cd27 pe cy7 Search Results


cd27 (o323; fitc, pe, apc pe-cy7  (Thermo Fisher)
90
Thermo Fisher cd27 (o323; fitc, pe, apc pe-cy7
(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 <t>CD27</t> + 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.
Cd27 (O323; Fitc, Pe, Apc Pe Cy7, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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autoreactivity source anti cd19 pe cy5  (Miltenyi Biotec)
95
Miltenyi Biotec autoreactivity source anti cd19 pe cy5
(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 <t>CD27</t> + 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.
Autoreactivity Source Anti Cd19 Pe Cy5, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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live/dead bv395 n/a  (Thermo Fisher)
90
Thermo Fisher live/dead bv395 n/a
(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 <t>CD27</t> + 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.
Live/Dead Bv395 N/A, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cd235a (pe-cy5  (Becton Dickinson)
90
Becton Dickinson cd235a (pe-cy5
(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 <t>CD27</t> + 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.
Cd235a (Pe Cy5, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti-cd27-pe-cy7 ebioscience cat #25-0279-42  (Thermo Fisher)
90
Thermo Fisher anti-cd27-pe-cy7 ebioscience cat #25-0279-42
(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 <t>CD27</t> + 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.
Anti Cd27 Pe Cy7 Ebioscience Cat #25 0279 42, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti-human cd27-pe/cy7 lg.7f9  (Thermo Fisher)
90
Thermo Fisher anti-human cd27-pe/cy7 lg.7f9
(A) Representative flow cytometry plots and summary data from eight CeD patients showing TG2 staining of IgA-switched B cells in peripheral blood before and after antigen-specific enrichment. GST was included as an irrelevant control antigen. The biotinylated antigens were attached to streptavidin (SA) that was conjugated to PE or <t>PE/Cy7,</t> allowing enrichment with anti-PE microbeads. (B) Representative flow cytometry plots showing identification of TG2-specific cells among all B cells after antigen-specific enrichment. Blood samples were obtained from patients with untreated CeD (UCeD), treated CeD (TCeD) or from healthy donors (HD). (C) Frequency of IgA-switched cells among all TG2-specific B cells in UCeD patients (n=27), TCeD patients (n=16) or HD (n=7) after antigen-specific enrichment. Mucosal healing in TCeD was evaluated by Marsh score classification, where 0 or 1 is considered normal. (D) Frequency of <t>CD27-cells</t> among TG2-specific and non-TG2-specific (other) IgA+ B cells in the same donors. Bar heights indicate medians, and difference between groups was evaluated by a Kruskal-Wallis H test with Dunn’s multiple comparisons correction. **p < 0.01, ***p < 0.001, ****p < 0.0001. (E) Correlation between frequency of IgA-switched cells and frequency of CD27- IgA-switched cells among TG2-specific B cells. Symbol colors indicate UCeD or TCeD as in (C). The data were fitted to a three-parameter dose-response curve, and the p value indicates Spearman correlation.
Anti Human Cd27 Pe/Cy7 Lg.7f9, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cd27 pe cy7  (Thermo Fisher)
86
Thermo Fisher cd27 pe cy7
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 + <t>CD27</t> - ) 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.
Cd27 Pe Cy7, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cd27 pe-cy7  (Becton Dickinson)
90
Becton Dickinson cd27 pe-cy7
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 + <t>CD27</t> - ) 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.
Cd27 Pe Cy7, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cd27 pe-cy7 antibody  (Thermo Fisher)
90
Thermo Fisher cd27 pe-cy7 antibody
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 + <t>CD27</t> - ) 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.
Cd27 Pe Cy7 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti-cd27-pe-cy7 o323  (Thermo Fisher)
90
Thermo Fisher anti-cd27-pe-cy7 o323
(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, <t>CD27,</t> 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.
Anti Cd27 Pe Cy7 O323, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti-cd3-percp  (Becton Dickinson)
90
Becton Dickinson anti-cd3-percp
(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, <t>CD27,</t> 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.
Anti Cd3 Percp, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cd27-pe-cy7  (Thermo Fisher)
90
Thermo Fisher cd27-pe-cy7

Cd27 Pe Cy7, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


(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.

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

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.

