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human tnfr1 tnfrsf1a antibody  (R&D Systems)


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    R&D Systems human tnfr1 tnfrsf1a antibody
    Human Tnfr1 Tnfrsf1a Antibody, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+human+tnf+ab/Human+TNF+RI%2FTNFRSF1A+Antibody/pm41571205-82-6-11
    Average 93 stars, based on 11 article reviews
    human tnfr1 tnfrsf1a antibody - by Bioz Stars, 2026-10
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    Related Articles

    Microscopy:

    Article Title: B cells inhibit bone formation in rheumatoid arthritis by suppressing osteoblast differentiation
    Article Snippet: BM-MPCs were then cultured in OB-inducing medium for another 14 days and mineralized bone nodules were counted after von Kossa staining. (5) For osteoclastogenic assays, BM cells were cultured with conditioned medium (1:50 dilution) from a M-CSF-producing cell line for 3 days in α-MEM with 10% FCS to enrich for osteoclast precursors (OCPs), which were then cultured with M-CSF-conditioned medium and RANKL (10ng/ml, R&D) for 2–3 days. .. After multinucleated cells were observed under a microscope, the cells were fixed, stained for TRAP activity to identify OCs (TRAP+ cells containing >3 nuclei) and counted . (6) For blocking experiments with neutralizing Abs, anti-mouse CCL3 Ab (AF-450-NA), anti-mouse TNF Ab (AF-410-NA), anti-human CCL3 Ab (MAB270), and anti-human TNF Ab (MAB610), were purchased from R & D systems and used according to the manufacturer’s instructions. (7) For human B cell stimulation, purified B cells were cultured with 5 μg/ml CpG 2006 (Oligos Etc.) plus 10 μg/ml Anti-Ig (A+G+M) (109-006-064, Jackson ImmunoResearch Laboratories) or PBS control for 4 h. At the end of the culture period, conditioned medium was collected for co-culture or ELISA. ..

    Staining:

    Article Title: B cells inhibit bone formation in rheumatoid arthritis by suppressing osteoblast differentiation
    Article Snippet: BM-MPCs were then cultured in OB-inducing medium for another 14 days and mineralized bone nodules were counted after von Kossa staining. (5) For osteoclastogenic assays, BM cells were cultured with conditioned medium (1:50 dilution) from a M-CSF-producing cell line for 3 days in α-MEM with 10% FCS to enrich for osteoclast precursors (OCPs), which were then cultured with M-CSF-conditioned medium and RANKL (10ng/ml, R&D) for 2–3 days. .. After multinucleated cells were observed under a microscope, the cells were fixed, stained for TRAP activity to identify OCs (TRAP+ cells containing >3 nuclei) and counted . (6) For blocking experiments with neutralizing Abs, anti-mouse CCL3 Ab (AF-450-NA), anti-mouse TNF Ab (AF-410-NA), anti-human CCL3 Ab (MAB270), and anti-human TNF Ab (MAB610), were purchased from R & D systems and used according to the manufacturer’s instructions. (7) For human B cell stimulation, purified B cells were cultured with 5 μg/ml CpG 2006 (Oligos Etc.) plus 10 μg/ml Anti-Ig (A+G+M) (109-006-064, Jackson ImmunoResearch Laboratories) or PBS control for 4 h. At the end of the culture period, conditioned medium was collected for co-culture or ELISA. ..

    Activity Assay:

    Article Title: B cells inhibit bone formation in rheumatoid arthritis by suppressing osteoblast differentiation
    Article Snippet: BM-MPCs were then cultured in OB-inducing medium for another 14 days and mineralized bone nodules were counted after von Kossa staining. (5) For osteoclastogenic assays, BM cells were cultured with conditioned medium (1:50 dilution) from a M-CSF-producing cell line for 3 days in α-MEM with 10% FCS to enrich for osteoclast precursors (OCPs), which were then cultured with M-CSF-conditioned medium and RANKL (10ng/ml, R&D) for 2–3 days. .. After multinucleated cells were observed under a microscope, the cells were fixed, stained for TRAP activity to identify OCs (TRAP+ cells containing >3 nuclei) and counted . (6) For blocking experiments with neutralizing Abs, anti-mouse CCL3 Ab (AF-450-NA), anti-mouse TNF Ab (AF-410-NA), anti-human CCL3 Ab (MAB270), and anti-human TNF Ab (MAB610), were purchased from R & D systems and used according to the manufacturer’s instructions. (7) For human B cell stimulation, purified B cells were cultured with 5 μg/ml CpG 2006 (Oligos Etc.) plus 10 μg/ml Anti-Ig (A+G+M) (109-006-064, Jackson ImmunoResearch Laboratories) or PBS control for 4 h. At the end of the culture period, conditioned medium was collected for co-culture or ELISA. ..

    Blocking Assay:

    Article Title: B cells inhibit bone formation in rheumatoid arthritis by suppressing osteoblast differentiation
    Article Snippet: BM-MPCs were then cultured in OB-inducing medium for another 14 days and mineralized bone nodules were counted after von Kossa staining. (5) For osteoclastogenic assays, BM cells were cultured with conditioned medium (1:50 dilution) from a M-CSF-producing cell line for 3 days in α-MEM with 10% FCS to enrich for osteoclast precursors (OCPs), which were then cultured with M-CSF-conditioned medium and RANKL (10ng/ml, R&D) for 2–3 days. .. After multinucleated cells were observed under a microscope, the cells were fixed, stained for TRAP activity to identify OCs (TRAP+ cells containing >3 nuclei) and counted . (6) For blocking experiments with neutralizing Abs, anti-mouse CCL3 Ab (AF-450-NA), anti-mouse TNF Ab (AF-410-NA), anti-human CCL3 Ab (MAB270), and anti-human TNF Ab (MAB610), were purchased from R & D systems and used according to the manufacturer’s instructions. (7) For human B cell stimulation, purified B cells were cultured with 5 μg/ml CpG 2006 (Oligos Etc.) plus 10 μg/ml Anti-Ig (A+G+M) (109-006-064, Jackson ImmunoResearch Laboratories) or PBS control for 4 h. At the end of the culture period, conditioned medium was collected for co-culture or ELISA. ..

