1d11 pan-tgfβ neutralizing antibody clone 1d11.16.8  (Bio X Cell)

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Elevated TGFβ signaling contributes to ocular anterior segment dysgenesis in Col4a1 mutant mice

doi: 10.1016/j.matbio.2022.05.001

Figure Lengend Snippet: (A) Schematic illustration of the 1D11 administration paradigm for histological analysis. (B) Representative images of H&E stained sections (left) showing whole eyes (top panels) and corneas (lower panels) from E18.5 mice and quantification graph (right) showing increased corneal stromal thickness in Col4a1+/G1344D mice treated with 1D11 compared to those that received the control IgG1 antibody. Scale bars = 200 μm (top) and 50 μm (bottom). n = 20 and 24 corneas from IgG1- and 1D11-treated Col4a1+/+ mice, and 24 and 30 corneas from IgG1- and 1D11-treated Col4a1+/G1344D mice, respectively. (C) Schematic illustration of the 1D11 administration paradigm for qPCR analyses. (D) qPCR analyses revealed that 1D11 treatment partially prevented the increased expression of TGFβ target genes in anterior segments from P0 Col4a1+/G1344D mice compared to their IgG1-treated counterparts. n = 5–6 samples per genotype. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001, two-way ANOVA and Tukey’s multiple comparison test.

Article Snippet: Timed-pregnant B6 females crossed with Col4a1 +/ G1344D males were injected intraperitoneally with the 1D11 pan-TGFβ neutralizing antibody (clone 1D11.16.8, BioXCell, West Lebanon, NH) or IgG1 isotype control antibody (clone MOPC-21, BioXCell) diluted in inVivoPure Dilution Buffer (pH 7.0, BioXCell) (20 mg/kg) every other day from E8.5 to E16.5 and animals were harvested at E18.5 or P0 for histological and molecular analyses, respectively.

Techniques: Staining, Control, Expressing, Comparison

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: Elevated TGFβ signaling contributes to ocular anterior segment dysgenesis in Col4a1 mutant mice

doi: 10.1016/j.matbio.2022.05.001

Figure Lengend Snippet: (A) Schematic illustration of the 1D11 administration paradigm for histological analysis. (B) Representative images of H&E stained sections (left) showing whole eyes (top panels) and corneas (lower panels) from E18.5 mice and quantification graph (right) showing increased corneal stromal thickness in Col4a1+/G1344D mice treated with 1D11 compared to those that received the control IgG1 antibody. Scale bars = 200 μm (top) and 50 μm (bottom). n = 20 and 24 corneas from IgG1- and 1D11-treated Col4a1+/+ mice, and 24 and 30 corneas from IgG1- and 1D11-treated Col4a1+/G1344D mice, respectively. (C) Schematic illustration of the 1D11 administration paradigm for qPCR analyses. (D) qPCR analyses revealed that 1D11 treatment partially prevented the increased expression of TGFβ target genes in anterior segments from P0 Col4a1+/G1344D mice compared to their IgG1-treated counterparts. n = 5–6 samples per genotype. Data are presented as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001, two-way ANOVA and Tukey’s multiple comparison test.

Article Snippet: 1D11 TGFβ neutralizing antibody treatment Timed-pregnant B6 females crossed with Col4a1 +/ G1344D males were injected intraperitoneally with the 1D11 pan-TGFβ neutralizing antibody (clone 1D11.16.8, BioXCell, West Lebanon, NH) or IgG1 isotype control antibody (clone MOPC-21, BioXCell) diluted in inVivoPure Dilution Buffer (pH 7.0, BioXCell) (20 mg/kg) every other day from E8.5 to E16.5 and animals were harvested at E18.5 or P0 for histological and molecular analyses, respectively.

