Journal: Journal of immunotherapy (Hagerstown, Md. : 1997)

Article Title: Blockade of BAFF Receptor BR3 on T cells Enhances Their Activation and Cytotoxicity

doi: 10.1097/CJI.0000000000000209

Figure Lengend Snippet: (A) Flow cytometric analysis of BR3 on purified human T cell subsets isolated from five different healthy donors. Bead-purified CD4+ and CD8+ cells were rested or activated with anti-CD3/CD28 for 24 hours. BR3 expression was analyzed on live T cells using anti-BR3 clone 1C11. Shown are the percent BR3+ cells within the live gate of total CD4+ or CD8+ T cells. Differences in percent BR3+ cells were significant between resting versus activated populations (p=0.009 and p=0.04 for CD4+ and CD8+ T cells, respectively) (B) Left: Representative overlays for BCMA+ versus BR3+ resting and activated CD8+ T cells. Right: Relative mRNA expression of BR3 versus BCMA in activated CD4+ and CD8+ cells as gauged by semi-quantitative PCR. (C) Left: Representative overlays for BAFF+ resting and activated CD4+ and CD8+ T cells (shaded histograms). Ig stained controls are not shaded. (D) Representative dot plots of CD25 expression on activated BR3+ versus BR3- T cells.

Article Snippet: BAFF receptor blocking antibodies anti-BR3 (cat# AF1162), anti-TACI (cat# AF174), and anti-BCMA (cat# AF193), were purchased from RnD Systems, Inc (Minneapolis, MN).

Techniques: Purification, Isolation, Expressing, Real-time Polymerase Chain Reaction, Staining

Journal: Journal of immunotherapy (Hagerstown, Md. : 1997)

Article Title: Blockade of BAFF Receptor BR3 on T cells Enhances Their Activation and Cytotoxicity

doi: 10.1097/CJI.0000000000000209

Figure Lengend Snippet: (A) Flow cytometric analysis of CD25 expression. Purified CD4+ and CD8+ T cells were incubated with goat polyclonal BR3, TACI, or BCMA specific neutralization antibodies or the goat IgG control and activated with plate-bound CD3/CD28 specific stimulatory antibodies for 24 hours. Anti-CD25 APC was used to detect CD25 expression which is gauged by percent positive CD25 cells. Five separate T cell donors were analyzed. (B) Flow cytometric analysis as for (A) measured by mean channel fluorescence of CD25. (C) Relative gene expression of CD25 using semi-quantitative PCR. Purified T cells from three healthy donors were incubated with either the anti-BR3 neutralization antibody or the goat IgG control and activated for 24 hours.

Article Snippet: BAFF receptor blocking antibodies anti-BR3 (cat# AF1162), anti-TACI (cat# AF174), and anti-BCMA (cat# AF193), were purchased from RnD Systems, Inc (Minneapolis, MN).

Techniques: Expressing, Purification, Incubation, Neutralization, Control, Fluorescence, Gene Expression, Real-time Polymerase Chain Reaction

Journal: Journal of immunotherapy (Hagerstown, Md. : 1997)

Article Title: Blockade of BAFF Receptor BR3 on T cells Enhances Their Activation and Cytotoxicity

doi: 10.1097/CJI.0000000000000209

Figure Lengend Snippet: (A) ELISA analysis of IFN-γ levels in culture supernatants after 24 hours of T cell activation in the presence of BR3, TACI, or BCMA blocking antibodies or the goat IgG control. (B) Relative gene expression of IFN-γ in CD4 and CD8 T cells after 24 hours of activation in the presence of anti-BR3 or the goat IgG control. (C) PCR analysis of granzymeB versus perforin gene expression in anti-BR3 treated cells. Purified T cell subsets of three healthy donors were analyzed for granzyme B/perforin mRNA expression. (D) ELISA analysis of granzyme B released into culture supernatants of activated CD4+ and CD8+ T cells treated with BR3, TACI, or BCMA blocking antibodies or the goat IgG control. Purified T cell subsets from five healthy donors were analyzed for secreted granzyme B levels.

