Journal: Cell

Article Title: Self-Recognition of an Inducible Host lncRNA by RIG-I Feedback Restricts Innate Immune Response.

doi: 10.1016/j.cell.2018.03.064

Figure Lengend Snippet: Figure 2. lnc-Lsm3b Inhibits RIG-I-Mediated Signaling in RNA Virus-Infected Macrophages (A–C) ELISA of IFN-a (A), IFN-b (B), and IL-6 (C) in the supernatants of peritoneal macrophages transfected with si-ctrl or si-lnc-Lsm3b for 48 hr before VSV, SeV, or HSV-1 infection for 16 hr. (D–F) ELISA of IFN-a (D), IFN-b (E), and IL-6 (F) in supernatants of lnc-Lsm3b+/+ and lnc-Lsm3b/ cells infected with VSV for indicated hours. (G and H) IFN-b (G) and NF-kB (H) promoter activity in HEK293T cells transfected with lnc-Lsm3b and RIG-I vectors upon VSV infection. (I) VSV titers by TCID50 assay in supernatants of lnc-Lsm3b+/+ and lnc-Lsm3b/ cells infected with VSV for 12 hr and 24 hr. (J) Immunoblot analysis of RIG-I signaling pathways in lnc-Lsm3b+/+ and lnc-Lsm3b/ cells infected with VSV for indicated hours. Data are shown as mean ± SD (n = 4) or typical photographs of one representative experiment. Similar results were obtained in three independent experiments. *p < 0.05; **p < 0.01. See also Figure S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER poly(I:C) HMW Biotin InvivoGen Cat #tlrl-picb TRIzol reagent Invitrogen Cat #15596-018 Mouse IFN-a Miltenyi Biotec Cat #130-093-130 Mouse IFN-b R&D Systems Cat #8234-MB-010 RNA FISH probe Biosearch technologies N/A GST-RIG-I This paper N/A His-RIG-I This paper N/A GST-CTD This paper N/A GST-CARD This paper N/A His-deltaCARD This paper N/A Critical Commercial Assays NorthernMax kit Thermo Fisher Scientific Cat #AM1940 RNeasy Mini Kit QIAGEN Cat #74104 miRNeasy Serum/Plasma Spike-In Control QIAGEN Cat #219610 MAXIscript SP6/T7 Kit Ambion Cat #AM1320 Streptavidin beads Thermo Fisher Scientific Cat #11206D Mouse IFN-a ELISA kit PBL Biomedical Laboratories Cat #42120-1 Mouse IFN-b ELISA kit PBL Biomedical Laboratories Cat #42400-1 Mouse IL-6 ELISA kit R&D Systems Cat #VAL604 Dual-Luciferase assay kit Promega Cat #TM040 SMARTer RACE 50/30 kit Clontech Cat #634858 ATPase/GTPase Activity Assay Kit Sigma Cat #MAK113 Deposited Data RIP-seq data This paper GEO: GSE106254 iCLIP data This paper GEO: GSE106254 Experimental Models: Cell Lines HEK293T cells American Type Culture Collection N/A L929 cells American Type Culture Collection N/A RAW264.7 cells American Type Culture Collection N/A lnc-Lsm3b / RAW264.7 cells This paper N/A Experimental Models: Organisms/Strains lnc-Lsm3b / mice This paper N/A IFNabR / mice The Jackson Laboratory JAX stock 000819 Recombinant DNA Plasmid: pSIF vector Provided by Dr. Qian Zhang from Second Military Medical University, Shanghai, China N/A Plasmid: pGEM-3zf vector Provided by Dr. Xiaozhong Peng from Peking Union Medical College, Beijing, China N/A cDNA lnc-Lsm3b This paper N/A cDNA lnc-Lsm3b mutants This paper N/A Software and Algorithms FastQC 0.11.5 Babraham Bioinfomatics http://www.bioinformatics.babraham.ac.uk/ projects/fastqc/ Samtools 0.1.19 John Marshall and Petr Danecek http://www.htslib.org/ HISAT2 2.0.4 The Center for Computational Biology http://ccb.jhu.edu/software/hisat2/index.shtml StringTie v1.3.3b The Center for Computational Biology http://ccb.jhu.edu/software/stringtie/ (Continued on next page) e2 Cell 173, 1–14.e1–e6, May 3, 2018 Please cite this article in press as: Jiang et al., Self-Recognition of an Inducible Host lncRNA by RIG-I Feedback Restricts Innate Immune Response, Cell (2018), https://doi.org/10.1016/j.cell.2018.03.064