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:

(A) Representative flow cytometry plots and summary data from eight CeD patients showing TG2 staining of IgA-switched B cells in peripheral blood before and after antigen-specific enrichment. GST was included as an irrelevant control antigen. The biotinylated antigens were attached to streptavidin (SA) that was conjugated to PE or PE/Cy7, allowing enrichment with anti-PE microbeads. (B) Representative flow cytometry plots showing identification of TG2-specific cells among all B cells after antigen-specific enrichment. Blood samples were obtained from patients with untreated CeD (UCeD), treated CeD (TCeD) or from healthy donors (HD). (C) Frequency of IgA-switched cells among all TG2-specific B cells in UCeD patients (n=27), TCeD patients (n=16) or HD (n=7) after antigen-specific enrichment. Mucosal healing in TCeD was evaluated by Marsh score classification, where 0 or 1 is considered normal. (D) Frequency of CD27-cells among TG2-specific and non-TG2-specific (other) IgA+ B cells in the same donors. Bar heights indicate medians, and difference between groups was evaluated by a Kruskal-Wallis H test with Dunn’s multiple comparisons correction. **p < 0.01, ***p < 0.001, ****p < 0.0001. (E) Correlation between frequency of IgA-switched cells and frequency of CD27- IgA-switched cells among TG2-specific B cells. Symbol colors indicate UCeD or TCeD as in (C). The data were fitted to a three-parameter dose-response curve, and the p value indicates Spearman correlation.

Journal: bioRxiv

Article Title: Generation of circulating autoreactive pre-plasma cells fueled by naïve B cells in celiac disease

doi: 10.1101/2023.10.20.563227

Figure Lengend Snippet: (A) Representative flow cytometry plots and summary data from eight CeD patients showing TG2 staining of IgA-switched B cells in peripheral blood before and after antigen-specific enrichment. GST was included as an irrelevant control antigen. The biotinylated antigens were attached to streptavidin (SA) that was conjugated to PE or PE/Cy7, allowing enrichment with anti-PE microbeads. (B) Representative flow cytometry plots showing identification of TG2-specific cells among all B cells after antigen-specific enrichment. Blood samples were obtained from patients with untreated CeD (UCeD), treated CeD (TCeD) or from healthy donors (HD). (C) Frequency of IgA-switched cells among all TG2-specific B cells in UCeD patients (n=27), TCeD patients (n=16) or HD (n=7) after antigen-specific enrichment. Mucosal healing in TCeD was evaluated by Marsh score classification, where 0 or 1 is considered normal. (D) Frequency of CD27-cells among TG2-specific and non-TG2-specific (other) IgA+ B cells in the same donors. Bar heights indicate medians, and difference between groups was evaluated by a Kruskal-Wallis H test with Dunn’s multiple comparisons correction. **p < 0.01, ***p < 0.001, ****p < 0.0001. (E) Correlation between frequency of IgA-switched cells and frequency of CD27- IgA-switched cells among TG2-specific B cells. Symbol colors indicate UCeD or TCeD as in (C). The data were fitted to a three-parameter dose-response curve, and the p value indicates Spearman correlation.

Article Snippet: Anti-human CCR9-BUV395 (clone C9MAB-1, BD), anti-human CD3-BV510 (clone OKT3, BioLegend), anti-human CD3-BV605 (clone UCHT1, BioLegend), anti-human CD14-BV510 (clone M5E2, BioLegend), anti-human CD14-BV605 (clone M5E2, BioLegend), anti-human CD11c-BUV805 (clone B-ly6, BD), anti-human CD11c-BV605 (clone 3.9, Biolegend), anti-human CD19-Pacific blue (clone HIB19, BioLegend), anti-human CD19-PerCP/Cy5.5 (clone HIB19, BioLegend), anti-human CD19-APC/Cy7 (clone HIB19, BioLegend), anti-human CD20-BUV496 (clone 2H7, BD), anti-human CD20-BV605 (clone 2H7, BioLegend), anti-human CD21-BV421 (clone 1048, BD), anti-human CD21-APC (clone Bu32, BioLegend), anti-human CD24-BV605 (clone ML5, BioLegend), anti-human CD27-BV421 (clone O323, BioLegend), anti-human CD27-BV711 (clone M-T271, BioLegend), anti-human CD27-PE/Cy7 (clone LG.7F9, eBioscience), anti-human CD27-APC (clone M-T271, BioLegend), anti-human CD27-APC/Cy7 (clone O323, BioLegend), anti-human CD38-BUV563 (clone 2B7, BD), anti-human CD38-BV605 (clone HIT2, BioLegend), anti-human CD38-FITC (clone HB7, eBioscience), anti-human CD38-PerCP-Cy5.5 (clone HIT2, Biolegend), anti-human CD62L-R718 (clone DREG-56, BD), anti-human CD86-BV785 (clone IT2.2, BioLegend), anti-human CD95-BUV737 (clone DX2, BD), anti-human CD95-Alexa Fluor 700 (clone DX2, BioLegend), anti-human CXCR3-PE-CF594 (clone 1C6/CXCR3, BD), anti-human CXCR4-BV605 (clone 12G5, BD), anti-human CXCR5-PE/Cy5 (clone J252D4, BioLegend), anti-human CXCR5-Alexa Fluor 647 (clone J252D4, BioLegend), anti-human IgA-FITC (clone IS11-8E10, Miltenyi), goat anti-human IgA-PE (Southern Biotech), anti-human IgA-APC (clone IS11-8E10, Miltenyi), anti-human IgA-APC-Vio770 (clone IS11-8E10, Miltenyi), goat anti-human IgD-FITC (Southern Biotech), anti-human IgD-PerCP/Cy5.5 (clone IA6-2, BioLegend), anti-human IgD-Alexa Fluor 700 (clone IA6-2, BioLegend), anti-human IgG-APC (clone M1310G05, BioLegend), anti-human IgM-BV510 (clone MHM-88, Biolegend), goat anti-human IgM-FITC (Southern Biotech), anti-human Integrin β7-BV650 (clone FIB504, BD), anti-human Integrin β7-APC (clone FIB504, BioLegend), rabbit anti-human TG2 (Pacific Immunology), goat anti-rabbit IgG-Alexa488 (Thermo Fisher), anti-biotin-PE (clone 1D4-C5, Biolegend).