    Cell Stimulation:

    Article Title: B cells inhibit bone formation in rheumatoid arthritis by suppressing osteoblast differentiation
    Article Snippet: BM-MPCs were then cultured in OB-inducing medium for another 14 days and mineralized bone nodules were counted after von Kossa staining. (5) For osteoclastogenic assays, BM cells were cultured with conditioned medium (1:50 dilution) from a M-CSF-producing cell line for 3 days in α-MEM with 10% FCS to enrich for osteoclast precursors (OCPs), which were then cultured with M-CSF-conditioned medium and RANKL (10ng/ml, R&D) for 2–3 days. .. After multinucleated cells were observed under a microscope, the cells were fixed, stained for TRAP activity to identify OCs (TRAP+ cells containing >3 nuclei) and counted . (6) For blocking experiments with neutralizing Abs, anti-mouse CCL3 Ab (AF-450-NA), anti-mouse TNF Ab (AF-410-NA), anti-human CCL3 Ab (MAB270), and anti-human TNF Ab (MAB610), were purchased from R & D systems and used according to the manufacturer’s instructions. (7) For human B cell stimulation, purified B cells were cultured with 5 μg/ml CpG 2006 (Oligos Etc.) plus 10 μg/ml Anti-Ig (A+G+M) (109-006-064, Jackson ImmunoResearch Laboratories) or PBS control for 4 h. At the end of the culture period, conditioned medium was collected for co-culture or ELISA. ..

    Purification:

    Article Title: B cells inhibit bone formation in rheumatoid arthritis by suppressing osteoblast differentiation
    Article Snippet: BM-MPCs were then cultured in OB-inducing medium for another 14 days and mineralized bone nodules were counted after von Kossa staining. (5) For osteoclastogenic assays, BM cells were cultured with conditioned medium (1:50 dilution) from a M-CSF-producing cell line for 3 days in α-MEM with 10% FCS to enrich for osteoclast precursors (OCPs), which were then cultured with M-CSF-conditioned medium and RANKL (10ng/ml, R&D) for 2–3 days. .. After multinucleated cells were observed under a microscope, the cells were fixed, stained for TRAP activity to identify OCs (TRAP+ cells containing >3 nuclei) and counted . (6) For blocking experiments with neutralizing Abs, anti-mouse CCL3 Ab (AF-450-NA), anti-mouse TNF Ab (AF-410-NA), anti-human CCL3 Ab (MAB270), and anti-human TNF Ab (MAB610), were purchased from R & D systems and used according to the manufacturer’s instructions. (7) For human B cell stimulation, purified B cells were cultured with 5 μg/ml CpG 2006 (Oligos Etc.) plus 10 μg/ml Anti-Ig (A+G+M) (109-006-064, Jackson ImmunoResearch Laboratories) or PBS control for 4 h. At the end of the culture period, conditioned medium was collected for co-culture or ELISA. ..

    Cell Culture:

    Article Title: B cells inhibit bone formation in rheumatoid arthritis by suppressing osteoblast differentiation
    Article Snippet: BM-MPCs were then cultured in OB-inducing medium for another 14 days and mineralized bone nodules were counted after von Kossa staining. (5) For osteoclastogenic assays, BM cells were cultured with conditioned medium (1:50 dilution) from a M-CSF-producing cell line for 3 days in α-MEM with 10% FCS to enrich for osteoclast precursors (OCPs), which were then cultured with M-CSF-conditioned medium and RANKL (10ng/ml, R&D) for 2–3 days. .. After multinucleated cells were observed under a microscope, the cells were fixed, stained for TRAP activity to identify OCs (TRAP+ cells containing >3 nuclei) and counted . (6) For blocking experiments with neutralizing Abs, anti-mouse CCL3 Ab (AF-450-NA), anti-mouse TNF Ab (AF-410-NA), anti-human CCL3 Ab (MAB270), and anti-human TNF Ab (MAB610), were purchased from R & D systems and used according to the manufacturer’s instructions. (7) For human B cell stimulation, purified B cells were cultured with 5 μg/ml CpG 2006 (Oligos Etc.) plus 10 μg/ml Anti-Ig (A+G+M) (109-006-064, Jackson ImmunoResearch Laboratories) or PBS control for 4 h. At the end of the culture period, conditioned medium was collected for co-culture or ELISA. ..

    Control:

    Article Title: B cells inhibit bone formation in rheumatoid arthritis by suppressing osteoblast differentiation
    Article Snippet: BM-MPCs were then cultured in OB-inducing medium for another 14 days and mineralized bone nodules were counted after von Kossa staining. (5) For osteoclastogenic assays, BM cells were cultured with conditioned medium (1:50 dilution) from a M-CSF-producing cell line for 3 days in α-MEM with 10% FCS to enrich for osteoclast precursors (OCPs), which were then cultured with M-CSF-conditioned medium and RANKL (10ng/ml, R&D) for 2–3 days. .. After multinucleated cells were observed under a microscope, the cells were fixed, stained for TRAP activity to identify OCs (TRAP+ cells containing >3 nuclei) and counted . (6) For blocking experiments with neutralizing Abs, anti-mouse CCL3 Ab (AF-450-NA), anti-mouse TNF Ab (AF-410-NA), anti-human CCL3 Ab (MAB270), and anti-human TNF Ab (MAB610), were purchased from R & D systems and used according to the manufacturer’s instructions. (7) For human B cell stimulation, purified B cells were cultured with 5 μg/ml CpG 2006 (Oligos Etc.) plus 10 μg/ml Anti-Ig (A+G+M) (109-006-064, Jackson ImmunoResearch Laboratories) or PBS control for 4 h. At the end of the culture period, conditioned medium was collected for co-culture or ELISA. ..