Techniques: Staining, Control, Expressing, Comparison

Journal: Cancer immunology research

Article Title: Activin A promotes regulatory T cell–mediated immunosuppression in irradiated breast cancer

doi: 10.1158/2326-6066.CIR-19-0305

Figure Lengend Snippet: Naïve CD4+ T cells isolated from spleens of Balb/c mice were cocultured with either untreated or 6 Gy-irradiated (B) TSA or (C) 4T1 breast cancer cells for 5 days. When applicable, blockade of activin A using follistatin-288 (FS) and/or TGFβ using 1D11 was performed. Percentages of naïve CD4+ T cells converted into Tregs are expressed as FoxP3+CD25+ of CD4+ live T cells (cell/mL)± SD; (A) Gating strategy. one-way ANOVA followed by Tukey post hoc comparisons; *P < 0.05; **P < 0.01; *** P < 0.001; and ****P < 0.0001. Experiment was done in duplicate.

Article Snippet: 1D11, a pan-isoform, TGFβ-neutralizing mouse monoclonal antibody (mAb)(200 μg/mouse; cat#BE0057), monoclonal anti-mouse CTLA-4 clone 9H10 (200 μg/mouse; cat#BE0131), and monoclonal anti-mouse PD-1 clone RMP1–14 (200 μg/mouse; cat#BE0146) were purchased from BioXCell.

Techniques: Isolation, Irradiation

Journal: Cancer immunology research

Article Title: Activin A promotes regulatory T cell–mediated immunosuppression in irradiated breast cancer

doi: 10.1158/2326-6066.CIR-19-0305

Figure Lengend Snippet: (A) In vivo experiment scheme. Syngeneic Balb/C mice received s.c. injection 4T1 or TSA cells. The pan TGFβ neutralizing antibody 1D11 was given every other day starting day 12. Radiation was selectively given to s.c. tumors in five daily fractions of 6 Gy (5×6Gy) on days (d)13, 14, 15, 16, and 17. On day 22, tumors were collected to analyze tumor-infiltrating Tregs, defined as CD25+FoxP3+ in CD45+CD4+ live cells. (B) Flow cytometry gating strategy. Percentages of FoxP3+CD25+ in CD4+ live T cells±SD in (C) TSA and (D) 4T1 tumors. Each dot represents one animal. One-way ANOVA followed by Tukey post hoc comparisons; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Experiment was done in duplicate with n=3 per group.

Article Snippet: 1D11, a pan-isoform, TGFβ-neutralizing mouse monoclonal antibody (mAb)(200 μg/mouse; cat#BE0057), monoclonal anti-mouse CTLA-4 clone 9H10 (200 μg/mouse; cat#BE0131), and monoclonal anti-mouse PD-1 clone RMP1–14 (200 μg/mouse; cat#BE0146) were purchased from BioXCell.

Techniques: In Vivo, Injection, Flow Cytometry

Journal: Cancer immunology research

Article Title: Activin A promotes regulatory T cell–mediated immunosuppression in irradiated breast cancer

doi: 10.1158/2326-6066.CIR-19-0305

Figure Lengend Snippet: (A) Murine breast cancer cells 4T1, TSA, and 67NR and (B) human breast cancer cells MDA-MB-231, 4175-TR, and MCF-7 were treated with the pan-isoform neutralizing TGFβ (1D11) antibody for 24hrs. and irradiated with various doses, as indicated. Twenty-four hours following radiation, secreted activin A was measured in supernatants by ELISA. Results are expressed in pg/mL±SD for 100,000 live cells. One-way ANOVA followed by Tukey post hoc comparisons; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Experiment was done in triplicate, with n=3/group.

Article Snippet: 1D11, a pan-isoform, TGFβ-neutralizing mouse monoclonal antibody (mAb)(200 μg/mouse; cat#BE0057), monoclonal anti-mouse CTLA-4 clone 9H10 (200 μg/mouse; cat#BE0131), and monoclonal anti-mouse PD-1 clone RMP1–14 (200 μg/mouse; cat#BE0146) were purchased from BioXCell.