Techniques: Flow Cytometry, Staining

(A) Detection of total and anti-TG2 IgA in culture supernatants of single TG2- specific (n=88) or other (n=88) IgA+ B cells sorted from PBMCs of a representative untreated CeD patient. The TG2 avidity index was calculated as the TG2-specific signal divided by the total IgA signal for cultures positive for total IgA (OD > 3x background, dashed line). (B) Comparison of antibody secretion (>10 ng/ml total IgA) in cultures of TG2-specific (n=106) or other (n=111) IgA+ B cells sorted from PBMCs of four untreated CeD patients. (C) Confirmation of TG2-reactivity in all cultures of TG2-specific B cells that were positive for total IgA. A TG2 avidity index >0.1 was considered TG2 positive. (D) Affinity of secreted antibodies in cultures of CD27+ or CD27- IgA+ B cells with confirmed TG2 reactivity. Bar heights indicate medians, and difference between groups was evaluated by a Mann-Whitney U test. ****p < 0.0001.

Journal: bioRxiv

Article Title: Generation of circulating autoreactive pre-plasma cells fueled by naïve B cells in celiac disease

doi: 10.1101/2023.10.20.563227

Figure Lengend Snippet: (A) Detection of total and anti-TG2 IgA in culture supernatants of single TG2- specific (n=88) or other (n=88) IgA+ B cells sorted from PBMCs of a representative untreated CeD patient. The TG2 avidity index was calculated as the TG2-specific signal divided by the total IgA signal for cultures positive for total IgA (OD > 3x background, dashed line). (B) Comparison of antibody secretion (>10 ng/ml total IgA) in cultures of TG2-specific (n=106) or other (n=111) IgA+ B cells sorted from PBMCs of four untreated CeD patients. (C) Confirmation of TG2-reactivity in all cultures of TG2-specific B cells that were positive for total IgA. A TG2 avidity index >0.1 was considered TG2 positive. (D) Affinity of secreted antibodies in cultures of CD27+ or CD27- IgA+ B cells with confirmed TG2 reactivity. Bar heights indicate medians, and difference between groups was evaluated by a Mann-Whitney U test. ****p < 0.0001.

Article Snippet: Anti-human CCR9-BUV395 (clone C9MAB-1, BD), anti-human CD3-BV510 (clone OKT3, BioLegend), anti-human CD3-BV605 (clone UCHT1, BioLegend), anti-human CD14-BV510 (clone M5E2, BioLegend), anti-human CD14-BV605 (clone M5E2, BioLegend), anti-human CD11c-BUV805 (clone B-ly6, BD), anti-human CD11c-BV605 (clone 3.9, Biolegend), anti-human CD19-Pacific blue (clone HIB19, BioLegend), anti-human CD19-PerCP/Cy5.5 (clone HIB19, BioLegend), anti-human CD19-APC/Cy7 (clone HIB19, BioLegend), anti-human CD20-BUV496 (clone 2H7, BD), anti-human CD20-BV605 (clone 2H7, BioLegend), anti-human CD21-BV421 (clone 1048, BD), anti-human CD21-APC (clone Bu32, BioLegend), anti-human CD24-BV605 (clone ML5, BioLegend), anti-human CD27-BV421 (clone O323, BioLegend), anti-human CD27-BV711 (clone M-T271, BioLegend), anti-human CD27-PE/Cy7 (clone LG.7F9, eBioscience), anti-human CD27-APC (clone M-T271, BioLegend), anti-human CD27-APC/Cy7 (clone O323, BioLegend), anti-human CD38-BUV563 (clone 2B7, BD), anti-human CD38-BV605 (clone HIT2, BioLegend), anti-human CD38-FITC (clone HB7, eBioscience), anti-human CD38-PerCP-Cy5.5 (clone HIT2, Biolegend), anti-human CD62L-R718 (clone DREG-56, BD), anti-human CD86-BV785 (clone IT2.2, BioLegend), anti-human CD95-BUV737 (clone DX2, BD), anti-human CD95-Alexa Fluor 700 (clone DX2, BioLegend), anti-human CXCR3-PE-CF594 (clone 1C6/CXCR3, BD), anti-human CXCR4-BV605 (clone 12G5, BD), anti-human CXCR5-PE/Cy5 (clone J252D4, BioLegend), anti-human CXCR5-Alexa Fluor 647 (clone J252D4, BioLegend), anti-human IgA-FITC (clone IS11-8E10, Miltenyi), goat anti-human IgA-PE (Southern Biotech), anti-human IgA-APC (clone IS11-8E10, Miltenyi), anti-human IgA-APC-Vio770 (clone IS11-8E10, Miltenyi), goat anti-human IgD-FITC (Southern Biotech), anti-human IgD-PerCP/Cy5.5 (clone IA6-2, BioLegend), anti-human IgD-Alexa Fluor 700 (clone IA6-2, BioLegend), anti-human IgG-APC (clone M1310G05, BioLegend), anti-human IgM-BV510 (clone MHM-88, Biolegend), goat anti-human IgM-FITC (Southern Biotech), anti-human Integrin β7-BV650 (clone FIB504, BD), anti-human Integrin β7-APC (clone FIB504, BioLegend), rabbit anti-human TG2 (Pacific Immunology), goat anti-rabbit IgG-Alexa488 (Thermo Fisher), anti-biotin-PE (clone 1D4-C5, Biolegend).

