Journal: European journal of immunology

Article Title: Toll like Receptor 2 engagement on CD4 + T cells promotes TH9 differentiation and function

doi: 10.1002/eji.201646846

Figure Lengend Snippet: TLR2, but no other TLR ligands increase TGF-β and IL-4 driven IL-9 cytokine secretion. Naive CD4+ T cells were activated with anti-CD3 and anti-CD28 mAbs in the presence of different TLR ligands (1μg/ml) under non-polarizing and TH9 polarizing conditions for 48 h. IL-9 (A) and IFN-γ (B) ELISA were measured by culture supernatants. Means ± SD of three independent experiments are shown. * p < 0.05, ** p < 0.01, NS denotes non-significant.

Article Snippet: IFN-γ was measured by sandwich ELISA (R&D Systems; MIF00) and IL-9 ELISA was done using the DuoSet ELISA kit (R&D Systems; DY409) by following manufactured protocol.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Toll like Receptor 2 engagement on CD4 + T cells promotes TH9 differentiation and function

doi: 10.1002/eji.201646846

Figure Lengend Snippet: TLR2 engagement on Antigen85B specific CD4+ T cells increases TH9 differentiation driven by TGF-β and IL-4. Naive Ag85B transgenic CD4+ T cells were co-incubated with TLR2 KO BMDM pulsed with Ag85B peptide (1μg/ml) and co-stimulated with or without TLR2 ligand (P3CSK4) under non-polarizing and TH9 polarizing conditions for 48 h. (A) CD4+ T cells were permeabilized and labeled with mAbs to IL-9 and IFN-γ and percentages of IL-9+ or IFN-γ+ cells were determined by flow cytometry. (B, C) IL-9 and IFN-γ were measured in culture supernatants by ELISA. Means ± SD of five independent experiments are shown. * p < 0.05, ** p < 0.01. NS denote non- significant.

Article Snippet: IFN-γ was measured by sandwich ELISA (R&D Systems; MIF00) and IL-9 ELISA was done using the DuoSet ELISA kit (R&D Systems; DY409) by following manufactured protocol.

Techniques: Transgenic Assay, Incubation, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Toll like Receptor 2 engagement on CD4 + T cells promotes TH9 differentiation and function

doi: 10.1002/eji.201646846

Figure Lengend Snippet: TLR2 engagement on human CD4+ T cells enhances IL9 mRNA and protein expression driven by polyclonal activation and TGF-β and IL-4. Naïve CD4+ T cells from three human donors were stimulated with anti-CD3 (10ug/ml) and anti-CD28 mAbs (1ug/ml) and exogenous TGF-β (5ng/ml) and IL-4 (10ng/ml), with or without P3CSK4 (2μg/ml) for 48h. (A) Relative expression of Il9 mRNA under non-polarizing and polarizing conditions with or without P3CSK4 (n=3) is shown. (B) IL-9 cytokine in culture supernatants were determined by ELISA. Means ± SD of three technical replicates for each donor are shown.

Article Snippet: IFN-γ was measured by sandwich ELISA (R&D Systems; MIF00) and IL-9 ELISA was done using the DuoSet ELISA kit (R&D Systems; DY409) by following manufactured protocol.

Techniques: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: European journal of immunology

Article Title: Critical role of IL-33 receptor ST2 in experimental cerebral malaria development.

doi: 10.1002/eji.201445206

Figure Lengend Snippet: Figure 6. ST2 deficiency does not affect lung or spleen inflammation after PbA sporozoite infection. WT- or ST2-deficient mice were infected with 1000 PbA sporozoites. (A) Lung inflammation with alveolar septae thickening with mononuclear cell infiltration, hemorrhage in alveoli, and interstitial edema on day 9 post infection in WT or ST2−/−mice; H&E staining. Magnification 20×; scale bar = 100 μm. Images are representative of two independent experiments. (B) Semiquantitative score of lung pathology, expressed in arbitrary unit (AUs). The bar graphs show the mean ± SEM of n = 9 mice per group and are pooled from two independent experiments. (C–F) PbA-induced spleen inflammation in WT or ST2−/−mice. The absolute numbers of CD8+ (C) and CD4+ (D) T cells, respectively, CD8+CD69+ and CD4+CD69+ T cells, CD8+CXCR3+ and CD4+CXCR3+ T cells per spleen are shown. IFN-γ (E) and IL-10 (F) levels were measured by ELISA in spleen homogenates on days 0 and 9 post infection. (C–F) Data are expressed as mean ± SEM of n = 5–6 mice per condition and are representative of two independent experiments; *p < 0.05; statistical analysis with Kruskal–Wallis test followed by Dunn’s comparison test.

Article Snippet: After centrifugation IL-33, IL-10, and IFN-γ levels were determined in supernatants using ELISA assay kits (Mouse DuoSet, R&D Systems, Minneapolis, USA).