Techniques: Virus, Infection, Enzyme-linked Immunosorbent Assay, Transfection, Activity Assay, TCID50 Assay, Western Blot, Protein-Protein interactions

Journal: Cell

Article Title: Self-Recognition of an Inducible Host lncRNA by RIG-I Feedback Restricts Innate Immune Response.

doi: 10.1016/j.cell.2018.03.064

Figure Lengend Snippet: Figure 3. lnc-Lsm3-Deficient Mice Produce More Type I IFNs (A) Survival of 6-week-old lnc-Lsm3b+/+ and lnc-Lsm3b/ mice given VSV (1 3 108 pfu/g body weight) via tail intravenous injection (i.v.) (n = 10). Kaplan-Meier method was used to evaluate survival curves. (B) ELISA of type I IFNs (IFN-a and IFN-b) production in sera from lnc-Lsm3b+/+ and lnc-Lsm3b/ mice (n = 5) at 18 hr and 24 hr after i.v. injection with VSV (5 3 107 pfu/g). (C) VSV loads by TCID50 assay and qPCR assay of VSV RNA copies in organs and peritoneal macrophages from mice in (B) (n = 4). (D) Pathology of lnc-Lsm3b+/+ and lnc-Lsm3b/ mice in response to VSV. H&E staining of lung sections from mice in (B). Scale bar, 80 mm. (E and F) qPCR analysis of Ifn-b (E) and lnc-Lsm3b (F) expression in organs and peritoneal macrophages from mice in (B) (n = 5). Data are shown as mean ± SD or typical photographs of one representative experiment. Similar results were obtained in three independent experiments. *p < 0.05; **p < 0.01. See also Figures S3 and S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER poly(I:C) HMW Biotin InvivoGen Cat #tlrl-picb TRIzol reagent Invitrogen Cat #15596-018 Mouse IFN-a Miltenyi Biotec Cat #130-093-130 Mouse IFN-b R&D Systems Cat #8234-MB-010 RNA FISH probe Biosearch technologies N/A GST-RIG-I This paper N/A His-RIG-I This paper N/A GST-CTD This paper N/A GST-CARD This paper N/A His-deltaCARD This paper N/A Critical Commercial Assays NorthernMax kit Thermo Fisher Scientific Cat #AM1940 RNeasy Mini Kit QIAGEN Cat #74104 miRNeasy Serum/Plasma Spike-In Control QIAGEN Cat #219610 MAXIscript SP6/T7 Kit Ambion Cat #AM1320 Streptavidin beads Thermo Fisher Scientific Cat #11206D Mouse IFN-a ELISA kit PBL Biomedical Laboratories Cat #42120-1 Mouse IFN-b ELISA kit PBL Biomedical Laboratories Cat #42400-1 Mouse IL-6 ELISA kit R&D Systems Cat #VAL604 Dual-Luciferase assay kit Promega Cat #TM040 SMARTer RACE 50/30 kit Clontech Cat #634858 ATPase/GTPase Activity Assay Kit Sigma Cat #MAK113 Deposited Data RIP-seq data This paper GEO: GSE106254 iCLIP data This paper GEO: GSE106254 Experimental Models: Cell Lines HEK293T cells American Type Culture Collection N/A L929 cells American Type Culture Collection N/A RAW264.7 cells American Type Culture Collection N/A lnc-Lsm3b / RAW264.7 cells This paper N/A Experimental Models: Organisms/Strains lnc-Lsm3b / mice This paper N/A IFNabR / mice The Jackson Laboratory JAX stock 000819 Recombinant DNA Plasmid: pSIF vector Provided by Dr. Qian Zhang from Second Military Medical University, Shanghai, China N/A Plasmid: pGEM-3zf vector Provided by Dr. Xiaozhong Peng from Peking Union Medical College, Beijing, China N/A cDNA lnc-Lsm3b This paper N/A cDNA lnc-Lsm3b mutants This paper N/A Software and Algorithms FastQC 0.11.5 Babraham Bioinfomatics http://www.bioinformatics.babraham.ac.uk/ projects/fastqc/ Samtools 0.1.19 John Marshall and Petr Danecek http://www.htslib.org/ HISAT2 2.0.4 The Center for Computational Biology http://ccb.jhu.edu/software/hisat2/index.shtml StringTie v1.3.3b The Center for Computational Biology http://ccb.jhu.edu/software/stringtie/ (Continued on next page) e2 Cell 173, 1–14.e1–e6, May 3, 2018 Please cite this article in press as: Jiang et al., Self-Recognition of an Inducible Host lncRNA by RIG-I Feedback Restricts Innate Immune Response, Cell (2018), https://doi.org/10.1016/j.cell.2018.03.064

