Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: HSV1 recombinant viruses expressing cGAS and/or STING exhibit restricted replication in human cancer cells (A) Schematic diagram of rHSV1 constructs. (B–N) 2 × 10 5 293T, hTERT, HT29, and SW48 cells infected with HSV1-Δγ34.5, HSV1-STING, HSV1-cGAS, and HSV1-STING-P2A-cGAS (HSV1-2A) at the MOI indicated. (B) Immunoblot analysis of cGAS, STING, phospho-STING, phospho-TBK1, phospho-IRF3, and β-actin 6 h post infection, (C) percentage of viable cells ( n = 4 biological replicates), and (D) virus titers ( n = 2 biological replicates). (E) Measurement by ELISA of the quantity of 2′3′ cGAMP in 5 × 10 5 293T cells 24 h post infection ( n = 3 biological replicates). (F) IFN-β-luciferase activity in 293T cells 24 h after plasmid transfection followed by 6 h of infection ( n = 3 technical replicates). (G, I, and K) Percentage of viable cells ( n = 3 biological replicates) and (H, J, and L) virus titers ( n = 3 biological replicates) on infected hTERT, HT29, and SW48 cells at MOI 1. (M) qPCR of Cxcl10 ( n = 2 biological replicates) and (N) ELISA analysis of human IFNβ production in hTERT, HT29, and SW48 cells 24 h after infection ( n = 6 biological replicates). Error bars indicate mean ± SEM; Student’s t test ∗ p < 0.05.

Article Snippet: TaqMan Probe: CXCL10 (mouse) , Thermo Fisher Scientific , Mm00445235_m1.

Techniques: Recombinant, Expressing, Construct, Infection, Western Blot, Virus, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Plasmid Preparation, Transfection

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: Recombinant HSV1 exhibits diminutive oncolytic activity yet retains in vivo anti-tumor properties dependent on extrinsic STING signaling (A) Immunoblot analysis of cGAS, STING, and β-actin in B16-OVA, B16-OVA cGAS KO (CKO), B16-OVA STING KO (SKO), and B16-OVA STING/cGAS KO (S/CKO) cells. (B and C) 2 × 10 5 B16 cells were infected with HSV1-Δγ34.5, HSV1-STING, HSV1-cGAS, and HSV1-STING-P2A-cGAS (HSV1-2A) at MOI 5 for 24 h. (B) Virus titers ( n = 2 biological replicates) and (C) percentage of viable cells were measured ( n = 2 biological replicates). (D–F) 2 × 10 5 293T and B16-OVA cells were infected at MOI 0.1 or 5 with HSV1- Δγ34.5-GFP. (D) The virus titer was determined by plaque assay ( n = 3 biological replicates), (E) the percentage of GFP + cells were measured by cytometry ( n = 3 biological replicates), and (F) the quantification of HSV1- Δγ34.5 genome was done by qPCR at 3 or 6 h, 24 h, and 48 h post infection ( n = 3 biological replicates). (G) B16 cells were infected with HSV1- Δγ34.5, HSV1-STING, HSV1-cGAS, or HSV1-2A at MOI 5 for 6 h. B16-OVA cells were treated with 3 μg/mL dsDNA90 as a control, and Cxcl10 was analyzed by qPCR ( n = 4 technical replicates). (H–Q) Wild-type (H–J: n = 6–8 mice per group; L–N: n = 4 mice per group), STING KO C57BL/6J ( n = 11–12 mice per group on 2 independent experiments), and BALB/c nude mice ( n = 7 mice by groups) were subcutaneously injected as indicated with B16-OVA, B16-OVA CKO, or B16-OVA S/CKO cells on the flank (5 × 10 5 cells/mouse). 5 × 10 6 PFU of replicating HSV1- Δγ34.5, HSV1-STING, HSV1-cGAS, or HSV1-2A was injected intratumorally (black arrows) three times. (H–L and O) The tumor volume was measured on the indicated days and calculated with the formula V = (length × width 2 )/2. At 16 or 17 days, the spleen and the tumors were extracted. (M and P) Digital photograph of tumors and (N and Q) ELISpot to measure IFNg release from CD8 + T cells. (R and S) Phagocytosis of B16-OVA and B16-OVA S/CKO cells by murine WT and STING KO macrophages. 1 × 10 6 cells were infected with HSV1-Δγ34.5 for 40 h at MOI 20 then irradiated by UV (120 mJ/cm) and incubated for 24 h. The irradiated cells were fed to macrophages (MØ) (2 × 10 5 cells). (R) Schematic representation and (S) ELISA analysis of IFN-β at 24 h in macrophages following engulfment of B16 ( n = 5 [WT macrophages] and 3 [STING macrophages] technical replicates). Error bars indicate mean ± SEM; Student’s t test and (H–L and O) ordinary one-way ANOVA test with Tukey’s multiple comparisons test ∗ p < 0.05.

Article Snippet: TaqMan Probe: CXCL10 (mouse) , Thermo Fisher Scientific , Mm00445235_m1.

Techniques: Recombinant, Activity Assay, In Vivo, Western Blot, Infection, Virus, Plaque Assay, Cytometry, Control, Injection, Enzyme-linked Immunospot, Irradiation, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: Nano-STAVs are readily opsonized by macrophages to stimulate STING signaling (A) Transmission electron microscopy image of nano-empty and nano-STAVs. (B and C) Western blot analysis of phosphorylated and total cGAS, STING, TBK1, and IRF3 proteins in WT or SKO murine macrophages (bone marrow-derived macrophages, BMDMs) and B16-OVA (2 × 10 5 cells) treated with nano-empty, nano-STAVs, or lipofectamine +/− STAVs at 1 μg/ml for 6 h. (D) macrophages and B16-OVA (5 × 10 4 cells) treated with nano-STAVs-Cy5 (red) (3 μg/mL) for 16 h, fixed and stained with DAPI (blue), and analyzed by microscopy confocal. (E and F) B16-OVA and macrophages (5 × 10 4 cells) were treated with nano-STAVs-Cy5 (red) (3 μg/mL) for 16 h, fixed and stained with EEA1-FITC, RAB7-FITC, CD63-FITC, LAMP1-FITC (green), and DAPI (blue), and analyzed by confocal microscopy. (G–N) (G–I and K–M) qPCR analysis of IFNb1, CXCL10, and CCL5 and (J and N) IFNb ELISA in B16-OVA, WT, and SKO macrophages (2 × 10 5 cells), treated with nano-empty, nano-STAVs, or lipofectamine +/− STAVs at 1 μg/ml for 6 h (qPCR) or 24 h (ELISA) ( n = 2 biological replicates). Error bars indicate mean ± SEM; Student’s t test ∗ p < 0.05.

Article Snippet: TaqMan Probe: CXCL10 (mouse) , Thermo Fisher Scientific , Mm00445235_m1.

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Derivative Assay, Staining, Microscopy, Confocal Microscopy, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: Murine and human tumor cells exposed to nano-STAVs activate APCs in trans in an STING-dependent manner, augmenting checkpoint therapeutic activity in vivo (A) Schematic representation of the phagocytosis of mouse and human cells by macrophages. 1 × 10 6 cells were treated with 1 μg/mL of nano-empty or nano-STAVs or transfected with lipofectamine + STAVs and irradiated by UV (120 mJ/cm). The irradiated cells were fed to murine or human macrophages (MØ) (2 × 10 5 cells) 24 h after UV irradiation. (B) Confocal microscopy analysis with nano-STAVs-cy5 (red) in CD11b + FITC murine macrophages (green). Cells were treated for 6 h with nanoparticles, and the phagocytosis was evaluated at 6 h. (C–H) (C, E, and G) RT-qPCR analysis of Cxcl10 at 6 h and (D, F, and H) IFN-β ELISA at 24 h in human and murine WT macrophages following engulfment of B16, SK-MEL-31, and SK-MEL-5 cells in presence or absence of nano-STAVs for 24 h ( n = 4 [mouse cell] and 3 [human cells] biological replicates). (I–L) Mice were subcutaneously injected with B16-OVA cells (5 × 10 5 cells/mouse) ( n = 13–17 mice per group on 2 independent experiments) on the right flank. On days 7, 10, and 13, after tumor inoculation, the mice were intratumorally injected with PBS, STAVs, nano-empty, or nano-STAVs (0.1 μg/mouse) and/or intraperitoneally with PD1 (50 μg/mouse) (black arrows). At day 19, the spleen was extracted to measure IFNg release from CD8 + T cells. (I) Schematic representation of experimental design. (J) The tumor volume was measured and calculated with the formula V = (length × width 2 )/2. (K) Digital photographs of tumors. (L) IFNg ELISpot. (C–H) Error bars indicate mean ± SEM; Student’s t test and (J and L) two-way ANOVA test with Tukey’s multiple comparisons test ∗ p < 0.05.