Techniques: Comparison, MANN-WHITNEY

(A-C) Representative flow cytometry plot (A) and summary data from seven untreated CeD patients showing staining of duodenal IgA+ plasma cells. The frequency of CD27- (B) and CD19+ (C) cells was evaluated among TG2-specific and non-TG2-specific (other) plasma cells. Bar heights indicate means, and difference between groups was evaluated by a paired t test. **p < 0.01, ***p < 0.001. ****p < 0.0001. (D-H) CITE-seq, V(D)J and transcriptome analysis of IgA+ plasma cells sorted from duodenal biopsies of four untreated CeD patients. Only cells with confirmed expression of a V(D)J sequence in connection with an IgA constant region were included in the analyses. (D) Cumulated CITE-seq data showing identification of TG2-binding and non-TG2-binding populations with and without surface CD27. (E) Examples of individual clonotypes spanning surface CD27+ and CD27- populations in a representative patient. (F) Number of expanded clonotypes consisting of only CD27+, only CD27- or both CD27+ and CD27- plasma cells as a function of the number of cells in each clone. (G) Examples of lineage trees showing TG2-specific clones comprising both CD27+ and CD27- cells. Colored circles represent IGHV sequences observed in individual cells, and numbers next to edges indicate mutations (nt). (H) Volcano plots showing differentially expressed genes between surface CD27+ and CD27- populations (upper panels) and verification of CD27 expression at the mRNA level (lower panels).

Journal: bioRxiv

Article Title: Generation of circulating autoreactive pre-plasma cells fueled by naïve B cells in celiac disease

doi: 10.1101/2023.10.20.563227

Figure Lengend Snippet: (A-C) Representative flow cytometry plot (A) and summary data from seven untreated CeD patients showing staining of duodenal IgA+ plasma cells. The frequency of CD27- (B) and CD19+ (C) cells was evaluated among TG2-specific and non-TG2-specific (other) plasma cells. Bar heights indicate means, and difference between groups was evaluated by a paired t test. **p < 0.01, ***p < 0.001. ****p < 0.0001. (D-H) CITE-seq, V(D)J and transcriptome analysis of IgA+ plasma cells sorted from duodenal biopsies of four untreated CeD patients. Only cells with confirmed expression of a V(D)J sequence in connection with an IgA constant region were included in the analyses. (D) Cumulated CITE-seq data showing identification of TG2-binding and non-TG2-binding populations with and without surface CD27. (E) Examples of individual clonotypes spanning surface CD27+ and CD27- populations in a representative patient. (F) Number of expanded clonotypes consisting of only CD27+, only CD27- or both CD27+ and CD27- plasma cells as a function of the number of cells in each clone. (G) Examples of lineage trees showing TG2-specific clones comprising both CD27+ and CD27- cells. Colored circles represent IGHV sequences observed in individual cells, and numbers next to edges indicate mutations (nt). (H) Volcano plots showing differentially expressed genes between surface CD27+ and CD27- populations (upper panels) and verification of CD27 expression at the mRNA level (lower panels).