Techniques: Injection, Enzyme-linked Immunosorbent Assay, TCID50 Assay, Staining, Expressing

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 1. Antitumor effect of intratumoral IFN-α gene transfer. (a) Growth of tumors injected with Ad-mIFN. Tumor volumes were measured at indicated days following the intratumoral injection of Ad-mIFN (n = 6) or Ad-AP (n = 8). Relative tumor volumes compared with those at day10 were presented. Data are shown as means ± standard deviation (s.d.). (b) ELISpot assay of IFN-γ-producing cells in response to stimulation of CT26 cells. Twenty-two days after tumor inoculation, splenocytes were isolated from mice injected with Ad-mIFN (n = 4) or Ad-AP (n = 3), and were cultured with CT26 or syngeneic lymphocytes. Data are presented as means ± s.d. (c) Intracellular cytokine staining of IFN-γ-producing cells in response to CT26 cells. The splenocytes from mice injected with Ad-mIFN (n = 4) or Ad-AP (n = 3) at day 22 were incubated with CT26 cells and stained by anti-mouse IFN-γ antibody. The activated cell fractions were analyzed by staining with anti-mouse CD8 antibody. Representative FACS plots (right panel) are shown.

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Injection, Standard Deviation, Enzyme-linked Immunospot, Isolation, Cell Culture, Staining, Incubation

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 2. Intratumoral IFN-α gene transfer reduced the frequency of Tregs in tumors. (a) Frequency of CD4+Foxp3+ Tregs per CD4+ T cells in tumors. Tumors injected with viruses were harvested at days 16, 22 and 28, and processed into single-cell suspension. The percentage of CD4+

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Injection, Suspension

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 3. Intratumoral IL-6 concentration was significantly increased by IFN-α gene transfer. (a) IL-6 concentration in tumors. Tumors injected with Ad-mIFN (n = 5) or Ad-AP (n = 5) were harvested at days 16, 22 and 28, and IL-6 concentration was measured by ELISA. (b) Relationship between IL-6 and IFN-α concentration in tumors. Tumors injected with Ad-mIFN (n = 5) or Ad-AP (n = 5) were harvested at day 16, and the concentrations of IL-6 and IFN-α were compared by ELISA. (c) IL-6 production from tumor CD11c+ cells. The CD11c+ and CD11c −cells were isolated from tumors injected with Ad-mIFN (n = 2) or Ad-AP (n = 2) at day 16, and 5 × 104 cells were plated in 96-well plates. After 48 h, supernatants were assayed for the measurement of IL-6 concentration by ELISA. (d) IL-6 production from splenic CD11c+ cells in response to a recombinant IFN-α protein. The CD11c+ and CD11c−cells isolated from naïve splenocytes, and 5 × 104 of CT26 cells were cultured in 96-well plates with depicted concentration of recombinant mouse IFN-α (Miltenyi Biotech). After 48 h, supernatants were assayed for the measurement of IL-6 concentration by ELISA (n = 2 for each group).

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Concentration Assay, Injection, Enzyme-linked Immunosorbent Assay, Isolation, Recombinant, Cell Culture

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 4. IL-6 receptor blockade suppressed IFN-α-mediated Treg reduction in tumors. (a) Schema of experiment. The 1000 μg of the monoclonal anti-IL-6 receptor antibody was intraperitoneally injected into the mice at days 7, 14 and 21 after tumor inoculation. Ad-mIFN or Ad-AP was injected once at day 10 after inoculation. (b) Frequency of CD4+Foxp3+ cells per CD4+ T cells in tumors treated with IL-6R ab. Tumors were harvested at day 22, and CD4+ T cells and CD4+Foxp3+ Tregs were analyzed by flow cytometry (n = 5 for the group of Ad-AP i.t. +IL-6R ab i.p., n = 4 for the other groups). (c) Ratio of CD8+ T cells to CD4+Foxp3+ Tregs in tumors. Frequency of CD8+ T cells within whole tumor cells (left panel). Frequency of CD4+Foxp3+ Tregs within whole tumor cells (middle panel). The number of CD8+ T cells was compared with that of CD4+Foxp3+ Tregs in tumors at day 16 (right panel) (n = 4 for the group of Ad-mIFN i.t.+IL-6R ab i.p., n = 6 for the other groups). IL-6R ab, anti-IL-6 receptor antibody; i.t., intratumoral injection; i.p., intraperitoneal administration; TDLNs, tumor-draining lymph nodes.