    Enzyme-linked Immunosorbent Assay:

    Article Title: B cells inhibit bone formation in rheumatoid arthritis by suppressing osteoblast differentiation
    Article Snippet: BM-MPCs were then cultured in OB-inducing medium for another 14 days and mineralized bone nodules were counted after von Kossa staining. (5) For osteoclastogenic assays, BM cells were cultured with conditioned medium (1:50 dilution) from a M-CSF-producing cell line for 3 days in α-MEM with 10% FCS to enrich for osteoclast precursors (OCPs), which were then cultured with M-CSF-conditioned medium and RANKL (10ng/ml, R&D) for 2–3 days. .. After multinucleated cells were observed under a microscope, the cells were fixed, stained for TRAP activity to identify OCs (TRAP+ cells containing >3 nuclei) and counted . (6) For blocking experiments with neutralizing Abs, anti-mouse CCL3 Ab (AF-450-NA), anti-mouse TNF Ab (AF-410-NA), anti-human CCL3 Ab (MAB270), and anti-human TNF Ab (MAB610), were purchased from R & D systems and used according to the manufacturer’s instructions. (7) For human B cell stimulation, purified B cells were cultured with 5 μg/ml CpG 2006 (Oligos Etc.) plus 10 μg/ml Anti-Ig (A+G+M) (109-006-064, Jackson ImmunoResearch Laboratories) or PBS control for 4 h. At the end of the culture period, conditioned medium was collected for co-culture or ELISA. ..



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    Figure 5. (A) The scheme illustrates the <t>multistep</t> <t>TNF-α</t> immunoassay reactions carried out on individual ZnO–Ag hybrid NRs. The immunoassay reactions led to the formation <t>of</t> <t>Alexa488-TNF-α</t> immunocomplex. (B) The plot displays normalized intensity with respect to TNF-α concentration for the junction (black triangle), AgMB (blue square), and ZnOMB (red circle). (C) The bar graph shows the average EF values for AgMB/B, ZnOMB/B, J/B, J/AgMB, and J/ZnOMB at varying TNF-α concentrations. (D,E) The plots show the average EF values as a function of TNF-α concentration. The data in (D) correspond to AgMB/B (blue square) and ZnOMB/B (red circle), whereas those in (E) belong to J/B (black triangle), J/AgMB (green square), and J/ZnOMB (orange circle). In (B,D,E), the dashed lines are the linear fits obtained through the data points.
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    Image Search Results


    Figure 5. (A) The scheme illustrates the multistep TNF-α immunoassay reactions carried out on individual ZnO–Ag hybrid NRs. The immunoassay reactions led to the formation of Alexa488-TNF-α immunocomplex. (B) The plot displays normalized intensity with respect to TNF-α concentration for the junction (black triangle), AgMB (blue square), and ZnOMB (red circle). (C) The bar graph shows the average EF values for AgMB/B, ZnOMB/B, J/B, J/AgMB, and J/ZnOMB at varying TNF-α concentrations. (D,E) The plots show the average EF values as a function of TNF-α concentration. The data in (D) correspond to AgMB/B (blue square) and ZnOMB/B (red circle), whereas those in (E) belong to J/B (black triangle), J/AgMB (green square), and J/ZnOMB (orange circle). In (B,D,E), the dashed lines are the linear fits obtained through the data points.

    Journal: Nanomaterials (Basel, Switzerland)

    Article Title: Individual ZnO-Ag Hybrid Nanorods for Synergistic Fluorescence Enhancement Towards Highly Sensitive and Miniaturized Biodetection.

    doi: 10.3390/nano15080617

    Figure Lengend Snippet: Figure 5. (A) The scheme illustrates the multistep TNF-α immunoassay reactions carried out on individual ZnO–Ag hybrid NRs. The immunoassay reactions led to the formation of Alexa488-TNF-α immunocomplex. (B) The plot displays normalized intensity with respect to TNF-α concentration for the junction (black triangle), AgMB (blue square), and ZnOMB (red circle). (C) The bar graph shows the average EF values for AgMB/B, ZnOMB/B, J/B, J/AgMB, and J/ZnOMB at varying TNF-α concentrations. (D,E) The plots show the average EF values as a function of TNF-α concentration. The data in (D) correspond to AgMB/B (blue square) and ZnOMB/B (red circle), whereas those in (E) belong to J/B (black triangle), J/AgMB (green square), and J/ZnOMB (orange circle). In (B,D,E), the dashed lines are the linear fits obtained through the data points.

    Article Snippet: Polyclonal anti-human TNF-α antibody (AB 210-NA, R&D Systems, Minneapolis, MN, USA) was conjugated with a fluorophore, Alexa488, using the Monoclonal Antibody Labeling kit (Invitrogen Molecular Nanomaterials 2025, 15, 617 4 of 17 Probes, Eugene, OR, USA) following the manufacturer’s protocol.

    Techniques: Concentration Assay

    Figure 6. The effect of the ZnO NR length was correlated to the %EF changes determined from the junction and Ag main body of individual ZnO–Ag hybrid NRs. (A) The plots display %EF values determined from the junction and Ag main body versus ZnO NR length for the TNF-α concentrations of 100 (black), 10 (blue), 1 (green) and 0.1 (red) pg/mL. The dashed lines represent linear fits through the data points. (B) The plot shows the combined regressions from %EF change versus ZnO NR length for all TNF-α concentrations in (A). The slope of the regressions increases with higher TNF-α concentration.