Article Snippet: TaqMan Probe: CXCL10 (mouse) , Thermo Fisher Scientific , Mm00445235_m1.

Techniques: Activity Assay, In Vivo, Transfection, Irradiation, Confocal Microscopy, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Injection, Enzyme-linked Immunospot

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: Nano-STAVs activity is augmented by type I IFN (A–D) Mice were subcutaneously injected with B16 OVA cells (5 × 10 5 cells/mouse) ( n = 17–18 mice per group on 2 independent experiments) on the right flank. On days 7, 10, and 13, after tumor inoculation, the mice were intratumorally injected with PBS, STAVs, nano-empty, or nano-STAVs (0.1 μg/mouse) and/or intraperitoneally with PD1 (50 μg/mouse) and/or IFNa (10,000 U/mouse) (black arrows). At day 17, the spleen was extracted to measure IFNg release from CD8 + T cells. (A) Schematic representation of experimental design. (B) The tumor volume was measured and calculated with the formula V = (length × width 2 )/2. (C) IFNg ELISpot. (D) Digital photographs of tumors. (E–J) qPCR analysis of CXCL10 and CCL5 in B16-OVA, B16-OVA-cGAS KO (CKO), and WT and SKO murine macrophages (2 × 10 5 cells), treated with IFNa at 1000 U/ml for 6 h ( n = 4 biological replicates). (K and L) Flow cytometry for H-2Kb and CD86 on macrophages (2 × 10 5 cells) following IFNa treatment at 1000 U/ml for 24 h ( n = 2 biological replicates). (M) Schematic representation of the phagocytosis of B16-OVA cells by macrophages. 1 × 10 6 B16-OVA cells were treated with IFNa at 1000 U/ml for 24 h and irradiated by UV (120 mJ/cm). The irradiated cells were fed to murine macrophages (MØ) (2 × 10 5 cells) previously treated or not with IFNa at 1000 U/ml for 24 h. (N) RT-qPCR analysis of Cxcl10 at 6 h ( n = 2 biological replicates). (O) IFN-β ELISA at 24 h of murine WT macrophages following engulfment of B16-OVA treated with IFNa ( n = 3 biological replicates). (P) Schematic representation of the phagocytosis of untreated B16-OVA cGAS KO cells (B16 CKO) by macrophages previously treated with IFNa at 1000 U/ml for 24 h. The conditions applied were the same as in (M). (Q) Flow cytometry for H-2Kb-SIINFEKL (OVA) on macrophages at 24 h following phagocytosis of B16-OVA cGAS KO ( n = 2 biological replicates). Error bars indicate mean ± SEM; Student’s t test and (B and C) two-way ANOVA test with Tukey’s multiple comparisons test ∗ p < 0.05.

Article Snippet: TaqMan Probe: CXCL10 (mouse) , Thermo Fisher Scientific , Mm00445235_m1.

Techniques: Activity Assay, Injection, Enzyme-linked Immunospot, Flow Cytometry, Irradiation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet: Nano-STAVs can facilitate the immunotherapeutic effects of radiation treatment in a head and neck tumor model (A–H) 2 × 10 5 MOC2 and Cal27 head and neck cell lines were treated with 1 μg/ml nano-empty, nano-STAVs, or lipofectamine +/− STAVs. (A and B) Western blot analysis of total and phosphorylated cGAS, STING, TBK1, IRF3 proteins, (C, D, F, and G) qPCR analysis of IFNb1 and CXCL10 after 6 h, and (E and H) IFNb ELISA 24 h after treatment ( n = 2 biological replicates). (I and J) MOC2 cells were irradiated with 10 Gy X-ray and then incubated for 24 h followed by treatment with nano-STAVs (1 μg/ml) for another 24 h. (I) For checking protein expression, MOC2 cells were collected and lysed with RIPA buffer and then analyzed by immunoblotting with indicated antibodies. (J) For phagocytosis assay, 2 × 10 6 MOC2 cells were phagocytosed with 2 × 10 6 dendritic cells (bone marrow-derived dendritic cells, BMDCs) for 6 h followed by isolating CD11c+ dendritic cells. Cxcl10 expression in dendritic cells was evaluated by qPCR analysis ( n = 2 biological replicates). (K and L) Immunocompetent C57BL/6J mice were subcutaneously injected with 5 × 10 5 MOC2 cells per mouse on the right flank and 2.5 × 10 5 MOC2 cells per mouse on left side. Right-side tumors were irradiated at days 4, 5 and 6 after tumor inoculation at 8 Gy (red arrows). On days 7, 10 and 13, after tumor inoculation, the mice were intratumorally injected on right side only with PBS, STAVs, nano-empty, or nano-STAVs (0.1 μg/mouse) and/or with PD1 (100 μg/mouse) (black arrows). The tumor volume was measured and calculated with the formula V = (length × width 2 )/2. (K) Schematic representation of experimental design. (L and M) (L) Immunocompetent WT ( n = 6 mice per group) or (M) STING KO C57BL/6J mice ( n = 6 mice per group). The tumor volumes are represented from day 21 or 24. (N and O) (N) Primary (right flank) and (O) abscopal effects (left flank) of the treatments ( n = 6 mice per group). Error bars indicate mean ± SEM; (C–J and O) Student’s t test and (N) two-way ANOVA test with Tukey’s multiple comparisons test ∗ p < 0.05.

Article Snippet: TaqMan Probe: CXCL10 (mouse) , Thermo Fisher Scientific , Mm00445235_m1.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Irradiation, Incubation, Expressing, Phagocytosis Assay, Derivative Assay, Injection

Journal: Cell Reports Medicine

Article Title: Exogenous non-coding dsDNA-dependent trans -activation of phagocytes augments anti-tumor immunity

doi: 10.1016/j.xcrm.2024.101528

Figure Lengend Snippet:

Article Snippet: TaqMan Probe: CXCL10 (mouse) , Thermo Fisher Scientific , Mm00445235_m1.

Techniques: Control, Staining, Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Knock-Out, Plasmid Preparation, Expressing, Software, Microscopy

Journal: Communications Biology

Article Title: Reshaping tumor microenvironment by regulating local cytokines expression with a portable smart blue-light controlled device

doi: 10.1038/s42003-024-06566-y

Figure Lengend Snippet: a Schematics of light-controlled gene expression system and plasmids constructions. b Regulation of gene expression with light intensities from 0 to 1000 μW cm -2 with a customized 24-well plate light-controllable hardware, and c the intensity-response curve of mRuby expression in P815-M-ILs cells at 48 h (flow cytometry assay). d Time-dependent mRNA expression levels of Ifng and Cxcl10 (qPCR assay, normalized to time zero) in P815-IFNG cells and P815-M cells (control group). e Time-dependent production of murine IFNG and CXCL10 proteins (ELISA assay) under the continuous illuminating (On), dark control (Off) and alternative illuminating group (12/12 h light/dark cycles, 12 h O/N). Flow cytometry data is a representative of three independent experiments, each with similar results. Other data are shown as mean ± sem ( n = 3). All statistical significant were computed against the control group, with * P < 0.05; ** P < 0.01.

Article Snippet: Cell supernatants from in vitro studies were collected at designed time points (0, 12, 24, 48, and 72 h after illumination) to quantify cytokines and chemokines with murine CXCL10 and IFNG ELISA kit (BOSTER, China).

Techniques: Gene Expression, Expressing, Flow Cytometry, Control, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: Reshaping tumor microenvironment by regulating local cytokines expression with a portable smart blue-light controlled device

doi: 10.1038/s42003-024-06566-y

Figure Lengend Snippet: a Timeline of in vivo experiment using DBA/2 mice with matched bilateral tumors (Left: P815-IFNG; Right: P815-M). b During illumination, representative BLI images of bilateral tumor-bearing mice at different times (Day 0, Day 3, and Day 6). c – d Representative BLI images of bilateral tumor-bearing mice were taken on different days (Day 0, Day 3, and Day 6) after illumination. Tumor size was quantified with average radiance value and compared with BLI signal at day 0 (the first day of illumination). e – g Blood analyses were performed after 6-day illumination, and the results show changes in the number of white blood cells (WBC) e , lymphocytes f , neutrophils g , monocytes ( h ) between light (2.5 mW cm -2 ), dark and control groups. i – l The concentration of murine INFG i , CXCL10 j , IL-6 k , and IL-10 ( l ) in plasma by the end of day 6 under illumination. Data are shown as mean ± s.e.m ( n = 4). Statistic significant was computed against the control group as * P < 0.05; ** P < 0.01.

Article Snippet: Cell supernatants from in vitro studies were collected at designed time points (0, 12, 24, 48, and 72 h after illumination) to quantify cytokines and chemokines with murine CXCL10 and IFNG ELISA kit (BOSTER, China).

