Journal: Journal of Investigative Dermatology

Article Title: Differential Production of Th1- and Th2-Type Chemokines by Mouse Langerhans Cells and Splenic Dendritic Cells

doi: 10.1111/j.0022-202x.2004.23607.x

Figure Lengend Snippet: Figure 1 RT-PCR analysis of CXCL10, CXCL9, and CXCL11 gene expression in freshly isolated Langerhans cells (fLC), 48 h cultured LC (cLC), interferon-c (IFN-c)-stimulated cLC (c-cLC), freshly isolated splenic dendritic cells (fDC), 48 h cultured splenic DC (cDC), and IFN-c- stimulated cDC (c-cDC). LC and splenic DC were purified and cul- tured for 48 h in the absence or presence of 100 ng per mL of IFN-g. Samples from fresh and cultured LC and splenic DC were collected and mRNA expression was analyzed using specific primers for each chemokine. CXCL10, CXCL9, and CXCL11 mRNA expression was hardly detectable in fLC. In cLC, mRNA expression of CXCL10 and CXCL11, but not CXCL9, was induced. CXCL9 mRNA was strongly expressed only in g-cLC. In addition, mRNA for CXCL10 and CXCL11 was also strongly expressed in g-cLC. In splenic DC, mRNA for these T helper 1(Th1)-type chemokines was almost undetectable both in fDC and cDC. When stimulated with IFN-g, CXCL10, CXCL9, and CXCL11 mRNA expression was induced. Data are representative of three inde- pendent experiments.

Article Snippet: Measurement of CXCL9, CXCL11, and CCL17 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL9, CXCL11, and CCL17 by ELISA using commercially available mouse CXCL9, CXCL11, and CCL17 immunoassay kits (R&D Systems), respectively, according to the manufacturer’s instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Gene Expression, Isolation, Cell Culture, Expressing

Journal: Journal of Investigative Dermatology

Article Title: Differential Production of Th1- and Th2-Type Chemokines by Mouse Langerhans Cells and Splenic Dendritic Cells

doi: 10.1111/j.0022-202x.2004.23607.x

Figure Lengend Snippet: Figure 2 Production of T helper 1-type chemokines during culture of Langerhans cells (LC) and splenic dendritic cells (DC). Purified LC and splenic DC were cultured with or without interferon-g (IFN-g), and the concentration of CXCL10, CXCL9, and CXCL11 was measured at different time points (0, 12, 24, 36, 48 h) in the supernatants by ELISA. Representative data of three independent experiments.

Article Snippet: Measurement of CXCL9, CXCL11, and CCL17 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL9, CXCL11, and CCL17 by ELISA using commercially available mouse CXCL9, CXCL11, and CCL17 immunoassay kits (R&D Systems), respectively, according to the manufacturer’s instructions.

Techniques: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Investigative Dermatology

Article Title: Differential Production of Th1- and Th2-Type Chemokines by Mouse Langerhans Cells and Splenic Dendritic Cells

doi: 10.1111/j.0022-202x.2004.23607.x

Figure Lengend Snippet: Figure 3 Chemotaxis of mCXCR3-transfected 2B4 T cells. Langerhans cells (LC) and splenic dendritic cells (DC) were cultured for 48 h with 100 ng per mL of interferon-g (IFN-g) and the supernatants were collected. The culture supernatants were preincubated with or without neutralizing anti-chemokine monoclonal antibody (mAb) indicated in the figure for 30 min, and assessed for chemotactic activity to CXCR3 transfectant. RPMI 10 medium alone served as a negative control. RPMI 10 medium containing recombinant chemokine served as a positive control. The supernatants of IFN-g-stimulated LC and splenic DC exhibited chemo- tactic activity to CXCR3 transfectant, which is mediated at least by CXCL10 and CXCL9. Mean (SD) (n ¼ 3). Data are representative of three independent experiments.

Article Snippet: Measurement of CXCL9, CXCL11, and CCL17 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL9, CXCL11, and CCL17 by ELISA using commercially available mouse CXCL9, CXCL11, and CCL17 immunoassay kits (R&D Systems), respectively, according to the manufacturer’s instructions.

