Journal: PLoS ONE

Article Title: Primary B-Cell Deficiencies Reveal a Link between Human IL-17-Producing CD4 T-Cell Homeostasis and B-Cell Differentiation

doi: 10.1371/journal.pone.0022848

Figure Lengend Snippet: (A) Correlation between Th17 frequency and frequency of switched-memory B cells (left), transitional B cells (middle) or CD21 low B cells (right), in CVID patients. Switched-memory B cells were defined as CD27 + IgD − cells, transitional B cells as CD38 high IgM high cells and CD21 low B cells were defined as CD21 low CD38 low cells within gated B cells (CD19 + ) after surface staining of whole blood samples. IL-17 expression was assessed at the single-cell level by intracellular staining following short-term stimulation of PBMC with PMA and ionomycin. (B) Th17 frequency in CVID individuals stratified according to their clinical manifestations, namely autoimmunity, lymphoid proliferation, splenomegaly, and adenopathies. (C) Th17 frequency in healthy controls and CVID patients grouped according to EUROclass. (D) Correlations between frequencies of activated CD4 T cells, defined by concurrent expression of HLA-DR and CD38, and of Th17 cells in CVID and healthy individuals. Each symbol represents one individual. Bars represent mean. Data were compared using Mann-Whitney test, and P values are shown. Correlation significance was assessed using Spearman coefficient test, and r and P values are shown.

Article Snippet: The following anti-human monoclonal antibodies were used, with clone and the directly conjugated fluorochrome specified in brackets: CD3 (SK7; peridinin chlorophyll protein (PerCP)), CD4 (SK3; PerCP), CD8 (SK1; PerCP and allophycocyanin (APC)-Cy7; RPA-T8; APC), CCR6 (11A9; phycoerythrin (PE)), CD38 (HB7; PE), CD45RA (L48; PE-Cy7), IgD (IA6-2; PE), IgM (G20-127; APC), HLA-DR (L243; fluorescein isothiocyanate (FITC)), IFN-γ (4S.B3; FITC), IL-2 (MQ1-17H12; PE), IL-4 (MP4-25D2; PE), from BD Biosciences, San Jose, CA; CD3 (UCHT1; APC-eFluor780), CD4 (RPA-T4, FITC, PerCP-Cy5.5 and PE-Cy7), CD8 (RPA-T8; FITC and PE), CD19 (HIB19; PerCP-Cy5.5 and PE-Cy7), CD27 (O323; FITC, PE, APC and PE-Cy7), CD45RO (UCHL1; PE), CD69 (FN50; FITC), CD45RA (HI100; FITC and APC), IL-17A (eBio64DEC17; PerCP-Cy5.5 and Alexa Fluor 647), TNF-α (MAb11; PE), from eBiosciences, San Diego, CA; CD4 (S3.5; PE) from Caltag, Buckingham, UK; CCR7 (150503; FITC), CXCR5 (51505.111; PE), from R&D Systems, Minneapolis, MN; CD21 (BL13; FITC) from IO Test, Beckman Coulter, Brea, CA.

Techniques: Staining, Expressing, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Primary B-Cell Deficiencies Reveal a Link between Human IL-17-Producing CD4 T-Cell Homeostasis and B-Cell Differentiation

doi: 10.1371/journal.pone.0022848

Figure Lengend Snippet: Correlation between the frequencies of switched-memory (CD27 + IgD − ) B cells and Th17 cells in healthy individuals. Each symbol represents one healthy individual. Correlation significance was assessed using Spearman coefficient test, and r and P values are shown.

Article Snippet: The following anti-human monoclonal antibodies were used, with clone and the directly conjugated fluorochrome specified in brackets: CD3 (SK7; peridinin chlorophyll protein (PerCP)), CD4 (SK3; PerCP), CD8 (SK1; PerCP and allophycocyanin (APC)-Cy7; RPA-T8; APC), CCR6 (11A9; phycoerythrin (PE)), CD38 (HB7; PE), CD45RA (L48; PE-Cy7), IgD (IA6-2; PE), IgM (G20-127; APC), HLA-DR (L243; fluorescein isothiocyanate (FITC)), IFN-γ (4S.B3; FITC), IL-2 (MQ1-17H12; PE), IL-4 (MP4-25D2; PE), from BD Biosciences, San Jose, CA; CD3 (UCHT1; APC-eFluor780), CD4 (RPA-T4, FITC, PerCP-Cy5.5 and PE-Cy7), CD8 (RPA-T8; FITC and PE), CD19 (HIB19; PerCP-Cy5.5 and PE-Cy7), CD27 (O323; FITC, PE, APC and PE-Cy7), CD45RO (UCHL1; PE), CD69 (FN50; FITC), CD45RA (HI100; FITC and APC), IL-17A (eBio64DEC17; PerCP-Cy5.5 and Alexa Fluor 647), TNF-α (MAb11; PE), from eBiosciences, San Diego, CA; CD4 (S3.5; PE) from Caltag, Buckingham, UK; CCR7 (150503; FITC), CXCR5 (51505.111; PE), from R&D Systems, Minneapolis, MN; CD21 (BL13; FITC) from IO Test, Beckman Coulter, Brea, CA.

