Journal: Clinical and Vaccine Immunology

Article Title: Association of Reduced Tumor Necrosis Factor Alpha, Gamma Interferon, and Interleukin-1β (IL-1β) but Increased IL-10 Expression with Improved Chest Radiography in Patients with Pulmonary Tuberculosis

doi: 10.1128/cvi.00381-09

Figure Lengend Snippet: FIG. 1. Schematic timeline of therapeutic regimen and clinical as- sessment. The workup for TB included sputum smear, chest radio- graphs, and plasma cytokine level determination.

Article Snippet: A mixture of antibodies for the detection of nine biotinylated anti-human cytokine antibodies (R&D Systems, Inc.) were diluted with PBSBT at 40 g/ml (1:200) and added to the cytokine arrays, which were kept at room temperature for 30 min.

Techniques: Clinical Proteomics

Journal: Frontiers in Immunology

Article Title: IL-9 Producing Tumor-Infiltrating Lymphocytes and Treg Subsets Drive Immune Escape of Tumor Cells in Non-Small Cell Lung Cancer

doi: 10.3389/fimmu.2022.859738

Figure Lengend Snippet: Increased IL-9 production by regulatory T cells (Tregs) of patients with NSCLC. (A) Immunohistochemistry staining of lung tissue sections for IL-9 (colored in brown) and Foxp3 (colored in blue). Representative microscopy images are shown for one healthy control patient (CN) and the CTR and TU lung region from one NSCLC patient. Images were taken at a 40x magnification while inserts show a magnification of 100x. (B) Total IL-9 and FoxP3 double-positive cells per area are shown for CN (n=10) and NSCLC CTR (n=35) and NSCLC TU (n=34) Data are shown as mean + SEM. using student´s two-tailed t-test *P,0.05; **P,0.01; ***P,0.001. (C) Correlation of IL-9 + cells and FoxP3 mRNA levels from the tumoral region of NSCLC patients. Correlation was done with IL-9 + cells from the TU of immunohistochemistry staining analysis and FoxP3 mRNA levels of the TU from the same patient. mRNA levels were detected by RT-qPCR and set in relation to HPRT mRNA level (n=16). (D) qPCR based analysis of IL-2 mRNA levels of CTR and TU set in relation to HPRT mRNA level (nCTR=12; nTU=11). (E) Experimental design for the in vitro culture of PBMCs. PBMCs from NSCLC patients or healthy control patients were isolated and cultured with different conditions for 4-5 days at 37°C and 5% CO 2 (500.000 cells/well). After harvesting the cells, the supernatant was used to perform ELISA and the cells were analyzed by flow cytometry. (F) Analysis of IL-9 concentration (pg/ml) in the supernatant of PBMC cell culture from healthy controls (n=3-5) and NSCLC patients by ELISA (n=4. (G) Representative flow cytometry analysis of CD25 high FoxP3 + cells (%) gated on CD3 + CD4 + lymphocytes (n=5). Representative dot-plots showing CD25 and FoxP3 staining of PBMCs from control patients and NSCLC patients after cell culture with different conditions (unstimulated; IL-4 (20 ng/ml) and TGFβ (20 ng/ml); Treg: IL-2 (2 ng/ml) and TGFβ (20 ng/ml); IL-9 (20 ng/ml)). (H) Quantification of CD25 high FoxP3 + Tregs (n HC =5, n NSCLC =5; IL9-condition: n HC =3, n NSCLC =3). For statistical analysis One-way ANOVA test was applied. *p < 0.05.

Article Snippet: Human ELISA Sets were purchased as followed: IL-9 ELISA (Human IL-9 DuoSet ELISA, R&D Systems, Cat# DY209-05), IL-21 ELISA (Human IL-21 DuoSet ELISA, R&D Systems, Cat# DY8879-05), IFNγ ELISA (BD OptEIATM Human IFN-γ ELISA Set, BD Biosciences, Cat# 555142).

Techniques: Immunohistochemistry, Staining, Microscopy, Control, Two Tailed Test, Quantitative RT-PCR, In Vitro, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Concentration Assay