Techniques: Chemotaxis Assay, Transfection, Cell Culture, Activity Assay, Negative Control, Recombinant, Positive Control

Journal: Journal of Investigative Dermatology

Article Title: Differential Production of Th1- and Th2-Type Chemokines by Mouse Langerhans Cells and Splenic Dendritic Cells

doi: 10.1111/j.0022-202x.2004.23607.x

Figure Lengend Snippet: Figure 7 Regulation of T helper 1-type chemokines produced by La- ngerhans cells (LC) and splenic dendritic cells (DC). LC (&) and splenic DC (’) were purified and cultured (1.5 106 cells per mL per 200 mL in each well) for 48 h with or without various stimuli. The supe- rnatants were collected and the concentration of CXCL10 (a), CXCL9 (b), and CXCL11 (c) was measured by ELISA. CXCL10 production by LC was induced by interferon-g (IFN-g), interleukin (IL)-12, lipopoly- saccharide (LPS), Staphylococcus aureus Cowen 1 (SAC), and poly- inosinic–polycytidylic acid (Poly(I:C)). In the case of CXCL9 and CXCL11, only IFN-g induced their production by LC. In the case of splenic DC, the production of CXCL10, CXCL9, and CXCL11 was in- duced by IFN-g, IL-18, LPS, and Poly(I:C). Results are the mean (SD) (n ¼ 4). Significant increase (po0.05) compared with the unstimulated group. Data are representative of four independent experiments.

Article Snippet: Measurement of CXCL9, CXCL11, and CCL17 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL9, CXCL11, and CCL17 by ELISA using commercially available mouse CXCL9, CXCL11, and CCL17 immunoassay kits (R&D Systems), respectively, according to the manufacturer’s instructions.

Techniques: Produced, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans

doi: 10.1101/2022.09.20.508420

Figure Lengend Snippet: (A) CXCL9, 10 and 11 can all bind and signal through the chemokine receptor CXCR3 that is typically found on T cells. (B) The EMBL-ELI expression atlas (human) was analysed for relatedness in expression of CXCR3 and its’ ligands CXCL9, 10 and 11 across all tissues or (C) in distinct tissues and diseases.

Article Snippet: Specific concentrations of CXCL9 were measured by enzyme-linked immunosorbent assay (ELISA), using the mouse CXCL9 ELISA kit (R&D Systems) in a 96-well high binding ELISA plate following the manufacturer’s instructions.

Techniques: Expressing

Journal: bioRxiv

Article Title: Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans

doi: 10.1101/2022.09.20.508420

Figure Lengend Snippet: (A) Schematic of the in vivo air pouch leukocyte recruitment model. (B) Analysis of chemokine concentration in the carrageenan inflamed air pouch. (C) Representative tSNE of all murine cells gated on live, single, CD45 + and built on CD4, CD8, F4/80, Ly6C, Ter119, CD3, TCRβ, CXCR3, Ly6G, CD11c, B220, CD11b, CD64, Siglec F, NK1.1 and TCRγδ. FlowSOM clusters are illustrated by gates. (D) tSNE analysis of air pouches injected with equimolar amounts of CXCL9, 10 and 11. (E) Quantification of all leukocytes (CD45 + ) and T cells within the air pouch following injection of CXCL9, 10 or 11. E analysed using a one-way ANOVA.

Article Snippet: Specific concentrations of CXCL9 were measured by enzyme-linked immunosorbent assay (ELISA), using the mouse CXCL9 ELISA kit (R&D Systems) in a 96-well high binding ELISA plate following the manufacturer’s instructions.

Techniques: In Vivo, Concentration Assay, Injection

Journal: bioRxiv

Article Title: Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans

doi: 10.1101/2022.09.20.508420

Figure Lengend Snippet: (A) Schematic of HS GAG structure, including sulphation points and the enzymes that produce them. (B) Normalised (relative to wild type) binding of labelled CXCL9, 10 or 11 to genetically modified CHO cells. (C) Normalised and absolute binding of CXCL9, 10 and 11 to CHO cells in which KS^ST1/2/3 have been genetically removed. (D and E) Normalised binding of CXCL9, 10 and 11 to CHO cells genetically engineered to express the enzymes regulating 3-O GAG sulphation. (F) EMBL-ELI expression atlas analysis of relatednessCXCL9, 10 and 11 and GAG sulphation gene expression. B and D, data plotted as mean from three separate pooled experiments. C and E data plotted as mean ± SEM from three separate pooled experiments and analysed using a one-way ANOVA.

Article Snippet: Specific concentrations of CXCL9 were measured by enzyme-linked immunosorbent assay (ELISA), using the mouse CXCL9 ELISA kit (R&D Systems) in a 96-well high binding ELISA plate following the manufacturer’s instructions.

