Journal: PLoS ONE

Article Title: Uptake and Presentation of Myelin Basic Protein by Normal Human B Cells

doi: 10.1371/journal.pone.0113388

Figure Lengend Snippet: PBMCs from healthy donors were incubated with or without 30 µg/ml MBP in medium containing normal autologous serum (30% v/v), or in pure medium. The resulting deposition of C3 and C1q on B cells was measured by flow cytometry after 5 min incubation (N = 3). Representative histogram plots show A) C3-deposition, and B) C1q-deposition on B cells. C) The binding of MBP was assessed using biotinylated MBP as probe and subsequent staining with streptavidin-PE. Blockade of CR1 or CR2 was achieved by pre-incubation of PBMCs with mAb3D9 and polyclonal sheep anti-human CR2 respectively. Monoclonal anti-glycophorin (GP)-A was used as negative control. D) Mean fluorescence intensity (MFI) values of 5–6 experiments are shown; background values (of samples with no MBP added) have been subtracted. Bars and error bars represent means and SEM. **p<0.01, ***p<0.001.

Article Snippet: Murine anti-human CR1 IgG1 antibody (mAb3D9) was kindly donated by Dr John O'Shea (Frederick Cancer Research and Development Center, Frederick, MD, USA), and polyclonal sheep anti-human CR2 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Incubation, Flow Cytometry, Binding Assay, Staining, Negative Control, Fluorescence

Journal: PLoS ONE

Article Title: Uptake and Presentation of Myelin Basic Protein by Normal Human B Cells

doi: 10.1371/journal.pone.0113388

Figure Lengend Snippet: PBMCs from healthy HLA-DR15+ donors were incubated for 18 h with MBP in media containing normal serum. Cells were stained with FITC anti-CD19 and biotinylated MK16, followed by streptavidin-PE. (A) The binding of MK16 at different serum concentrations is shown as mean fluorescence (MFI) values normalised to that of 10% serum, (N = 4). B) Before addition of serum (30% v/v), different concentrations of the complement inhibitory compound sodium polyanethole sulphonate (SPS) were added. MFI values are shown, normalised to samples without SPS, (N = 6). (C) The PBMCs were pre-incubated with the anti-CR1 mAb3D9 or polyclonal sheep anti-human CR2, or both, before addition of serum (30% v/v) and MBP. Anti-glycophorin (GP)-A was used as negative control. Data are shown as means±SEM, (N = 4–6). **p<0.01.

Article Snippet: Murine anti-human CR1 IgG1 antibody (mAb3D9) was kindly donated by Dr John O'Shea (Frederick Cancer Research and Development Center, Frederick, MD, USA), and polyclonal sheep anti-human CR2 was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Incubation, Staining, Binding Assay, Fluorescence, Negative Control

Journal: Clinical and Translational Medicine

Article Title: Single‐cell transcriptome sequencing of B‐cell heterogeneity and tertiary lymphoid structure predicts breast cancer prognosis and neoadjuvant therapy efficacy

doi: 10.1002/ctm2.1346

Figure Lengend Snippet: Correlation analysis between TIL‐B and mature tertiary lymphoid structures (TLS). (A) Among the 200 patients included in the BRCA dataset from the TCGA research consortium, a subset of 18 patients who exhibited TLS positivity were identified. H&E images of characteristic TLS in these patients. (B) Co‐immunofluorescence subimages depicting the co‐localization of CD23 and CD21 within CD23‐positive TLS were obtained from a collection of 70 BC samples that we compiled. (C) In 9 TLS‐positive patients of 70 BC patients, representative images of H&E staining and IHC staining of CD4, CD8, CD20, CD23 and BCL6 expression in TLS‐positive BC patients. (D) Analysis of DEGs in 200 TCGA BRCA cases categorized as TLS‐negative and TLS‐positive. Source data are available at: https://portal.gdc.cancer.gov . (E and F) H&E (E) and CD23 IHC (F) images of the typical TLS‐positive case in 70 BC patients. (G) Different tumour types exhibit FCER2 expression, according to TIMER2. * p < .05;** p < .01;*** p < .001. (H) Utilizing the EPIC and TIDE algorithms within TIMER2, the relationship between FCER2 expression and tumour infiltrating immune cells in BC was computed. (I‐J) Kaplan–Meier Survival curve of FCER2(I), co‐acting with FCER2 and B cells(J) in BC by TIMER2. TIL‐B, tumor‐infiltrating B cells.

Article Snippet: 1% bovine serum albumin for 10 min at room temperature, followed by overnight incubation at 4°C with the following primary antibodies: monoclonal antibody against FCER2 (CD23; 1:400; 60208‐2‐Ig; Proteintech), anti‐CD21 (1:400; 24374‐1‐AP; Proteintech), anti‐CD20 (1:400; 60271‐1‐Ig; Proteintech), anti‐CD4 (1:400; 67786‐1‐Ig; Proteintech), anti‐CD8 (1:400; 66868‐1‐Ig; Proteintech) and anti‐BCL6 (1:400; 66340‐1‐Ig; Proteintech).

