mouse th17 cell differentiation kit  (R&D Systems)

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: (A) Treatment with the α2B/2C agonist (AGN-762) after the development of chronic DED effectively ameliorates the disease, assessed by corneal fluorescein staining (CFS) score. ****, p < 0.0001 vs untreated or vehicle group; n = 30 eyes (15 mice) /group from two independent experiments. (B) Corneal inflammation was assessed at the end of treatment by quantifying mature MHC-II + CD11b + cells using flow cytometry and protein levels of IL-1β and IL-6 using ELISA. The upper panel shows the gating strategy for flow cytometry analysis. (C) Conjunctival inflammation was assessed by quantifying infiltrating CD4 + T cells using flow cytometry and protein levels of IL-17A, IL-17F, and IFN-γ using ELISA. The upper panel of the flow plots shows the gating strategy for flow cytometry analysis. (D) Draining lymph node (DLN) Th17 response was determined by flow cytometry. The upper panel shows the gating strategy for flow cytometry analysis. (E) The suppressive function of Treg cells on cell proliferation was quantified using the BrdU incorporation assay and compared with the proliferative responses in the absence of Treg (0 % suppression). For flow cytometry of corneal and conjunctival tissues, samples were pooled from 5 eyes in each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; data shown were from one representative experiment out of two performed and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Article Snippet: Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, BrdU Incorporation Assay

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: (A) A 7-day treatment with the α2B/2C agonist (AGN-762) in chronic DED (day 21 – 28) effectively maintains therapeutic efficacy for months (day 28 – 56), and suppresses disease exacerbation in treated animals upon re-challenged with desiccating stress (day 56 – 63). Disease severity was assessed by corneal fluorescein staining (CFS) score. ***, p < 0.001; ****, p < 0.0001 vs untreated or vehicle group; n = 20 eyes (10 mice) /group from two independent experiments. (B) Corneal inflammation was assessed at day 63 by quantifying mature MHC-II + CD11b + cells using flow cytometry and protein levels of IL-1β and IL-6 using ELISA. The same gating strategy for flow cytometry analysis was used as shown in . (C) Conjunctival inflammation was assessed at day 63 by quantifying protein levels of IL-17A, IL-17F, and IFN-γ using ELISA. (D) Draining lymph node Th17 response was determined by flow cytometry at day 63. The same gating strategy for flow cytometry analysis was used as shown in . (E) The suppressive function of Treg cells on cell proliferation was quantified using the BrdU incorporation assay and compared with the proliferative responses in the absence of Treg (0 % suppression). For flow cytometry of corneal and conjunctival tissues, samples were pooled from 5 eyes in each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s., not significant; data shown were from one representative experiment out of two performed and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Article Snippet: Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, BrdU Incorporation Assay

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: (A) Flow cytometry plots show the purity of freshly sorted Teff cells defined as CD4 + CD25 − Foxp3 − cells that were subjected to cell cultures in vitro. (B) Flow cytometric analysis of CD4 + CD25 − Teff after 24 h culturing with AGN-762, with representative dot plots shown (the upper panel shows the gating strategy), and frequencies of IL-17A + IFN-γ − Th17, IL-17A + IFN-γ + Th17/1, and IL-17A − IFN-γ + Th1 summarized in the bar graphs. (C) ELISA assay on the culture supernatants for the levels of IL-17A and IFN-γ. Data shown were from one representative experiment out of two performed. *, p < 0.05; data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Article Snippet: Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Flow Cytometry, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: (A) Treg cells were isolated from DED mice and in vitro cultured with 1 μM AGN-762 or vehicle for 24 h. The freshly sorted effector CD4 + T cells from DED (Teff) along with normal Treg, DED-Treg pre-treated with the vehicle, or DED-Treg pre-treated with AGN-762 were adoptively transferred to Rag1 −/− mice, which were subsequently exposed to desiccating stress for 6 days. Disease severity was assessed by corneal fluorescein staining (CFS) score, and summarized from two independent experiments. n = 10–14 eyes (5–7 mice) /group. (B) Flow cytometric analysis of T cell response in the draining lymph nodes of Rag1 −/− recipients at day 6, with representative dot plots shown on the left (the upper panel shows the gating strategy) and frequencies of IL-17-producing CD4 + T cells (including both IL-17A + IFN-γ − Th17 and IL-17A + IFN-γ + Th17/1) summarized from two independent experiments on the right. *, p < 0.05; **, p < 0.01; data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Article Snippet: Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Isolation, In Vitro, Cell Culture, Staining

