Journal: Frontiers in Immunology

Article Title: Identification and Characterization of Post-activated B Cells in Systemic Autoimmune Diseases

doi: 10.3389/fimmu.2019.02136

Figure Lengend Snippet: Increased Syk(Y 352 ) phosphorylation reflecting BCR responsiveness of CD27 + memory B cells from HD, SLE, RA, and pSS upon co-stimulation with CD40L. PBMCs from HD (black), SLE (red), RA (blue), and pSS (green) patients were treated with CD40L and subsequently stimulated with anti-IgG/IgM F(ab) 2 . (A) Representative histograms of pSyk(Y 352 ) in CD27 − (top row) and CD27 + memory (bottom row) B cells in the presence (colored areas) or absence (gray areas) of prior CD40L co-stimulation. (B) pSyk(Y 352 ) in CD27 − (filled boxes) and CD27 + memory (clear boxes) B cells with [n(HD/SLE/RA/pSS) = 11/7/5/5] and without [n(HD/SLE/RA/pSS) = 30/18/16/15] CD40 co-stimulation. Box whisker plots represent median (line), mean (plus), and the range from minimum to maximum (ANOVA with DMCT, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001; paired t -test, # p ≤ 0.05, ## p ≤ 0.01, ### p ≤ 0.001).

Article Snippet: For chronic BCR stimulation experiments, cells were pre-incubated with anti-IgG/IgM (2 μg/ml), CpG (0.5 μg/ml) or RPMI for 24, 48, or 72 h (37°C, 5% CO 2 ) and subsequently stimulated with anti-IgG/IgM or RPMI as a control for 5 min. For co-stimulation experiments, cells were pre-incubated with recombinant human IL-4 (20 ng/ml) or IL-21 (20 ng/ml) or CD40L (500 ng/ml, human CD40L Multimer kit, Miltenyi Biotec) or combinations thereof for 48 h (37°C, 5% CO 2 ) and subsequently stimulated with anti-IgG/IgM or RPMI as a control for 5 min.

Techniques: Phospho-proteomics, Whisker Assay

Journal: Frontiers in Immunology

Article Title: Identification and Characterization of Post-activated B Cells in Systemic Autoimmune Diseases

doi: 10.3389/fimmu.2019.02136

Figure Lengend Snippet: Proliferation of AID B cells upon CD40 stimulation or combined stimulation of BCR and TLR9 is comparable to HD. B cells from HD (black), SLE (red), RA (blue), and pSS (green) patients were stimulated with anti-IgG/IgM, CD40L, or CpG and the combinations CD40L/CpG, CpG/anti-IgG/IgM, or CD40L/CpG/anti-IgG/IgM. (A) Representative CFSE histograms of HD, SLE, RA, and pSS B cells after 5 days of culture for the indicated stimulus (lines) compared to background control (gray). (B) Percentages of CFSE low B cells [n(HD/SLE/RA/pSS) = 17/19/9/10 (IL-2/IL-10 control); 11/10/6/5 (IgG/IgM); 10/7/4/5 (CD40); 16/18/7/9 (TLR9); 10/7/4/5 (TLR9/CD40); 16/19/7/9 (IgG/IgM/TLR9); 10/7/4/5 (IgG/IgM/TLR9/CD40)]. (C) Resulting frequency of CD27 + CD38 + B cells upon activation in culture [n(HD/SLE/RA/pSS) = 18/18/11/10 (IL-2/IL-10 control); 12/8/6/5 (IgG/IgM); 12/8/7/7 (CD40); 17/18/9/10 (TLR9); 12/8/5/6 (TLR9/CD40); 17/18/8/10 (IgG/IgM/TLR9); 12/8/5/6 (IgG/IgM/TLR9/CD40)]. Bars shown represent mean ± SD (ANOVA with DMCT, * p ≤ 0.05, ** p ≤ 0.01; # p ≤ 0.05, ## p ≤ 0.01, ### p ≤ 0.001).