Techniques:

Journal: Frontiers in Immunology

Article Title: Mesenchymal stromal cells induced regulatory B cells are enriched in extracellular matrix genes and IL-10 independent modulators

doi: 10.3389/fimmu.2022.957797

Figure Lengend Snippet: MSC induce regulatory B cells enriched in transitional B cell phenotypes and secrete IL-10. (A) Graphical protocol for the generation of in vitro induced regulatory B cells (iBreg) and activated B cells and downstream analysis including gating strategy of flow cytometry samples. (B) Representative FACS plots showing the percentage of Transitional B (CD19 + CD24 Hi CD38 Hi ) cells from alive B cells (CD19 + 7AAD - ). (C) Summarized data for the frequency of Transitional B cells, showing a significant increase at day 7 (D) Representative FACS plots showing the percentage of naïve B cells (CD19 + CD27 - ) from alive B cells (CD19 + 7AAD - ). (E) Summarized data for the frequency of Naïve B cells showing a significant increase in the presence of MSC. (F) Supernatant cytokine quantification by ELISA of IL-10. IL-10 production increased after day 5, with a peak production observed at day 7. (G) Supernatant cytokine quantification by ELISA of TNFα. TNFα production was absent in iBreg and activated B cells. Data shows at least three independent experiments in each group. Cells from 2 MSC donors and 3 Tonsil donors were used. Error bars represent SD. Two-way ANOVA was performed to determine statistical significance. *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: To asses surface marker expression, iBreg cultures were labelled for flow cytometry analysis with the following antibodies: IgD-APC-Cy7 (BioLegend, clone IA6-2, San Diego, CA, USA), CD19-BV510 (BD Horizon, clone SJ25C1), CD24-APC (Invitrogen, clone eBioSN3), CD27-PE-Cy7 (Invitrogen, clone 0323), CD38-PE (Invitrogen, clone HB7) and 7AAD (BD Pharmingen).

Techniques: In Vitro, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Phenotypic, Genomic and Functional Characterization Reveals No Differences between CD138 ++ and CD138 low Subpopulations in Multiple Myeloma Cell Lines

doi: 10.1371/journal.pone.0092378

Figure Lengend Snippet: (A) Dot plots showing the percentage of CD138 ++ and CD138 low cells in MM cell lines. (B) Top: summary of the expression of CD19, CD20, CD27, CD38, CD45 and CD56 in CD138 ++ and CD138 low subpopulations in MM cell lines. Code: – (negative); −/+ (heterogeneity); dim (weak positive); + (positive); ++ (high positive). Bottom: representative dot plots corresponding to non-stained RPMI-8226 cells (negative control; light grey) and stained CD138 ++ (black) and CD138 low (dark grey) RPMI-8226 cells. (C) Expression of CD138 by real-time quantitative PCR in CD138 ++ and CD138 low RPMI-8226 subpopulations. Relative values were calculated by the 2 −ΔCt method (ΔCt = Ct (Gene) −Ct (GAPDH) ). The GAPDH gene was used as a control gene. Results are expressed as the means ± SEM (n = 3). (D–K) Confocal images corresponding to the immunocytochemistry for CD138 (red) in CD138 ++ and CD138 low RPMI-8226 and NCI-H929 cells. Nuclear DNA was stained with DAPI (blue). Magnification of the lens, 63x. Specific “4× zoom” was made in E, G, I, K.

Article Snippet: The origin of the antibodies used in immunocytochemistry and flow cytometry was as follows: anti-CD138-APC (clone B-B4), used for immunocytochemistry and flow cytometry, from Miltenyi Biotec (Auburn, CA); anti-CD20-FITC (clone L27), anti-CD138-PerCP-Cy5 (clone MI15), anti-CD56-APC (clone NCAM16.2), anti-CD45-AmCyan (clone 2D1) and anti-CD38-PE (clone HB7) from BD Biosciences (San Jose, CA, USA); anti-CD19-PacificBlue (clone HIB19) and anti-CD27-PE-Cy7 (clone O323) antibodies from eBioscience (San Diego CA, USA); anti-CD38-AlexaFluor700 antibody (clone HIT2) from Exbio (Vestec, Czech Republic) and anti-CD138-FITC (clone B-A38) from Cytognos S.L. (Salamanca, Spain).

Techniques: Expressing, Staining, Negative Control, Real-time Polymerase Chain Reaction, Immunocytochemistry

Journal: Cell Reports

Article Title: Targeting RSV-neutralizing B cell receptors with anti-idiotypic antibodies

doi: 10.1016/j.celrep.2024.114811

Figure Lengend Snippet:

Article Snippet: Cells were then washed with EasySep buffer and resuspended in 200μL EasySep buffer containing CD19-BV711 (BioLegend) at a 1:200 dilution, CD27-PE-Cy7 (eBioscience) at a 1:600 dilution, CD14-FITC (BD Pharmingen) at a 1:60 dilution, CD3-FITC (BD Pharmingen) at a 1:60 dilution, CD20-AF700 (BioLegend) at a 1:300 dilution, IgD-PerCP-Cy5.5 (eBioscience) at a 1:120 dilution, IgM-BV605 (BioLegend) at a 1:120 dilution, Fixable Viability Dye, V500 (eBioscience) at a 1:300 dilution, and i) biotinylated murine anti-idiotypic mAbs conjugated to Total SeqC-0960 APC streptavidin (BioLegend), Total SeqC-0959 APC streptavidin (BioLegend), Total SeqC-0958 APC streptavidin (BioLegend), Total SeqC-0957 APC streptavidin (BioLegend), or Total SeqC-0956 APC streptavidin (BioLegend), ii) recombinant anti-idiotypic mAbs labeled with APC and PE (separately labeled pools) using Zenon Human IgG Labeling Kits (ThermoFisher Scientific) murine anti-idiotypic antibodies labeled with Zenon-PE.

Techniques: Recombinant, Virus, Control, Labeling, Amplification, Binding Assay, Clone Assay, Transfection, Gel Extraction, Polymerase Chain Reaction, Nested PCR, Sequencing, Expressing, Plasmid Preparation, Software, Transmission Assay, Microscopy