Journal: Mucosal immunology

Article Title: Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function

doi: 10.1016/j.mucimm.2024.11.002

Figure Lengend Snippet: (A) DED-Treg cells were pre-treated with 1 μM AGN-762 or vehicle for 24 h and then subjected to suppressive function assay in the presence of anti-IL-10 neutralizing antibody or isotype IgG control. (B) Treg cells were isolated from DED mice and in vitro cultured with 1 μM AGN-762 for 24 h. The freshly sorted effector CD4 + T cells from DED (Teff) along with DED-Treg pre-treated with AGN-762 were adoptively transferred to Rag1 −/− mice, which were subsequently exposed to desiccating stress for 6 days. The recipients received treatment with anti-IL-10 antibody or control IgG immediately after adoptive transfer (day 0) as well as at day 3. Disease severity was assessed by corneal fluorescein staining (CFS) score, and summarized from two independent experiments. n = 16 eyes (8 mice) /group (C) Flow cytometric analysis of T cell response in the draining lymph nodes of Rag1 −/− recipients at day 6, with representative dot plots shown on the left and frequencies of total T cells and IL-17-producing CD4 + T cells (including both IL-17A + IFN-γ − Th17 and IL-17A + IFN-γ + Th17/1) summarized from two independent experiments on the right. *, p < 0.05; **, p < 0.01; data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test (A) or unpaired t test (B and C).

Article Snippet: Th17 cell polarization were performed using Mouse Th17 cell differentiation kit (CDK017, R&D system) according to the manufacturer’s instructions.

Techniques: Functional Assay, Control, Isolation, In Vitro, Cell Culture, Adoptive Transfer Assay, Staining

Journal: Communications biology

Article Title: STAT3 and SOX-5 induce BRG1-mediated chromatin remodeling of RORCE2 in Th17 cells.

doi: 10.1038/s42003-023-05735-9

Figure Lengend Snippet: Fig. 1 STAT3 and SOX-5 activate RORCE2 in Th17 cells. a The deletion of the STAT3-BS sequence in RORCE2 by CRISPR/Cas9 (top) and confirmation by sequencing (bottom). b–d ChIP‒qPCR assays were performed on WT and STAT3-BS−/−Th17-polarized cells with the indicated antibodies. e ChIP‒qPCR was performed on WT and SOX-5-BS−/−Th17-polarized cells with the indicated antibodies. STAT3-SOX-5-BS indicates the location covering the STAT3 binding site and SOX-5 binding site in RORCE2. CNS9 (+5802 to +7963 bp from the RORC TSS) indicates a cis-regulatory element distal to STAT3-BS in RORCE2 and can be bound by STAT3, as the negative control for ChIP‒qPCR in this study. f, g 3C-qPCR (f) and ChIP-loop assays (with an anti-STAT3 antibody) (g) were performed to evaluate the interaction between RORCE2 and the RORγt gene promoter in WT and STAT3-BS−/−Th17-polarized cells. Means ± SEMs are shown, n = 5 biologically independent animals (b–e), n = 3 biologically independent animals (f, g). D = differentiation.

Article Snippet: Th17, Th1 and Th2 polarizations were then conducted following the instructions of the CellXVivo mouse Th17 cell differentiation kit (R&D Systems, CDK017), the CellXVivo mouse Th1 cell differentiation kit (R&D Systems, CDK018) and the CellXVivo mouse Th2 cell differentiation kit (R&D Systems, CDK019), respectively.

Techniques: Sequencing, CRISPR, Binding Assay, Negative Control

Journal: Communications biology

Article Title: STAT3 and SOX-5 induce BRG1-mediated chromatin remodeling of RORCE2 in Th17 cells.

doi: 10.1038/s42003-023-05735-9

Figure Lengend Snippet: Fig. 2 STAT3-BS deficiency of RORCE2 downregulates RORγt expression and Th17 cell differentiation in vivo. a The relative mRNA levels of RORγt in CD4+ T cells from the spleens of WT and STAT3-BS−/−mice were quantified by RT‒qPCR. b, c RORγt+ Th17 cell frequency in splenic CD4+ T cells from WT and STAT3-BS−/−mice and mean fluorescence intensity (MFI) of RORγt in RORγt+ Th17 cells were analyzed by flow cytometry (b) and statistically evaluated (c). d The relative mRNA levels of IL-17A in CD4+ T cells from the spleens of the WT and STAT3-BS−/−mice. e, f IL-17A+ Th17 cell frequencies among splenic CD4+ T cells of the WT and STAT3-BS−/−mice. g, h RORγt+ Th17 cell frequencies among CD4+ CD45+ Lin+ T cells were determined by flow cytometry in LPLs of the small intestine from the WT and STAT3-BS−/−mice and RORγt MFI in Th17 cells. i, j IL-17A+ Th17 cell frequencies among CD4+ CD45+ Lin+ T cells from LPLs of the small intestine from the WT and STAT3-BS−/−mice. Means ± SEMs are shown, n = 5 biologically independent animals (a, c, d, f), n = 3 biologically independent animals (h, j).