Article Snippet: For chronic BCR stimulation experiments, cells were pre-incubated with anti-IgG/IgM (2 μg/ml), CpG (0.5 μg/ml) or RPMI for 24, 48, or 72 h (37°C, 5% CO 2 ) and subsequently stimulated with anti-IgG/IgM or RPMI as a control for 5 min. For co-stimulation experiments, cells were pre-incubated with recombinant human IL-4 (20 ng/ml) or IL-21 (20 ng/ml) or CD40L (500 ng/ml, human CD40L Multimer kit, Miltenyi Biotec) or combinations thereof for 48 h (37°C, 5% CO 2 ) and subsequently stimulated with anti-IgG/IgM or RPMI as a control for 5 min.

Techniques: Control, Activation Assay

Journal: Frontiers in Immunology

Article Title: Identification and Characterization of Post-activated B Cells in Systemic Autoimmune Diseases

doi: 10.3389/fimmu.2019.02136

Figure Lengend Snippet: Common reduction of receptor-type PTP expression upon co-stimulation with CD40L/IL-4. Differential expression of selected genes related to receptor-type and non receptor-type PTPs in SLE vs. HD CD20 + B cells (6 SLE, 7 HD), MS vs. HD CD19 + B cells (10 MS, 10 HD), SLE vs. HD CD4 + (53 SLE, 41 HD), and CD8 + T cells (22 SLE, 31 HD) (lines 1–4 are from publicly available data). Differential gene expression from un-stimulated SLE vs. HD and CD40L/IL-4 stimulated for SLE vs. un-stimulated SLE or HD CD19 + B cells, respectively [n(HD/SLE) = 1/2], (lines 4–7).

Article Snippet: For chronic BCR stimulation experiments, cells were pre-incubated with anti-IgG/IgM (2 μg/ml), CpG (0.5 μg/ml) or RPMI for 24, 48, or 72 h (37°C, 5% CO 2 ) and subsequently stimulated with anti-IgG/IgM or RPMI as a control for 5 min. For co-stimulation experiments, cells were pre-incubated with recombinant human IL-4 (20 ng/ml) or IL-21 (20 ng/ml) or CD40L (500 ng/ml, human CD40L Multimer kit, Miltenyi Biotec) or combinations thereof for 48 h (37°C, 5% CO 2 ) and subsequently stimulated with anti-IgG/IgM or RPMI as a control for 5 min.

Techniques: Expressing, Quantitative Proteomics, Gene Expression

Journal: Blood

Article Title: Differential induction of plasma cells by isoforms of human TACI

doi: 10.1182/blood-2014-05-575845

Figure Lengend Snippet: Expression of TACI isoforms in primary human B cells. (A) Qualitative analysis of TACI cDNA expression in freshly isolated B cells from 2 healthy donors (HD1 and HD2). TACI sequences were amplified from mRNA by RT-PCR and examined by gel electrophoresis. Two bands correspond to each of the 2 isoforms. (B) TACI immunoblot from the same healthy donors expressing the 2 TACI protein bands. β-actin was used as loading control. (C) Quantitative analysis of CD27+, naïve IgD+, and total CD19+ B cells of TACI isoforms from freshly isolated peripheral blood mononuclear cells further cultured with or without ODN for 3 days. (D) Quantitative analysis of mRNA levels of TACI isoforms in freshly isolated B cells from spleens. Follicular (Fo) and marginal zone (MZ) B cells were cultured for 3 days with the addition of ODN or interleukin-21 (IL21) and CD40L. For panels C and D, data represent 3 different donors; error bars represent standard error of the mean. *P < .05; 2-tailed unpaired Student t test.

Article Snippet: For B-cell activation, cells were cultured with or without oligodeoxynucleotide (ODN) 2006 (300 ng/mL), or interleukin-21 (100 ng/mL; PeproTech) plus CD40L (500 ng/mL; PeproTech) for 3 days, as described previously.