Techniques: Infection, Staining, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Journal of medical primatology

Article Title: Cloning and functional testing of rhesus macaque ( Macaca mulatta ) IL-9 and IL-33

doi: 10.1111/jmp.12464

Figure Lengend Snippet: (A) RT-PCR amplification of IL-9 and IL-33 from rhesus PBMC and CD4+ T cells. (B) Expression of IL-9 by CD4+ T cells. PBMC from two rhesus macaques were stimulated with PMA and ionomycin for four hours, permeabilized, stained with anti-human IL-9 antibody, and analyzed by flow cytometry. Plots are gated on live, CD19-CD20-CD8-CD3+CD4+ T cells. Nucleotide alignment of rhesus observed (Rmobsv), predicted (Rmpre), mouse, and human (Hum) IL-9 (C) and IL-33 (D). The boxes indicate codon differences between the rhesus observed and human.

Article Snippet: 20 ng/ml of human recombinant IL-9 (cat # 209-ILB/CF, R&D systems) and 20 ng/ml of IL-33 (cat #200-33, Peprotech) were used as positive controls and pUNO-1 empty vector transfected cell supernatant (no dilution) was used as a negative control to treat the MO7e and D10.G4.T1.

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Expressing, Staining, Flow Cytometry

Journal: Journal of medical primatology

Article Title: Cloning and functional testing of rhesus macaque ( Macaca mulatta ) IL-9 and IL-33

doi: 10.1111/jmp.12464

Figure Lengend Snippet: Amino acid alignments of mouse, human, predicted, and synthesized (syn) IL-9 (A) and IL-33 (B) proteins.

Article Snippet: 20 ng/ml of human recombinant IL-9 (cat # 209-ILB/CF, R&D systems) and 20 ng/ml of IL-33 (cat #200-33, Peprotech) were used as positive controls and pUNO-1 empty vector transfected cell supernatant (no dilution) was used as a negative control to treat the MO7e and D10.G4.T1.

Techniques: Synthesized

Journal: Journal of medical primatology

Article Title: Cloning and functional testing of rhesus macaque ( Macaca mulatta ) IL-9 and IL-33

doi: 10.1111/jmp.12464

Figure Lengend Snippet: HEK293T cells were transfected with cloned rhesus IL-9 and IL-33 and supernatant was collected to measure the production of IL-9 (A) and IL-33 (B) protein by ELISA. Decreasing titrations of recombinant human cytokine included as positive control, supernatant from empty vector control transfected cells (pUNO1) included as negative control. Bars represent mean ± SEM of triplicate samples.

Article Snippet: 20 ng/ml of human recombinant IL-9 (cat # 209-ILB/CF, R&D systems) and 20 ng/ml of IL-33 (cat #200-33, Peprotech) were used as positive controls and pUNO-1 empty vector transfected cell supernatant (no dilution) was used as a negative control to treat the MO7e and D10.G4.T1.

Techniques: Transfection, Clone Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Positive Control, Plasmid Preparation, Negative Control

Journal: Journal of medical primatology

Article Title: Cloning and functional testing of rhesus macaque ( Macaca mulatta ) IL-9 and IL-33

doi: 10.1111/jmp.12464

Figure Lengend Snippet: (A) Human MO7e cells were cultured with rhesus macaque IL-9 (RmIL-9), in the absence or presence of IL-9 neutralizing antibody for 3 days. (B) Mouse D10.G4.T1 cells were cultured with rhesus macaque IL-33 (RmIL-33) in the absence or presence of IL-33 neutralizing antibody for 3 days. Recombinant human IL-9 and IL-33 were used as positive control and empty vector pUNO1 as negative control. Bars represent mean ± SEM of triplicate samples. * indicates p<0.05 compared to pUNO1.

Article Snippet: 20 ng/ml of human recombinant IL-9 (cat # 209-ILB/CF, R&D systems) and 20 ng/ml of IL-33 (cat #200-33, Peprotech) were used as positive controls and pUNO-1 empty vector transfected cell supernatant (no dilution) was used as a negative control to treat the MO7e and D10.G4.T1.

Techniques: Cell Culture, Recombinant, Positive Control, Plasmid Preparation, Negative Control

Journal: Journal of medical primatology

Article Title: Cloning and functional testing of rhesus macaque ( Macaca mulatta ) IL-9 and IL-33

doi: 10.1111/jmp.12464

Figure Lengend Snippet: Rhesus BLCL-C162 cells were cultured for 3 days with rhesus macaque IL-9 or IL-33 in the presence or absence of neutralizing antibody. * indicates p<0.05 compared to pUNO1.

Article Snippet: 20 ng/ml of human recombinant IL-9 (cat # 209-ILB/CF, R&D systems) and 20 ng/ml of IL-33 (cat #200-33, Peprotech) were used as positive controls and pUNO-1 empty vector transfected cell supernatant (no dilution) was used as a negative control to treat the MO7e and D10.G4.T1.