Techniques: In Vivo, Control, Concentration Assay, Clinical Proteomics

Journal: The Journal of Experimental Medicine

Article Title: Role for NF-κB in herpes encephalitis pathology in mice genocopying an inborn error of IRF3-IFN immunity

doi: 10.1084/jem.20250064

Figure Lengend Snippet: Comparative analysis of systemic and CNS responses to HSV-1 infection in vivo and in vitro . (A–C) Analysis of viral replication on day 4 after HSV-1 ocular infection (2 × 10 6 PFU/eye) of WT ( n = 12) and Irf3 WT/R278Q heterozygous ( n = 12) mice. UI controls were included (WT UI, n = 3; Irf3 WT/R278Q UI, n = 3). Viral titer in (A) eyes, (B) TG, and (C) brain stem homogenates quantified by plaque assay. (D–I) Systemic cytokine levels measured in serum from WT and Irf3 R278Q/R278Q mice on day 5 after HSV-1 infection. Data points represent cytokine level (pg/ml) in serum from individual mice measured by mesoscale. (J–N) CNS response in WT and Irf3 R278Q/R278Q mice to systemic LPS stimulation in vivo . Gene expression of Ifnb , Tnfa , Il1b , Il6 , and Ccl2 in whole-brain homogenates from mice treated with 5 mg/kg LPS or saline by i.p. injection. Gene expression was measured by RT-qPCR, and data were normalized to β-actin ( Actb ). (O) Validation of microglia depletion by qPCR of Iba1 expression in MBCs treated with 0.5 μM PLX5622 (PLX) or untreated (UT). (P and Q) Expression of Ccl2 in WT mixed brain cultures treated with (P) 0.5 μM PLX5622 (PLX) or mock treated for microglia depletion, or (Q) NF-κB activation inhibitors BMS-345541 2 µM, PDTC 25 µM, or saline, and infected with HSV-1 at MOI 1.0 for 24 h. (R and S) Isg15 and Cxcl10 infection-dose response analysis in murine microglia analyzed by RT-qPCR 24 h after infection with HSV-1 at increasing MOI. For all panels where statistical analyses were performed, the analysis was two-tailed two-way ANOVA for difference of means, followed by two-tailed unpaired t test of means, error bars; SD. P values <0.05 were considered statistically significant. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: Quantitative PCR was performed using the following TaqMan Gene Expression Assays (Applied Biosystems): ACTB (Hs01060665_g1), 18S (Hs03003631_g1), IFNB (Hs01077958_s1), IL6 (Hs00174131_m1), TNFA (Hs00174128_m1), IL1B (Hs01555410), MX1 (Hs00895598_m1), CXCL10 (Hs00171042), and ISG15 (Hs01921425). mRNA levels of interest were normalized to the housekeeping gene ACTB or 18S (as indicated) using the ΔΔCt method.

Techniques: Infection, In Vivo, In Vitro, Plaque Assay, Gene Expression, Saline, Injection, Quantitative RT-PCR, Biomarker Discovery, Expressing, Activation Assay, Two Tailed Test

Journal: Animal Cells and Systems

Article Title: CXCL10 promotes melanoma angiogenesis and tumor growth

doi: 10.1080/19768354.2024.2402024

Figure Lengend Snippet: Host-deficiency of CXCL10 decreases melanoma tumor growth and angiogenesis in vivo. B16-FL cells were subcutaneously injected into WT and Cxcl10 -/- mice. Tumor burden and tumor angiogenesis were then analyzed. (A) Serum CXCL10 levels at day 21 in non-tumor-bearing or B16-FL tumor-bearing WT and Cxcl10 -/- mice. (B) A schematic diagram of experimental procedures (upper panel). Representative bioluminescence images on days 5, 10, and 21 after injection of B16-FL cells (bottom panel). (C) Bioluminescence imaging analysis of tumor burden on indicated days. (D) Gross of tumor-feeding vessels at 10 days after tumor formation. Quantification of tumor-feeding vessels is shown. (E) IHC analyses of tumor tissues with an anti-CD31 antibody (upper images) and H&E staining (bottom images) at 5 days after tumor formation (left panel). Quantification of microvessel number (right panel) is shown. Scale bar, 50 μm (CD31) and 100 μm (H&E). (F) TCGA correlation analyses between Cxcl10 mRNA expression and CD31 mRNA expression from TCGA-SKCM (skin cutaneous melanoma) data ( n = 468). Data are presented as mean ± SD of three independent experiments ( A, C, D, E ). *, P < 0.05; **, P < 0.01; ***, P < 0.001, based on the Student’s t-test.

Article Snippet: Serum protein levels of mouse CXCL10 were measured using a Mouse CXCL10 DuoSet ELISA kit (#DY466; R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: In Vivo, Injection, Imaging, Staining, Expressing

Journal: Animal Cells and Systems

Article Title: CXCL10 promotes melanoma angiogenesis and tumor growth

doi: 10.1080/19768354.2024.2402024

Figure Lengend Snippet: CXCL10 induces expression of pro-angiogenic factors in B16F10 melanoma cells and enhances B16F10-mediated angiogenesis in vitro . (A) Relative mRNA expression levels of vegf , pdgf-b , fgf2 , hgf, and angpt2 in B16F10 cells treated with or without CXCL10 (100 ng/ml) for 24 h. (B) GEO analysis of Cxcl10 mRNA expression in normal ( n = 122) and melanoma tumor ( n = 58) tissues from GSE13355 data. (C and D) TCGA correlation analyses between Cxcl10 mRNA expression and vegf , pdgf-b , fgf2 , hgf, or angpt2 mRNA expression from TCGA-SKCM (skin cutaneous melanoma) data (C; n = 468) and TCGA-UM (uveal melanoma) data (D; n = 80), respectively. (E) HUVECs were treated with or without conditioned medium (CM) from control B16F10 cells or CXCL10-treated B16F10 cells in the presence or absence of VEGF (20 ng/mL). After 4 h, tube formation of HUVECs was observed. Representative images were acquired under an optical microscope (50×) and tube number (/field) was quantified. (F) HUVECs were treated with or without CM from control B16F10 cells or CXCL10-treated B16F10 cells in the presence or absence of VEGF (20 ng/mL). After 24 and 48 h, the cells were analyzed by a WST-8 assay. Data are presented as mean ± SD of three independent experiments ( A, E, F ). *, P < 0.05; **, P < 0.01; ***, P < 0.001, based on the Student’s t-test.

Article Snippet: Serum protein levels of mouse CXCL10 were measured using a Mouse CXCL10 DuoSet ELISA kit (#DY466; R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Expressing, In Vitro, Control, Microscopy

Journal: Animal Cells and Systems

Article Title: CXCL10 promotes melanoma angiogenesis and tumor growth

doi: 10.1080/19768354.2024.2402024

Figure Lengend Snippet: CXCL10 induces activation of pro-angiogenic and pro-growth signals in B16F10 melanoma cells. (A) Serum-starved B16F10 cells treated with or without CXCL10 (100 ng/mL) for the indicated period of time. Immunoblotting analyses were performed with indicated antibodies. (B) Serum-starved B16F10 cells treated with or without CXCL10 (100 ng/mL) for 48 h. Immunoblotting analyses were performed with indicated antibodies. (C) Serum-starved indicated cancer cells treated with or without CXCL10 (100 ng/mL), TGF-β (20 ng/mL) or IL-1 (10 ng/mL) for 48 h. Immunoblotting analyses were performed with indicated antibodies.

Article Snippet: Serum protein levels of mouse CXCL10 were measured using a Mouse CXCL10 DuoSet ELISA kit (#DY466; R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: Activation Assay, Western Blot

Journal: Animal Cells and Systems

Article Title: CXCL10 promotes melanoma angiogenesis and tumor growth

doi: 10.1080/19768354.2024.2402024

Figure Lengend Snippet: CXCL10 induces B16F10 melanoma tumor growth in an in vitro 3D culture. B16F10 melanoma cells were cultured on top of a thin layer of Matrigel for 10 days with or without CXCL10 (100 ng/ml). After culturing, colonies were measured (A) and counted (B). Data are presented as mean ± SD of three independent experiments. *, P < 0.05 based on Student’s t-test. Scale bar, 100 μm.