Techniques: Expressing, Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Western Blot, Control, Cell Culture

Journal: Human Vaccines & Immunotherapeutics

Article Title: Genomic, proteomic, and immunologic associations with a durable complete remission of measurable metastatic melanoma induced by a patient-specific dendritic cell vaccine

doi: 10.1080/21645515.2019.1680239

Figure Lengend Snippet: Serum marker levels in relation to normal control values at week-0 baseline week-0 (1 week before first vaccine injection) and week-4 (1 week after third weekly vaccine injection) and percent change.

Article Snippet: Control MLR groups included PBMC alone, non-antigen-loaded DC, PBMCs treated with phorbol-12-myristate-13-acetate (PMA, 50 ng/ml, Sigma-Aldrich, St. Louis, MO) and ionomycin (1 μg/ml, Sigma-Aldrich, St. Louis, MO), PBMCs co-cultured with antigen-loaded DC in the presence of costimulatory molecule CD40L (500 ng/ml, Thermo Fisher Scientific, Waltham, MA) and anti-human CD28/CD49d antibodies (500 ng/ml, BD Biosciences, San Jose, CA), and PBMCs co-cultured with antigen-loaded DC in the presence of anti-IL-12 antibody (LEAF, 100 μg/ml, BioLegend, San Diego, CA).

Techniques: Marker, Control, Injection, Produced, Activation Assay

Journal: Human Vaccines & Immunotherapeutics

Article Title: Genomic, proteomic, and immunologic associations with a durable complete remission of measurable metastatic melanoma induced by a patient-specific dendritic cell vaccine

doi: 10.1080/21645515.2019.1680239

Figure Lengend Snippet: Rankings of serum markers by baseline levels relative to control values, and greatest percent changes after three vaccine injections.

Article Snippet: Control MLR groups included PBMC alone, non-antigen-loaded DC, PBMCs treated with phorbol-12-myristate-13-acetate (PMA, 50 ng/ml, Sigma-Aldrich, St. Louis, MO) and ionomycin (1 μg/ml, Sigma-Aldrich, St. Louis, MO), PBMCs co-cultured with antigen-loaded DC in the presence of costimulatory molecule CD40L (500 ng/ml, Thermo Fisher Scientific, Waltham, MA) and anti-human CD28/CD49d antibodies (500 ng/ml, BD Biosciences, San Jose, CA), and PBMCs co-cultured with antigen-loaded DC in the presence of anti-IL-12 antibody (LEAF, 100 μg/ml, BioLegend, San Diego, CA).

Techniques: Control, Marker

Journal: Journal of Nanobiotechnology

Article Title: G2-S16 dendrimer microbicide does not interfere with the vaginal immune system

doi: 10.1186/s12951-019-0496-9

Figure Lengend Snippet: Effect of G2-S16 on B cell population and B cell activation. Not activated and PBMCs activated with IL-4 and CD40L were treated with G2-S16 (10 µM) for 48 h and protein expression was determined by flow cytometry and the percentage of positive cells was analyzed for each marker. a The B cell population was identified as CD3 − CD19 + cells. b We studied the standard activation markers for the B cell population: CD25, CD71, CD86 and HLA-DR

Article Snippet: For B cell studies, PBMCs were stimulated with 50 ng/mL interleukin 4 (rhIL-4; Immunotools) and 500 ng/mL CD40 ligand (CD40L, Invitrogen).