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay

Journal: Cancer immunology research

Article Title: Activin A promotes regulatory T cell–mediated immunosuppression in irradiated breast cancer

doi: 10.1158/2326-6066.CIR-19-0305

Figure Lengend Snippet: (A) Experiment scheme. 4T1shInhba or 4T1shNS were injected s.c. into Balb/C mice on day 0. On day 8, mice were fed with doxycycline to silence the expression of the Inhba gene in 4T1 cells. Starting on day 12, 1D11 was given every other day. Radiation was selectively given to s.c. tumors in five daily fractions of 6 Gy (5×6Gy) on days (d)13, 14, 15, 16, and 17. When applicable, anti-CTLA-4 was administered on day 17, 20, and 23 and anti-PD-1 was given on days 18, 22, 26, and 30. (B) Individual and (C) mean tumor growth and survival of mice bearing 4T1shNS or 4T1shInhba tumors. (B-C) Tumor growth: n=10/group; ****p<0.0001; two-way ANOVA followed by Tukey post hoc comparisons. Survival: n=10/group; n.s. non-significant; *p<0.05; ****p<0.0001; Log-rank (Mantel-Cox) test. (D) Tumor-free survival and survival of mice from 4T1shInhba+RT+1D11 (n=5; dashed purple), 4T1shInhba+RT+1D11+anti-PD-1 (n=8; dashed orange), and 4T1shInhba+RT+1D11+anti-CTLA4 (n=10; green) that cleared tumors and were rechallenged at day 100 with a tumorigenic inoculum of 4T1 cells, together with a group of naïve mice (n=5; grey). *p<0.05; **p<0.01; ***p<0.001; Log-rank (Mantel-Cox) test.

Article Snippet: 1D11, a pan-isoform, TGFβ-neutralizing mouse monoclonal antibody (mAb)(200 μg/mouse; cat#BE0057), monoclonal anti-mouse CTLA-4 clone 9H10 (200 μg/mouse; cat#BE0131), and monoclonal anti-mouse PD-1 clone RMP1–14 (200 μg/mouse; cat#BE0146) were purchased from BioXCell.

Techniques: Injection, Expressing

Journal: Cancer immunology research

Article Title: Activin A promotes regulatory T cell–mediated immunosuppression in irradiated breast cancer

doi: 10.1158/2326-6066.CIR-19-0305

Figure Lengend Snippet: (A) Experiment scheme. 4T1shInhba or 4T1shNS were injected s.c. into Balb/C mice on day 0. On day 8, mice were fed with doxycycline to silence the expression of the Inhba gene in 4T1 cells. Starting on day 12, 1D11 was given every other day. Radiation was selectively given to s.c. tumors in five daily fractions of 6 Gy (5×6Gy) on days (d)13, 14, 15, 16, and 17. (B-D) On day 22, tumors and tumor-draining lymph nodes (TDLNs) were collected for immune analysis in 4T1 tumor-bearing animals. (B) Frequency of Tregs [FoxP3+CD25+ in live CD45+ CD4+ T cells (cell/μL ± SD)] in 4T1 tumors (n=3/group), (C) IFNγ production by TDLN cells in response to the CD8+ T-cell epitope AH1 (full circles) or control peptide MCMV (open circles). Horizontal lines indicate the mean of antigen-specific (solid lines) or control (dashed lines) IFNγ concentration and (D) 4T1 tumor-infiltrating CD8:Treg ratios. (B-D) Each symbol represents one animal. One-way ANOVA followed by Tukey post hoc comparisons; n.s. non-significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Experiment was done in duplicate with n=3 per group. (E) Mean and (F) individual tumor growth and (G) survival of mice bearing 4T1shNS or 4T1shInhba tumors treated with RT and/or TGFβ blockading antibody 1D11. (E-F) Two-way ANOVA followed by Tukey post hoc comparisons; n.s. non-significant; *p<0.05. Experiment was done in duplicate with n=7/group. (G) Survival by Log-rank (Mantel-Cox) test; **p<0.01. Experiment was done in duplicate with n=7/group.