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Injection, Cytometry

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 5. IL-6 receptor blockade partially attenuated IFN-α-mediated tumor growth suppression. (a) Growth of tumors treated with IL-6R ab. Tumor volumes in mice treated with the viruses and/or IL-6R ab were measured at the indicated days (n = 5 for the group of Ad-AP i.t.+IL-6R ab i.p., n = 6 for the other groups). Relative tumor volumes compared with those at day 10 were presented. (b) ELISpot assay of IFN-γ- producing cells in mice treated with IL-6R ab. The splenocytes were isolated from mice as shown in Figure 4a at day 28, and the cells were cultured with CT26 or syngeneic splenocytes (n = 5 for the group of Ad-AP i.t.+IL-6R ab i.p., n = 6 for the other groups).

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Enzyme-linked Immunospot, Isolation, Cell Culture

Journal: Cancer gene therapy

Article Title: Type I IFN gene delivery suppresses regulatory T cells within tumors.

doi: 10.1038/cgt.2014.60

Figure Lengend Snippet: Figure 6. Intratumoral IFN-α expression increased the number of Th17 cells in tumors. (a) Expression of RORγt and Foxp3 genes in tumors. The tumors injected with viruses were harvested at day 16 and were subjected to real-time PCR analysis (Ad-mIFN: n = 4, Ad-AP: n = 3). (b) Expression of IL-17 in tumors. The tumors were harvested at day 28, and subjected to RT-PCR for IL-17 expression (n = 3 for the group of Ad-AP i.t.+PBS i.p., n = 2 for the group of Ad-mIFN i.t.+PBS i.p., n = 3 for the group of Ad-mIFN i.t.+IL-6R ab i.p.). (c) Intracellular cytokine staining of IL-17A in CD4+ T cells. The tumors and tumor-draining lymph nodes were harvested at day 22, and IL-17A expressions were analyzed by flow cytometry (n = 3 for the group of Ad-AP i.t.+PBS i.p., n = 4 for the group of Ad-mIFN i.t.+PBS i.p., n = 4 for the group of Ad-AmIFN i.t.+IL-6R ab i.p.) (upper panel). Representative FACS plots of tumors (lower left panel) and tumor-draining lymph nodes (lower right panel) were shown.

Article Snippet: The amounts of cytokines in cell culture supernatants and tumors were assayed with antibodies for IL-6 and mouse IFN-α (Quantikine; R&D systems, Minneapolis, MN, USA) in accordance with the manufacturer’s recommendations.

Techniques: Expressing, Injection, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Staining, Cytometry

Journal: Nature immunology

Article Title: TANK is a negative regulator of Toll-like receptor signaling and critical for preventing autoimmune nephritis

doi: 10.1038/ni.1771

Figure Lengend Snippet: ( a , b ) BM-DCs from wild-type and Tank −/− mice were infected with NDV for 24 h. The concentrations of IFN-α ( a ) and IL-6 ( b ) in the culture supernatants were measured by ELISA. ( c , d ) Flt3L-DCs from wild-type and Tank −/− mice were stimulated with 0.1 or 1μM CpG-DNA for 24 h. The concentrations of IFN-α ( c ) and IL-6 ( d ) in the culture supernatants were measured by ELISA. ( e , f ) Peritoneal macrophages from wild-type and Tank −/− mice were stimulated with MALP-2 (10 ng/ml), poly I:C (100 μg/ml), LPS (100 ng/ml), R-848 (10 nM) or CpG-DNA (1 μM) for 24 h. The concentrations of IL-6 (e) and TNF (f) in the culture supernatants were measured by ELISA. (g, h) Wild-type ( n = 5) and Tank −/− ( n = 5) mice were intraperitoneally injected with 30 nmol of R-848. Sera were collected and the concentrations of IL-6 (g) and IFN-α (h) were determined by ELISA. Data represent the means ± s.d. of triplicate assays. Similar results were obtained in three independent experiments. *, P < 0.05, **, P < 0.01 and ***, P < 0.005, versus Tank −/− mice.