Article Snippet: BAFF receptor blocking antibodies anti-BR3 (cat# AF1162), anti-TACI (cat# AF174), and anti-BCMA (cat# AF193), were purchased from RnD Systems, Inc (Minneapolis, MN).

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Blocking Assay, Control, Gene Expression, Purification, Expressing

Journal: Journal of immunotherapy (Hagerstown, Md. : 1997)

Article Title: Blockade of BAFF Receptor BR3 on T cells Enhances Their Activation and Cytotoxicity

doi: 10.1097/CJI.0000000000000209

Figure Lengend Snippet: (A) Flow cytometric analysis of BR3, CD25, and IFN-γ expression after transfection of CD4+ T cells with the pGIPZ control shRNA plasmid versus BR3 specific shRNA p685. BR3 expression was measured in cells rested 24 hours post-transfection. CD25 was measured in T cells activated for 24 hours and is noted as mean channel fluoresence. IFN-γ was analyzed by intracellular flow cytometry in T cells activated for 24 hours and is denoted as percent + IFN-γ cells in the live gate. (B) Flow cytometric analysis of BR3 and granzyme B in CD8+ cells transfected with the pGIPZ control or BR3-specific shRNA construct p835. BR3 expression was measured in cells rested 24 hours post-transfection. Granzyme B was measured by intracellular flow cytometry in CD8+ cells activated for 24 hours.

Article Snippet: BAFF receptor blocking antibodies anti-BR3 (cat# AF1162), anti-TACI (cat# AF174), and anti-BCMA (cat# AF193), were purchased from RnD Systems, Inc (Minneapolis, MN).

Techniques: Expressing, Transfection, Control, shRNA, Plasmid Preparation, Flow Cytometry, Construct

Journal: Journal of immunotherapy (Hagerstown, Md. : 1997)

Article Title: Blockade of BAFF Receptor BR3 on T cells Enhances Their Activation and Cytotoxicity

doi: 10.1097/CJI.0000000000000209

Figure Lengend Snippet: (A) Representative dot plot of flow cytometric analysis of CD25 expression in CRTAM+ versus CRTAM− activated CD4+ T cells with and without anti-BR3 blockade. (B) Representative overlays of CD25 expression in CRTAM+ versus CRTAM− CD4+ and CD8+ T cells. (C) Mean channel fluorescence of CD25 expression of CD4+CRTAM+ and CD8+CRTAM+ cells treated goat IgG, anti-BR3, anti-TACI, or anti-BCMA blocking antibodies, or without any treatment (No Tx). Differences between controls and anti-BR3 were statistically significant for both CD4+ and CD8+ T cells (p<0.05).

Article Snippet: BAFF receptor blocking antibodies anti-BR3 (cat# AF1162), anti-TACI (cat# AF174), and anti-BCMA (cat# AF193), were purchased from RnD Systems, Inc (Minneapolis, MN).

Techniques: Expressing, Fluorescence, Blocking Assay

Journal: Journal of immunotherapy (Hagerstown, Md. : 1997)

Article Title: Blockade of BAFF Receptor BR3 on T cells Enhances Their Activation and Cytotoxicity

doi: 10.1097/CJI.0000000000000209

Figure Lengend Snippet: (A) Redirected killing of CD4+ and CD8+ T cells as measured by lysis of P-815 cells. Lysis was measured by LDH release. BR3 neutralization enhanced the lysis mediated by both CD4+ and CD8+ cells (p<0.01). (B) Representative dot plots of granzyme B mediated apoptosis as measured by cleaved PARP-1. CD4+ T cells were co-cultured with HeLa or A375 cells at a 25:1 ratio for 18 hours with subsequent intracellular staining of tumor cells for cleaved PARP-1. (C) Percent c-PARP-1 + tumor cells co-cultured with activated CD4+ T cells with and without anti-BR3. Of the PBMC donors screened, CD4+ T cells from Donor 1 were able to mediate apoptosis in HeLa and A375 cells. Those from Donor 2 mediated apoptosis of HeLa cells. Anti-BR3 mediated increases in cleaved PARP-1 were statistically significant (p<0.05). Data represent five separate experiments.

