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

Article Title: Age-specific Adjuvant Synergy: Dual TLR7/8 and Mincle Activation of Human Newborn Dendritic Cells Enables Th1-polarization

doi: 10.4049/jimmunol.1600282

Figure Lengend Snippet: Adult and newborn DCs were stimulated with R848 (50 μM), TDB (100 μg/ml), or both (A+B). This restored TNF production to adult-like levels in newborn cells and enhanced production IL-1α, IL-1β and IL-18 (n=6, Paired Student’s t-test, *p<0.05, **p<0.01 and ***p<0.001). Naïve (CD4+CD45RA+CD45RO−) T cells were isolated, cultured in 10% (vol/vol) autologous plasma, and activated for 6 days with anti-CD3/CD28 beads, in the presence of culture supernatants of autologous MoDCs activated with agonists as indicated. After 6 days, IFN-γ- and IL-4-producing cells were quantified by flow cytometry following the addition of Brefeldin A. Mean relative percentage of IFN-γ producing cells (C), mean relative percentage of IL-4 producing cells (D), and ratio of IFN-γ producing cells over IL-4 producing cells (E) show an increase in Th1 polarization after treatment with culture supernatants from R848+TDB-activated MoDCs (n=4, Mean+SEM, paired Student’s t-test, *p<0.05, **p<0.01 and ***p<0.001). Representative images of flow cytometric quantification of IFN-γ and IL-4 producing cells are shown. Depicted are control samples of a representative newborn and adult whose T cells were activated with anti-CD3/28 in the presence of supernatant from unstimulated autologous MoDCs. (F–G).

Article Snippet: This was supplemented with 50 ng/ml recombinant human (rh) IL-4 and 100 ng/ml rhGM-CSF (R&D Systems; Minneapolis, MN, USA).

Techniques: Isolation, Cell Culture, Flow Cytometry

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

Article Title: Age-specific Adjuvant Synergy: Dual TLR7/8 and Mincle Activation of Human Newborn Dendritic Cells Enables Th1-polarization

doi: 10.4049/jimmunol.1600282

Figure Lengend Snippet: Adult and newborn MoDCs were stimulated for 18 hours with GLA-AF (1000 ng/ml), BGP (100 μg/ml) or the combination. Secreted cytokines were measured by multiplexing bead array (A,B) (n=3–6, Paired Student’s t-test, *p<0.05, **p<0.01 and ***p<0.001). IFN-γ- and IL-4-producing cells were quantified by flow cytometry following treatment of autologous naïve CD4+ T cells with MoDC culture supernatant for 6 days and subsequent addition of Brefeldin A. Mean relative percentage of IFN-γ producing cells (C), mean relative percentage of IL-4 producing cells (D), and ratio of IFN-γ producing cells over IL-4 producing cells (E) show an increase in Th1 polarization after treatment with culture supernatants from R848+TDB-activated MoDCs (n=4, mean+SEM, paired Student’s t-test, *p<0.05, **p<0.01 and ***p<0.001). The expression of a panel of 80 innate immune pathway-related genes was measured by qRT-PCR and the mean (n=3) depicted as a volcano plot (F). Inflammasome activation was confirmed by incubation of cells that were treated as indicated with FITC-labeled Caspase-1 substrate FAM-YVAD-FMK (representative experiment shown, n=3) (G) Activation of NF-κB was confirmed by detection of IκBa degradation in lysates from cells treated as indicated (representative experiment shown, n=3) (H).

Article Snippet: This was supplemented with 50 ng/ml recombinant human (rh) IL-4 and 100 ng/ml rhGM-CSF (R&D Systems; Minneapolis, MN, USA).

Techniques: Multiplexing, Flow Cytometry, Expressing, Quantitative RT-PCR, Activation Assay, Incubation, Labeling