Techniques: Binding Assay, Genetically Modified, Expressing, Gene Expression

Journal: bioRxiv

Article Title: Chemokines form complex signals during inflammation and disease that can be decoded by extracellular matrix proteoglycans

doi: 10.1101/2022.09.20.508420

Figure Lengend Snippet: (A) CXCL9, 10 and 11 all bind to the same receptor with different affinities and biased signalling outcomes and are found in over-lapping expression patterns during inflammation and disease. (B) Differential GAG interactions means that CXCL9 is more likely to be retained on GAGs on the cell surface or within the ECM, with CXCL10 and CXCL11 being more likely to be present in their soluble state.

Article Snippet: Specific concentrations of CXCL9 were measured by enzyme-linked immunosorbent assay (ELISA), using the mouse CXCL9 ELISA kit (R&D Systems) in a 96-well high binding ELISA plate following the manufacturer’s instructions.

Techniques: Expressing

Journal: OncoImmunology

Article Title: Tumor-intrinsic RGS1 potentiates checkpoint blockade response via ATF3-IFNGR1 axis

doi: 10.1080/2162402x.2023.2279800

Figure Lengend Snippet: Figure 2. RGS1 is a modulator of IFNγ-JAK-STAT1 signaling. a Differentially expressed cancer-related gene sets (H_Hallmarks) with high RGS1 expression in the KIRC cohort, LUAD cohort, LUSC cohort, and SKCM cohort from TCGA. NES, normalized enrichment score. FDR q, false discovery rate q value. b – d GSEA output of genes in the HALLMARK_INTERFERON_GAMMA_RESPONSE by RGS1 high and low expression groups from the KIRC cohort (b), LUAD cohort (c) and LUSC cohort (d) in the TCGA database. ES, enrichment score. e – l Analysis of IFNγ-STAT1 signaling in 786O and Renca cells. Cell lysates of 786O and Renca cells stably expressing nc or shRGS1 (e, g) and Vector or RGS1-OE (i, k) were analyzed by western blotting using RGS1, STAT1, P-STAT1 (Y701), IFNGR1, and IRF1 antibodies. Tublin was used as an internal control. f, j IFNγ-inducible gene expression in 786O cells. mRNA expression of IRF1, IRF9, STAT1, and IFNGR1 were detected by real-time qPCR. Actin was used as an internal control. h, l IFNγ-inducible gene expression in Renca cells. mRNA expression of Irf1, Irf9, and Stat1 were detected by real-time qPCR. Actin was used as an internal control. Cells in e, f, i, j were stimulated with 10 ng/ml human recombinant IFNγ or 0.1% BSA negative control for 2 h. Cells in g, h, k, l were stimulated with 5 ng/ml mouse recombinant IFNγ or 0.1% BSA negative control for 2 h. m, n IFNγ-induced CXCL9 secretion. Renca (m) and LLC (n) cells were cultured in serum-free medium and treated with 5 ng/ml IFNγ for 24 h. The concentration of CXCL9 was analyzed using an ELISA kit. o, p Cell surface levels of IFNGR1 in nc or shRGS1 (o) and Vector or RGS1-OE (p) 786O cells (pre-gated with FSC-A vs. SSC-A, and FSC-A vs. FSC-H). Cells were treated with 10 ng/ml IFNγ for 2 h. Right, quantification of the mean fluorescence intensity (MFI). Unpaired t-test was performed with GraphPad Prism 9. All data are representative of three independent experiments. Data in the bar graphs represent mean ± S.D., n = 3. *p < .05, **p < .01, ***p < .001.

Article Snippet: The concentrations of mouse CXCL9/MIG, human cAMP level and the activity of PKA in the supernatants were analyzed using CXCL9 ELISA kit (R&D Systems, MCX900), cAMP ELISA Kit (Elabscience, E-EL-0056c) and PKA Colorimetric Activity Kit (Thermo Fisher scientific, EIAPKA) respectively, according to the manufacturer’s protocols.