    Journal: Nanomaterials (Basel, Switzerland)

    Article Title: Individual ZnO-Ag Hybrid Nanorods for Synergistic Fluorescence Enhancement Towards Highly Sensitive and Miniaturized Biodetection.

    doi: 10.3390/nano15080617

    Figure Lengend Snippet: Figure 6. The effect of the ZnO NR length was correlated to the %EF changes determined from the junction and Ag main body of individual ZnO–Ag hybrid NRs. (A) The plots display %EF values determined from the junction and Ag main body versus ZnO NR length for the TNF-α concentrations of 100 (black), 10 (blue), 1 (green) and 0.1 (red) pg/mL. The dashed lines represent linear fits through the data points. (B) The plot shows the combined regressions from %EF change versus ZnO NR length for all TNF-α concentrations in (A). The slope of the regressions increases with higher TNF-α concentration.

    Article Snippet: Polyclonal anti-human TNF-α antibody (AB 210-NA, R&D Systems, Minneapolis, MN, USA) was conjugated with a fluorophore, Alexa488, using the Monoclonal Antibody Labeling kit (Invitrogen Molecular Nanomaterials 2025, 15, 617 4 of 17 Probes, Eugene, OR, USA) following the manufacturer’s protocol.

    Techniques: Concentration Assay

    Figure 7. The effect of the ZnO NR length was correlated to the %EF changes determined from the junction and ZnO NR main body of individual ZnO–Ag hybrid NRs. (A) The plots display %EF changes of the junction to ZnO NR main body versus ZnO NR length for the TNF-α concentrations of 100 (black), 10 (blue), 1 (green) and 0.1 (red) pg/mL. The dashed lines represent the linear fits through the data points. (B) The plot shows the combined regressions from %EF change versus ZnO NR length for all TNF-α concentrations in (A). The slope of the regressions increases with higher TNF-α concentration.

    Journal: Nanomaterials (Basel, Switzerland)

    Article Title: Individual ZnO-Ag Hybrid Nanorods for Synergistic Fluorescence Enhancement Towards Highly Sensitive and Miniaturized Biodetection.

    doi: 10.3390/nano15080617

    Figure Lengend Snippet: Figure 7. The effect of the ZnO NR length was correlated to the %EF changes determined from the junction and ZnO NR main body of individual ZnO–Ag hybrid NRs. (A) The plots display %EF changes of the junction to ZnO NR main body versus ZnO NR length for the TNF-α concentrations of 100 (black), 10 (blue), 1 (green) and 0.1 (red) pg/mL. The dashed lines represent the linear fits through the data points. (B) The plot shows the combined regressions from %EF change versus ZnO NR length for all TNF-α concentrations in (A). The slope of the regressions increases with higher TNF-α concentration.

    Article Snippet: Polyclonal anti-human TNF-α antibody (AB 210-NA, R&D Systems, Minneapolis, MN, USA) was conjugated with a fluorophore, Alexa488, using the Monoclonal Antibody Labeling kit (Invitrogen Molecular Nanomaterials 2025, 15, 617 4 of 17 Probes, Eugene, OR, USA) following the manufacturer’s protocol.

    Techniques: Concentration Assay

    Soluble factors released during CD3xHER2 bsAb‐mediated T‐cell killing of HER2+ cells include cytokines and induce bystander killing. (A) Alamar Blue analysis of HER2 + (left) and HER2 − (right) cells 3 days after exposure to CM obtained from BT474 HER2 + cells in combination with T‐cells with or without CD3xHER2 bsAbs as indicated. Data are normalized to cell proliferation in a normal culture medium (CTR). The graph represents three independent experiments, each performed with 2D cultures in six wells of a 96‐well plate. Mean ± SEM is shown. Two‐way ANOVA followed by Dunnett's multiple comparisons test was performed. *** p < 0.001. (B) Transmission images showing HER2 + and HER2 − tumor cells exposed for 3 days to CM from the co‐culture of BT474 HER2 + tumor cells and T‐cells with or without CD3xHER2 bsAbs. Note cell rounding and debris in the presence of CM from co‐cultures with bsAbs indicating cytotoxicity. Scale bar = 50 µm. (C) ELISA analysis of IFNγ and TNFα levels in CM from 3‐day co‐cultures of BT474 HER2 + cells and T‐cells with or without CD3xHER2 bsAbs. Data from two T‐cell donors are combined, each performed in duplicate. An unpaired two‐tailed t ‐test was performed. ** p < 0.01; *** p < 0.001.

    Journal: European Journal of Immunology

    Article Title: CD3xHER2 bsAb‐Mediated Activation of Resting T‐cells at HER2 Positive Tumor Clusters Is Sufficient to Trigger Bystander Eradication of Distant HER2 Negative Clusters Through IFNγ and TNFα

    doi: 10.1002/eji.202451589

    Figure Lengend Snippet: Soluble factors released during CD3xHER2 bsAb‐mediated T‐cell killing of HER2+ cells include cytokines and induce bystander killing. (A) Alamar Blue analysis of HER2 + (left) and HER2 − (right) cells 3 days after exposure to CM obtained from BT474 HER2 + cells in combination with T‐cells with or without CD3xHER2 bsAbs as indicated. Data are normalized to cell proliferation in a normal culture medium (CTR). The graph represents three independent experiments, each performed with 2D cultures in six wells of a 96‐well plate. Mean ± SEM is shown. Two‐way ANOVA followed by Dunnett's multiple comparisons test was performed. *** p < 0.001. (B) Transmission images showing HER2 + and HER2 − tumor cells exposed for 3 days to CM from the co‐culture of BT474 HER2 + tumor cells and T‐cells with or without CD3xHER2 bsAbs. Note cell rounding and debris in the presence of CM from co‐cultures with bsAbs indicating cytotoxicity. Scale bar = 50 µm. (C) ELISA analysis of IFNγ and TNFα levels in CM from 3‐day co‐cultures of BT474 HER2 + cells and T‐cells with or without CD3xHER2 bsAbs. Data from two T‐cell donors are combined, each performed in duplicate. An unpaired two‐tailed t ‐test was performed. ** p < 0.01; *** p < 0.001.

