO in unstimulated T cells, T cells stimulated with SEB and T cells co-cultured with naïve CD21 pos B cells of HD or naive CD21 low B cells of a patient in the absence or presence of SEB. Graph shows the proportion of PD-1 positive memory T cells in T-cell cultures stimulated as indicated to unstimulated T cells. Subsets marked with green stars were significantly different to the SEB-stimulated T-cell controls. Differences were considered significant at *p < 0.05. The different diseases were marked by different symbols as indicated in the legend. **p < 0.01.

Article Snippet: Anti-CD19 APC-Cy7, anti-CD38 PerCp-Cy5.5, anti-CD4 PE-Cy7, anti-CD25 PerCP-Cy5.5, anti-CD86 APC, anti-CD80 BV421, anti-CD45RA BV605, anti-ICOS PE, anti-CD21 PE-Cy7, anti-CD80 BV421, anti-CD27 PerCp-Cy5.5, anti-PD-1 APC, anti-CD45RA APC-Cy7, anti-HLA-DR BV605, anti-CD11c AF700, anti-CD86 BV711, anti-CD95 BV650, anti-IgD BV786, anti-FCRL5 APC, anti-CXCR3 BV421, anti-CCR6 BV605, anti-CD19 BV650, anti-CD19 BV605, anti-PD-L1 BV421, anti-PD-L2 PE, anti-ICOS-L PE, anti-OX40L PE, anti-IL-10 PE, anti-IL-10 APC, anti-TNF-α APC-Cy7 (all obtained from BioLegend); anti-IgD FITC and anti-IgD PE (both from Southern Biotech); anti-CD27 PE (Dako); and anti-CD21 PE-Cy7, anti-CD69 FITC, anti-HLA-DR FITC, anti-CD27 BV605, anti-CD40 FITC, anti-CD40L PE, anti-ICAM-1 PE, anti-CXCR5 BUV395 and anti-CD27 BUV395 (all from BD Biosciences).

Techniques: Expressing, Cell Culture

Journal: Frontiers in Immunology

Article Title: The Antigen Presenting Potential of CD21 low B Cells

doi: 10.3389/fimmu.2020.535784

Figure Lengend Snippet: (A) Cytokines measured in supernatants of T-B co-cultures. Production of TNF-α, IL-2, IFN-γ, IL-4, and IL-10 was measured in T-B co-cultures with different B-cell subsets of AI patients and HD. Subsets marked in green were significantly different to the SEB-stimulated T-cell control. (B) Intracellular cytokine staining of TNF-α and IL-10 versus CD19 in T-B co-cultures with and without SEB and T-cell SEB control. Representative FACS Plots of co-cultures with naïve B cells of HD are displayed. Data are representative for 1–3 times for each CD21 low B-cell subpopulation of patients and 2–4 times of each CD21 pos B-cell subpopulations of HD in 4 independent experiments. Differences were considered significant at *p < 0.05 and **p < 0.01.

Article Snippet: Anti-CD19 APC-Cy7, anti-CD38 PerCp-Cy5.5, anti-CD4 PE-Cy7, anti-CD25 PerCP-Cy5.5, anti-CD86 APC, anti-CD80 BV421, anti-CD45RA BV605, anti-ICOS PE, anti-CD21 PE-Cy7, anti-CD80 BV421, anti-CD27 PerCp-Cy5.5, anti-PD-1 APC, anti-CD45RA APC-Cy7, anti-HLA-DR BV605, anti-CD11c AF700, anti-CD86 BV711, anti-CD95 BV650, anti-IgD BV786, anti-FCRL5 APC, anti-CXCR3 BV421, anti-CCR6 BV605, anti-CD19 BV650, anti-CD19 BV605, anti-PD-L1 BV421, anti-PD-L2 PE, anti-ICOS-L PE, anti-OX40L PE, anti-IL-10 PE, anti-IL-10 APC, anti-TNF-α APC-Cy7 (all obtained from BioLegend); anti-IgD FITC and anti-IgD PE (both from Southern Biotech); anti-CD27 PE (Dako); and anti-CD21 PE-Cy7, anti-CD69 FITC, anti-HLA-DR FITC, anti-CD27 BV605, anti-CD40 FITC, anti-CD40L PE, anti-ICAM-1 PE, anti-CXCR5 BUV395 and anti-CD27 BUV395 (all from BD Biosciences).

Techniques: Staining