Article Snippet: Anti-human CCR9-BUV395 (clone C9MAB-1, BD), anti-human CD3-BV510 (clone OKT3, BioLegend), anti-human CD3-BV605 (clone UCHT1, BioLegend), anti-human CD14-BV510 (clone M5E2, BioLegend), anti-human CD14-BV605 (clone M5E2, BioLegend), anti-human CD11c-BUV805 (clone B-ly6, BD), anti-human CD11c-BV605 (clone 3.9, Biolegend), anti-human CD19-Pacific blue (clone HIB19, BioLegend), anti-human CD19-PerCP/Cy5.5 (clone HIB19, BioLegend), anti-human CD19-APC/Cy7 (clone HIB19, BioLegend), anti-human CD20-BUV496 (clone 2H7, BD), anti-human CD20-BV605 (clone 2H7, BioLegend), anti-human CD21-BV421 (clone 1048, BD), anti-human CD21-APC (clone Bu32, BioLegend), anti-human CD24-BV605 (clone ML5, BioLegend), anti-human CD27-BV421 (clone O323, BioLegend), anti-human CD27-BV711 (clone M-T271, BioLegend), anti-human CD27-PE/Cy7 (clone LG.7F9, eBioscience), anti-human CD27-APC (clone M-T271, BioLegend), anti-human CD27-APC/Cy7 (clone O323, BioLegend), anti-human CD38-BUV563 (clone 2B7, BD), anti-human CD38-BV605 (clone HIT2, BioLegend), anti-human CD38-FITC (clone HB7, eBioscience), anti-human CD38-PerCP-Cy5.5 (clone HIT2, Biolegend), anti-human CD62L-R718 (clone DREG-56, BD), anti-human CD86-BV785 (clone IT2.2, BioLegend), anti-human CD95-BUV737 (clone DX2, BD), anti-human CD95-Alexa Fluor 700 (clone DX2, BioLegend), anti-human CXCR3-PE-CF594 (clone 1C6/CXCR3, BD), anti-human CXCR4-BV605 (clone 12G5, BD), anti-human CXCR5-PE/Cy5 (clone J252D4, BioLegend), anti-human CXCR5-Alexa Fluor 647 (clone J252D4, BioLegend), anti-human IgA-FITC (clone IS11-8E10, Miltenyi), goat anti-human IgA-PE (Southern Biotech), anti-human IgA-APC (clone IS11-8E10, Miltenyi), anti-human IgA-APC-Vio770 (clone IS11-8E10, Miltenyi), goat anti-human IgD-FITC (Southern Biotech), anti-human IgD-PerCP/Cy5.5 (clone IA6-2, BioLegend), anti-human IgD-Alexa Fluor 700 (clone IA6-2, BioLegend), anti-human IgG-APC (clone M1310G05, BioLegend), anti-human IgM-BV510 (clone MHM-88, Biolegend), goat anti-human IgM-FITC (Southern Biotech), anti-human Integrin β7-BV650 (clone FIB504, BD), anti-human Integrin β7-APC (clone FIB504, BioLegend), rabbit anti-human TG2 (Pacific Immunology), goat anti-rabbit IgG-Alexa488 (Thermo Fisher), anti-biotin-PE (clone 1D4-C5, Biolegend).

Techniques: Flow Cytometry, Staining, Expressing, Sequencing, Binding Assay, Clone Assay

(A) Representative flow cytometry histograms comparing expression of surface markers associated with a memory B cell or plasmablast phenotype on TG2-specific and non-TG2-specific (other) IgA+ B cells in peripheral blood of an untreated CeD patient. (B and C) Representative flow cytometry plot (B) and summary data from 12 patients (C) showing expression of CD38 and CD27 on IgA+ B cells in blood. Bar heights represent medians, and difference between groups was evaluated by a Wilcoxon signed rank test. ***p < 0.001. (D) ELISPOT detection of IgA- secreting cells among circulating IgA+ B cells with the indicated combinations of surface CD27 and CD38. Numbers indicate the number of sorted cells that were placed in culture in the individual wells. (E) UMAP plot based on scRNA-seq data obtained from TG2-specific and other IgA+ B cells sorted from PBMCs of two untreated CeD patients. Based on gene expression and amount of Ig transcripts, two main clusters were assigned as memory B cells (MBC) and plasmablasts (PB). (F) Expression of genes typically associated with B-cell differentiation in the two clusters.

Journal: bioRxiv

Article Title: Generation of circulating autoreactive pre-plasma cells fueled by naïve B cells in celiac disease

doi: 10.1101/2023.10.20.563227

Figure Lengend Snippet: (A) Representative flow cytometry histograms comparing expression of surface markers associated with a memory B cell or plasmablast phenotype on TG2-specific and non-TG2-specific (other) IgA+ B cells in peripheral blood of an untreated CeD patient. (B and C) Representative flow cytometry plot (B) and summary data from 12 patients (C) showing expression of CD38 and CD27 on IgA+ B cells in blood. Bar heights represent medians, and difference between groups was evaluated by a Wilcoxon signed rank test. ***p < 0.001. (D) ELISPOT detection of IgA- secreting cells among circulating IgA+ B cells with the indicated combinations of surface CD27 and CD38. Numbers indicate the number of sorted cells that were placed in culture in the individual wells. (E) UMAP plot based on scRNA-seq data obtained from TG2-specific and other IgA+ B cells sorted from PBMCs of two untreated CeD patients. Based on gene expression and amount of Ig transcripts, two main clusters were assigned as memory B cells (MBC) and plasmablasts (PB). (F) Expression of genes typically associated with B-cell differentiation in the two clusters.