Article Snippet: Serum protein levels of mouse CXCL10 were measured using a Mouse CXCL10 DuoSet ELISA kit (#DY466; R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

Techniques: In Vitro, Cell Culture

Journal: The Journal of infectious diseases

Article Title: The CXC chemokine MIG/CXCL9 is important in innate immunity against Streptococcus pyogenes.

doi: 10.1086/510857

Figure Lengend Snippet: Figure 1. Presence of chemokines at the tonsillar surface during streptococcal pharyngitis and their production by inflamed epithelial cells in vitro. A, Large amounts of monokine induced by interferon (IFN)–g (MIG)/CXCL9 in tonsil fluid during streptococcal pharyngitis. Swabs were used to collect fluid from the surface of tonsils, and the chemokine content was determined by ELISA. Samples were taken either from patients with pharyngitis caused by Streptococcus pyogenes (np8) or from healthy control subjects (np8). B, Parallel between production of MIG/CXCL9, IFN-inducible protein (IP)–10/CXCL10, and IFN-inducible T cell a-chemoattractant (I-TAC)/CXCL11 by inflamed pharyngeal cells in vitro and that in vivo. A human pharyngeal epithelium cell line (Detroit 562) was used to investigate the production of MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 on stimulation with proinflammatory cytokines. The cell line was grown to confluence and subsequently stimulated with IFN-g (100 U/mL) and tumor necrosis factor–a (10 ng/mL) for 96 h. The content of MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 in the cell culture medium was determined by ELISA. Data are values from 3 independent experiments. C–E, Increased production of MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 by inflamed pharyngeal mean SE cells in response to S. pyogenes. Detroit 562 cells were incubated with IFN-g (100 U/mL) in the absence (white boxes) or presence (black circles) of heat-killed S. pyogenes bacteria of the AP1 strain ( cfu/mL). The concentration of MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 was determined 8 1 10 in the cell culture medium by ELISA at the time points indicated. Data are values from 3 separate experiments. mean SE

Article Snippet: Recombinant human IFN-g, TNFa, growth-related oncogene (GRO)–a/CXCL1, interleukin (IL)– 8/CXCL8, MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 peptides; polyclonal goat antibodies against MIG/CXCL9, IP-10/ CXCL10, and I-TAC/CXCL11; and ELISAs for the detection of MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 were obtained from R&D Systems.

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Control, In Vivo, Cell Culture, Incubation, Bacteria, Concentration Assay

Journal: The Journal of infectious diseases

Article Title: The CXC chemokine MIG/CXCL9 is important in innate immunity against Streptococcus pyogenes.

doi: 10.1086/510857

Figure Lengend Snippet: Figure 2. Analysis of the bactericidal activity of chemokines. A–C, Antibacterial activity of recombinant monokine induced by interferon (IFN)–g (MIG)/CXCL9, IFN-inducible protein (IP)–10/CXCL10, and IFN-inducible T cell a-chemoattractant (I-TAC)/CXCL11 against Streptococcus pyogenes (AP1 strain). Bacteria (50 mL; cfu/mL) were incubated with the chemokines at the concentrations indicated for 2 h at 37C. Bacterial killing (in 6 2 10 percentage) was determined by comparing the no. of bacterial colonies after exposure to chemokines with the no. obtained after incubation in buffer alone. Data are values from 4–7 separate experiments. D, Bacterial killing in the absence or presence of sodium chloride (150 mmol/L). mean SE S. pyogenes (AP1 strain) (50 mL; cfu/mL) was incubated with MIG/CXCL9, IP-10/CXCL10, or I-TAC/CXCL11 (0.2 mmol/L) as described for panels 6 2 10 A–C. Data are values from 4 separate experiments. E, Structure prediction of MIG/CXCL9 showing an NH2-terminal region containing 3 mean SE antiparallel b strands and an extended a-helix in the COOH-terminal part of the molecule. Two peptides were synthesized spanning cationic regions in the NH2-terminal (red) and COOH-terminal (green) regions. The sequences of the peptides and their isoelectric points (pI) are indicated in the figure. F, Separate incubation of the NH2- and COOH-terminal peptides with S. pyogenes (AP1 strain) (50 mL; cfu/mL) for 2 h at 37C. Bacterial killing 6 2 10 was determined by comparing the no. of colony-forming units after incubation in medium alone. Data are values from 3 separate mean SE experiments.

Article Snippet: Recombinant human IFN-g, TNFa, growth-related oncogene (GRO)–a/CXCL1, interleukin (IL)– 8/CXCL8, MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 peptides; polyclonal goat antibodies against MIG/CXCL9, IP-10/ CXCL10, and I-TAC/CXCL11; and ELISAs for the detection of MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 were obtained from R&D Systems.

Techniques: Activity Assay, Recombinant, Bacteria, Incubation, Synthesized

Journal: The Journal of infectious diseases

Article Title: The CXC chemokine MIG/CXCL9 is important in innate immunity against Streptococcus pyogenes.

doi: 10.1086/510857

Figure Lengend Snippet: Figure 3. Inhibition of the antibacterial, but not the chemotactic, activity of monokine induced by interferon (IFN)–g (MIG)/CXCL9, IFN-inducible protein (IP)–10/CXCL10, and IFN-inducible T cell a-chemoattractant (I-TAC)/CXCL11 by the streptococcal inhibitor of complement (SIC) protein. A, Incubation of Streptococcus pyogenes (strain AP1) (50 mL; cfu/mL) with MIG/CXCL9 (0.2 mmol/L) in the absence or presence of SIC at the 6 2 10 concentrations indicated. Data are values from 4 separate experiments. B, Determination of the antibacterial activity of MIG/CXCL9, IP- mean SE 10/CXCL10, and I-TAC/CXCL11 (all at 0.2 mmol/L) against S. pyogenes (strain AP1) (50 mL; cfu/mL) in the absence or presence of SIC (1 mmol/ 6 2 10 L). Student’s t test for paired observations was used to calculate the P values (* ; *** ). C, Injection of MIG/CXCL9 at different P ! .05 P ! .005 concentrations (31–250 nmol/L) over SIC immobilized on a BIAcore CM5 sensor chip. The injection was started at (indicated by the left arrow) t p 0 and was replaced by running buffer after the binding plateaus (indicated by the right arrow at 100 s), resulting in dissociation of bound peptide. Binding parameters were determined, and the dissociation rate constant was calculated to be 145 nmol/L. Data from 1 representative experiment are shown. D, Investigation of the effect of SIC on the chemotactic activity of the chemokines, by use of a chemotactic assay with cells transfected with CXC chemokine receptor 3 (CXCR3), the MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 receptor. The chemokines (50 nmol/L) were preincubated with buffer alone or with SIC (1.6 mmol/L) before the chemotactic assay. Cells were allowed to migrate in a modified Boyden chamber with a filter pore size of 5 mm for 5 h. Data are values from 3 separate experiments. E, Electron micrographs showing S. pyogenes exposed to MIG/CXCL9 mean SE in the absence (left) or presence (right) of SIC. MIG/CXCL9 caused protrusions and leakage of cellular content from the bacteria (left; arrows), which was not seen with SIC present (right). MIG/CXCL9 was labeled with 5-nm colloidal gold, and the left insert shows MIG/CXCL9 in association with lysed bacteria. The insert on the right electron micrograph shows gold-labeled MIG/CXCL9 associated with M proteins on the surface of intact bacteria. RU, resonance units.

Article Snippet: Recombinant human IFN-g, TNFa, growth-related oncogene (GRO)–a/CXCL1, interleukin (IL)– 8/CXCL8, MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 peptides; polyclonal goat antibodies against MIG/CXCL9, IP-10/ CXCL10, and I-TAC/CXCL11; and ELISAs for the detection of MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11 were obtained from R&D Systems.

Techniques: Inhibition, Activity Assay, Incubation, Injection, Binding Assay, Chemotaxis Assay, Transfection, Pore Size, Bacteria, Labeling

Journal: Cancer Science

Article Title: EBV ‐positive pyothorax‐associated lymphoma expresses CXCL9 and CXCL10 chemokines that attract cytotoxic lymphocytes via CXCR3

doi: 10.1111/cas.15782

Figure Lengend Snippet: Expression analysis of CXCR3 ligand genes in PAL cell lines. Relative mRNA expression levels were calculated using the 2 − Δ C t values or 2 − ΔΔ C t method with B2M used as the housekeeping control. (A) Expression of CXCL9 , CXCL10 , and CXCL11 in six PAL cell lines and 10 EBV‐negative DLBCL cell lines. The graph includes the average values in PBMCs obtained from 12 healthy donors as the control. (B) Effects of the blockade of the signaling pathways on the expression of CXCL9 and CXCL10 . Cells were treated with various inhibitors of signaling pathways or DMSO as a control. (C) Effects of cytokines on the expression of CXCL9 and CXCL10 . Cells were treated with various inflammatory cytokines: IL‐6 at 10 ng/mL, TNF‐α at 50 ng/mL, and IFN‐γ at 100 ng/mL, or PBS as a control. Data are shown as the mean ± SEM of three independent experiments. Significant differences are shown as * P < 0.05.

Article Snippet: The primers and fluorogenic probes for CXCL9 (Hs00171065_m1), CXCL10 (Hs01124252_g1), CXCL11 (Hs00171138_m1), and β2‐microglobulin ( B2M ; Hs00187842_m1) were based on the information provided for TaqMan Gene Expression Assays (Thermo Fisher Scientific).