Article Snippet: BAFF receptor blocking antibodies anti-BR3 (cat# AF1162), anti-TACI (cat# AF174), and anti-BCMA (cat# AF193), were purchased from RnD Systems, Inc (Minneapolis, MN).

Techniques: Lysis, Neutralization, Cell Culture, Staining

Journal: Frontiers in Immunology

Article Title: Identification of tumor associated neutrophils-related genes in triple-negative breast cancer for predicting prognosis and therapeutic response through integrated single-cell analysis

doi: 10.3389/fimmu.2025.1613529

Figure Lengend Snippet: Identification of candidate genes. (A) Differences in survival between the high and low neutrophil group. (B) RSF analysis and expression levels of genes related to prognosis. (C) Expression levels and distributions of RASGRP4, COX20, CD47, TIMM10B, LY86, GRAP, TNFRSF13C and ATP6V0D1 in different tumor subtypes. (D-G) Kaplan–Meier graphs displaying the survival potential of patients with TNBC, grouped by the expression levels of significant genes.

Article Snippet: The primary antibodies used in the experiment included antibodies against MPO (ZSGB-Bio, ZA-0197, diluted1:200), TIMM10B (Proteintech, 10907-1, diluted 1:200), GRAP (Proteintech, 14505-1, diluted 1:200), and TNFRSF13C (Proteintech, 22582-1, diluted 1:200), RASGRP4 (Abcam, 96293, diluted 1:100).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Identification of tumor associated neutrophils-related genes in triple-negative breast cancer for predicting prognosis and therapeutic response through integrated single-cell analysis

doi: 10.3389/fimmu.2025.1613529

Figure Lengend Snippet: Correlations between TIMM10B, GRAP, TNFRSF13C and RASGRP4 and drug sensitivity. (A-D) Correlations between key genes and the IC50 of chemotherapeutic agents.

Article Snippet: The primary antibodies used in the experiment included antibodies against MPO (ZSGB-Bio, ZA-0197, diluted1:200), TIMM10B (Proteintech, 10907-1, diluted 1:200), GRAP (Proteintech, 14505-1, diluted 1:200), and TNFRSF13C (Proteintech, 22582-1, diluted 1:200), RASGRP4 (Abcam, 96293, diluted 1:100).

Techniques:

Journal: Frontiers in Immunology

Article Title: Identification of tumor associated neutrophils-related genes in triple-negative breast cancer for predicting prognosis and therapeutic response through integrated single-cell analysis

doi: 10.3389/fimmu.2025.1613529

Figure Lengend Snippet: GSEA and GSVA of the four genes. (A-D) . GSEA revealed the enriched signaling pathways associated with TIMM10B, GRAP, TNFRSF13C and RASGRP4. (E-H) . Analysis of key genes using GSVA. The x-axis illustrates the t value of the GSVA score, and the y-axis depicts KEGG pathways; blue highlights upregulated pathways, whereas green highlights downregulated pathways. |NES| ≥ 1 and FDR < 0.25.

Article Snippet: The primary antibodies used in the experiment included antibodies against MPO (ZSGB-Bio, ZA-0197, diluted1:200), TIMM10B (Proteintech, 10907-1, diluted 1:200), GRAP (Proteintech, 14505-1, diluted 1:200), and TNFRSF13C (Proteintech, 22582-1, diluted 1:200), RASGRP4 (Abcam, 96293, diluted 1:100).