    Article Snippet: Cytokines were depleted from CM using neutralizing Abs against IFNγ (AF‐285‐NA, R&D systems, RRID: AB_354445) or TNFα (MAB225‐100, R&D systems, RRID: AB_2204150).

    Techniques: Transmission Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

    Bystander killing after bsAb‐induced, T‐cell‐mediated antitumor cytotoxicity involves paracrine signaling through IFNγ and TNFα. (A) Alamar Blue analysis of HER2 + and HER2 − cells 5 days after exposure to 0.1–10 µg/mL IFNγ. Data are normalized to cell proliferation without IFNγ treatment (CTR). The graph represents three independent experiments, each performed with 2D cultures in six wells of a 96‐well plate. Mean ± SEM is shown. Two‐way ANOVA followed by Dunnett's multiple comparisons test was performed. * p < 0.05; ** p < 0.01. (B) Alamar Blue analysis of HER2 + and HER2 − cells 5 days after exposure to 0.01–10 µg/mL TNFα or a combination of 10 µg/mL IFNγ and 10 µg/mL TNFα. Data are normalized to cell proliferation without TNFα treatment (CTR). The graph represents three independent experiments, each performed with 2D cultures in three wells of a 96‐well plate. Mean ± SEM is shown. Two‐way ANOVA followed by Dunnett's multiple comparisons test was performed. * p < 0.05; ** p < 0.01; *** p < 0.001. (C) Alamar Blue analysis of HER2 + and HER2 − cells 72 h after exposure to CM collected from the co‐culture of BT474 HER2 + tumor cells and T‐cells with CD3xHER2, with or without 10 µg/mL neutralizing Abs against IFNγ, TNFα, or a combination. Data are normalized to cell proliferation without treatment (CTR). Data with T‐cells from two donors are combined, each performed with 2D cultures in three wells of a 96‐well plate. Mean ± SEM is shown. Two‐way ANOVA followed by Dunnett's multiple comparisons test was performed. * p < 0.05; ** p < 0.01; *** p < 0.001.

    Journal: European Journal of Immunology

    Article Title: CD3xHER2 bsAb‐Mediated Activation of Resting T‐cells at HER2 Positive Tumor Clusters Is Sufficient to Trigger Bystander Eradication of Distant HER2 Negative Clusters Through IFNγ and TNFα

    doi: 10.1002/eji.202451589

    Figure Lengend Snippet: Bystander killing after bsAb‐induced, T‐cell‐mediated antitumor cytotoxicity involves paracrine signaling through IFNγ and TNFα. (A) Alamar Blue analysis of HER2 + and HER2 − cells 5 days after exposure to 0.1–10 µg/mL IFNγ. Data are normalized to cell proliferation without IFNγ treatment (CTR). The graph represents three independent experiments, each performed with 2D cultures in six wells of a 96‐well plate. Mean ± SEM is shown. Two‐way ANOVA followed by Dunnett's multiple comparisons test was performed. * p < 0.05; ** p < 0.01. (B) Alamar Blue analysis of HER2 + and HER2 − cells 5 days after exposure to 0.01–10 µg/mL TNFα or a combination of 10 µg/mL IFNγ and 10 µg/mL TNFα. Data are normalized to cell proliferation without TNFα treatment (CTR). The graph represents three independent experiments, each performed with 2D cultures in three wells of a 96‐well plate. Mean ± SEM is shown. Two‐way ANOVA followed by Dunnett's multiple comparisons test was performed. * p < 0.05; ** p < 0.01; *** p < 0.001. (C) Alamar Blue analysis of HER2 + and HER2 − cells 72 h after exposure to CM collected from the co‐culture of BT474 HER2 + tumor cells and T‐cells with CD3xHER2, with or without 10 µg/mL neutralizing Abs against IFNγ, TNFα, or a combination. Data are normalized to cell proliferation without treatment (CTR). Data with T‐cells from two donors are combined, each performed with 2D cultures in three wells of a 96‐well plate. Mean ± SEM is shown. Two‐way ANOVA followed by Dunnett's multiple comparisons test was performed. * p < 0.05; ** p < 0.01; *** p < 0.001.

    Article Snippet: Cytokines were depleted from CM using neutralizing Abs against IFNγ (AF‐285‐NA, R&D systems, RRID: AB_354445) or TNFα (MAB225‐100, R&D systems, RRID: AB_2204150).

