Journal: Scientific Reports

Article Title: Interleukin-10 deficiency impairs regulatory T cell-derived neuropilin-1 functions and promotes Th1 and Th17 immunity

doi: 10.1038/srep24249

Figure Lengend Snippet: ( A ) Implantation of IL-10-deficient mice with B16/F10 cells resulted in enhanced CD4 + Foxp3 + Tregs in the spleen and tumor on day +15, as assessed by flow cytometry. ( B ) Measurement of TGF-β1 in NP-40-processed B16/F10 tumor tissues (described in the Methods section) obtained from WT and IL-10-deficient tumor-bearing mice. The TGF-β1 cytokine was measured using Luminex (Millipore Merck). ( C ) Among CD4 + T cells from IL10 −/− tumor-bearing mice, splenic TGF-β1 + Foxp3 + cells were increased compared with WTB16/F10 mice; however, differences in the tumor tissues and TDLNs were not significant. ( D ) IL10 deficiency down-regulated Nrp-1 protein expression in tumor tissues. Representative photomicrographs of B16/F10 tumors harvested on day 15. Left panel, representative immunohistochemistry images obtained using a microscope with a 20X objective. Right panel, statistical analysis of the mean IOD for tumor cell proliferation using Image-Pro Plus software. Nonlinear regression was performed using GraphPad Prism (San Diego, CA, USA). ( E ) Spleen Nrp-1-expressing CD4 + Foxp3 + T cells from IL10 −/− tumor-bearing mice were decreased compared with WT B16/F10 mice. Panels ( A,B,D,E ) Evaluated using Student’s t-test to determine statistical significance with *P < 0.05, **P < 0.01, ***P < 0.001between experimental groups. The data are expressed as the mean values ± SEM from n = 3–4 mice. Panel ( C ) Evaluated using ANOVA, ***P < 0.001, ±SEM from n = 3–4 mice.

Article Snippet: Similar groups of mice were treated i.v. with either a rabbit anti-mouse TGF-β pan-specific polyclonal rabbit antibody (3 mg/kg; R&D Systems AB-100-NA) or an isotype-specific polyclonal rabbit IgG (3 mg/kg; R&D Systems AB-105-C) .

Techniques: Flow Cytometry, Luminex, Expressing, Immunohistochemistry, Microscopy, Software

Journal: Scientific Reports

Article Title: Interleukin-10 deficiency impairs regulatory T cell-derived neuropilin-1 functions and promotes Th1 and Th17 immunity

doi: 10.1038/srep24249

Figure Lengend Snippet: ( A ) A high-affinity neutralizing anti-Nrp-1 antibody (described in the Methods section) was used to treat WT or IL10 −/− B16/F10 tumor-bearing mice. The B16/F10 tumor mass volume was monitored every three days after implantation. ( B ) The majority of B16/F10 tumor-derived CD4 + CD8a − lymphocytes were increased in the IL10 −/− mice after treatment with the anti-Nrp-1 polyclonal Ab, as assessed by flow cytometry. ( C ) Use of anti-Nrp-1 in IL10 −/− mice with B16/F10 tumors allowed the augmentation of a CD4 + CD69 + -activated T cell population in the tumor microenvironment by flow cytometry. ( D ) Neutralization of Nrp-1 in IL10 −/− mice implanted with B16/F10 tumors augmented CD4 + IL17A + (Th17) T cells in the tumor microenvironment compared with WT mice with B16/F10 tumors alone, as assessed by flow cytometry. ( E , F ) Numbers within quadrants represent the percentages of positive cells for a given marker within the lymphocyte gate . Anti-Nrp-1 augmented TDLN or tumor-derived CD4 + IFN-γ + T cells in WT or IL10 −/− B16/F10 mice based on flow cytometry. The bar graph shows data from a representative experiment (mean ± SEM; N = 3–4 mice per group) in which two separate experiments were performed. *P < 0.05, **P < 0.01, ***P < 0.001; assessed by pairwise multiple comparison procedures (Tukey test) and ANOVA.

Article Snippet: Similar groups of mice were treated i.v. with either a rabbit anti-mouse TGF-β pan-specific polyclonal rabbit antibody (3 mg/kg; R&D Systems AB-100-NA) or an isotype-specific polyclonal rabbit IgG (3 mg/kg; R&D Systems AB-105-C) .

