Journal: Journal of Immunotherapy

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

doi: 10.1097/cji.0000000000000209

Figure Lengend Snippet: FIGURE 1. Analysis of BR3 and BAFF on CD4+ and CD8+ resting and activated T cells. A, Flow cytometric analysis of BR3 on purified human T-cell subsets isolated from 5 different healthy donors. Bead-purified CD4+ and CD8+ cells were rested or activated with anti-CD3/ anti-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 semiquantitative PCR. C, Left: representative overlays for BAFF+ 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. PCR indicates polymerase chain reaction.

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. All 3 are goat polyclonal antibodies.

Techniques: Purification, Isolation, Expressing, Staining, Polymerase Chain Reaction

Journal: Journal of Immunotherapy

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

doi: 10.1097/cji.0000000000000209

Figure Lengend Snippet: FIGURE 2. CD25 expression increases with anti-BR3 blockade. A, Flow cytometric analysis of CD25 expression. Purified CD4+ and CD8+ T cells were incubated with goat polyclonal anti-BR3, anti-TCI, or anti-BCMA and anti-CD3/anti-CD28 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 semiquantitative PCR. Purified T cells from 3 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. All 3 are goat polyclonal antibodies.

Techniques: Expressing, Purification, Incubation, Fluorescence, Gene Expression, Neutralization, Control

Journal: Journal of Immunotherapy

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

doi: 10.1097/cji.0000000000000209

Figure Lengend Snippet: FIGURE 3. IFN-γ and granzyme B expression increases with anti-BR3 blockade. 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 granzyme B versus perforin gene expression in anti-BR3–treated cells. Purified T-cell subsets of 3 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 5 healthy donors were analyzed for secreted granzyme B levels. ELISA indicates enzyme-linked immunosorbent assay.

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. All 3 are goat polyclonal antibodies.

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

Journal: Journal of Immunotherapy

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

doi: 10.1097/cji.0000000000000209

Figure Lengend Snippet: FIGURE 4. Down-modulation of BR3 gene expression via shRNA. 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 after 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 after 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. All 3 are goat polyclonal antibodies.

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

Journal: Journal of Immunotherapy

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

doi: 10.1097/cji.0000000000000209

Figure Lengend Snippet: FIGURE 5. Anti-BR3 increases cytolytic T-cell activation. 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). MCF indicates mean channel fluorescence.

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. All 3 are goat polyclonal antibodies.

Techniques: Activation Assay, Expressing, Fluorescence, Blocking Assay

Journal: Journal of Immunotherapy

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

doi: 10.1097/cji.0000000000000209

Figure Lengend Snippet: FIGURE 6. Anti-BR3 enhances the cytolytic function of CD4+ and CD8+ T cells. A, Redirected killing of CD4+ and CD8+ T cells as measured by lysis of P-815 cells. Lysis was measured by lactose dehydrogenase 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 cocultured 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 cocultured with activated CD4+ T cells with and without anti-BR3. Among 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 5 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. All 3 are goat polyclonal antibodies.

Techniques: Lysis, Neutralization, Staining

Journal: International Journal of Molecular Sciences

Article Title: Modelling Microglial Innate Immune Memory In Vitro: Understanding the Role of Aerobic Glycolysis in Innate Immune Memory

doi: 10.3390/ijms24108967

Figure Lengend Snippet: BAFF pre-treatment produces training in culture. ( A ) BV2 cells were treated with H 2 O or B-cell-activating factor (BAFF) (10 ng/mL) for 3 h followed by washout. After 24 h of incubation, the cells were treated with H 2 O or Lipopolysaccharide (LPS) (25 ng/mL) for 3 h or 24 h prior to analysis. Created with BioRender.com. ( B ) RT qPCR assessment of inflammatory gene expression of Il1b , Tnfa and Cxcl16 in BV2 treated with a priming stimulus of BAFF or H 2 O followed by a second stimulus of H 2 O or LPS. n = 3 independent experiments, 2–3 replicates per experiment. ( C ) RT qPCR assessment of anti-inflammatory gene expression of Il10 and Il6 in BV2 treated with a priming stimulus of BAFF or H 2 O followed by a second LPS stimulus. n = 3 independent experiments, 2 replicates per experiment. ( D ) Phagocytosis of pH-rodo E. coli -tagged beads assessed at 24 h after second stimulus of LPS or H 2 O. n = 3 independent experiments, 2 replicates per experiment. ( E ) Release of NO measured 24 h after second LPS treatment, measured through Griess reagent assay. n = 3 independent experiments, 2 replicates per experiment. qPCR shown as bar graph of Log 2 (Fold Change) + SEM Tukey’s post hoc significances denoted by (ns p > 0.05, ** p < 0.002, *** p < 0.0002, **** p < 0.0001). All statistics are reported in .