    Techniques: Co-Culture Assay

    Neutralizing antibodies against IFNγ and TNFα block bystander killing of HER2‐ tumoroids after T‐cell‐mediated killing of HER2+ tumoroids via bsAbs. (A) Schematic representation of the experimental design based on Model 3 (see Figure ), with or without neutralizing antibodies. (B) Maximum projections of 3D confocal imaging showing HER2 + and HER2 − tumoroids co‐printed as described in (A) and treated with or without the indicated concentrations of an IFNγ neutralizing antibody. Images were taken 6, 18, and 30 h after exposure to CD3xHER2 bsAbs. Green = BT474 WT, Lifeact‐EGFP‐transduced; blue = BT474 HER2 KO, Hoechst33342‐labeled; yellow = T‐cells, CellTracker Deep Red‐labeled; red = dead cells, PI stained. Scale bar = 100 µm. (C) Quantification of T‐cell mediated tumor killing upon exposure to CD3xHER2 bsAbs in the presence or absence of IFNγ neutralizing antibody as shown in (B). Graphs show fold change in HER2 + and HER2 − cell numbers at the indicated time points relative to 6 h. The graph represents three independent experiments, each performed with 2 co‐cultures, with each co‐culture generated in an individual well. Mean ± SEM is shown. Two‐way ANOVA followed by Dunnett's multiple comparisons test was performed. *** p < 0.001. ns = non‐significant. (D) Maximum projections of 3D confocal imaging of HER2 + and HER2 − tumoroids co‐printed as described in (A) and treated with or without 10 µg/mL TNFα neutralizing antibody, or a combination of 10 µg/mL IFNγ and 10 µg/mL TNFα neutralizing antibodies. A co‐culture without bsAb treatment serves as a negative control. Images were taken over a 50 h time course ( note : T‐cell signals were excluded from these images). Green = BT474 WT, Lifeact‐EGFP‐transduced; blue = BT474 HER2 KO, Hoechst33342‐labeled; red = dead cells, PI stained. Scale bar = 100 µm. (E) Quantification of T‐cell‐mediated tumor killing over time as shown in (D). Graphs show fold change in HER2 + and HER2 − cell numbers at the indicated time points relative to 0 h. The graph represents two independent experiments, each performed with 1–2 co‐cultures, with each co‐culture generated in an individual well. Mean ± SEM is shown. Two‐way ANOVA followed by Dunnett's multiple comparisons test was performed. * p < 0.05; ** p < 0.01; *** p < 0.001 compared with CTR. ns = non‐significant.

    Journal: European Journal of Immunology

    Article Title: CD3xHER2 bsAb‐Mediated Activation of Resting T‐cells at HER2 Positive Tumor Clusters Is Sufficient to Trigger Bystander Eradication of Distant HER2 Negative Clusters Through IFNγ and TNFα

    doi: 10.1002/eji.202451589

    Figure Lengend Snippet: Neutralizing antibodies against IFNγ and TNFα block bystander killing of HER2‐ tumoroids after T‐cell‐mediated killing of HER2+ tumoroids via bsAbs. (A) Schematic representation of the experimental design based on Model 3 (see Figure ), with or without neutralizing antibodies. (B) Maximum projections of 3D confocal imaging showing HER2 + and HER2 − tumoroids co‐printed as described in (A) and treated with or without the indicated concentrations of an IFNγ neutralizing antibody. Images were taken 6, 18, and 30 h after exposure to CD3xHER2 bsAbs. Green = BT474 WT, Lifeact‐EGFP‐transduced; blue = BT474 HER2 KO, Hoechst33342‐labeled; yellow = T‐cells, CellTracker Deep Red‐labeled; red = dead cells, PI stained. Scale bar = 100 µm. (C) Quantification of T‐cell mediated tumor killing upon exposure to CD3xHER2 bsAbs in the presence or absence of IFNγ neutralizing antibody as shown in (B). Graphs show fold change in HER2 + and HER2 − cell numbers at the indicated time points relative to 6 h. The graph represents three independent experiments, each performed with 2 co‐cultures, with each co‐culture generated in an individual well. Mean ± SEM is shown. Two‐way ANOVA followed by Dunnett's multiple comparisons test was performed. *** p < 0.001. ns = non‐significant. (D) Maximum projections of 3D confocal imaging of HER2 + and HER2 − tumoroids co‐printed as described in (A) and treated with or without 10 µg/mL TNFα neutralizing antibody, or a combination of 10 µg/mL IFNγ and 10 µg/mL TNFα neutralizing antibodies. A co‐culture without bsAb treatment serves as a negative control. Images were taken over a 50 h time course ( note : T‐cell signals were excluded from these images). Green = BT474 WT, Lifeact‐EGFP‐transduced; blue = BT474 HER2 KO, Hoechst33342‐labeled; red = dead cells, PI stained. Scale bar = 100 µm. (E) Quantification of T‐cell‐mediated tumor killing over time as shown in (D). Graphs show fold change in HER2 + and HER2 − cell numbers at the indicated time points relative to 0 h. The graph represents two independent experiments, each performed with 1–2 co‐cultures, with each co‐culture generated in an individual well. Mean ± SEM is shown. Two‐way ANOVA followed by Dunnett's multiple comparisons test was performed. * p < 0.05; ** p < 0.01; *** p < 0.001 compared with CTR. ns = non‐significant.

    Article Snippet: Cytokines were depleted from CM using neutralizing Abs against IFNγ (AF‐285‐NA, R&D systems, RRID: AB_354445) or TNFα (MAB225‐100, R&D systems, RRID: AB_2204150).

    Techniques: Blocking Assay, Imaging, Labeling, Staining, Co-Culture Assay, Generated, Negative Control