Techniques: Derivative Assay, Flow Cytometry, Neutralization, Marker, Comparison

Journal: Scientific Reports

Article Title: Interleukin-10 deficiency impairs regulatory T cell-derived neuropilin-1 functions and promotes Th1 and Th17 immunity

doi: 10.1038/srep24249

Figure Lengend Snippet: ( A,B ) Measurement of B16/F10 TDLN and tumor-derived CD4 + Foxp3 + Treg cells in IL10 −/− B16/F10 mice or WT B16/F10 mice after treatment with an anti-Nrp-1 polyclonal Ab by flow cytometry. TDLN and tumor tissue samples were processed as described in the Methods section. ( C ) Measurement of B16/F10 tumor-derived Nrp-1 + expression in gated CD4 + Foxp3 + T cells from IL10 −/− B16/F10 mice or WT B16/F10 mice after treatment with the anti-Nrp-1 polyclonal Ab by flow cytometry using micro-dissected and digested tumor tissues. ( D–F ) Measurement of TGF-β, IL6 and VEGF expression in NP-40-processed B16/F10 tumor tissues (described in the Methods section) obtained from mice treated with anti-Nrp-1. The cytokines were measured using Luminex (Millipore Merck). ( G ) Blood vessels stained with an anti-CD31 antibody. The images were obtained using a microscope with a 10X objective. ( H ) Compared with the control group, the tumor tissues of mice treated with the anti-Nrp-1 antibody showed much fewer Ki-67-positive cells. Left panel, representative immunohistochemistry images obtained using a microscope with a 20X objective. Right panel, statistical analysis of the mean IOD for blood vessels and tumor cell proliferation performed using Image-Pro Plus software. Nonlinear regressions were performed using GraphPad Prism (San Diego, CA, USA). The data shown are from one representative experiment of two independent experiments with the same results. The results were evaluated using ANOVA for the determination of statistical significance with *P < 0.05, **P < 0.01, ***P < 0.001 between experimental groups. The data are expressed as the mean values ± SEM from n = 4–6 mice.

Article Snippet: Similar groups of mice were treated i.v. with either a rabbit anti-mouse TGF-β pan-specific polyclonal rabbit antibody (3 mg/kg; R&D Systems AB-100-NA) or an isotype-specific polyclonal rabbit IgG (3 mg/kg; R&D Systems AB-105-C) .

Techniques: Derivative Assay, Flow Cytometry, Expressing, Luminex, Staining, Microscopy, Control, Immunohistochemistry, Software

Journal: Scientific Reports

Article Title: Interleukin-10 deficiency impairs regulatory T cell-derived neuropilin-1 functions and promotes Th1 and Th17 immunity

doi: 10.1038/srep24249

Figure Lengend Snippet: ( A ) A neutralizing anti-TGF-β antibody (described in the Methods section) was used to treat WT or IL10 −/− B16/F10 tumor-bearing mice. The tumor mass volume was monitored every three days after implantation. ( B ) TGF-β1 from NP-40-processed B16/F10 tumor tissues was measured using Luminex. Dual inhibition of IL-10 and TGF-β resulted in significantly lower levels of tumor-secreted VEGF protein in B16/F10 tumor-bearing mice. ( C ) Tumor-derived WT B16/F10-derived Nrp-1 levels were elevated compared with IL10 −/− B16/F10 mice treated with anti-TGF-β or IL10 −/− B16/F10 mice by flow cytometry. ( D ) VEGF from NP-40-processed B16/F10 tumor tissues measured using Luminex. ( E ) The absence of IL-10 or blocked TGF-β antibody had a more pronounced effect on CD4 + CD8a − T cells in B16/F10 tumors. ( F ) Anti-TGF-β in IL10 −/− B16/F10 mice allowed for the expansion of tumor IFN-γ-expressing CD4 + (Th1) populations measured by flow cytometry. ( G ) Anti-TGF-β did not allow the expansion of tumor CD4 + IL17A + (Th17) populations in IL10 −/− B16/F10 mice measured by flow cytometry. The data shown are from one representative experiment of two independent experiments with same results and were evaluated using ANOVA for the determination of statistical significance with *P < 0.05, **P < 0.01, ***P < 0.001between experimental groups. The data are expressed as the mean values ± SEM from n = 4–6 mice.