Techniques: Activation Assay, Expressing, Flow Cytometry, Marker

Journal: Cancer Immunology, Immunotherapy

Article Title: Support of BCP-ALL-cells by autologous bone marrow Th-cells involves induction of AID expression but not widespread AID off-target mutagenesis

doi: 10.1007/s00262-020-02835-x

Figure Lengend Snippet: IL-13, TGFβ and CD40L mediate Th-cell-induced AICDA expression in BCP-ALL-cells. a Primary BCP-ALL-cells were stimulated with CIT ± IFNγ for 3d. AICDA expression was determined using qRT-PCR after preamplification of target genes. Shown are mean ± SD of dCT values normalized to the geometric mean of HMBS , TBP , GUSB and YWAHZ . Different symbols represent different patients. p values were calculated using a mixed-effect analysis with Sidak correction for multiple comparison. p > 0.05 not significant (n.s.), p < 0.01** b Xenograft ALL671 BMMCs were stimulated using CIT ± IFNγ for 5d. Total AID protein expression was assessed using western blotting. β-actin was used as loading control. Western blot of primary cells was performed once. c RS4;11, TOM1 and SD1 cells were stimulated for 3d using different combinations of CD40L, IL-13 and TGFβ. AICDA expression was determined using qRT-PCR. Shown are mean ± SD of ddCT values normalized to the geometric mean of HMBS and GUSB and to unstimulated cells. Dots represent different replicates. d p values in the table were calculated from dCT values using three-way ANOVA with Geisser-Greenhouse correction for sphericity. e SD1 cells were stimulated using CD40L and IL-13 for 5d. AID protein expression was analyzed using western blotting. α-tubulin was used as loading control. Shown is a representative result of five replicates

Article Snippet: Stimulation with HA-tagged recombinant human CD40 Ligand (CD40L) (500 ng/ml) was complemented with anti-HA antibody (200 ng/ml, both R&D Systems) enabling multimer formation.

Techniques: Expressing, Quantitative RT-PCR, Comparison, Western Blot

Journal: Cancer Immunology, Immunotherapy

Article Title: Support of BCP-ALL-cells by autologous bone marrow Th-cells involves induction of AID expression but not widespread AID off-target mutagenesis

doi: 10.1007/s00262-020-02835-x

Figure Lengend Snippet: Th-cell-induced AICDA is transcriptionally regulated via canonical NF-κB, Stat6 and Smad2/3 signaling in BCP-ALL-cells. a , b RS4;11 and SD1 cell lines were stimulated for 2 h with CD40L, IL-13 and TGFβ. a Localization of transcription factors in cytoplasmic and nuclear fraction was analyzed using western blotting. α-Tubulin and Lamin A/C were used as a loading and fractionation controls. RS4;11 cells do not express Lamin A/C. b Phosphorylation of Stat6 was analyzed using western blotting. β-actin was used as a loading control. c Transcription factors were knocked-down in RS4;11 cells by electroporation of siRNA for 48 h and stimulated with CD40L, IL-13 or TGFβ for 24 h. AICDA expression was analyzed using qRT-PCR. Shown are ddCT normalized to HMBS and siCtrl/unstimulated condition. p values were calculated using a mixed-effect analysis by comparing siCtrl and with targeting siRNA with same stimulation. Sidak correction for multiple comparison was used. p > 0.05 not significant (n.s.), p < 0.05*, p < 0.01**, n.a. not analyzed

Article Snippet: Stimulation with HA-tagged recombinant human CD40 Ligand (CD40L) (500 ng/ml) was complemented with anti-HA antibody (200 ng/ml, both R&D Systems) enabling multimer formation.

Techniques: Western Blot, Fractionation, Electroporation, Expressing, Quantitative RT-PCR, Comparison

Journal: Journal of Translational Medicine

Article Title: TSLP promoting B cell proliferation and polarizing follicular helper T cell as a therapeutic target in IgG4-related disease

doi: 10.1186/s12967-022-03606-1

Figure Lengend Snippet: The effect of TSLP on B cells in IgG4-RD. CD19+ B cells from IgG4-RD patients were stimulated with IL-4, anti-IgM, CD40L, with or without TSLP. Proliferation quantified by Ki-67 ( A , B ), apoptosis measured by 7-AAD and Annexin-V ( C , D ), and B cell subsets ( E , F ) were assessed on 5 days. Supernatant IgG and IgG4 levels on 7 days were quantified by ELISA ( G ). *p < 0.05