    SEB-stimulated T cells secrete inflammatory cytokines and induce alterations of both permeability and morphology in Caco-2 cells. (A–E) Human T cells isolated from the peripheral blood of healthy donors (HD) were unstimulated (T) or stimulated for different times with 0.1 μg ml -1 SEB (T SEB). IFN-γ (A) , IL-6 (B) , TNF-α (C) , IL-17A (D) and IL-22 (E) levels in culture supernatant were measured by ELISA. Data show the mean ± SEM (standard error of the mean) of different HD (n = 5). Statistical significance was calculated by one-way ANOVA. Mean (pg ml -1 ) ± SEM values (n = 5): IFN-γ (A) 24 h, T = 20.74 ± 17.93, T SEB = 708.3 ± 486.1; 48 h, T = 5.29 ± 3.29, T SEB = 1505 ± 698; 72 h, T = 4.34 ± 2.52, T SEB = 1983 ± 903.5; IL-6 (B) 24 h, T = 86.96 ± 49.57, T SEB = 408.7 ± 267.0; 48 h, T = 74.8 ± 51.27, T SEB = 516.2 ± 243.7; 72 h, T = 129.4 ± 100.3, T SEB = 812.7 ± 394.7; TNF-α (C) 24 h, T = 38.55 ± 35.91, T SEB = 1522 ± 587.7; 48 h, T = 35.81 ± 33.88, T SEB = 1792 ± 712.7; 72 h, T = 69.56 ± 41.9, T SEB = 2285 ± 767.3; IL-17A (D) 24 h, T = 0 ± 0, T SEB = 218.8 ± 96.34; 48 h, T = 3.51 ± 3.51, T SEB = 1448 ± 609.3; 72 h, T = 9.07 ± 9.07, T SEB = 2252 ± 944.3; IL-22 (E) 24h, T = 28.82 ± 10.46, T SEB = 1665 ± 758; 48 h, T = 48.88 ± 14.99, T SEB = 4852 ± 1363; 72 h, T = 55.07 ± 14.38, T SEB = 7929 ± 2055. (F) Caco-2 cells were cultured in 24 trans-well plates for 72 h with medium alone (CTR) or T cells (T), or T cells stimulated with 0.1 μg ml -1 SEB (T SEB). Cell death was analysed by flow cytometry by quantifying the ability of cells to incorporate propidium iodide (PI). Data show the mean % PI positive cells ± SEM of different HD (n = 5). Statistical significance was calculated by one-way ANOVA. Mean (%) ± SEM values (n = 5); CTR = 14.06 ± 3.65, T = 14.38 ± 4.38, T SEB = 24.22 ± 4.55. (*) p < 0.05, (**) p < 0.01, (****) p < 0.0001, NS = not significant. (G) Caco-2 cells were cultured in 24 trans-well plates for the indicated times with medium alone (CTR) or T cells (T), or T cells stimulated with 0.1 μg ml -1 SEB (T SEB) for 24, 48 or 72 h. After fixing and permeabilization, F-actin was stained with 594-conjugated phalloidin (red) and analysed by confocal microscopy. Nucleus was stained with DAPI (blue). The white arrows indicate the tight parallel bundles or stress-like fibers. Scale bar = 20 μm.

    Journal: Frontiers in Immunology

    Article Title: Targeting staphylococcal enterotoxin B binding to CD28 as a new strategy for dampening superantigen-mediated intestinal epithelial barrier dysfunctions

    doi: 10.3389/fimmu.2024.1365074

    Figure Lengend Snippet: SEB-stimulated T cells secrete inflammatory cytokines and induce alterations of both permeability and morphology in Caco-2 cells. (A–E) Human T cells isolated from the peripheral blood of healthy donors (HD) were unstimulated (T) or stimulated for different times with 0.1 μg ml -1 SEB (T SEB). IFN-γ (A) , IL-6 (B) , TNF-α (C) , IL-17A (D) and IL-22 (E) levels in culture supernatant were measured by ELISA. Data show the mean ± SEM (standard error of the mean) of different HD (n = 5). Statistical significance was calculated by one-way ANOVA. Mean (pg ml -1 ) ± SEM values (n = 5): IFN-γ (A) 24 h, T = 20.74 ± 17.93, T SEB = 708.3 ± 486.1; 48 h, T = 5.29 ± 3.29, T SEB = 1505 ± 698; 72 h, T = 4.34 ± 2.52, T SEB = 1983 ± 903.5; IL-6 (B) 24 h, T = 86.96 ± 49.57, T SEB = 408.7 ± 267.0; 48 h, T = 74.8 ± 51.27, T SEB = 516.2 ± 243.7; 72 h, T = 129.4 ± 100.3, T SEB = 812.7 ± 394.7; TNF-α (C) 24 h, T = 38.55 ± 35.91, T SEB = 1522 ± 587.7; 48 h, T = 35.81 ± 33.88, T SEB = 1792 ± 712.7; 72 h, T = 69.56 ± 41.9, T SEB = 2285 ± 767.3; IL-17A (D) 24 h, T = 0 ± 0, T SEB = 218.8 ± 96.34; 48 h, T = 3.51 ± 3.51, T SEB = 1448 ± 609.3; 72 h, T = 9.07 ± 9.07, T SEB = 2252 ± 944.3; IL-22 (E) 24h, T = 28.82 ± 10.46, T SEB = 1665 ± 758; 48 h, T = 48.88 ± 14.99, T SEB = 4852 ± 1363; 72 h, T = 55.07 ± 14.38, T SEB = 7929 ± 2055. (F) Caco-2 cells were cultured in 24 trans-well plates for 72 h with medium alone (CTR) or T cells (T), or T cells stimulated with 0.1 μg ml -1 SEB (T SEB). Cell death was analysed by flow cytometry by quantifying the ability of cells to incorporate propidium iodide (PI). Data show the mean % PI positive cells ± SEM of different HD (n = 5). Statistical significance was calculated by one-way ANOVA. Mean (%) ± SEM values (n = 5); CTR = 14.06 ± 3.65, T = 14.38 ± 4.38, T SEB = 24.22 ± 4.55. (*) p < 0.05, (**) p < 0.01, (****) p < 0.0001, NS = not significant. (G) Caco-2 cells were cultured in 24 trans-well plates for the indicated times with medium alone (CTR) or T cells (T), or T cells stimulated with 0.1 μg ml -1 SEB (T SEB) for 24, 48 or 72 h. After fixing and permeabilization, F-actin was stained with 594-conjugated phalloidin (red) and analysed by confocal microscopy. Nucleus was stained with DAPI (blue). The white arrows indicate the tight parallel bundles or stress-like fibers. Scale bar = 20 μm.