Article Snippet: Anti-human CCR9-BUV395 (clone C9MAB-1, BD), anti-human CD3-BV510 (clone OKT3, BioLegend), anti-human CD3-BV605 (clone UCHT1, BioLegend), anti-human CD14-BV510 (clone M5E2, BioLegend), anti-human CD14-BV605 (clone M5E2, BioLegend), anti-human CD11c-BUV805 (clone B-ly6, BD), anti-human CD11c-BV605 (clone 3.9, Biolegend), anti-human CD19-Pacific blue (clone HIB19, BioLegend), anti-human CD19-PerCP/Cy5.5 (clone HIB19, BioLegend), anti-human CD19-APC/Cy7 (clone HIB19, BioLegend), anti-human CD20-BUV496 (clone 2H7, BD), anti-human CD20-BV605 (clone 2H7, BioLegend), anti-human CD21-BV421 (clone 1048, BD), anti-human CD21-APC (clone Bu32, BioLegend), anti-human CD24-BV605 (clone ML5, BioLegend), anti-human CD27-BV421 (clone O323, BioLegend), anti-human CD27-BV711 (clone M-T271, BioLegend), anti-human CD27-PE/Cy7 (clone LG.7F9, eBioscience), anti-human CD27-APC (clone M-T271, BioLegend), anti-human CD27-APC/Cy7 (clone O323, BioLegend), anti-human CD38-BUV563 (clone 2B7, BD), anti-human CD38-BV605 (clone HIT2, BioLegend), anti-human CD38-FITC (clone HB7, eBioscience), anti-human CD38-PerCP-Cy5.5 (clone HIT2, Biolegend), anti-human CD62L-R718 (clone DREG-56, BD), anti-human CD86-BV785 (clone IT2.2, BioLegend), anti-human CD95-BUV737 (clone DX2, BD), anti-human CD95-Alexa Fluor 700 (clone DX2, BioLegend), anti-human CXCR3-PE-CF594 (clone 1C6/CXCR3, BD), anti-human CXCR4-BV605 (clone 12G5, BD), anti-human CXCR5-PE/Cy5 (clone J252D4, BioLegend), anti-human CXCR5-Alexa Fluor 647 (clone J252D4, BioLegend), anti-human IgA-FITC (clone IS11-8E10, Miltenyi), goat anti-human IgA-PE (Southern Biotech), anti-human IgA-APC (clone IS11-8E10, Miltenyi), anti-human IgA-APC-Vio770 (clone IS11-8E10, Miltenyi), goat anti-human IgD-FITC (Southern Biotech), anti-human IgD-PerCP/Cy5.5 (clone IA6-2, BioLegend), anti-human IgD-Alexa Fluor 700 (clone IA6-2, BioLegend), anti-human IgG-APC (clone M1310G05, BioLegend), anti-human IgM-BV510 (clone MHM-88, Biolegend), goat anti-human IgM-FITC (Southern Biotech), anti-human Integrin β7-BV650 (clone FIB504, BD), anti-human Integrin β7-APC (clone FIB504, BioLegend), rabbit anti-human TG2 (Pacific Immunology), goat anti-rabbit IgG-Alexa488 (Thermo Fisher), anti-biotin-PE (clone 1D4-C5, Biolegend).

Techniques: Flow Cytometry, Expressing, Enzyme-linked Immunospot, Cell Differentiation

(A) Examples of lineage trees showing clonal relationships between circulating TG2-specific memory B cells (MBC) and plasmablasts (PB) with or without surface CD27. (B and C) Quantification of overlap (B) and examples of lineage trees (C) showing clonal relationships between TG2-specific gut plasma cells (PCs) and IgA+ B cells in blood of six untreated CeD patients. PCs were defined as either short-lived (CD19+CD45+) or intermediate-lived (CD19-CD45+) based on flow cytometry staining. (D) Frequency of heavy and light chain variable region mutations introduced by somatic hypermutation (SHM) among TG2-specific and non-TG2-specific (other) IgA+ blood B cells or gut plasma cells of six untreated CeD patients. (E and F) Comparison of mutation levels between CD27+ and CD27- IgA cells in blood (E) and gut biopsies (F). Centers indicate medians, and statistical significance was evaluated by a Mann-Whitney U test. *p < 0.05.