Article Snippet: B cells were activated in the presence of 50 ng/ml TSLP (peprotech) or PBS, 10 µg/ml anti-human IgM (Invitrogen), 500 ng/ml recombinant human CD40L (Abcam), 100 ng/ml IL-4 (peprotech).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of Translational Medicine

Article Title: TSLP promoting B cell proliferation and polarizing follicular helper T cell as a therapeutic target in IgG4-related disease

doi: 10.1186/s12967-022-03606-1

Figure Lengend Snippet: TSLP-activated B cells polarized Naïve T cells into Tfh cells though OX40L. CD19+ B cells from HC or IgG4-RD patients were stimulated with or without TSLP for 3 days and then were co-cultured with CD4+ Naïve T cells from HC with anti-CD3 and anti-CD28 for 5 days. Representative FACS ( A ) and summary graphs ( B ) of Tfh gated as CXCR5+PD-1+, Treg gated as CD25+foxp-3+, and IL-4+ in CD4+ T cells. C TSLP-B cells were directly or indirectly (transwell) co-cultured with CD4+ Naïve T cells, with anti-CD3 and anti-CD28 for 5 days and Tfh were measured by FACS. B cells were stimulated with anti-IgM, CD40L, with or without TSLP for 3 days, and representative FACS and summary graphs ( D ) showed the changes of co-stimulatory molecules on B cells. E B cells or TSLP-B cells were co-cultured with CD4+ Naïve T cells with anti-CD3, anti-CD28, anti-OX40L antibody or isotype control for 5 days and Tfh were measured by FACS. TSLP-B, TSLP activated B cells

Article Snippet: B cells were activated in the presence of 50 ng/ml TSLP (peprotech) or PBS, 10 µg/ml anti-human IgM (Invitrogen), 500 ng/ml recombinant human CD40L (Abcam), 100 ng/ml IL-4 (peprotech).

Techniques: Cell Culture, Control

Journal: Journal of Translational Medicine

Article Title: TSLP promoting B cell proliferation and polarizing follicular helper T cell as a therapeutic target in IgG4-related disease

doi: 10.1186/s12967-022-03606-1

Figure Lengend Snippet: TSLP activated JAK-STAT signaling pathway of B cells in IgG4-RD. CD19+ B cells from HC and IgG4-RD patients were stimulated with or without TSLP for 3 days and were detected by RNA-sequencing. A Venn diagram showed overlap of differential expression genes (DEGs) compared between TSLP-B and B cells from IgG4-RD patients (PA) and HC. B Volcano plot of DEGs of TSLP-B cells (n = 4) and B cells (n = 4) from IgG4-RD patients showed downregulated genes (left, n = 76), and upregulated genes (right, n = 81). C Heatmap of DEGs (n = 157) between TSLP-B and B cells from IgG4-RD patients. D KEGG enrichment of DEGs in metabolism (blue) and environmental information processing (red) of TSLP-B and B cells from IgG4-RD patients. E CD19+ B cells from HC were activated with anti-IgM, CD40L for 72 h, then were stimulated with or without TSLP, with or without anti-TSLP antibody for 60 min. Activated JAK-STAT family molecules were detected by western blotting with β-actin serving as an internal control. F , G CD19+ B cells from HC or IgG4-RD patients were activated with anti-IgM, CD40L for 72 h, and representative western blot ( F ) and summary graphs ( G ) of phosphorylated forms of JAK-STAT were shown

Article Snippet: B cells were activated in the presence of 50 ng/ml TSLP (peprotech) or PBS, 10 µg/ml anti-human IgM (Invitrogen), 500 ng/ml recombinant human CD40L (Abcam), 100 ng/ml IL-4 (peprotech).

Techniques: RNA Sequencing Assay, Expressing, Western Blot, Control