Techniques: Expressing, Control, Protein-Protein interactions

Journal: Cancer Science

Article Title: EBV ‐positive pyothorax‐associated lymphoma expresses CXCL9 and CXCL10 chemokines that attract cytotoxic lymphocytes via CXCR3

doi: 10.1111/cas.15782

Figure Lengend Snippet: Secretion of CXCL9 and CXCL10 by PAL and EBV‐negative DLBCL cell lines in vitro. The cells were seeded into 24‐well plates at 5 × 10 5 /ml and cultured for 3 days. Concentrations of CXCL9 and CXCL10 in the cell‐free culture supernatants were measured by ELISA. Data are shown as the mean ± SEM of three independent experiments.

Article Snippet: The primers and fluorogenic probes for CXCL9 (Hs00171065_m1), CXCL10 (Hs01124252_g1), CXCL11 (Hs00171138_m1), and β2‐microglobulin ( B2M ; Hs00187842_m1) were based on the information provided for TaqMan Gene Expression Assays (Thermo Fisher Scientific).

Techniques: In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cancer Science

Article Title: EBV ‐positive pyothorax‐associated lymphoma expresses CXCL9 and CXCL10 chemokines that attract cytotoxic lymphocytes via CXCR3

doi: 10.1111/cas.15782

Figure Lengend Snippet: Expression of CXCL9 and CXCL10 in primary PAL tissues. (A) Specimens of PAL and DLBCL, NOS, were stained with hematoxylin and eosin. Immunohistochemistry was performed on the sections using isotype control IgG antibody, anti‐CXCL9 antibody, and anti‐CXCL10 antibody. Samples from representative patients are shown. (B) Immunofluorescent double staining for CD20 (red) and CXCL9/CXCL10 (green). (C) Immunofluorescent double staining for LMP1 (red) and CXCL9/CXCL10 (green). (D) Immunofluorescent double staining for EBNA2 (red) and CXCL9/CXCL10 (green). The nuclei were counterstained with DAPI (blue). Box plots depict the percentages of the double staining‐positive cells in PAL tissues ( n = 5) and DLBCL, NOS, tissues ( n = 6). For the semiquantitative assessment of protein expression, the percentage of positive cells was calculated in more than five randomly selected higher‐power fields that included over 100 cells each, and the mean was used as the labeling index for each lesion. Data are shown as the mean ± SD. Significant differences are shown as ** P < 0.01.

Article Snippet: The primers and fluorogenic probes for CXCL9 (Hs00171065_m1), CXCL10 (Hs01124252_g1), CXCL11 (Hs00171138_m1), and β2‐microglobulin ( B2M ; Hs00187842_m1) were based on the information provided for TaqMan Gene Expression Assays (Thermo Fisher Scientific).

Techniques: Expressing, Staining, Immunohistochemistry, Control, Double Staining, Labeling

Journal: Cancer Science

Article Title: EBV ‐positive pyothorax‐associated lymphoma expresses CXCL9 and CXCL10 chemokines that attract cytotoxic lymphocytes via CXCR3

doi: 10.1111/cas.15782

Figure Lengend Snippet: Induction of CXCR3‐mediated cell migration by secretions from PAL cells. (A) Culture supernatants of OPL‐2 and OPL‐7 cells were tested for chemotactic activity using CXCR3‐expressing B300‐19 cells. Serially diluted culture supernatants were also tested for chemotactic activity. Cell migration is expressed as a percentage of the input cells. Recombinant CXCL9/CXCL10 and medium alone were used as positive and negative controls, respectively. (B) Effect of anti‐CXCL9 antibody (1 μg/ml), anti‐CXCL10 antibody (1 μg/ml), and AMG487 (1 μM) on CXCR3‐mediated cell migration. (C) Cell migration of human PBMCs induced by the culture supernatants from OPL‐2 cells was measured. The cells that migrated into the lower wells were stained for CD3, CD4, CD8, CD56, and CXCR3, and quantified by flow cytometry. Results from three to four separate experiments are shown as the mean ± SEM. Significant differences are shown as * P < 0.05 and ** P < 0.01.

Article Snippet: The primers and fluorogenic probes for CXCL9 (Hs00171065_m1), CXCL10 (Hs01124252_g1), CXCL11 (Hs00171138_m1), and β2‐microglobulin ( B2M ; Hs00187842_m1) were based on the information provided for TaqMan Gene Expression Assays (Thermo Fisher Scientific).

Techniques: Migration, Activity Assay, Expressing, Recombinant, Staining, Flow Cytometry

Journal: Cancer Science

Article Title: EBV ‐positive pyothorax‐associated lymphoma expresses CXCL9 and CXCL10 chemokines that attract cytotoxic lymphocytes via CXCR3

doi: 10.1111/cas.15782

Figure Lengend Snippet: In vivo recruitment of cytotoxic effector lymphocytes via CXCR3 secreted by PAL cells. (A) OPL‐2, OPL‐7, Nu‐DUL‐1, or WSu‐DLCL‐2 cells (5 × 10 5 ) were injected intraperitoneally into BALB/c mice. After 24 h, cells were isolated from the peritoneal cavity and the cell numbers were counted. The cells were stained for CD4, CD8, CD49b, and IFN‐γ. The CD4 + IFN‐γ + cells, CD8 + IFN‐γ + cells, and CD49b + NK cells were counted by flow cytometry using the CD45 gate. Total cell number was calculated using the following formula: total cell number = number of total peritoneal exudate cells enumerated by cell counting × the ratio of CD4 + IFN‐γ + cells, CD8 + IFN‐γ + cells, or CD49b + cells analyzed by flow cytometry. Control mice were injected with medium alone. The data are expressed as the SEM of the results obtained from three to 10 mice. (B) The CD4 + IFN‐γ + cells, CD8 + IFN‐γ + cells, and CD49b + cells were analyzed further for CXCR3 expression by flow cytometry. Representative data are shown from at least three independent experiments. (C) OPL‐2 or OPL‐7 cells (5 × 10 5 ) were injected intraperitoneally into BALB/c mice with or without AMG487 at a concentration of 0.5 or 5.0 mg/kg. (D) OPL‐2 or OPL‐7 cells (5 × 10 5 ) were injected intraperitoneally into BALB/c mice with 10 μg of isotype control, anti‐CXCL9 antibody, or anti‐CXCL10 antibody. The data are expressed as the SEM of the results obtained from three to four mice. Significant differences are shown as * P < 0.05.

Article Snippet: The primers and fluorogenic probes for CXCL9 (Hs00171065_m1), CXCL10 (Hs01124252_g1), CXCL11 (Hs00171138_m1), and β2‐microglobulin ( B2M ; Hs00187842_m1) were based on the information provided for TaqMan Gene Expression Assays (Thermo Fisher Scientific).

Techniques: In Vivo, Injection, Isolation, Staining, Flow Cytometry, Cell Counting, Control, Expressing, Concentration Assay

Journal: Cell metabolism

Article Title: T-bet + B cells accumulate in adipose tissue and exacerbate metabolic disorder during obesity.