Techniques: Cell Culture

Journal: Cell Reports

Article Title: Mechanical force receptor Piezo1 regulates TH9 cell differentiation

doi: 10.1016/j.celrep.2024.115136

Figure Lengend Snippet: Figure 6. Piezo1 deficiency inhibits TH9 cell differentiation through the SIRT3-SDHA-OXPHOS metabolism pathway (A) The oxygen consumption rate (OCR) was examined as a readout for OXPHOS in CD4+ T cells isolated from WT and Piezo1/ mice for 5 days under TH9 cell- inducing conditions. (B) Mean fluorescent intensity (MFI) of SDHA in CD4+ T cells isolated from WT and Piezo1/ mice for 5 days under TH9 cell-inducing conditions. (C) SDH/complex II activities of T cells isolated from WT and Piezo1/ mice for 5 days under TH9 cell-inducing conditions by ELISA. (D) Mass spectrometry analysis of the metabolite abundance of fumarate (FA), succinate (SC), and malate (MA) in CD4+ T cells isolated from WT and Piezo1/

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER GsMTx4 MedChemExpress, USA Cat#HY-P1410 Ionomycin Sigma-Aldrich, USA Cat#I0634 Yoda1 MedChemExpress, USA Cat#HY-18723 Critical commercial assays Co-IP buffer Thermo Fisher Scientific, USA Cat#88804 CD4+ T cell isolation kit Miltenyi Biotec, USA Cat#130-104-453 ECL kit Beyotime Biotechnology, China Cat#P10018AS Enhanced BCA protein assay kit Beyotime Biotechnology, China Cat#P0009 Foxp3/transcription factor staining buffer set BD Bioscience, USA Cat#560133 Mito stress test kit Seahorse Bioscience, USA Cat#101706-100 NE-PER nuclear and cytoplasmic extraction reagents Thermo Fisher Scientific, USA Cat#78835 RIPA lysis buffer Beyotime Biotechnology, China Cat#P0013B RNeasy mini kit Qiagen, Germany Cat#74106 PrimeScriptTM RT reagent kit TakaRa, Japan Cat#TakaRa_RR037A SuperReal preMix plus SYBR Green Tiangen, Germany Cat#FP205-02 Dual-Luciferase assay system Promega, USA Cat#E1910 Mouse IL-9 DuoSet ELISA R&D system, USA Cat#DY409 Human IL-9 DuoSet ELISA R&D system, USA Cat#DY209-05 Deposited data Raw and analyzed data This paper GEO:GSE268658 Experimental models: Cell lines Mouse melanoma cell line B16.F10 China cell bank ATCC, China B16F10 Mouse lymphoma cell line EL-4 China cell bank ATCC, China EL4 HEK293T ATCC, USA Cat#CRL-11268 Phoenic-Eco packaging cells Allele Biotechnology, USA Cat#ABP-RVC-10001 Experimental models: Organisms/strains Mouse: C57BL/6J (CD45.1) Beijing Weitonglihua Experimental Animal Center, China NA Mouse: C57BL/6J (CD45.2) Beijing Weitonglihua Experimental Animal Center, China NA Mouse: C57BL/6J-Rag1 / GemPharmatech, China Cat#T004753 Mouse: Piezo1flox/flox GemPharmatech, China Cat#T009627 Mouse: Cd4-Cre GemPharmatech, China Cat#T067676 Mouse: Piezo1 / GemPharmatech, China Cat#T012739 Mouse: Sirt3flox/flox GemPharmatech, China Cat#T006658 Mouse: Hif1aflox/flox GemPharmatech, China Cat#T007329 Mouse: C57BL/6J (GFP) GemPharmatech, China Cat#T054573 Oligonucleotides siRNA targeting sequence: SDHA siRNA, 50-UAAAUCUUCCCAUCUUCAGUU-30 This paper NA siRNA targeting sequence: Piezo1 siRNA, 50-CACCGGCATCTACGTCAAATA-30 This paper NA Piezo1 (Mm01241549_m1) Applied Biosystems, USA Cat#4331182 Piezo1 (Hs00207230_m1) Applied Biosystems, USA Cat#4331182 IL-9 (Mm00434305_m1) Applied Biosystems, USA Cat#4331182 IL-9 (Hs00174125_m1) Applied Biosystems, USA Cat#4331182 Sirt3 (Mm00452131_m1) Applied Biosystems, USA Cat#4331182 Sirt3 (Hs00202030_m1) Applied Biosystems, USA Cat#4331182 (Continued on next page) Cell Reports 44, 115136, January 28, 2025 17

Techniques: Cell Differentiation, Isolation, Enzyme-linked Immunosorbent Assay, Mass Spectrometry