Article Snippet: Th17, Th1 and Th2 polarizations were then conducted following the instructions of the CellXVivo mouse Th17 cell differentiation kit (R&D Systems, CDK017), the CellXVivo mouse Th1 cell differentiation kit (R&D Systems, CDK018) and the CellXVivo mouse Th2 cell differentiation kit (R&D Systems, CDK019), respectively.

Techniques: Expressing, Cell Differentiation, In Vivo, Cytometry

Journal: Communications biology

Article Title: STAT3 and SOX-5 induce BRG1-mediated chromatin remodeling of RORCE2 in Th17 cells.

doi: 10.1038/s42003-023-05735-9

Figure Lengend Snippet: Fig. 3 STAT3-BS deficiency of RORCE2 results in impaired Th17 cell polarization in vitro. Naïve CD4+ T cells from the spleens of WT or STAT3-BS−/−

Article Snippet: Th17, Th1 and Th2 polarizations were then conducted following the instructions of the CellXVivo mouse Th17 cell differentiation kit (R&D Systems, CDK017), the CellXVivo mouse Th1 cell differentiation kit (R&D Systems, CDK018) and the CellXVivo mouse Th2 cell differentiation kit (R&D Systems, CDK019), respectively.

Techniques: In Vitro

Journal: Communications biology

Article Title: STAT3 and SOX-5 induce BRG1-mediated chromatin remodeling of RORCE2 in Th17 cells.

doi: 10.1038/s42003-023-05735-9

Figure Lengend Snippet: Fig. 4 STAT3-BS deficiency in RORCE2 alleviates the severity of EAE. a The mean daily clinical scores of EAE for WT and STAT3-BS−/−mice are shown. b Representative staining image of hematoxylin-eosin (HE) and luxol fast blue (LFB) in the spinal cord sections at 30 days after immunization. Scale bars, 500 or 100 µm. c–e Flow cytometry analysis of mononuclear cells from the spinal cord of EAE-induced WT and STAT3-BS−/−mice at 30 days after immunization. Th17 cell frequencies among CD4+ T cells (c, d). The numbers of infiltrating CD4+ T cells in the spinal cord (e). f Inguinal lymph node cells were extracted at day 8 after immunization and restimulation with MOG peptide for 3 days. IL-17A, IFNγ, and IL-4 production were examined by ELISA. Means ± SEMs are shown, n = 5 biologically independent animals (d, f), n = 3 biologically independent animals (e).

Article Snippet: Th17, Th1 and Th2 polarizations were then conducted following the instructions of the CellXVivo mouse Th17 cell differentiation kit (R&D Systems, CDK017), the CellXVivo mouse Th1 cell differentiation kit (R&D Systems, CDK018) and the CellXVivo mouse Th2 cell differentiation kit (R&D Systems, CDK019), respectively.

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Communications biology

Article Title: STAT3 and SOX-5 induce BRG1-mediated chromatin remodeling of RORCE2 in Th17 cells.

doi: 10.1038/s42003-023-05735-9

Figure Lengend Snippet: Fig. 5 The binding of STAT3 and SOX-5 on RORCE2 is essential for the recruitment of BRG1. a ATAC-seq data at the RORC locus in STAT3fl/flTh17 cells versus STAT3fl/fl-CD4Cre Th17 cells. b A chromatin accessibility assay was performed on SOX-5-BS and STAT3-BS in RORCE2 in the WT and STAT3-BS−/−Th17-polarized cells. c The expression of BRG1 and GAPDH proteins in the WT, STAT3-BS−/−and SOX-5-BS−/−Th17-polarized cells was detected by Western blotting. d, e ChIP‒qPCR assays of BRG1 were performed in the WT, STAT3-BS−/−and SOX-5-BS−/−Th17-polarized cells. CNS9 (+5802 to +7963 bp from the RORC TSS) indicates a cis-regulatory element distal to STAT3-BS in RORCE2 and can be bound by STAT3, as the negative control for ChIP‒qPCR in this study. f Transfected HeLa cells were subjected to IP with the indicated antibody. Input and IP proteins were then subjected to IB with the indicated antibody. g Whole-cell lysates from Th17 cells were subjected to IP with an anti-BRG1 antibody or control mouse IgG and to IB with an anti-STAT3 or anti-SOX-5 antibody. Input proteins (input) were also subjected to IB with an anti-STAT3 or anti-SOX-5 antibody. Means ± SEMs are shown, n = 5 biologically independent animals (b, d, e). D = differentiation.

Article Snippet: Th17, Th1 and Th2 polarizations were then conducted following the instructions of the CellXVivo mouse Th17 cell differentiation kit (R&D Systems, CDK017), the CellXVivo mouse Th1 cell differentiation kit (R&D Systems, CDK018) and the CellXVivo mouse Th2 cell differentiation kit (R&D Systems, CDK019), respectively.

Techniques: Binding Assay, Expressing, Western Blot, Negative Control, Transfection, Control