Techniques: Immunofluorescence, Staining, Immunohistochemistry, Expressing

Journal: Clinical and Translational Medicine

Article Title: Single‐cell transcriptome sequencing of B‐cell heterogeneity and tertiary lymphoid structure predicts breast cancer prognosis and neoadjuvant therapy efficacy

doi: 10.1002/ctm2.1346

Figure Lengend Snippet: Different tertiary lymphoid structures (TLS) states in breast cancer (BC) display metabolic variability and survival variations. (A) A bar graph depicting the distribution of immune cells that have infiltrated tumours in TLS‐positive and TLS‐negative groups among 9 BC patients. (B) Genetic analysis of immune cell differences in TLS‐positive and TLS‐negative groups among 9 BC patients. (C) Heatmap of metabolic pathway scores in TLS‐positive and TLS‐negative groups among 9 BC patients. (D) Heatmap showing average metabolic gene expression in TLS‐positive and TLS‐negative groups among 9 BC patients. (E) To ascertain the expression correlation between CD23 and the biomarkers citrate synthase (CS), HK2, lactate dehydrogenase A (LDHA), and IDH3G in BC, the cBioportal tool ( https://www.cbioportal.org ) was utilized, employing the TCGA dataset (Firehose Legacy dataset; encompassing 1108 BC samples) for this purpose. (F) Kaplan–Meier survival curves depicting the disparity in BC outcomes between cohorts with high and low APOD expression, as analyzed through GEPIA. (G) Imaging before and after neoadjuvant chemotherapy of TLS‐positive patients with significant effectiveness. (H) Imaging before and after neoadjuvant immunotherapy of TLS‐positive patients with significant effectiveness.

Article Snippet: 1% bovine serum albumin for 10 min at room temperature, followed by overnight incubation at 4°C with the following primary antibodies: monoclonal antibody against FCER2 (CD23; 1:400; 60208‐2‐Ig; Proteintech), anti‐CD21 (1:400; 24374‐1‐AP; Proteintech), anti‐CD20 (1:400; 60271‐1‐Ig; Proteintech), anti‐CD4 (1:400; 67786‐1‐Ig; Proteintech), anti‐CD8 (1:400; 66868‐1‐Ig; Proteintech) and anti‐BCL6 (1:400; 66340‐1‐Ig; Proteintech).

Techniques: Gene Expression, Expressing, Imaging

Journal: Clinical and Translational Medicine

Article Title: Single‐cell transcriptome sequencing of B‐cell heterogeneity and tertiary lymphoid structure predicts breast cancer prognosis and neoadjuvant therapy efficacy

doi: 10.1002/ctm2.1346

Figure Lengend Snippet: The potential significance of tertiary lymphoid structures (TLS) and TLS‐specific markers for breast cancer (BC) prognosis. After matching with Fitness score matching (PSM), survival analysis and prognostic factor analysis were performed. For survival analysis, the Kaplan–Meier method and the log‐rank test were employed. For prognostic factors, COX proportional hazard regression model was used. The two‐sided log rank test was used to determine the p values. (A) Kaplan–Meier survival curves for the disease‐free survival (DFS) (left) and OS (right) of 920 TCGA BRCA patients based on single gene expression (CD20, CD23 and CD8). (B) The impact of CD20, CD23, CD8, CD4 and BCL6 on 920 TCGA BRCA patients’ prognosis. The forest map shows HRs (center pink and blue squares) and 95% confidence interval (horizontal ranges), and PSM matching has been made for factors such as molecular typing, lymph node status, tumour size, diagnosis age and histological grading of BC. (C) Kaplan–Meier survival curves for the DFS of 70 BC patients based on TLS expression.

Article Snippet: 1% bovine serum albumin for 10 min at room temperature, followed by overnight incubation at 4°C with the following primary antibodies: monoclonal antibody against FCER2 (CD23; 1:400; 60208‐2‐Ig; Proteintech), anti‐CD21 (1:400; 24374‐1‐AP; Proteintech), anti‐CD20 (1:400; 60271‐1‐Ig; Proteintech), anti‐CD4 (1:400; 67786‐1‐Ig; Proteintech), anti‐CD8 (1:400; 66868‐1‐Ig; Proteintech) and anti‐BCL6 (1:400; 66340‐1‐Ig; Proteintech).

Techniques: Gene Expression, Biomarker Discovery, Expressing

Journal: Journal of Animal Science and Biotechnology

Article Title: Investigation of HCAR2 antagonists as a potential strategy to modulate bovine leukocytes

doi: 10.1186/s40104-024-00999-5

Figure Lengend Snippet: Depiction of gating strategy used for flow cytometric analysis of bovine circulating immune cells. First, debris were gated out, then we captured single cells and stained for live cells. Then, we set gates for each cell population of interest (CD3 + , CD21 + , CD172a + , and granulocytes) to investigate HCAR2 expression within each population

Article Snippet: Antibodies used in this panel were CD172a (HR-BOV2049, WSU Antibody Center, Pullman, WA, USA) conjugated to PE/R-PE (abcam102918, Cambridge, UK); CD3 (Bu-BOV2009, WSU Antibody Center) conjugated to PE-Cy7 (ab102903, Cambridge, UK); CD21 with FITC fluorophore (MCA1424F, Bio-Rad); and HCAR2 with AlexaFluor 647 (51-4779-42, ThermoFisher Scientific).

Techniques: Staining, Expressing