Techniques: Protein-Protein interactions

Journal: Frontiers in Immunology

Article Title: Identification of tumor associated neutrophils-related genes in triple-negative breast cancer for predicting prognosis and therapeutic response through integrated single-cell analysis

doi: 10.3389/fimmu.2025.1613529

Figure Lengend Snippet: Cell communication and quasitemporal analysis. (A) Circus plot illustrating the greater total number of significantly interacting pairs between neutrophils and immune cells as estimated by CellPhoneDB (P<0.05). (B) Bubble diagram of the cell communication network between ligands and neutrophils and other cell subtypes as well as with neutrophil itself themselves. (C-E) Trajectory analysis of the potential relatedness between the two groups according to pseudotime, cell type and group. (F-H) Changes in the expression of TIMM10B, GRAP, TNFRSF13C and RASGRP4 over pseudotime.

Article Snippet: The primary antibodies used in the experiment included antibodies against MPO (ZSGB-Bio, ZA-0197, diluted1:200), TIMM10B (Proteintech, 10907-1, diluted 1:200), GRAP (Proteintech, 14505-1, diluted 1:200), and TNFRSF13C (Proteintech, 22582-1, diluted 1:200), RASGRP4 (Abcam, 96293, diluted 1:100).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Identification of tumor associated neutrophils-related genes in triple-negative breast cancer for predicting prognosis and therapeutic response through integrated single-cell analysis

doi: 10.3389/fimmu.2025.1613529

Figure Lengend Snippet: RASGRP4, TIMM10B and TNFRSF13C exhibited protumorigenic activities. (A) Representative images of colonies of MDA-MB-231 cells treated with control or gene-overexpressing neutrophils. (B) Relative quantified number of colonies. (C) Representative images of migrated cells after cocultured with control or gene-overexpressing cells. (D) Quantification of the relative number of migrated cells.

Article Snippet: The primary antibodies used in the experiment included antibodies against MPO (ZSGB-Bio, ZA-0197, diluted1:200), TIMM10B (Proteintech, 10907-1, diluted 1:200), GRAP (Proteintech, 14505-1, diluted 1:200), and TNFRSF13C (Proteintech, 22582-1, diluted 1:200), RASGRP4 (Abcam, 96293, diluted 1:100).

Techniques: Control

Journal: Frontiers in Immunology

Article Title: Identification of tumor associated neutrophils-related genes in triple-negative breast cancer for predicting prognosis and therapeutic response through integrated single-cell analysis

doi: 10.3389/fimmu.2025.1613529

Figure Lengend Snippet: Clinical relevance of TIMM10B, GRAP, TNFRSF13C and RASGRP4. (A) Polychromatic immunofluorescence staining showing the distribution of MPO, TIMM10B, GRAP, TNFRSF13C and RASGRP4 expression. Scale bar (upper panel), 200 µm. Scale bar (bottom panel), 50 µm. (B) IHC scores of MPO, TIMM10B, GRAP, TNFRSF13C and RASGRP4 in adjacent tissue (AT), stage I-II (I-II) and III TNBC. (C) Correlations between MPO and the four candidate genes in stage I-II (I-II) TNBC. (D) Correlations between MPO and the four candidate genes in stage III (III) TNBC. (E) Representative images of coimmunostaining for MPO and four target genes in adjacent tissue from the stage I-II and III TNBC groups. Scale bar, 20 µm. (F) Percentage of TIMM10B-, GRAP-, TNFRSF13C- and RASGRP4-positive cells in AT and stage I-II and III TNBC. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant.

Article Snippet: The primary antibodies used in the experiment included antibodies against MPO (ZSGB-Bio, ZA-0197, diluted1:200), TIMM10B (Proteintech, 10907-1, diluted 1:200), GRAP (Proteintech, 14505-1, diluted 1:200), and TNFRSF13C (Proteintech, 22582-1, diluted 1:200), RASGRP4 (Abcam, 96293, diluted 1:100).

Techniques: Immunofluorescence, Staining, Expressing