Journal: Science Advances

Article Title: STAT3 haploinsufficiency is associated with autosomal dominant hyper-IgE syndrome

doi: 10.1126/sciadv.adw2464

Figure Lengend Snippet: ( A ) STAT3 expression was evaluated by Western blot in Stat3 +/+ and Stat3 +/− cells derived from the spleen, thymus, and bone marrow (BM). Tubulin was used as loading control. Each lane represents one mouse. ( B ) A quantification of the STAT3/loading control ratio after normalization is shown for independently performed experiments as in (A). The mean ± SD is plotted. ( C ) IgE serum levels in Stat3 +/+ and Stat3 +/− mice were measured by ELISA. ( D ) Flow cytometry to identify IgG1 + and IgE + cells from IgM − IgD − cells 4 days after culturing Stat3 +/+ and Stat3 +/− B220 + splenocytes with IL-4 (12.5 ng/ml) and anti-CD40 antibody (62.5 ng/ml) in the absence or presence of IL-21 (25 ng/ml). Numbers in dot plots represent cell frequencies. ( E ) Mean (±SD) frequencies of the IgG1 + and IgE + cells as gated in (D). ( F ) Levels of IgG1 and IgE in the supernatant of cell cultures as done in (D) were measured by ELISA. B220 + splenocytes from Stat3 +/+ and Stat3 +/− mice were stimulated for different time points with IL-21 (50 ng/ml) ( G ), IL-10 (50 ng/ml) ( H ), and IL-6 (50 ng/ml) ( I ), and the phosphorylation of STAT3 (p-STAT3) was evaluated by flow cytometry. Mean (±SD) of mean fluorescent intensity (MFI) is plotted. Data are representative of at least three independent experiments. P values by unpaired t test (C) or two-way ANOVA followed by Bonferroni’s or Tukey’s multiple comparisons test (B and E to I).

Article Snippet: Cells were cultured in complete B cell medium [RPMI with 10% FBS, 10 mM Hepes, GlutaMAX, penicillin-streptomycin (Gibco), and 50 μM β-mercaptoethanol (Sigma-Aldrich), supplemented with IL-4 (12.5 ng/ml; PeproTech) and anti-CD40 antibody (62.5 ng/ml; Miltenyi Biotec), in the absence or presence of IL-21 (25 ng/ml; PeproTech)].

Techniques: Expressing, Western Blot, Derivative Assay, Control, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Phospho-proteomics

Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: Pembrolizumab does not substantially affect B cell activation and antibody production ex vivo (A-C) Human naïve B cells were isolated from PBMCs and cultured under indicated conditions for 7 days; Pembrolizumab or isotype control IgG4 was added on Day 2, n = 8. (A) Expression of CD38 and CD27 on B cells. Right, percentages of CD27 + CD38 - , CD27 + CD38 + B cells. (B) Expression of CD138 on CD27 + CD38 + B cells. Right, percentage of CD138 + CD27 + CD38 + B cells. (C) Different immunoglobulin isotypes were measured in the culture supernatant of (A-B) by multiplex assay. (D-F) B cells from HuPD-1 mice were isolated and cultured with LPS, IL4, BAFF, or ODN2006, anti-IgM, IL-21, IL-4 or R848, anti-IgM, anti-CD40, IL-21, IFN-γ for 3 days, pembrolizumab or isotype control was added on Day 1. (D) Expression of IgG2c on activated B cells. Right, percentage of IgG2c + B cells, n = 5. (E) Expression of IgG1 on activated B cells. Right, percentage of IgG1 + B cells, n = 5. (F) Different immunoglobulin isotypes in the supernatant of were measured by multiplex assay, n = 5. Data in graphs represent mean ± SEM, Significance was tested by Two-way ANOVA.

Article Snippet: Condition 1: 2.5 μg/mL anti-human Ig (M + G + A) (Jackson Immunoresearch, Cat# 109-006-064), 2.5 μg/mL CpG ODN (Invivogen, Cat# tlrl-2006-1), 10 μg/mL anti-human CD40 (BioXcell, Cat# BE0189), 20 ng/mL rhIL-21 (Peprotech, Cat# 200-21-50UG), 10 ng/mL rhIL-4 (Biolegend, Cat# 574004), 10 ng/mL rhIL-2 (Peprotech, Cat#200-02-250UG).