Journal: Frontiers in Immunology

Article Title: IL-9 Producing Tumor-Infiltrating Lymphocytes and Treg Subsets Drive Immune Escape of Tumor Cells in Non-Small Cell Lung Cancer

doi: 10.3389/fimmu.2022.859738

Figure Lengend Snippet: Increased IL-9 production by T cells in the tumoral lung region of NSCLC patients. (A) Representative photographic image of the resected lung of one NSCLC patient as a showcase of defined regions implemented for our human study cohort. Lung tissue samples were dissected from the tumoral area (TU), the peri-tumoral area (PT) surrounding the tumor and from the control area (CTR) consisting out of healthy lung tissue. (B) IL-9 immunohistochemistry (IHC) on paraffin-embedded tissue arrays obtained from the lung of tumor-free control patients (CN) or the CTR and TU lung regions of non-small cell lung cancer (NSCLC) patients (nCN=10; nCTR=15; nTU=17) (scale bar=50 µm). (C, D) Proteins were isolated from lung tissue samples of control patients (CN), the CTR and the TU of NSCLC ADC patients (classified by grading of tumor cell differentiation (G1, G2 and G3) and Western blot was performed. Detected protein levels of IL-9 were normalized on total protein of the samples (CN: n=3, CTR: n=10, TU:n=10). (E) Proteins were isolated from tissue samples of control patients that underwent surgery due to disease unrelated to tumor and from cells of the adenocarcinoma cell line A549. Analysed data are shown as mean ± SEM (nCN=1; nLu-Infl (Lung inflammation)=1; CTR-Lu=CTR, n=2; nA549 = 1) or single values. (F) Flow cytometry analysis of IL-9+ cells (shown in percentage) gated on Epcam+ cells of cells isolated from the CTR, the PT and the TU of a NSCLC patient. (G) Double IHC for IL-9 (brown) and CD3 (blue) on lung tissue obtained from the CTR and TU regions of an adenocarcinoma ( ADC) patient (scale bar=50 µm ). (H) ELISA analysis of IL-9 levels (pg/ml), (I) IL-21 levels (pg/ml), (J) IFNγ (pg/ml) in supernatants obtained from total cells isolated from the control (CTR), peri-tumoral (PT) and tumoral (TU) region of adenocarcinoma (ADC) patients and cultured with anti(α)-CD3/CD28 antibodies for 24h (IL-21: nCTR=6, nPT=4, nTU=5; IFNγ: nCTR=3; nPT=3; nTU=3). (K) TNF-alpha mRNA level from total lung cells was normalized on HPRT mRNA level (nCTR=12, nPT=11, nTU=12). N values are given per group. Bar charts indicate mean values +/- s.e.m. using student´s two-tailed t-test *P,0.05; **P,0.01; ***P,0.001.

Article Snippet: Human ELISA Sets were purchased as followed: IL-9 ELISA (Human IL-9 DuoSet ELISA, R&D Systems, Cat# DY209-05), IL-21 ELISA (Human IL-21 DuoSet ELISA, R&D Systems, Cat# DY8879-05), IFNγ ELISA (BD OptEIATM Human IFN-γ ELISA Set, BD Biosciences, Cat# 555142).

Techniques: Control, Immunohistochemistry, Isolation, Cell Differentiation, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: IL-9 Producing Tumor-Infiltrating Lymphocytes and Treg Subsets Drive Immune Escape of Tumor Cells in Non-Small Cell Lung Cancer

doi: 10.3389/fimmu.2022.859738

Figure Lengend Snippet: Targeted deletion of IL-9 resulted in lung tumor rejection in BL6 syngenic mice. (A) In vitro studies of murine LL/2-luc-M38 (LL/2) lung carcinoma cells treated with/out recombinant IL-9 for 24h. Flow cytometry analysis of the percentage of Ki67 expressing LL/2 cells after stimulation with or without IL-9 for 24h and representative histograms (left) (n unst. = 3; n30 ng/ml=3; n100 ng/ml=3). (B) Annexin V/PI flow cytometry analysis of LL/2 cells after stimulation with or without IL-9 for 24h and representative dot plots (n unst. = 3; n30 ng/ml=3; n100 ng/ml=3). (C) Cell count by using Neubauer chamber. Bar charts indicate mean values ± SEM using student´s two-tailed t -test (b–g) or ordinary one-way ANOVA (i–j) (*p<0.05, **p<0.01, ***p<0.001). (D) qPCR based analysis of Spi1 encoding PU.1 (n naive =3; n LL/2 = 4) and (E) Il9r (n naive =7; n LL/2 = 10) in relation to Hprt mRNA level in CD4 + T cells isolated cells from the lung of naïve and lung tumor-bearing (LL/2) WT mice. (F) Experimental design for the induction of lung tumor development in BL/6 WT, BL/6-IL-9 +/- and BL/6 IL-9 -/- mice which were intravenously (i.v.) with LL/2-luc-M38 (LL/2) lung carcinoma cells on day 0. The experiment ended on day 16. (G) Monitored body weight during tumor development. (H) Quantification of the tumor-infiltrated area of the lung (left) and representative H&E staining of the lung of WT, IL-9 +/- and IL-9 -/- mice (right) (n WT =10; n IL-9+/-= 10 n IL-9-/- =10). (I) Circles represent mice per group tumor free (circle sectors in white) and mice with lung bearing tumor (sectors in grey). (J) ELISA analysis of IL-21 levels (pg/ml) in supernatant obtained from cells isolated from the lung of wild-type and IL-9 -/- tumor-bearing mice. Lung cells were cultured for 4 days unstimulated or re-stimulated with aCD3/aCD28 antibodies (n WT =5, n IL9-/- =6).