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Western Blot, Control, Gene Expression, Recombinant, Negative Control, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: OncoImmunology

Article Title: Tumor-intrinsic RGS1 potentiates checkpoint blockade response via ATF3-IFNGR1 axis

doi: 10.1080/2162402x.2023.2279800

Figure Lengend Snippet: Figure 5. RGS1 is associated with T cell infiltration in RCC and NSCLC mouse models. a, d Representative images and quantification (right) of immunohistochemistry staining of IFNGR1, ATF3 and CXCL9 expression in harvested CTRL and ShRgs1 Renca (a) or LLC (d) subcutaneous tumor sections. The percentages of positively stained area (right) were analyzed using Image J software. Scale bar, 100 µm. b, e T cell infiltration and quantification (below) of Renca (b) or LLC (e) subcutaneous tumor. Paraffin-embedded tissue sections of murine tumors were immunohistochemically stained with antibodies against CD3, CD4, CD8 and PD1. The percentages of positively stained cells (below) were analyzed using Image J software. Scale bar, 100 µm. c, f Representative immunofluorescence images of CD8 and PD1 staining in ShRgs1 or CTRL Renca (c) or LLC (f) tumor sections. White arrows indicate merge of CD8 and PD1 fluorescence signals. Scale bar, 50 µm. Data in the graphs represent mean ± S.D, n = 5. *p < .05, **p < .01, ***p < .001.

Article Snippet: The concentrations of mouse CXCL9/MIG, human cAMP level and the activity of PKA in the supernatants were analyzed using CXCL9 ELISA kit (R&D Systems, MCX900), cAMP ELISA Kit (Elabscience, E-EL-0056c) and PKA Colorimetric Activity Kit (Thermo Fisher scientific, EIAPKA) respectively, according to the manufacturer’s protocols.

Techniques: Immunohistochemistry, Staining, Expressing, Software, Immunofluorescence, Fluorescence

Journal: bioRxiv

Article Title: CD11c+ Tbet+ B cells constrain obesity- and vaccination-induced germinal center B cells and T helper cells

doi: 10.1101/2025.09.01.673552

Figure Lengend Snippet: Representative flow cytometry contour plots of PD-1 x CXCR5 expression within CD25-CD4+ T cells isolated from NCD (left) and HFD (right) mouse spleen (top) and liver (bottom) ( A ). Blue gates represent PD-1 HI CXCR5+ cells, yellow gates represent PD-1 HI CXCR5-cells, and black gates represent PD-1-cells ( A ). Frequency and absolute number of PD-1 HI CXCR5+ T FH cells ( B , blue), or PD-1 HI CXCR5-T PH cells ( C , yellow) within the CD25-CD4+ T cell compartments of spleen (top) and liver (bottom) of NCD (grey) and HFD (blue/yellow) mice. Quantification of Bcl-6 ( D ), ICOS ( E ), and T-bet ( F ) protein expression (gMFI) in splenic (left) or hepatic (right) CD25-CD4+ T cell subsets. Representative flow cytometry contour plots depicting relative expression of IL-21 and IFNg by PD-1 HI CXCR5+ T FH cells or PD-1 HI CXCR5-T PH cells from spleen (top) or liver (bottom) that are also CXCR3+ (blue/red) or CXCR3-(black) ( G ). Summary of frequency of IL-21 ( H,J; left) or IFNg ( I,K ; right) producing CD4+ PD-1 HI CXCR5+ T FH (blue) or PD-1 HI CXCR5-T PH (red) cells categorized by CXCR3+ expression as noted in spleen ( H, I ) or liver ( J,K ) from HFD-fed mice. Data pooled from 2 independent experiments with 4 female mice/group, Bar ± SEM; [ Student’s t-test (two-tailed) (B,C); one-way ANOVA (D-F; H-K)]; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: C57BL/6J WT female mice were injected IP with 0.25 mg of anti-mouse CXCR3 (CD183) and anti-mouse CXCL9 (MIG) or hamster IgG (BioXCell) every other day for 14 days after completing 12 weeks on HFD.

Techniques: Flow Cytometry, Expressing, Isolation, Two Tailed Test

Journal: Methods in Molecular Biology

Article Title: Allergy

doi: 10.1007/978-1-4939-9591-2

Figure Lengend Snippet: Fig. 1 Different types of immunoelectrophoresis of cat hair extract (Fel d) using a polyclonal rabbit antibody raised against the same extract (a Fel d). (a) CIE of Fel d extract, (b) FRIE following a purification (immunoab- sorption) of the major allergen Fel d 1, (c) CIE of purified Fel d 1 against a Fel d, (d) CLIE with Fel d 1 in inter- mediate gel for identification of the allergen, (e) TCIE with Fel d extract and Fel d 1 showing a double peak, (f) CIIE with monospecific Fel d 1 in intermediate gel

Article Snippet: Detection: Biotinylated goat anti-human MIG polyclonal (R&D Systems #BAF392 or PeproTech).

Techniques: Immunoelectrophoresis, Purification