Article Snippet: 1D11, a pan-isoform, TGFβ-neutralizing mouse monoclonal antibody (mAb)(200 μg/mouse; cat#BE0057), monoclonal anti-mouse CTLA-4 clone 9H10 (200 μg/mouse; cat#BE0131), and monoclonal anti-mouse PD-1 clone RMP1–14 (200 μg/mouse; cat#BE0146) were purchased from BioXCell.

Techniques: Injection, Expressing, Control, Concentration Assay

Journal: Cancer immunology research

Article Title: Activin A promotes regulatory T cell–mediated immunosuppression in irradiated breast cancer

doi: 10.1158/2326-6066.CIR-19-0305

Figure Lengend Snippet: (A) Experiment scheme. Half of the mice with tetracycline-inducible Inhba in TSA (TSAKI Inhba) in the primary tumor and parental TSA in the secondary tumor were given doxycycline. In some mice, five daily fractions of 6Gy were selectively administered to the primary tumor combined with TGFβ blockade. On day (d)22, three mice per group were euthanized to harvest tumors and TDLNs for immune analysis. (B) Percentages of FoxP3+CD25+ in live CD45+CD4+ T cells (%±SD) in TSA tumors (n=3/group). (C) IFNγ secretion by TDLN cells in response to the CD8+ T-cell epitope AH1 (full circles) or control peptide MCMV (open circles) (n=3/group). (B-C) Each symbol represents one animal. One-way ANOVA followed by Tukey post hoc comparisons; n.s. non-significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. (D) Mean tumor growth of the irradiated and (E) abscopal tumors in mice treated with isotype (dashed back line), isotype+doxycycline (grey line), 1D11 (dashed green), 1D11+doxycycline (orange line), RT+isotype (blue line), RT+isotype+doxycycline (dotted back line), RT+1D11 (red line), or RT+1D11+doxycycline (dashed purple line). (D-E) Longitudinal primary tumor growth data were analyzed using two-way ANOVA to assess overall group differences followed by Tukey post hoc comparisons; n.s. non-significant, ****p<0.0001; comparison of abscopal tumor growth, two-way ANOVA followed by Tukey’s post hoc comparisons; non-significant; #p<0.05; ###p<0.001; ####p<0.0001. Duplicate experiment with n=7/group.

Article Snippet: 1D11, a pan-isoform, TGFβ-neutralizing mouse monoclonal antibody (mAb)(200 μg/mouse; cat#BE0057), monoclonal anti-mouse CTLA-4 clone 9H10 (200 μg/mouse; cat#BE0131), and monoclonal anti-mouse PD-1 clone RMP1–14 (200 μg/mouse; cat#BE0146) were purchased from BioXCell.

Techniques: Control, Irradiation, Comparison

Journal: Cancer immunology research

Article Title: Activin A promotes regulatory T cell–mediated immunosuppression in irradiated breast cancer

doi: 10.1158/2326-6066.CIR-19-0305

Figure Lengend Snippet: Naïve CD4+ T cells isolated from spleens of Balb/c mice were cocultured with either untreated or 6 Gy-irradiated (B) TSA or (C) 4T1 breast cancer cells for 5 days. When applicable, blockade of activin A using follistatin-288 (FS) and/or TGFβ using 1D11 was performed. Percentages of naïve CD4+ T cells converted into Tregs are expressed as FoxP3+CD25+ of CD4+ live T cells (cell/mL)± SD; (A) Gating strategy. one-way ANOVA followed by Tukey post hoc comparisons; *P < 0.05; **P < 0.01; *** P < 0.001; and ****P < 0.0001. Experiment was done in duplicate.