Techniques: Activation Assay, Ex Vivo, Isolation, Cell Culture, Control, Expressing, Multiplex Assay

Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: PD-1 inhibition does not have substantial impact on B cells ex vivo (A) Human naïve B cells were isolated from healthy donor PBMCs and stimulated with different conditions; Pembrolizumab (Keytruda) or isotype control IgG4 was added into the culture media on day 2, mean fluorescence intensity (MFI) of CD86 on B cells was measured, n = 8. (B-F) B cells were isolated from HuPD-1 mice, labeled with CTV, and cultured under 3 different conditions: LPS, rmIL-4, BAFF or Anti-IgM, CpG ODN, rmIL-21, rmIL-4, 100 rhIL-2 or R848, anti-CD40, anti-IgM, rmIL-21, rmIFN-γ for 3 days. Pembrolizumab or isotype control IgG4 was added on day 1, n = 5. (B) Summary of human PD-1 MFI on B cells from different groups. (C) Expression of CD138 on activated B cells. Right, percentage of CD138 + B cells. (D) Summaries of activation marker CD69 and CD86 MFIs on B cells from different groups. (E) Summaries of CD71 and CD98 MFIs on B cells from different groups. (F) Representative flow cytometry plot of CTV dilution on B cells. Right, a summary of the proliferation index of B cells from different groups. Data in graphs represent mean ± SEM, Significance was tested by Two-way ANOVA.

Article Snippet: Condition 1: 2.5 μg/mL anti-human Ig (M + G + A) (Jackson Immunoresearch, Cat# 109-006-064), 2.5 μg/mL CpG ODN (Invivogen, Cat# tlrl-2006-1), 10 μg/mL anti-human CD40 (BioXcell, Cat# BE0189), 20 ng/mL rhIL-21 (Peprotech, Cat# 200-21-50UG), 10 ng/mL rhIL-4 (Biolegend, Cat# 574004), 10 ng/mL rhIL-2 (Peprotech, Cat#200-02-250UG).

Techniques: Inhibition, Ex Vivo, Isolation, Control, Fluorescence, Labeling, Cell Culture, Expressing, Activation Assay, Marker, Flow Cytometry

Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: Inflammatory signatures enriched in irAE patients reduce antibody production (A-F) Beads based multiplex assays were used to measure plasma concentration of IL-6, and IL-12p70 (A), TNF-α, IFN-γ and IL-1β (B), HC (n = 19), irAE (n = 34), RAC (n = 45), ICI (n = 9). IP-10 (CXCL10), CXCL11, and CXCL9 (C, HC, n = 17; irAE, n = 33; RAC, n = 46; ICI, n = 17), CCL20 (D), CX3CL1 (E), and CCL2 (F). (G-J) Human naïve B cells were isolated and cultured with 0.5 μg/mL anti-human CD40, 2.5 μg/mL anti-human Ig (M+G+A), and 20 ng/mL rhIL-21 with 100 ng/mL IFN-α, 100 ng/mL IL-6, 100 ng/mL IL-12, control, or the combination of IFN-α, IL-6, and IL-12 for 7 days. Cells and culture supernatants were analyzed. (G) Representative flow plot of CD38 and CD138 expression on CD27 hi CD38 hi ASCs. Right, a summary of the percentage of CD138 + ASCs, n = 6. (H) Expression of CD11c and CD27 on CD27 - IgD - ASCs. Right, percentage of CD11c + IgD - CD27 - B cells, n = 6. (I) Expression of active-caspase-3 in B cells. Right, percentage of active-caspase-3 + B cells from different groups, n = 3. (J) Different immunoglobulin isotype levels in the culture supernatants from G-H were measured by the multiplex assay, n = 6. Data in graphs represent mean ± SEM, Significance was tested by One-way ANOVA (A-I), and paired Student’s t-test (J).

Article Snippet: Condition 1: 2.5 μg/mL anti-human Ig (M + G + A) (Jackson Immunoresearch, Cat# 109-006-064), 2.5 μg/mL CpG ODN (Invivogen, Cat# tlrl-2006-1), 10 μg/mL anti-human CD40 (BioXcell, Cat# BE0189), 20 ng/mL rhIL-21 (Peprotech, Cat# 200-21-50UG), 10 ng/mL rhIL-4 (Biolegend, Cat# 574004), 10 ng/mL rhIL-2 (Peprotech, Cat#200-02-250UG).

Techniques: Multiplex Assay, Clinical Proteomics, Concentration Assay, Isolation, Cell Culture, Control, Expressing

Journal: Scientific reports

Article Title: Distinct gene expression patterns for CD14++ and CD16++ monocytes in preeclampsia.

doi: 10.1038/s41598-022-19847-5

Figure Lengend Snippet: Figure 2. Immunophenotyping of the patient’s monocytes. Representative histograms of gated monocytes stained with antibodies to CD40, CD80, CD86, CD163, CD206 or CX3CR1 (magenta-filled histogram) and control samples (empty contour) for three groups of patients (a). Percents of positively stained cells are indicated above the gate bars. Levels of CD40+, CD80+, CD86+, CD163+, CD206+ and CX3CR1+ monocytes in three groups: the data are listed as mean ± SD (b): eoPE early-onset PE (n = 12), loPE late-onset PE (n = 10), control (n = 11).