Article Snippet: Human ELISA Sets were purchased as followed: IL-9 ELISA (Human IL-9 DuoSet ELISA, R&D Systems, Cat# DY209-05), IL-21 ELISA (Human IL-21 DuoSet ELISA, R&D Systems, Cat# DY8879-05), IFNγ ELISA (BD OptEIATM Human IFN-γ ELISA Set, BD Biosciences, Cat# 555142).

Techniques: In Vitro, Recombinant, Flow Cytometry, Expressing, Cell Counting, Two Tailed Test, Isolation, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Frontiers in Immunology

Article Title: IL-9 Producing Tumor-Infiltrating Lymphocytes and Treg Subsets Drive Immune Escape of Tumor Cells in Non-Small Cell Lung Cancer

doi: 10.3389/fimmu.2022.859738

Figure Lengend Snippet: Later treatment with anti-IL9 antibodies during tumor development, protected from experimental induced tumor. (A) Experimental design: BL/6 WT mice were intravenously injected with LL/2 cells on day 0. Mice were then treated intraperitoneally (i.p.) with anti (α)-IL9 antibody or IgG2a isotype control on day 6, 9, 10 and 13 after tumor induction. The experiment ended on day 20. (B, C) Tumor load was analyzed via bioluminescence-based imaging system on the indicated days. Representative in vivo images of lung tumor load analysis at day 11, 14, 17 and 19 in WT mice treated with αIL9 antibody or IgG2a isotype control (c). (b)Quantification of lung tumor load (Total flux=photons/second) (Day10-20 n αIL-9+LL/2 = 5; n IgG2a+LL/2 = 5). (D) FACS analysis of lung pSTAT5+CD4+CD25+ lung cells after 24 hours culture with anti_CD3/CD28 antibodies (n-αIL-9+LL/2 = 5; n-IgG2a+LL/2 = 5). (E) ELISA analysis of IFNγ levels (pg/ml) in supernatants obtained from cells isolated from the lung of wild-type (WT) lung tumor-bearing mice treated with/out anti(α)-IL9 antibody or IgG2a isotype control and cultured for 24h at 37°C (nIgG2a= 5; nαIL-9 = 5). N values are given per group. Bar charts indicate mean values ± SEM using student´s two-tailed t-test *p<0.05, **p<0.01, ***p<0.001. (F) Experimental design: Balb/c WT mice were intravenously injected with L1C2 cells on day 0. Mice were then treated intraperitoneally (i.p.) with anti (α)-IL9 antibody or IgG2a isotype control on day 9, 11, 14 and 16 after tumor induction. Tumor load was analyzed via immune histology on the indicated days. The experiment ended on day 21. (G, H) Representative H&E staining of WT mice untreated or treated with αIL9 antibody or IgG2a isotype control on day 9, 16 and 21 (top, scale bar=500 µm; bottom, scale bar=50 µm) and quantification of the number of pre-tumoral lesions in the in the lung on day 21 ( nIgG2a = 5; n αIL-9 = 5). (I) Flow cytometry analysis of percentage of CD4+CD25+ cells gated on lymphocytes in non-treated or with αIL9 antibody or IgG2a isotype control treated mice. (J) ELISA analysis of IL-10 levels (pg/ml) in supernatant obtained from cells isolated from the lung of wild-type (WT) lung tumor-bearing mice treated with/out anti(α)-IL9 antibody cultured for 24h at 37°C (n untr. = 5; nIgG2a = 5; n αIL-9 = 5). N values are given per group. Bar charts indicate mean values ± SEM using student´s two-tailed t -test *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Human ELISA Sets were purchased as followed: IL-9 ELISA (Human IL-9 DuoSet ELISA, R&D Systems, Cat# DY209-05), IL-21 ELISA (Human IL-21 DuoSet ELISA, R&D Systems, Cat# DY8879-05), IFNγ ELISA (BD OptEIATM Human IFN-γ ELISA Set, BD Biosciences, Cat# 555142).

Techniques: Injection, Control, Imaging, In Vivo, Enzyme-linked Immunosorbent Assay, Isolation, Cell Culture, Two Tailed Test, 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: (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