Article Snippet: 1D11, a pan-isoform, TGFβ-neutralizing mouse monoclonal antibody (mAb)(200 μg/mouse; cat#BE0057), monoclonal anti-mouse CTLA-4 clone 9H10 (200 μg/mouse; cat#BE0131), and monoclonal anti-mouse PD-1 clone RMP1–14 (200 μg/mouse; cat#BE0146) were purchased from BioXCell.

Techniques: Isolation, Irradiation

Journal: Cancer immunology research

Article Title: Activin A promotes regulatory T cell–mediated immunosuppression in irradiated breast cancer

doi: 10.1158/2326-6066.CIR-19-0305

Figure Lengend Snippet: (A) In vivo experiment scheme. Syngeneic Balb/C mice received s.c. injection 4T1 or TSA cells. The pan TGFβ neutralizing antibody 1D11 was given every other day starting day 12. Radiation was selectively given to s.c. tumors in five daily fractions of 6 Gy (5×6Gy) on days (d)13, 14, 15, 16, and 17. On day 22, tumors were collected to analyze tumor-infiltrating Tregs, defined as CD25+FoxP3+ in CD45+CD4+ live cells. (B) Flow cytometry gating strategy. Percentages of FoxP3+CD25+ in CD4+ live T cells±SD in (C) TSA and (D) 4T1 tumors. Each dot represents one animal. One-way ANOVA followed by Tukey post hoc comparisons; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Experiment was done in duplicate with n=3 per group.

Article Snippet: 1D11, a pan-isoform, TGFβ-neutralizing mouse monoclonal antibody (mAb)(200 μg/mouse; cat#BE0057), monoclonal anti-mouse CTLA-4 clone 9H10 (200 μg/mouse; cat#BE0131), and monoclonal anti-mouse PD-1 clone RMP1–14 (200 μg/mouse; cat#BE0146) were purchased from BioXCell.

Techniques: In Vivo, Injection, Flow Cytometry

Journal: Cancer immunology research

Article Title: Activin A promotes regulatory T cell–mediated immunosuppression in irradiated breast cancer

doi: 10.1158/2326-6066.CIR-19-0305

Figure Lengend Snippet: (A) Murine breast cancer cells 4T1, TSA, and 67NR and (B) human breast cancer cells MDA-MB-231, 4175-TR, and MCF-7 were treated with the pan-isoform neutralizing TGFβ (1D11) antibody for 24hrs. and irradiated with various doses, as indicated. Twenty-four hours following radiation, secreted activin A was measured in supernatants by ELISA. Results are expressed in pg/mL±SD for 100,000 live cells. One-way ANOVA followed by Tukey post hoc comparisons; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Experiment was done in triplicate, with n=3/group.

Article Snippet: 1D11, a pan-isoform, TGFβ-neutralizing mouse monoclonal antibody (mAb)(200 μg/mouse; cat#BE0057), monoclonal anti-mouse CTLA-4 clone 9H10 (200 μg/mouse; cat#BE0131), and monoclonal anti-mouse PD-1 clone RMP1–14 (200 μg/mouse; cat#BE0146) were purchased from BioXCell.

Techniques: Irradiation, Enzyme-linked Immunosorbent Assay

Journal: Cancer immunology research

Article Title: Activin A promotes regulatory T cell–mediated immunosuppression in irradiated breast cancer