doi: 10.1016/j.cmet.2022.07.002

Figure Lengend Snippet: Figure 5. T-bet-expressing B cells secrete CXCL10 and contribute to metabolic disorder in obesity (A) Representative flow cytometry histograms and dot plot summaries of cell size (FSC-A), intracellular T-bet protein expression, and surface CD69, CD86, CD21, and CD1d protein expression of isolated WT splenic B cells cultured in vitro with (red dots/histograms) or without (gray dots/histograms) 2.5 mg/mL R848 for 48 h. (B) ELISA quantification of CXCL10 in supernatants from WT splenic B cells stimulated with (black) or without (white) R848 as in (A). (C) ELISA measured CXCL10 in day 4 serum of WT mice with (black) or without (white) i.p. injection of 50 mg R848 on day 0 and day 3. (D and E) GTT and glucose area under curve (AUC) for WT mice fed the NCD (D, circles) or HFD (E, squares) with (black) or without (white) transfer of B cells enriched for T-bet+ CD11c+ B cells. Donor B cells were from WT mice treated with R848 and NP-KLH in vivo prior to isolation/transfer. See also Figure S5. Bar graphs show GTT AUC 4 days after transfer. Data are from one experiment with at least 4 mice/group (A), pooled from three experiments (B and C), or from 2 experiments with 2–4 mice/group (D and E). Symbol centers or bar heights indicate individual values or group means, respectively, and data show mean ± SEM; Student’s t test; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER NP-KLH Biosearch Technologies N-5060-5 DMEM Fisher MT10014CV BSA Lee Biosolutions 100-12 PBS Fisher BP243820 Critical commercial assays CXCL10 ELISA R&D systems DY466-05 Foxp3/Transcription Factor Staining Buffer Set eBioscience Cat# 00-5523-00 LIVE/DEAD Fixable Near-IR Dead Cell Staining Kit Life Technologies Cat# L10119 B cell isolation kit StemCell 19854 Melon Gel IgG Spin ThermoFisher 45206 Pierce Protein Concentrators PES, 50K MWCO ThermoFisher 88538 Experimental models: Organisms/strains Mouse: Wild type C57BL/6J The Jackson Laboratory Strain Code: JAX 000664 Mouse: WT Congenic (CD45.1) B6.SJL-Ptprca Pepcb/BoyJ The Jackson Laboratory Strain Code: JAX 002014 Mouse: GREAT C.129S4(B6)-Ifngtm3.1Lky/J Markus Mohrs Strain code: JAX 017580 Mouse: Va14tg C57BL/6-Tg(Cd4-TcraDN32D3)1Aben/J Michael Brenner; The Jackson Laboratory Strain Code: JAX 014639 Mouse: B6.SJL B1-8hi B6.129P2 (C)-Ighttm12Cgn/J Michel Nussenzweig Strain code: JAX 12642 Mouse: C57BL/6 Ja18 / Michael Brenner N/A Mouse: CD1d / B6.129S6-Del(3Cd1d2-Cd1d1)1Sbp/J Mark Exley; The Jackson Laboratory Strain Code: JAX 008881 Mouse: Tbetfl/fl B6.129-Tbx21tm2Srnr/J mice Nu Zhang; The Jackson Laboratory Strain Code: JAX 022741 Mouse: CD19Cre C.Cg-Cd19tm1(cre)Cgn Ighb/J The Jackson Laboratory Strain Code: JAX 004126 Mouse: T-betfl/fl CD19cre/+; C.Cg-Cd19tm1(cre)Cgn Ighb/J crossed with B6.129-Tbx21tm2Srnr/J mice This paper N/A Mouse: CXCL10 / C.129S4(B6)-Cxcl10tm1Adl/J The Jackson Laboratory Strain code: JAX 006087 Oligonucleotides Primer Tbx21 Forward: 50-AACTGTGTTCCCGAGGTGTC-30 N/A N/A Primer Tbx21 Reverse: 30- GGAGCCCACAAGCCATTACA-30 N/A N/A Software and algorithms GraphPad Prism v7 GraphPad https://www.graphpad.com/scientific- software/prism/ FlowJo (version 10.1) FlowJo, LLC https://www.flowjo.com MS Excel 2016 Microsoft N/A Diva BD Biosciences http://www.bdbiosciences.com/us/ instruments/clinical/software/flowcytometryacquisition/bd-facsdivasoftware/m/333333/ overview Other High Fat Diet (HFD; 60% kcal fat) Research Diets D12492 FACS analyzer BD Bioscience BD FACSCelesta FACS sorter BD Bioscience BD FACSAriaIIIb 100 mm cell strainer Fisher 22-363-549; 08-771-19 40 mm cell strainer Fisher 22-363-547; 08-771-1 Crysostat microtome Leica VT1000S Glucose strips Bayer Contour 7097C Glucometer Bayer Contour 9545C Cell Metabolism 34, 1121–1136.e1–e6, August 2, 2022 e3

Techniques: Expressing, Cytometry, Isolation, Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay, Injection, In Vivo

Journal: Nature immunology

Article Title: Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING

doi: 10.1038/ni.2350

Figure Lengend Snippet: VLPs induce a type I IFN response. ( a ) Structural contents of VLPs and virions visualized with cryo-EM (top) and immunoblotting against VP5 and gD (bottom). Images are from one representative of two independent experiments. ( b ) PCs from C57BL6 mice were treated with either L-particles or PREPs (MOI 15) for 4 hours. RNA from two independent experiments was collected and analyzed by Nanostring technology. Numbers indicate fold increase against untreated controls. Data represent means from two independent experiments. Green indicates an increase of >3 fold, red a decrease of >3 fold, and yellow no change (<3 fold). ( c,d ) RNA from samples prepared as in ( b ) was analyzed by QPCR after treatment with PREPs ( c ) or L-particles ( d ) (5–15 MOI). ( e,f ) CXCL10 expression in cultures of BMDCs ( e ) and hMDMs ( f ) treated with VLPs (MOI 15) for 4 hours. Data in c, d, e , and f represent means and s.e.m. of five independent experiments. ( g ) BMDCs were treated with VLPs or dilutions of infectious HSV1 stock for 24 hours. Culture supernatants were analyzed for CXCL10 using ELISA. ( h, i ) Vials of PREPs and L-particles were treated with DNase I for 20 min or left untreated. Part of each vial was analyzed for viral DNA content by QPCR ( h ), while the remaining were used to treat PCs followed by analysis of CXCL10 mRNA expression ( i ). ( j ) PCs from Ifnar1 −/− mice were treated as described above and analyzed for CXCL10 expression. Data in g–j represent means and s.e.m. of two independent experiments.

Article Snippet: Cells were then stained with DAPI and anti-CXCL10 Ab (G15, Santa Cruz).

Techniques: Cryo-EM Sample Prep, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING

doi: 10.1038/ni.2350

Figure Lengend Snippet: VLPs increase TLR7 and TLR9 sensitivity and induce leukocyte recruitment and activation in vivo . ( a ) PCs from C57BL6 mice were pretreated with LPS (20 ng/ml) and subsequently treated with either media (M), HSV (MOI 3), VLP (MOI 15), or p(dAdT) (2 µg/ml). Supernatants were harvested 24 h later and analyzed for IL-1β protein. ( b ) BMDCs were pre-stimulated with LPS for 3 hours. Cells were then either left untreated or stimulated with either VLPs or HSV-1 for 16 hours. Supernatants were subsequently analyzed for IL-1β by immunoblotting. ( c ) PCs were treated with VLPs or HSV-1 for 6 hours. Whole-cell extracts were analyzed for LC3 II by immunoblotting (bottom) or for LC3 foci formation by confocal microscopy (top). ( d ) PCs were pre-stimulated with VLPs for two hours. Cells were then treated with suboptimal concentration of ODN1829 (0.5µM) or ssRNA40 (1µg/mL) for an additional 4 hours. Cells were then analyzed for CXCL10 expression by QPCR. Data in a–d show one representative of two independent experiments. ( e–h ) Eight-week-old mice were injected intraperitoneally either with PBS (M), VLPs, or ΔgB VLPs both suspended in PBS (25×10 6 particles per animal). Injections were repeated after 12 hours. After 24 hours cells were harvested from the peritoneal cavity. Cell numbers and the expression of surface markers were quantified by flow cytometry using count beads. Flow-charts show representative stainings using cells from mice injected with VLPs. Data in f–h show one representative of two independent experiments each with 7 mice in each experimental group. *Indicates P<0.05 using a students t-test.

Article Snippet: Cells were then stained with DAPI and anti-CXCL10 Ab (G15, Santa Cruz).

Techniques: Activation Assay, In Vivo, Western Blot, Confocal Microscopy, Concentration Assay, Expressing, Injection, Flow Cytometry

Journal: Nature immunology

Article Title: Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING

doi: 10.1038/ni.2350

Figure Lengend Snippet: Fusion induces type I IFN and CXCL10 expression. ( a–c ) VLPs prepared from wt ( a ) or the fusion deficient HSV-1 mutants ΔgH ( b ) and ΔgB( c ) were used to treat PCs (MOI 15) from C57BL6 mice for 4 hours. Expressions of CXCL10, TNF and IFNβ were analyzed by QPCR. Data in a–c represent means and s.e.m. of four independent experiments. ( d ) Wt, ΔgH and ΔgB VLPs were used to treat hMDMs as in panel a and analyzed for CXCL10 expression by QPCR. ( e–f ) HEK293 cells were transfected with expression plasmids for wt HIV Env, F522 Env (FY), or the ΔKS Env. After two days, the HEK293 cells were transferred to cultures with hMDMs. RNA was isolated 8 h later and analyzed for IFNβ and CXCL10 by QPCR. Data in d–f represent means and s.e.m. of data obtained using four independent hMDM donors. ( g–h ) PCs from wt or Myd88 −/− mice ( g ) or an immortalized MyD88 and TRIF double deficient macrophage cell-line ( h ) were treated with cationic liposomes for 4 hours. RNA was isolated and analyzed for CXCL10 and TNF by QPCR. Data in g–h represent means and s.e.m. of five independent experiments. ( i ) BMDCs were treated with PtdCho, DOTAP, or LR-DOPE-DOTAP liposomes for 4 hours. BMDCs were then analyzed for CXCL10 expression by QPCR. Data in i are represent means and s.e.m of two independent experiments.

Article Snippet: Cells were then stained with DAPI and anti-CXCL10 Ab (G15, Santa Cruz).