Article Snippet: BV2 microglia were treated with lipopolysaccharides (LPS) from Escherichia coli (SigmaAldrich #L5418, St. Louis, MI, USA), recombinant mouse B-cell activating factor (BAFF) (R&D Systems, #8876-BF, Minneapolis, MN, USA) or sodium oxamate (Alfa Aesar #A16532-06, Haverhill, MA, USA).

Techniques: Incubation, Quantitative RT-PCR, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Modelling Microglial Innate Immune Memory In Vitro: Understanding the Role of Aerobic Glycolysis in Innate Immune Memory

doi: 10.3390/ijms24108967

Figure Lengend Snippet: LPS but not BAFF induces rapid induction of lactate production. ( A ) BV2 cells are treated with Lipopolysaccharide (LPS) (25 ng/mL) or B-cell-activating factor (BAFF) (10 ng/mL) for 3 h prior to analysis. RT-qPCR analysis of inflammatory cytokines for inflammatory genes. n = 3–4 independent experiments, 2–4 replicates per experiment. ( B ) BV2 cells are treated with LPS (25 ng/mL) or BAFF (10 ng/mL) for 3 h prior to analysis. RT-qPCR analysis of anti-inflammatory cytokines Il10 and Il6. n = 3–4 independent experiments, 2–4 replicates per experiment. ( C ) Intracellular L-lactate in BV2 cells treated with LPS (25 ng/mL) or BAFF (10 ng/mL) recorded 24 h after treatment. n = 3 independent experiments, 2 replicates per experiment. qPCR shown as bar graph of Log 2 (Fold Change) + SEM Tukey’s post hoc significances denoted by (ns p > 0.05, ** p < 0.01, **** p < 0.0001). All statistics are reported in .

Article Snippet: BV2 microglia were treated with lipopolysaccharides (LPS) from Escherichia coli (SigmaAldrich #L5418, St. Louis, MI, USA), recombinant mouse B-cell activating factor (BAFF) (R&D Systems, #8876-BF, Minneapolis, MN, USA) or sodium oxamate (Alfa Aesar #A16532-06, Haverhill, MA, USA).

Techniques: Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Modelling Microglial Innate Immune Memory In Vitro: Understanding the Role of Aerobic Glycolysis in Innate Immune Memory

doi: 10.3390/ijms24108967

Figure Lengend Snippet: Sodium oxamate prevents tolerized aerobic metabolism. ( A ) BV2 cells are treated with Lipopolysaccharide (LPS) (25 ng/mL) or H 2 O for 3 h prior to washout; 24 h after, the cells are treated with LPS (25 ng/mL) or H 2 O for 24 h prior to analysis. L-lactate levels are depicted as a fold change relative to the H 2 O H 2 O control. n = 3 independent experiments, 2 replicates per experiment. ( B ) BV2 cells are treated with B-cell-activating factor (BAFF) (10 ng/mL) or H 2 O for 3 h prior to washout; 24 h afterward, the cells are treated with LPS (25 ng/mL) or H 2 O for 24 h prior to analysis. L-lactate levels are depicted as a fold change relative to the H 2 O H 2 O control. n = 3 independent experiments, 2 replicates per experiment. ( C ) BV2 cells are treated with sodium oxamate (10 mM) or vehicle and LPS (25 ng/mL) or H 2 O for 3 h prior to washout; 24 h afterward, the cells are treated with LPS (25 ng/mL) or H 2 O for 24 h prior to analysis. L-lactate levels are depicted as a fold change relative to the H 2 O H 2 O control. n = 3 independent experiments, 2 replicates per experiment. Graphs shown as bar graph + SEM Tukey’s post hoc significances denoted by (ns p > 0.05, * p < 0.05, *** p < 0.001, **** p < 0.0001). All statistics are reported in .

Article Snippet: BV2 microglia were treated with lipopolysaccharides (LPS) from Escherichia coli (SigmaAldrich #L5418, St. Louis, MI, USA), recombinant mouse B-cell activating factor (BAFF) (R&D Systems, #8876-BF, Minneapolis, MN, USA) or sodium oxamate (Alfa Aesar #A16532-06, Haverhill, MA, USA).

Techniques: Control