Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells 1

doi: 10.4049/jimmunol.1102038

Figure Lengend Snippet: PBMCs were stimulated with Influenza virus (panel A) or HSV (panel B) for different time periods, and then stained for pDC markers and intracellular expression of IFN-α and IFN-λ expression. Data are representative of 2 separate experiments. C) PBMC were stimulated with HSV-1 for varying times, with BFA added two hours before each time point. Data are representative of two separate donors. D) Kinetics of expression of IFN-α (left panel) and IFN-λ (right panel). Enriched pDC were stimulated with HSV-1 (without the addition at BFA) and supernatants were collected at the indicated time points and tested by ELISA for IFN-α and IFN-λ. Data for two separate donors are shown. E) Summary of IFN-α and –λ in supernatants of purified pDC stimulated with HSV for 18 hr. Data are mean ± 1 SD for 4 separate donors. F) ELISA-determined concentration of IFN-λ in the supernatants of purified pDC after treatment with CpGA or CpGB for 18 hr. Data are mean ± 1 SD for 4 separate donors.

Article Snippet: For some experiments, PE- or FITC-labeled anti-IFN-α (Miltenyi Biotec) was used.

Techniques: Virus, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Purification, Concentration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells 1

doi: 10.4049/jimmunol.1102038

Figure Lengend Snippet: A, B) Co-expression of IFN-α and –λ: After stimulation of PBMC with HSV for 7hr, pDC were stained and the cells were permeabilized and stained for the co-expression of IFN-α and IFN-λ1/3 (Panel A), and for IFN-α and IFN-λ2 (panel B) and for IFN-λ1/3 and IFN-λ2 (panel C). Data are representative of three separate experiments for each panel.

Article Snippet: For some experiments, PE- or FITC-labeled anti-IFN-α (Miltenyi Biotec) was used.

Techniques: Expressing, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells 1

doi: 10.4049/jimmunol.1102038

Figure Lengend Snippet: A) PBMC were stimulated with purified HSV-GFP with final stimulation conditions containing <5 pg/ml of IFN-λ, then stained for IFN-λ expression in pDC as described in Figure 2. Data re representative of two similar experiments. B) HSV-1 was pelleted in an ultracentrifuge then PBMC were stimulated with pelleted virus (MOI of 1) or supernatant from the centrifuged virus for 7 hr and intracellular expression of IFN-α and IFN-λ1/3 were determined as described in Figure 3. C) Purified pDC were incubated with HSV for 14 hr with or without a 30 min pre-treatment with IFN-α receptor neutralizating antibody (αRAb, 12.5µg/ml). The amount of IFN-λ (i) and IFN-α (ii) in supernatants was measured by ELISA, with mock and IFN-α receptor neutralization antibody (12.5µg/ml) treatment alone as controls. Data are mean ± 1 SD (n=3). D) Purified pDC (98–99% pure) were treated with 10,000 IU rIFN-α for the times shown; as a control, pDC were stimulated for 24 with HSV-1 or without HSV (MOCK). Supernatants were collected and the levels of IFN-λ were determined by ELISA. Data are representative of two similar experiments.

Article Snippet: For some experiments, PE- or FITC-labeled anti-IFN-α (Miltenyi Biotec) was used.

Techniques: Purification, Staining, Expressing, Virus, Incubation, Enzyme-linked Immunosorbent Assay, Neutralization

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells 1

doi: 10.4049/jimmunol.1102038

Figure Lengend Snippet: A) Cross-linking of CD4 or BDCA-2 on pDC inhibits IFN-λ and IFN-α production of PDC: CD4 or BDCA-2 on pDC within PBMC was cross-linked with anti-CD4 or anti-BDCA-2 microbeads, and then PBMC were simulated for 7 hr with HSV. The expression of IFN-α (i) or IFN-λ1 (ii) by pDC was measured by flow cytometry as described above. Data represent mean ± 1 SD for 6 separate experiments. * P<0.05 vs. no crosslinking, ** P<0.01 vs. no crosslinking as determined by ANOVA. B) A TLR9 inhibitor inhibits HSV- but not Flu-induced IFN-λ production of pDC. IFN-λ1 production by pDC in PBMC was measured by flow cytometry after stimulation with HSV (i) and Flu (ii), with or without a TLR9 inhibitory ODN (5.42 µg/ml). Data represent mean ± 1 SD for 6 separate donors. ** P<0.01 vs. sample without CpG inhibitor as determined by ANOVA. C) HSV replication is not required for the induction of IFN-λ in pDC. PBMC were simulated with HSV or UV-HSV. The expression of IFN-λ1 of PDC in PBMC was measured by flow cytometry as described above. Data represent mean ± 1 SD for 3 separate donors.

Article Snippet: For some experiments, PE- or FITC-labeled anti-IFN-α (Miltenyi Biotec) was used.