Techniques: Expressing, Transfection, Isolation, Liposomes

Journal: Nature immunology

Article Title: Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING

doi: 10.1038/ni.2350

Figure Lengend Snippet: Fusion leads to activation of the PLC-PI3K pathway upstream CXCL10 expression. CaSki cells ( a,b,h ) or BMDCs ( b ) were pre-incubated with Fura-2 for 60 min. Cells were stimulated with VLPs (MOI 15) and HSV (MOI 3) as indicated and levels of free calcium were measured and presented as either ( a ) kinetics or ( b,h ) peak calcium levels. ( c ) CaSki cells were pre-incubated with 2-APB (100 µM) 1 hour before stimulation. ( d ) BMDCs were pretreated with 2-APB for 1 hour before stimulation with VLPs and analysis of CXCL10 expression by QPCR. ( e ) BMDCs were stimulated with VLPs or liposomes (0.1 mg/ml) for either 15 or 30 min. Cell lysates were then analyzed for pAKT and total AKT by immunoblotting (top). This was also performed with BMDCs pre-incubated with either U73122 (50 µM) for both VLPs and liposome treatments or with Ly294002 (5 µM) for VLP stimulations. ( g ) BMDCs were pre-incubated with indicated inhibitors for 45 min. before stimulation with VLPs or IFN-γ for 4 hours and subsequent RNA analysis by QPCR. ( h ) CaSKi cells were incubated with indicated inhibitors before stimulation with VLPs. Calcium concentrations were then subsequently analyzed. Data show one representative of two independent experiments. (mean and s.e.m.).

Article Snippet: Cells were then stained with DAPI and anti-CXCL10 Ab (G15, Santa Cruz).

Techniques: Activation Assay, Expressing, Incubation, Liposomes, Western Blot

Journal: Nature Communications

Article Title: Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation

doi: 10.1038/ncomms9164

Figure Lengend Snippet: ( a ) Quantification of microglial activation (IBA-1), T-cell infiltration (CD3) and demyelination (LFB/PAS) 7 days after fibrinogen injection in the corpus callosum of Itgam −/− or control WT mice ( n =6). Data are presented as mean±s.e.m. * P <0.05, ** P <0.01 (non-parametric Mann–Whitney U -test). ( b ) Fibrinogen-induced gene expression of Cxcl10 and Ccl2 is reduced in the corpus callosum of Itgam −/− mice compared with WT control. Results are mean±s.e.m. of 6–7 mice per group, ** P <0.01, **** P <0.0001 (two-way ANOVA and Bonferroni's multiple comparisons test). ( c ) Fibrinogen-induced gene expression of T-bet , IFN- γ and IL-12p40 is reduced in the corpus callosum of Itgam −/− mice compared with WT control ( n =5–8 mice). Data are presented as mean±s.e.m. ** P <0.01, *** P <0.001 (two-way ANOVA and Bonferroni's multiple comparisons test). ANOVA, analysis of variance; d, days; LFB, Luxol fast blue.

Article Snippet: Primary antibodies were rabbit anti-Iba-1 (1:1,000, Wako), rabbit anti-CD3 (1:1,000, Dako), rabbit anti-GFAP (1:500, Sigma), mouse anti-MBP (1:100, Covance), rat anti-mouse MHC class II (1:200, BMA Biomedicals) or goat anti-mouse CXCL10 (1:200, R&D system).

Techniques: Activation Assay, Injection, Control, MANN-WHITNEY, Gene Expression

Journal: Nature Communications

Article Title: Blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation

doi: 10.1038/ncomms9164

Figure Lengend Snippet: ( a ) Fibrinogen-induced local T-cell activation (OX-40) in the corpus callosum of WT mice is significantly reduced in Itgam −/− mice ( n =4). Representative images are shown. Data are presented as mean±s.e.m. ** P <0.01, *** P <0.001 (one-way ANOVA and Bonferroni's multiple comparisons test). Scale bar, 100 μm. ( b ) In vivo pharmacologic blockade of CD11b by intracerebroventricular delivery of anti-CD11b antibody reduces fibrinogen-induced Cxcl10 and Ccl2 gene expression, compared with isotype IgG control antibody. Data are presented as mean±s.e.m. ( n =5 per group). * P <0.05, ** P <0.01, *** P <0.001 (one-way ANOVA and Bonferroni's multiple comparisons test). ( c ) Quantification of infiltrated CD3 + T cells and RFP + macrophages in the corpus callosum 7 days after injection of fibrinogen in WT mice treated with anti-CD11b or IgG isotype control antibody. Data are presented as mean±s.e.m. (CD3, n =6–7 mice per group; RFP, n =7 mice per group). * P <0.05, ** P <0.01 (non-parametric Mann–Whitney U -test). ( d ) Proposed model for the role of fibrin, the final product of the coagulation cascade, in the development of CNS autoimmunity. On BBB disruption, fibrinogen extravagates into the CNS and is converted to fibrin upon activation of coagulation. Fibrin, the high-affinity plasma-derived ligand for CD11b/CD18, activates CNS resident innate immune cells (microglia and perivascular macrophages) to stimulate chemokine release leading to recruitment of peripheral inflammatory macrophages/monocytes and T cells. Fibrin also induces antigen-presenting properties and provides instructive signals (such as IL-12) for inducing Th1-cell differentiation. Fibrin-induced microglial activation, recruitment of peripheral macrophages and T-cell activation lead to inflammatory demyelination. ANOVA, analysis of variance.

Article Snippet: Primary antibodies were rabbit anti-Iba-1 (1:1,000, Wako), rabbit anti-CD3 (1:1,000, Dako), rabbit anti-GFAP (1:500, Sigma), mouse anti-MBP (1:100, Covance), rat anti-mouse MHC class II (1:200, BMA Biomedicals) or goat anti-mouse CXCL10 (1:200, R&D system).

Techniques: Activation Assay, In Vivo, Gene Expression, Control, Injection, MANN-WHITNEY, Coagulation, Disruption, Clinical Proteomics, Derivative Assay, Cell Differentiation

Journal: Nature communications

Article Title: Non-redundant Requirement for CXCR3 Signaling during Tumoricidal T Cell Trafficking across Tumor Vascular Checkpoints

doi: 10.1038/ncomms8458

Figure Lengend Snippet: ( a ) Cognate chemokine ligands for CXCR3, CCR5, and CCR2 (i.e., CXCL9/CXCL10, CCL5, and CCL2, respectively) were quantified by ELISA in B16-OVA tumor extracts (tumor volume ~ 400–500 mm 3 ) or in normal skin fom tumor-free mice. Data (mean ± s.e.m.) are from ≥3 independent experiments (n ≥2 mice per group). ( b ) Transwell assays were performed where fluorescently-labeled WT OT-I T cells were admixed with equivalent numbers of chemokine receptor-deficient effector cells and tested for migration to the indicated recombinant chemokines. WT and chemokine receptor-deficient cells were also pretreated with the global G-protein inhibitor pertussis toxin (PTX) and migration was quantified by flow cytometry. Background migration (absence of chemokine) was subtracted from all values. Data (mean ± s.e.m.) are represented as migration relative to WT and are from ≥3 independent experiments. ( a, b ) * P < 0.05; ns, not significant; unpaired two-tailed Student’s t -test.

Article Snippet: Briefly, yellow-green fluorescent sulfate microspheres (Ex/Em 505/515, 1.0 μm diameter, Life Technologies) were labeled with 10 μg/mL of Ab specific for mouse chemokines including anti-CXCL10 (cat# MAB466-500 clone 134013), anti-CXCL9 (cat# AF392 affinity-purified polyclonal Ab), anti-CCL5 (cat# MAB478 clone 53405), anti-CCL2 (cat# BAF479 biotinylated affinity purified polyclonal goat Ab), or isotype-matched control Abs (R&D Systems) and injected i.v. into tumor-bearing mice.

Techniques: Enzyme-linked Immunosorbent Assay, Labeling, Migration, Recombinant, Flow Cytometry, Two Tailed Test

Journal: Nature communications

Article Title: Non-redundant Requirement for CXCR3 Signaling during Tumoricidal T Cell Trafficking across Tumor Vascular Checkpoints

doi: 10.1038/ncomms8458

Figure Lengend Snippet: ( a ) Chemokine receptor profile on CD8 + T cells (activated ex vivo) from PBL of 2 normal human donors. Gray-filled histograms represent isotype control Ab staining. ( b ) Concentration of human and murine chemokines in tumor extracts from 2 human melanoma xenografts (M537 and M888) was determined by Luminex and ELISA, respectively. Data (mean ± s.e.m.) are from 4 independent experiments (n ≥4 mice per group) for human chemokines and from ≥2 independent experiments (n ≥3 mice per group) for murine chemokines. * P < 0.05; * * P < 0.006, M537 versus M888 tumors. ( c ) Left, schematic for short-term (1 h) competitive homing studies. The groups used for adoptive cell transfer (ACT) included untreated (Untx) human effector T cells from donor 1 or donor 2 that were comixed at a 1:1 ratio with untreated cells, PTX-pretreated cells, α-CXCR3 Ab-pretreated cells, or cells where CXCR3 was desensitized by exposure to recombinant CXCL10 prior to transfer into mice (CXCR3 dsn). Right, ratio of adoptively transferred T cells relative to untreated cells in tumors of M537 or M888 tumor-bearing SCID mice following short-term competitive homing assays is shown. PTX, pertussis toxin; α-CXCR3, CXCR3 blocking Ab. Data (mean ± s.e.m.) are from ≥2 independent experiments (n = 2 mice per group). * P < 0.02; ns, not significant. ( b–c ) Data analyzed by unpaired two-tailed Student’s t -test.