Journal: bioRxiv

Article Title: Generation of circulating autoreactive pre-plasma cells fueled by naïve B cells in celiac disease

doi: 10.1101/2023.10.20.563227

Figure Lengend Snippet: (A) Examples of lineage trees showing clonal relationships between circulating TG2-specific memory B cells (MBC) and plasmablasts (PB) with or without surface CD27. (B and C) Quantification of overlap (B) and examples of lineage trees (C) showing clonal relationships between TG2-specific gut plasma cells (PCs) and IgA+ B cells in blood of six untreated CeD patients. PCs were defined as either short-lived (CD19+CD45+) or intermediate-lived (CD19-CD45+) based on flow cytometry staining. (D) Frequency of heavy and light chain variable region mutations introduced by somatic hypermutation (SHM) among TG2-specific and non-TG2-specific (other) IgA+ blood B cells or gut plasma cells of six untreated CeD patients. (E and F) Comparison of mutation levels between CD27+ and CD27- IgA cells in blood (E) and gut biopsies (F). Centers indicate medians, and statistical significance was evaluated by a Mann-Whitney U test. *p < 0.05.

Article Snippet: Anti-human CCR9-BUV395 (clone C9MAB-1, BD), anti-human CD3-BV510 (clone OKT3, BioLegend), anti-human CD3-BV605 (clone UCHT1, BioLegend), anti-human CD14-BV510 (clone M5E2, BioLegend), anti-human CD14-BV605 (clone M5E2, BioLegend), anti-human CD11c-BUV805 (clone B-ly6, BD), anti-human CD11c-BV605 (clone 3.9, Biolegend), anti-human CD19-Pacific blue (clone HIB19, BioLegend), anti-human CD19-PerCP/Cy5.5 (clone HIB19, BioLegend), anti-human CD19-APC/Cy7 (clone HIB19, BioLegend), anti-human CD20-BUV496 (clone 2H7, BD), anti-human CD20-BV605 (clone 2H7, BioLegend), anti-human CD21-BV421 (clone 1048, BD), anti-human CD21-APC (clone Bu32, BioLegend), anti-human CD24-BV605 (clone ML5, BioLegend), anti-human CD27-BV421 (clone O323, BioLegend), anti-human CD27-BV711 (clone M-T271, BioLegend), anti-human CD27-PE/Cy7 (clone LG.7F9, eBioscience), anti-human CD27-APC (clone M-T271, BioLegend), anti-human CD27-APC/Cy7 (clone O323, BioLegend), anti-human CD38-BUV563 (clone 2B7, BD), anti-human CD38-BV605 (clone HIT2, BioLegend), anti-human CD38-FITC (clone HB7, eBioscience), anti-human CD38-PerCP-Cy5.5 (clone HIT2, Biolegend), anti-human CD62L-R718 (clone DREG-56, BD), anti-human CD86-BV785 (clone IT2.2, BioLegend), anti-human CD95-BUV737 (clone DX2, BD), anti-human CD95-Alexa Fluor 700 (clone DX2, BioLegend), anti-human CXCR3-PE-CF594 (clone 1C6/CXCR3, BD), anti-human CXCR4-BV605 (clone 12G5, BD), anti-human CXCR5-PE/Cy5 (clone J252D4, BioLegend), anti-human CXCR5-Alexa Fluor 647 (clone J252D4, BioLegend), anti-human IgA-FITC (clone IS11-8E10, Miltenyi), goat anti-human IgA-PE (Southern Biotech), anti-human IgA-APC (clone IS11-8E10, Miltenyi), anti-human IgA-APC-Vio770 (clone IS11-8E10, Miltenyi), goat anti-human IgD-FITC (Southern Biotech), anti-human IgD-PerCP/Cy5.5 (clone IA6-2, BioLegend), anti-human IgD-Alexa Fluor 700 (clone IA6-2, BioLegend), anti-human IgG-APC (clone M1310G05, BioLegend), anti-human IgM-BV510 (clone MHM-88, Biolegend), goat anti-human IgM-FITC (Southern Biotech), anti-human Integrin β7-BV650 (clone FIB504, BD), anti-human Integrin β7-APC (clone FIB504, BioLegend), rabbit anti-human TG2 (Pacific Immunology), goat anti-rabbit IgG-Alexa488 (Thermo Fisher), anti-biotin-PE (clone 1D4-C5, Biolegend).

Techniques: Flow Cytometry, Staining, Comparison, Mutagenesis, MANN-WHITNEY

(A-C) Representative flow cytometry plots showing staining of B cells from peripheral blood of a healthy donor before (d0) or after (d9) nine days of culture with transfected fibroblasts expressing human BAFF, CD40L and IL-21. The cultured B cells were analyzed for activation (A), class-switching (B) and similarity to TG2-specific IgA+ B cells in CeD (C). To induce IgA-switching, cultures of naïve B cells were supplemented with retinoic acid (RA). (D and E) Induction of a TG2-specific B-cell response in treated patients undergoing a 14-days oral gluten challenge. The frequency of IgA-switched TG2-specific B cells in peripheral blood was followed during and after the challenge by flow cytometry (D). In the two patients, who responded to the challenge, expression of surface CD27 and CD38 by the TG2-specifc cells was assessed at the peak of the response (E).