Techniques: Expressing, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells 1

doi: 10.4049/jimmunol.1102038

Figure Lengend Snippet: A) PBMC were treated with either rIFN-α (1000 IU/ml) or IFN-λ1 (10 ng/ml) for 40 minutes; the cells were collected and immediately surface stained with CD123-PE and BDCA2-APC to identify the pDC population, then permeabilized for intracellular staining for the detection of phosphorylated Stat1. Histogram overlays comparing mock (filled) and stimulated pDC (lines). Data are representative of two independent experiments. B) Functional activation of pDC by IFN-λ1 priming: PBMC were primed with IFN-λ1 (10 ng/ml) for 3hr, and then cells were stimulated by HSV for another 4 hr. The percentage and mean fluorescence intensity (MFI) of IFN-α positive (left panels) and IFN-λ positive (right panels) pDC were detected by flow cytometry. Data are mean ± 1 SD for 5 separate donors. C) PBMC were treated with IFN-λ1 (25ng/ml) for 6 hr, then PDC, monocytes, B cells, NK cells, CD4+ T cells, and CD8+ T cells were labeled with specific markers. The expression of HLA-ABC in these subpopulations was measured by flow cytometry with anti-HLA-ABC antibody. (Filled: isotype control; Open: anti-HLA-ABC antibody.) Data are representative of 3 different donors. D) PBMC were treated with IFN-α (1000 U/ml) or IFN-λ1 (25 ng/ml) for 7 hr, and stained with anti-CD83-FITC or anti-HLA-ABC-FITC vs. isotype controls. The mean fluorescence intensities (MFI) of HLA-ABC (i) and CD83 (ii) of PDC after IFN-α or IFN-λ treatment was compared with that of mock for n=3 donors. *p<0.05.

Article Snippet: For some experiments, PE- or FITC-labeled anti-IFN-α (Miltenyi Biotec) was used.

Techniques: Staining, Functional Assay, Activation Assay, Fluorescence, Flow Cytometry, Labeling, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Type III IFNs are produced by and stimulate human plasmacytoid dendritic cells 1

doi: 10.4049/jimmunol.1102038

Figure Lengend Snippet: A) Enriched pDC were treated with 1 µM dexamethasone (Dex) for 1 hr prior to stimulation with HSV-1, HIV-1 or highly purified GFP-HSV for another 18 hr. IFN-λ in supernatants was determined by ELISA. Data represent mean and range for two separate donors. B) Exogenous IFN-λ inhibits apoptosis of pDC. PBMC were treated with Dex, with or without IFN-α (10,000 U/ml) or IFN-λ1 (10ng/ml) for 6 hr. Active caspase-3 in pDC was stained intracellularly in pDC. Data are representative of 3 different donors. C) Annexin V/PI staining was carried out for pDC within PBMC treated with 1 µM Dex in the presence or absence of rIFN-α (100 ng/ml) or –λ1 (10 or 100 ng/ml). Data are representative of three experiments.

Article Snippet: For some experiments, PE- or FITC-labeled anti-IFN-α (Miltenyi Biotec) was used.

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Staining

Journal: PLoS ONE

Article Title: Plasmacytoid Dendritic Cells Are Inefficient in Activation of Human Regulatory T Cells

doi: 10.1371/journal.pone.0044056

Figure Lengend Snippet: A: Production of IFN-α by activated pDC. Supernatants of activated pDC (CpG/IL-3) or cDC (cytokine cocktail) cultures were harvested. Production of IFN-α was investigated by ELISA. Each circle resembles one individual experiment, bar represents mean; n.d.: not detectable, n = 4. B: Neutralization of cytokines in cocultures of Teff, Treg and pDC. Teff plus Treg (1∶1) were stimulated with allogeneic pDC at a ratio of 1∶5 in absence (Ø) or presence of neutralizing antibodies against IFN-α, IL-1β, IL-6 or TNF-α (10 µg/ml each). Proliferation in cocultures (mean ± SD of triplicates) is normalized to untreated cocultures (set to 100%). One representative experiment out of three is shown. C: Addition of supernatants from standard cultures. CD4 + Teff and Treg (1∶1) were stimulated with anti-CD3 mAb (0.5 µg/ml) plus irradiated TC-depleted PBMC. Supernatants from these cocultures were collected on day 3 and titrated (1∶2) to pDC-stimulated CD4 + Teff, Treg or cocultures. Proliferation was assessed on day 3 by 3 H-Tdr-incorporation. Diagrams show mean values (± SD) of three independent experiments. Proliferation of untreated and treated cocultures was normalized to untreated or treated Teff-proliferation (set to 100%).

Article Snippet: For determination of IFN-α production, activated 10 6 /ml pDC (stimulated with CPG plus IL-3) and cDC (stimulated with the cytokine cocktail) were cultured for 24 h. Supernatants were harvested and IFN-α was detected by using a commercial available IFN-α ELISA (Diaclone, Besancon, France, detection limit 3.16 pg/ml) as indicated by the manufacturer.

Techniques: Enzyme-linked Immunosorbent Assay, Neutralization, Irradiation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Intratumoral delivered novel circular mRNA encoding cytokines for immune modulation and cancer therapy

doi: 10.1016/j.omtn.2022.09.010

Figure Lengend Snippet: cmRNA mediated expression of different kinds of proteins in vitro and in vivo (A) Images show the bioluminescence of firefly luciferase at 6 h, 24 h, and 9 days after intramuscular injection of 10 μg of the LNP-cmRNA complex. (B) Detection of membrane protein IL-15 CD8α and IL-15 GPI expression by FACS. (C) ELISA detection of the secreted form of RBD antigen in the culture supernatant of 293T cells at 24 h post transfection with 0.5 μg of cmRNA, and in mouse serum at 24 h post intramuscular injection with 10 μg of LNP-cmRNA complex. ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Article Snippet: The expression of human IFN-a2b, IL-15, GM-CSF, and IL-12sc were determined using commercial ELISA kits (Human IFN-α ELISA kit, Beyotime; Human IL-15 ELISA kit, Abcam; Human GM-CSF ELISA kit, Proteintech; ELISA MAX Deluxe Set Human IL-12 [p70], BioLegend).

Techniques: Expressing, In Vitro, In Vivo, Luciferase, Injection, Membrane, Enzyme-linked Immunosorbent Assay, Transfection