Article Snippet: Similar groups of mice were treated i.v. with either a rabbit anti-mouse TGF-β pan-specific polyclonal rabbit antibody (3 mg/kg; R&D Systems AB-100-NA) or an isotype-specific polyclonal rabbit IgG (3 mg/kg; R&D Systems AB-105-C) .

Techniques: Luminex, Inhibition, Derivative Assay, Flow Cytometry, Expressing

Journal: Scientific Reports

Article Title: Interleukin-10 deficiency impairs regulatory T cell-derived neuropilin-1 functions and promotes Th1 and Th17 immunity

doi: 10.1038/srep24249

Figure Lengend Snippet: ( A,B ) Percentages of the different T cell subpopulations gated on lymphocytes (FSC/SSC). IL-10 was more important than TGF-β for increasing the CD4 + Foxp3 + iTreg cell population in the tumor and spleen microenvironment, as measured by flow cytometry. ( C ) The frequency of Nrp-1–expressing Foxp3 + Treg cells from WT or IL10 −/− tumor-bearing mice blocked with or without TGF-β1 was determined within tumors or spleens by flow cytometry gating on CD4 + Foxp3 + cells . These data show that TGF-β increased the ability of tumor-derived Nrp-1 + CD4 + Foxp3 + T cells to support tumor growth. Panel ( A ) Evaluated using Student’s t-test to determine statistical significance with *P < 0.05 between experimental groups. The data are expressed as the mean values ± SEM from n = 3–4 mice. Panels ( B , C ) Evaluated using ANOVA, *P < 0.05, ±SEM from n = 3–4 mice.

Article Snippet: Similar groups of mice were treated i.v. with either a rabbit anti-mouse TGF-β pan-specific polyclonal rabbit antibody (3 mg/kg; R&D Systems AB-100-NA) or an isotype-specific polyclonal rabbit IgG (3 mg/kg; R&D Systems AB-105-C) .

Techniques: Flow Cytometry, Expressing, Derivative Assay

Journal:

Article Title: The immunomodulator FTY720 and its phosphorylated derivative activate the Smad signalling cascade and upregulate connective tissue growth factor and collagen type IV expression in renal mesangial cells

doi: 10.1038/sj.bjp.0706452

Figure Lengend Snippet: Effect of a TGF-βR type I kinase inhibitor on FTY720-, phospho-FTY- and TGF-β-stimulated CTGF expression in mesangial cells. Cells were pretreated for 30 min with either vehicle (−) or the indicated concentrations (in μM) of the TGF-βRI inhibitor prior to stimulation with either FTY720 (FTY; 3 μM), phospho-FTY720 (p-FTY; 3 μM) or TGF-β2 (20 ng ml−1) for 4 h. Thereafter, cell lysates were subjected to Western blot analysis using antibodies against CTGF or GAPDH at dilutions of 1 : 1000. Bands corresponding to CTGF were densitometrically evaluated. Results are means±s.d. (n=3), *P<0.05, **P<0.01, considered statistically significant when compared to the stimulated values in the absence of the inhibitor.

Article Snippet: FTY720, phospho-FTY720 and TGF- β 2 were kindly donated by Novartis Pharma Ltd, Basel, Switzerland; phospho-specific antibodies against p42/p44-MAPK, JNK, p38-MAPK, phospho-Smad-1(Ser 463/465 ) and phospho-Smad-2(Ser 465/467 ) and total Smad-2 were from Cell signalling, Frankfurt am Main, Germany; the TGF- β RI kinase inhibitor and the total polyclonal anti-human Smad-1 antibody (directed against amino-acid residues 147–258 of human Smad-1) were from Merck Biosciences, Schwalbach, Germany; the total Smad-3 antibody (FL-425) and the collagen type IV (H-234) antibody were from Santa Cruz, Heidelberg, Germany; the neutralizing pan-specific TGF- β antibody and the TGF- β ELISA were from Biosource Deutschland GmbH, Solingen, Germany; [ 35 S]methionine/cysteine pro-mixture (specific activity >1000 Ci mmol −1 ), [ α - 32 P]dCTP (specific activity 3000 Ci mmol −1 ), protein A sepharose 4B, anti-rabbit and anti-mouse horseradish peroxidase-linked IgGs and Hyperfilm were purchased from Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany; all cell culture nutrients were from Gibco, Karlsruhe, Germany.

Techniques: Expressing, Western Blot