doi: 10.1158/2326-6066.CIR-19-0305

Figure Lengend Snippet: (A) Experiment scheme. 4T1shInhba or 4T1shNS were injected s.c. into Balb/C mice on day 0. On day 8, mice were fed with doxycycline to silence the expression of the Inhba gene in 4T1 cells. Starting on day 12, 1D11 was given every other day. Radiation was selectively given to s.c. tumors in five daily fractions of 6 Gy (5×6Gy) on days (d)13, 14, 15, 16, and 17. (B-D) On day 22, tumors and tumor-draining lymph nodes (TDLNs) were collected for immune analysis in 4T1 tumor-bearing animals. (B) Frequency of Tregs [FoxP3+CD25+ in live CD45+ CD4+ T cells (cell/μL ± SD)] in 4T1 tumors (n=3/group), (C) IFNγ production by TDLN cells in response to the CD8+ T-cell epitope AH1 (full circles) or control peptide MCMV (open circles). Horizontal lines indicate the mean of antigen-specific (solid lines) or control (dashed lines) IFNγ concentration and (D) 4T1 tumor-infiltrating CD8:Treg ratios. (B-D) Each symbol represents one animal. One-way ANOVA followed by Tukey post hoc comparisons; n.s. non-significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Experiment was done in duplicate with n=3 per group. (E) Mean and (F) individual tumor growth and (G) survival of mice bearing 4T1shNS or 4T1shInhba tumors treated with RT and/or TGFβ blockading antibody 1D11. (E-F) Two-way ANOVA followed by Tukey post hoc comparisons; n.s. non-significant; *p<0.05. Experiment was done in duplicate with n=7/group. (G) Survival by Log-rank (Mantel-Cox) test; **p<0.01. Experiment was done in duplicate with n=7/group.

Article Snippet: 1D11, a pan-isoform, TGFβ-neutralizing mouse monoclonal antibody (mAb)(200 μg/mouse; cat#BE0057), monoclonal anti-mouse CTLA-4 clone 9H10 (200 μg/mouse; cat#BE0131), and monoclonal anti-mouse PD-1 clone RMP1–14 (200 μg/mouse; cat#BE0146) were purchased from BioXCell.

Techniques: Injection, Expressing, Concentration Assay

Journal: Cancer immunology research

Article Title: Activin A promotes regulatory T cell–mediated immunosuppression in irradiated breast cancer

doi: 10.1158/2326-6066.CIR-19-0305

Figure Lengend Snippet: (A) Experiment scheme. Half of the mice with tetracycline-inducible Inhba in TSA (TSAKI Inhba) in the primary tumor and parental TSA in the secondary tumor were given doxycycline. In some mice, five daily fractions of 6Gy were selectively administered to the primary tumor combined with TGFβ blockade. On day (d)22, three mice per group were euthanized to harvest tumors and TDLNs for immune analysis. (B) Percentages of FoxP3+CD25+ in live CD45+CD4+ T cells (%±SD) in TSA tumors (n=3/group). (C) IFNγ secretion by TDLN cells in response to the CD8+ T-cell epitope AH1 (full circles) or control peptide MCMV (open circles) (n=3/group). (B-C) Each symbol represents one animal. One-way ANOVA followed by Tukey post hoc comparisons; n.s. non-significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. (D) Mean tumor growth of the irradiated and (E) abscopal tumors in mice treated with isotype (dashed back line), isotype+doxycycline (grey line), 1D11 (dashed green), 1D11+doxycycline (orange line), RT+isotype (blue line), RT+isotype+doxycycline (dotted back line), RT+1D11 (red line), or RT+1D11+doxycycline (dashed purple line). (D-E) Longitudinal primary tumor growth data were analyzed using two-way ANOVA to assess overall group differences followed by Tukey post hoc comparisons; n.s. non-significant, ****p<0.0001; comparison of abscopal tumor growth, two-way ANOVA followed by Tukey’s post hoc comparisons; non-significant; #p<0.05; ###p<0.001; ####p<0.0001. Duplicate experiment with n=7/group.

Article Snippet: 1D11, a pan-isoform, TGFβ-neutralizing mouse monoclonal antibody (mAb)(200 μg/mouse; cat#BE0057), monoclonal anti-mouse CTLA-4 clone 9H10 (200 μg/mouse; cat#BE0131), and monoclonal anti-mouse PD-1 clone RMP1–14 (200 μg/mouse; cat#BE0146) were purchased from BioXCell.