Journal: bioRxiv

Article Title: Generation of circulating autoreactive pre-plasma cells fueled by naïve B cells in celiac disease

doi: 10.1101/2023.10.20.563227

Figure Lengend Snippet: (A-C) Representative flow cytometry plots showing staining of B cells from peripheral blood of a healthy donor before (d0) or after (d9) nine days of culture with transfected fibroblasts expressing human BAFF, CD40L and IL-21. The cultured B cells were analyzed for activation (A), class-switching (B) and similarity to TG2-specific IgA+ B cells in CeD (C). To induce IgA-switching, cultures of naïve B cells were supplemented with retinoic acid (RA). (D and E) Induction of a TG2-specific B-cell response in treated patients undergoing a 14-days oral gluten challenge. The frequency of IgA-switched TG2-specific B cells in peripheral blood was followed during and after the challenge by flow cytometry (D). In the two patients, who responded to the challenge, expression of surface CD27 and CD38 by the TG2-specifc cells was assessed at the peak of the response (E).

Article Snippet: Anti-human CCR9-BUV395 (clone C9MAB-1, BD), anti-human CD3-BV510 (clone OKT3, BioLegend), anti-human CD3-BV605 (clone UCHT1, BioLegend), anti-human CD14-BV510 (clone M5E2, BioLegend), anti-human CD14-BV605 (clone M5E2, BioLegend), anti-human CD11c-BUV805 (clone B-ly6, BD), anti-human CD11c-BV605 (clone 3.9, Biolegend), anti-human CD19-Pacific blue (clone HIB19, BioLegend), anti-human CD19-PerCP/Cy5.5 (clone HIB19, BioLegend), anti-human CD19-APC/Cy7 (clone HIB19, BioLegend), anti-human CD20-BUV496 (clone 2H7, BD), anti-human CD20-BV605 (clone 2H7, BioLegend), anti-human CD21-BV421 (clone 1048, BD), anti-human CD21-APC (clone Bu32, BioLegend), anti-human CD24-BV605 (clone ML5, BioLegend), anti-human CD27-BV421 (clone O323, BioLegend), anti-human CD27-BV711 (clone M-T271, BioLegend), anti-human CD27-PE/Cy7 (clone LG.7F9, eBioscience), anti-human CD27-APC (clone M-T271, BioLegend), anti-human CD27-APC/Cy7 (clone O323, BioLegend), anti-human CD38-BUV563 (clone 2B7, BD), anti-human CD38-BV605 (clone HIT2, BioLegend), anti-human CD38-FITC (clone HB7, eBioscience), anti-human CD38-PerCP-Cy5.5 (clone HIT2, Biolegend), anti-human CD62L-R718 (clone DREG-56, BD), anti-human CD86-BV785 (clone IT2.2, BioLegend), anti-human CD95-BUV737 (clone DX2, BD), anti-human CD95-Alexa Fluor 700 (clone DX2, BioLegend), anti-human CXCR3-PE-CF594 (clone 1C6/CXCR3, BD), anti-human CXCR4-BV605 (clone 12G5, BD), anti-human CXCR5-PE/Cy5 (clone J252D4, BioLegend), anti-human CXCR5-Alexa Fluor 647 (clone J252D4, BioLegend), anti-human IgA-FITC (clone IS11-8E10, Miltenyi), goat anti-human IgA-PE (Southern Biotech), anti-human IgA-APC (clone IS11-8E10, Miltenyi), anti-human IgA-APC-Vio770 (clone IS11-8E10, Miltenyi), goat anti-human IgD-FITC (Southern Biotech), anti-human IgD-PerCP/Cy5.5 (clone IA6-2, BioLegend), anti-human IgD-Alexa Fluor 700 (clone IA6-2, BioLegend), anti-human IgG-APC (clone M1310G05, BioLegend), anti-human IgM-BV510 (clone MHM-88, Biolegend), goat anti-human IgM-FITC (Southern Biotech), anti-human Integrin β7-BV650 (clone FIB504, BD), anti-human Integrin β7-APC (clone FIB504, BioLegend), rabbit anti-human TG2 (Pacific Immunology), goat anti-rabbit IgG-Alexa488 (Thermo Fisher), anti-biotin-PE (clone 1D4-C5, Biolegend).

Techniques: Flow Cytometry, Staining, Transfection, Expressing, Cell Culture, Activation Assay

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.

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

(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.

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