Article Snippet: Briefly, yellow-green fluorescent sulfate microspheres (Ex/Em 505/515, 1.0 μm diameter, Life Technologies) were labeled with 10 μg/mL of Ab specific for mouse chemokines including anti-CXCL10 (cat# MAB466-500 clone 134013), anti-CXCL9 (cat# AF392 affinity-purified polyclonal Ab), anti-CCL5 (cat# MAB478 clone 53405), anti-CCL2 (cat# BAF479 biotinylated affinity purified polyclonal goat Ab), or isotype-matched control Abs (R&D Systems) and injected i.v. into tumor-bearing mice.

Techniques: Ex Vivo, Control, Staining, Concentration Assay, Luminex, Enzyme-linked Immunosorbent Assay, Recombinant, Blocking Assay, Two Tailed Test

Journal: Nature immunology

Article Title: Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING.

doi: 10.1038/ni.2350

Figure Lengend Snippet: Figure 1 VLPs induce a type I interferon response. (a) Structural contents of VLPs and virions visualized by cryo-electron microscopy (top) and immunoblot analysis of VP5 and gD (below). Original magnification (top), ×10,000. (b) Multiplex platform analysis of RNA expression (genes, left margin) by PCs treated for 4 h with L-particles (L-part) or PREPs (MOI, 15), presented relative to expression in untreated control cells (green, >300%; yellow, between 33% and 300%; red, <33%). (c,d) Quantitative PCR analysis of mRNA encoding CXCL10 (Cxcl10), TNF (Tnf) and IFN-β (Ifnb) in PCs left untreated (UT) or treated for 4 h with PREPs (c) or L-particles (d) at an MOI of 5 or 15 (wedges); results are presented relative to those of untreated cells. (e,f) Expression of mRNA encoding CXCL10 in cultures of mouse BMDCs (e) and human MDMs (f) left untreated or treated for 4 h with VLPs (MOI, 15); results are presented relative to those of untreated cells. (g) Enzyme-linked immunosorbent assay of CXCL10 in supernatants of BMDCs treated for 24 h with VLPs or dilutions of infectious HSV-1 stock. (h) Quantitative PCR analysis of HSV-1 DNA in serial dilutions (horizontal axis) of HSV-1 stock (HSV) or PREPs and L-particles left untreated (− DNase) or treated for 20 min with DNase I (+ DNase), presented as the threshold cycle (CT). (i) Expression of mRNA encoding CXCL10 in PCs left untreated or treated with PREPs and L-particles treated as in h, presented relative to expression in untreated cells. (j) Expression of mRNA encoding CXCL10 in PCs from wild-type mice (WT) and mice deficient in the IFN-α receptor chain 1 (Ifnar1−/−), treated (and presented) as in c (MOI, 15). Data are from one experiment representative of two independent experiments (a) or are from two (b,g–j) or five (c–f) independent experiments (mean and s.e.m.).

Article Snippet: Cells were then stained with DAPI (4,6-diamidino-2-phenylindole) and antibody to CXCL10 (G15; Santa Cruz).

Techniques: Cryo-Electron Microscopy, Western Blot, Multiplex Assay, RNA Expression, Expressing, Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING.

doi: 10.1038/ni.2350

Figure Lengend Snippet: Figure 2 VLPs enhance the sensitivity of TLR7 and TLR9 and induce the recruitment and activation of leukocytes in vivo. (a) IL-1β protein in supernatants of C57BL/6 mouse PCs pretreated with lipopolysaccharide (20 ng/ml), then treated for 24 h with medium alone (M), HSV (MOI, 3), VLPs (MOI, 15) or poly(dA:dT) (pdAdT); 2 µg/ml). (b) Immunoblot analysis of pro-IL-1β and IL-1β in supernatants of BMDCs prestimulated for 3 h with lipopolysaccharide, then left untreated or stimulated for 16 h with VLPs or HSV-1. (c) Formation of LC3 foci in PCs left untreated or treated for 6 h with HSV-1 or VLPs, assessed by confocal microscopy (top) and immunoblot analysis of LC3 II in whole-cell extracts (below). GAPDH (glyceraldehyde phosphate dehydrogenase) serves as a loading control. (d) Quantitative PCR analysis of mRNA encoding CXCL10 in PCs without prestimulation (VLP −) or prestimulated for 2 h with VLPs (VLP +), then left untreated (Ligand −) or treated for an additional 4 h (Ligand +) with a suboptimal concentration of CpG (oligodeoxynucleotide 1826; 0.5 µM) or ssRNA40 (1 µg/ml); results are presented relative to those of untreated cells. (e) Flow cytometry of cells from the peritoneal cavity of an 8-week-old mouse injected intraperitoneally VLPs suspended in PBS (25 × 106 particles), followed by another injection after 12 h and collection of cells after 24 h, to assess expression of the natural killer cell marker NK1.1 through the use of count beads (FL2, fluorescence channel 2). (f) Total natural killer (NK) cells in the peritoneal cavities of 8-week-old mice injected intraperitoneally (as in e) with PBS (M), wild-type (WT) VLPs or ∆gB VLPs (suspended as in e). (g) Flow cytometry of cells from the peritoneal cavity of an 8-week-old mouse injected as in e, to assess expression of the activation marker CD69 (as in e). (h) Frequency of CD69+ cells in the peritoneal cavities of 8-week-old mice injected as in f. *P < 0.05 (Student’s t-test). Data are from one experiment representative of two independent experiments (a–d) or two independent experiments with seven mice per group (e–h).

Article Snippet: Cells were then stained with DAPI (4,6-diamidino-2-phenylindole) and antibody to CXCL10 (G15; Santa Cruz).

Techniques: Activation Assay, In Vivo, Western Blot, Confocal Microscopy, Control, Real-time Polymerase Chain Reaction, Concentration Assay, Flow Cytometry, Injection, Expressing, Marker, Fluorescence

Journal: Nature immunology

Article Title: Virus-cell fusion as a trigger of innate immunity dependent on the adaptor STING.

doi: 10.1038/ni.2350

Figure Lengend Snippet: Figure 5 Fusion leads to activation of the PLC-γ–PI(3)K pathway upstream of CXCL10 expression. (a,b) Free calcium (Ca2+) in CaSki cells (a) or BMDCs (b) preincubated for 60 min with the calcium indicator Fura-2, then mock treated (Mock) or stimulated with HSV (MOI, 3), wild-type VLPs (MOI, 15) or ∆gB VLPs (MOI, 15), presented as kinetics (a) or peak calcium concentration (b). (c) Peak calcium concentration in CaSki cells without preincubation (Ctrl) or preincubated with 2-APB (100 µM) 1 h, then left untreated or stimulated with wild-type VLPs or HSV-1. (d) Quantitative PCR analysis of mRNA encoding CXCL10 in mouse BMDCs left untreated (UT), given no pretreatment and stimulated with wild-type VLPs (VLP) or pretreated for 1 h with 2-APB before being stimulated with VLPs (VLP 2-APB); results are presented relative to those of untreated cells. (e) Immunoblot analysis of phosphorylated (p-) and total Akt in lysates of BMDCs left unstimulated (0) or stimulated for 15 or 30 min with VLPs or liposomes (0.1 mg/ml; top) or given no pretreatment (−) or preincubated with U73122 (U73; 50 µM) or Ly294002 (Ly; 5 µM), then stimulated with VLPs or liposomes (bottom). (f) Quantification of phosphorylated Akt in BMDCs left untreated or stimulated with VLPs or liposomes. (g) Quantitative PCR analysis of mRNA encoding CXCL10 in BMDCs left untreated (UT) or given no pretreatment (−) or preincubated for 45 min with Ly294002 or U73122, then stimulated for 4 h with VLPs or or IFN-γ; results are presented relative to those of untreated cells. (h) Peak calcium concentration in CaSKi cells given no pretreatment (Ctrl) or preincubated with Ly294002 or U73122 (key), then left untreated or stimulated with VLPs or HSV-1. Data are from one experiment representative of two independent experiments (mean and s.e.m.).

Article Snippet: Cells were then stained with DAPI (4,6-diamidino-2-phenylindole) and antibody to CXCL10 (G15; Santa Cruz).

Techniques: Activation Assay, Expressing, Concentration Assay, Real-time Polymerase Chain Reaction, Western Blot, Liposomes