Techniques: Irradiation

Journal: Oncotarget

Article Title: Chronic NF-κB activation links COPD and lung cancer through generation of an immunosuppressive microenvironment in the lungs

doi: 10.18632/oncotarget.6562

Figure Lengend Snippet: A. Number of CD45+/CD68+ macrophages identified by flow cytometry in lungs of IKTA mice at baseline (week 0) or after dox treatment (0.1 g/L, n = 3-6 mice per group per time point, *p < 0.05 compared to the baseline). B. Representative photomicrograph of alveolar macrophages collected from IKTA after 3 weeks of dox treatment. C–I. mRNA expression for M1 markers (TNFα, CCL3) and M2 markers (Ym1, Fizz1, Arginase-1, TGFβ, IL-10) by alveolar macrophages isolated from WT and IKTA mice after 3 weeks of continuous treatment with dox. *p < 0.05. J. Total TGFβ concentration in BAL and K. IL-10 concentration in whole lung homogenates collected from WT and IKTA mice after 3 weeks of dox treatment. ND - not detected, *p < 0.05. L. CD11b + lung macrophages (MΦ) collected from WT and IKTA mice after 3 weeks of dox treatment reduce proliferation of CD4 + T cells stimulated by allogeneic bone marrow-derived dendritic cells (DC).

Article Snippet: To investigate contribution of TGFβ, IL-10 and retinoic acids in generation of Foxp3+ Tregs by alveolar macrophages, cells were co-cultured in the presence of pan-TGFβ neutralizing antibodies (10 μg/ml, clone 1D11, Genzyme Corp) [ ], anti-IL-10 antibodies (10 ug/ml, clone JES5-2A5, Biolegend) or IgG1 isotype control antibodies (Biolegend, San Diego, CA, USA) or a RALDH inhibitor, 4-(diethylamino)benzaldehyde (DEAB; 15 μM, Sigma-Aldrich) [ ], added to culture media at day 0.

Techniques: Flow Cytometry, Expressing, Isolation, Concentration Assay, Derivative Assay

Journal: Oncotarget

Article Title: Chronic NF-κB activation links COPD and lung cancer through generation of an immunosuppressive microenvironment in the lungs

doi: 10.18632/oncotarget.6562

Figure Lengend Snippet: A. Representative FACS plots and B. CD4 + /CD25 + /Foxp3 + Tregs generated ex vivo by syngeneic alveolar macrophages (AM) from naive CD4+ − T cells (T cell: AM ratio is 2:1) alone or after treatment with antibodies to TGFβ (10 ug/ml, 1D11) or IL-10 (10 ug/ml, JES5-2A5), or in presence of an inhibitor of retinoic acid synthesis (DEAB, 15 uM). Alveolar macrophages were collected from WT and IKTA mice after 3 weeks of continuous treatment with dox (0.1g/L). Prior to co-culture CD4+ naive T cells were activated through T cell receptors (TCR stimul) using CD3/CD28 antibody cocktail. Analysis of CD4+CD25+Foxp3+ Tregs was performed in triplicate after 3 days of co-culture, *p < 0.05. C–D. mRNA expression of RALDH1 and RALDH2 by alveolar macrophages from WT and IKTA mice after 3 weeks of continuous treatment with dox (0.1g/L). *p < 0.05.

Article Snippet: To investigate contribution of TGFβ, IL-10 and retinoic acids in generation of Foxp3+ Tregs by alveolar macrophages, cells were co-cultured in the presence of pan-TGFβ neutralizing antibodies (10 μg/ml, clone 1D11, Genzyme Corp) [ ], anti-IL-10 antibodies (10 ug/ml, clone JES5-2A5, Biolegend) or IgG1 isotype control antibodies (Biolegend, San Diego, CA, USA) or a RALDH inhibitor, 4-(diethylamino)benzaldehyde (DEAB; 15 μM, Sigma-Aldrich) [ ], added to culture media at day 0.

Techniques: Generated, Ex Vivo, Co-Culture Assay, Expressing