    Article Snippet: Inflammatory cytokines neutralising Abs (NAbs) used were mouse anti-TNF-α (#MAB610), anti-IFN-γ (#MAB2852), anti-IL6 (#MAB206), anti-IL-17A (#MAB317) and anti-IL-22 (#MAB7822) (R&D Systems, USA).

    Techniques: Permeability, Isolation, Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry, Staining, Confocal Microscopy

    Inflammatory cytokines produced by SEB-stimulated T cells mediate Caco-2 cell dysfunctions. (A) Caco-2 cells were cultured in 24 trans-well plates for 72 h with medium alone (CTR) or T cells (T), or T cells stimulated with 0.1 μg ml -1 SEB (T SEB) in the presence of 2.5 μg ml -1 isotype control or 2.5 μg ml -1 anti-IL-6, anti-TNF-α, anti-IFN-γ, anti-IL-17A Abs neutralising Abs (NAbs). After fixing and permeabilization Caco-2 cells were stained with anti-ZO-1 followed Alexa Fluor 647 Abs (white), anti-E-CAD followed by Alexa Fluor 488 Abs (green), and F-actin was stained with Alexa Fluor 594 phalloidin (red). Nucleus was stained with DPI (blue). Scale bar = 20 μm. (B, C) ZO-1 and CDH1 (B) , and SNAIL-1, TWIST-1 and ZEB-1 (C) mRNA levels in Caco-2 cells cultured for 72 h with medium alone or with T cells from HD (n =3) or SEB-stimulated T cells (T SEB) in the presence of isotype control or cytokine NAbs. Values, normalized to GAPDH, were expressed as fold inductions (F.I.) over the basal level of Caco-2 cultured with medium alone. Data show the mean F.I. ± SEM and statistical significance was calculated by one-way ANOVA. Mean (F.I.) ± SEM values (n = 3); (B) ZO-1, T = 1.05 ± 0.09, T SEB = 0.34 ± 0.15, T SEB NAbs = 1.57 ± 0.18; CDH1, T = 0.73 ± 0.10, T SEB = 0.28 ± 0.07, T SEB NAbs = 0.65 ± 0.09; (C) SNAIL-1, T = 1.73 ± 0.40, T SEB = 9.04 ± 2.18, T SEB NAbs = 3.30 ± 0.87; TWIST-1, T = 1.75 ± 0.80, T SEB = 6.60 ± 0.76, T SEB NAbs = 1.13 ± 0.25; ZEB-1, T = 1.88 ± 1.30, T SEB = 18.13 ± 2.91, T SEB NAbs = 4.78 ± 2.53. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001. NS, not significant.

    Journal: Frontiers in Immunology

    Article Title: Targeting staphylococcal enterotoxin B binding to CD28 as a new strategy for dampening superantigen-mediated intestinal epithelial barrier dysfunctions

    doi: 10.3389/fimmu.2024.1365074

    Figure Lengend Snippet: Inflammatory cytokines produced by SEB-stimulated T cells mediate Caco-2 cell dysfunctions. (A) Caco-2 cells were cultured in 24 trans-well plates for 72 h with medium alone (CTR) or T cells (T), or T cells stimulated with 0.1 μg ml -1 SEB (T SEB) in the presence of 2.5 μg ml -1 isotype control or 2.5 μg ml -1 anti-IL-6, anti-TNF-α, anti-IFN-γ, anti-IL-17A Abs neutralising Abs (NAbs). After fixing and permeabilization Caco-2 cells were stained with anti-ZO-1 followed Alexa Fluor 647 Abs (white), anti-E-CAD followed by Alexa Fluor 488 Abs (green), and F-actin was stained with Alexa Fluor 594 phalloidin (red). Nucleus was stained with DPI (blue). Scale bar = 20 μm. (B, C) ZO-1 and CDH1 (B) , and SNAIL-1, TWIST-1 and ZEB-1 (C) mRNA levels in Caco-2 cells cultured for 72 h with medium alone or with T cells from HD (n =3) or SEB-stimulated T cells (T SEB) in the presence of isotype control or cytokine NAbs. Values, normalized to GAPDH, were expressed as fold inductions (F.I.) over the basal level of Caco-2 cultured with medium alone. Data show the mean F.I. ± SEM and statistical significance was calculated by one-way ANOVA. Mean (F.I.) ± SEM values (n = 3); (B) ZO-1, T = 1.05 ± 0.09, T SEB = 0.34 ± 0.15, T SEB NAbs = 1.57 ± 0.18; CDH1, T = 0.73 ± 0.10, T SEB = 0.28 ± 0.07, T SEB NAbs = 0.65 ± 0.09; (C) SNAIL-1, T = 1.73 ± 0.40, T SEB = 9.04 ± 2.18, T SEB NAbs = 3.30 ± 0.87; TWIST-1, T = 1.75 ± 0.80, T SEB = 6.60 ± 0.76, T SEB NAbs = 1.13 ± 0.25; ZEB-1, T = 1.88 ± 1.30, T SEB = 18.13 ± 2.91, T SEB NAbs = 4.78 ± 2.53. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, (****) p < 0.0001. NS, not significant.

    Article Snippet: Inflammatory cytokines neutralising Abs (NAbs) used were mouse anti-TNF-α (#MAB610), anti-IFN-γ (#MAB2852), anti-IL6 (#MAB206), anti-IL-17A (#MAB317) and anti-IL-22 (#MAB7822) (R&D Systems, USA).

    Techniques: Produced, Cell Culture, Control, Staining