Article Snippet: The ELISA kit for mouse IFN-α was purchased from PBL Biomedical Laboratories.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Injection

Journal: Nature medicine

Article Title: Host-cell sensors for Plasmodium activate innate immunity against liver-stage infection.

doi: 10.1038/nm.3424

Figure Lengend Snippet: Figure 2 Plasmodium-mediated type I IFN induction requires Mavs, and Mda5 senses Plasmodium RNA. (a) Schematic representation of type I IFN signaling pathways leading to the induction of IFN-α and IFN-β (IFN-α/β) transcription. cGAS, cyclic GMP-AMP synthase. (b) Gene expression analysis of 5 representative ISGs in livers of WT, Irf7−/−, Irf3−/− and Irf7−/−; Irf3−/− mice as well as bone marrow (BM)-chimeric mice (WT mice with Irf3−/− BM and Irf3−/− mice with WT BM) 42 h after infection with 5 × 104 P. berghei sporozoites. (c–g) Gene expression analysis of 5 representative ISGs in livers of WT, Myd88−/−, Trif−/− and Myd88−/−; Trif−/− (c), Tlr3−/− and Tlr4−/− (d), Mavs−/− (e) Rig-I−/− (f) and Mda5−/− (g) mice 42 h after infection with 5 × 104 P. berghei sporozoites. Statistical analysis was performed for each individual gene; the least significant P value is shown, **P < 0.05. (h) ISG expression in livers of WT and Mda5−/− mice 4 h after hydrodynamic injection with 50 µg per mouse of P. berghei RNA. Expression of individual genes from sporozoite-infected livers was tested against mock-injected control samples. The least significant P value of all samples is shown, ***P < 0.01. Complete statistics in Supplementary Table 2. Data are expressed as means ± s.e.m.

Article Snippet: Rat monoclonal antibody to mouse IFN-α and IFN-β (PBL Biomedical Laboratories clone RMMA-1 for IFN-α and clone RMMB-1 for IFN-β) was used as capture antibody (2 μg per ml for coating), rabbit polyclonal antibody to mouse IFN-α and IFN-β (PBL Biomedical Laboratories 32100-1 for IFN-α and 32400-1 for IFN-β) was used at 80 neutralizing units per ml for detection and HRP-conjugated donkey anti-rabbit IgG (1:3,000) (Bio-Rad 170-6515) was used as secondary reagent.

Techniques: Protein-Protein interactions, Gene Expression, Infection, Expressing, Injection, RNA Expression, Control

Journal: bioRxiv

Article Title: Epstein-Barr virus induced 3 attributes to TLR7-mediated splenomegaly and bicytopenia

doi: 10.1101/2024.06.10.598101

Figure Lengend Snippet: Ebi3 deficiency did not alter the development and function of plasmacytoid dendritic cells (pDCs). (A) Representative flow cytometric analysis of the percentage of pDCs (CD11c + B220 + ) and conventional dendritic cells (cDCs) (CD11c + B220 - ) in the spleen of wild-type (WT) or KO mice. Numbers in the plots indicate the percentage of cells in each gate. (B) Percentages of pDCs (CD11c + B220 + Gr-1 + ) or cDCs (CD11c + B220 - CD11b + or CD11c + B220 - CD11b - ) in the spleen of WT or KO mice. (C) Infa gene expression in ear skin sample of Vaseline- or imiquimod (IMQ)-treated WT or KO mice. Gene expression was analyzed with quantitative polymerase chain reaction (qPCR). (D) IFN-α concentration in serum of Vaseline- or IMQ-treated WT or KO mice. IFN-α concentrations were quantified using enzyme-linked immunosorbent assay (ELISA). (E, F) Bone marrow-derived pDCs were stimulated with 200 ng/ml of IMQ for 6 hours. (E) Ifna gene expression in the bone-marrow derived pDCs. Expression levels were analyzed by qPCR and normalized to that of Hprt. (F) The concentrations of IFN-α in the culture supernatants. All data are expressed as the mean + standard deviation.

Article Snippet: Concentrations of IFN-α in culture supernatants or mouse serum were measured using Mouse IFN-alpha All Subtype Quantikine ELISA Kit (MFNAS0, R&D Systems) according to the manufacturer’s protocols.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Expressing, Standard Deviation

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: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

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: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

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: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

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: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

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: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

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: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

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: Mouse IFN Beta ELISA Kit , PBL Assay Science , 42400–2.

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