Article Snippet: For surface immunophenotype marker staining, the cells we resuspended in autoMACS® Rinsing Solution (1 × 105 cells in 100 μL) with 1% BSA and stained with anti-CD16 (A07766, Beckman Coulter), anti-CD14 (130-110-518, Miltenyi Biotec), anti-CD45 (A07785, Beckman Coulter), anti-CD206 (130-095-131, Miltenyi Biotec), anti-CD86 (130-116-160, Miltenyi Biotec), anti-CD163 (130-097-630, Miltenyi Biotec), anti-HLA-DR (130-111-790, Miltenyi Biotec), CD40 (130-110-947, Miltenyi Biotec), CD80 (130-117- 683, Miltenyi Biotec) and anti-CX3CR1 (12-6099-42, eBioscience).

Techniques: Staining, Control

Journal: Cancer cell

Article Title: Dendritic cell paucity leads to dysfunctional immune surveillance in pancreatic cancer

doi: 10.1016/j.ccell.2020.02.008

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-human CD40 (5C3) , Fluidigm , #3165005B.

Techniques: Recombinant, Cell Stimulation, Enzyme-linked Immunosorbent Assay, CyQUANT Assay, LDH Cytotoxicity Assay, Expressing, Staining, Derivative Assay, Software

Journal: PLoS Medicine

Article Title: Inhibition of Endothelial Activation: A New Way to Treat Cerebral Malaria?

doi: 10.1371/journal.pmed.0020245

Figure Lengend Snippet: HBEC-5i were left unstimulated or were activated with LMP-420 alone, TNF alone, TNF with LMP-420, LT alone, or LT with LMP-420. After an overnight incubation, cells were detached and analyzed by flow cytometry after immunolabeling for ICAM-1, VCAM-1, and CD40. Results are expressed as dotplots of mean fluorescence intensity of surface expression of the antigens measured in six experiments (Dunn's test, ** corresponds to p < 0.01).

Article Snippet: HBEC-5i were then harvested and labeled by indirect labeling using mouse anti-human CD54 (ICAM-1 [84H10]) and CD106 (VCAM-1 [1G1]) antibodies (Beckman-Coulter Immunotech, Marseille, France), CD40 monoclonal antibody (mAb) (B-B20, Diaclone, Besançon, France), and CD36 mAb (FA6–152, gift from L. Edelman, Institut Pasteur Paris) as the first step.

Techniques: Incubation, Flow Cytometry, Immunolabeling, Fluorescence, Expressing

Journal: bioRxiv

Article Title: Single-cell RNA Profiling Identifies Diverse Cellular Responses to EWSR1-FLI1 Down-regulation in Ewing Sarcoma

doi: 10.1101/750539

Figure Lengend Snippet: A , Discriminative gene-features that define subclusters B , ( VISNE PLOTS ) show the low percentage of isolated cells that were considered to express all three proteins at high levels in EWS xenograft tissues. C, (VISNE PLOTS) Visualization plots of exponentially proliferating EW8 cells highlight coexpression patterns of newly identified stem-like cell surface markers (CELSR2, CD40) with SOX2, MYC, FGFR1, and CCND1. D , Heterogeneous expression of CELSR2 in tissue sections of EWS xenografts. Percentgae of cells scoring positive was assessed from scoring 3-high powered fields (40X). E , 7-day self-renewal assayTumor-sphere formation was quantitated and measured from a total of 2400 cells expressing high or low CD40 (Total=4800 cells); (p-value=0.00, unpaired t test). F , Quantitation of primary and secondary spheroid colonies formed by CD40-positive and CD-40 negatice EW8 cells. (mean ± SD) G , Patient overall survival relative to expression of CD40 and SOX2 ‘stem-like’ markers in Cohort COG GSE63155.

Article Snippet: The primary antibodies used were human Cyclin B1-153Eu (Fluidigm; 3153009A), Cyclin D-141Pr (Cell Signaling;2978BF), CD271-149Sm (Fluidigm;3149017B), CD40-142Nd (Fluidigm; 3142010B), CD79a-158Gd (Cell Signaling;13333BF), CD49b-161Dy (Fluidigm; 3161012B), CD49e-160Gd (Fluidigm; 3160015B), CD63-150Nd (Fluidigm; 3150021B), CDKN2A-155Gd (Abcam;Ab186932), CELSR2-165Ho (Abcam; Ab189045), FGFR1-159Tb (Cell Signaling;9740BF), MKI67-147Sm (Cell Signaling; 9449BF), MYC-176Yb (Fluidigm;3176012B), TGFBR2-170Er (Abcam;Ab 78419),TGFB1-163Dy (Fluidigm;3163010B), SOX2-169Tm (Cell Signaling;3579BF).

Techniques: Isolation, Expressing, Quantitation Assay