Journal: Frontiers in Immunology
Article Title: Synergistic Effects of Interferon-γ and Vitamin D 3 Signaling in Induction of ILT-3 high PDL-1 high Tolerogenic Dendritic Cells
doi: 10.3389/fimmu.2019.02627
Figure Lengend Snippet: γ/D 3 DCs exhibit an exceptional tolerogenic phenotype. (A) Immature DCs were left untreated or treated with IFN-γ (500 U/ml), vit D 3 (10 ng/ml), or IFN-γ + vit D 3 at same concentrations. After 24 h, the DCs were washed extensively and re-stimulated using multiple CD40 ligand for an additional 24 h. Afterwards, the cell supernatants were analyzed for the presence of IL-1β, IL-6, IL-8, IL-10, IL-12p70, and TNF-α. Shown are mean ± SD of four independent experiments. (B) The ratio between the capacity of D 3 DCs and γ/D 3 DCs to produce IL-10 vs. IL-12p70 was calculated by dividing mean values of both cytokines, determined in cell culture supernatants after 24 h stimulation with CD40 ligand. (C) Immature DCs were treated with various concentrations of vit D 3 (0.1 ng/ml, 1.0 ng/ml, or 10.0 ng/ml) in the presence or absence of 500 U/ml of IFN-γ. After 24 h, surface expression of ILT-3 and PDL-1 was determined using flow cytometry. Shown are mean ± SD of MFI values calculated on the basis of five independent experiments. (D) Dendritic cells were treated with vit D 3 or IFN-γ + vit D 3 for 24 h, followed by an addition 24 h exposure to LPS (20 ng/ml). Subsequently, expression of surface molecules ILT-3 and PDL-1 was analyzed and individual samples were compared to their counterpart, which was not stimulated with LPS. Shown are mean ± SD of MFIs of three independent experiments. (E) Dendritic cells were treated IFN-γ, vit D 3 , or combination of both and stained after 24 h for surface expression of different inhibitory molecules. Shown is one representative out of five independent experiments performed. The numbers on histograms represent MFI values. Statistical significance between individual pairs was calculated using Student's unpaired t -test (ns, non-significant; * p < 0.05; ** p < 0.01).
Article Snippet: For flow cytometry analysis of DC phenotype we used the following monoclonal antibodies (mAbs): FITC-labeled anti-CD1a (clone: HI149), anti-CD1c (clone: L161), anti-CD14 (clone: 63D3), anti-CD16 (clone: 3G8), and anti-HLA I (clone: W6/32) (all from Biolegend, CA, USA); Alexa Fluor 488-labeled anti-CD4 (clone: RPA-T4) (Biolegend); PE-labeled anti-CD11b (clone: ICRF44), anti-CD11c (clone: 3.9), anti-DC-SIGN (clone: 9E9A8), anti-CD80 (clone: 2D10), anti-CD83 (clone: HB15e), anti-CD86 (clone: BU63) (all from Biolegend), and anti-HLA II (clone: AC122), anti-ILT-3 (clone: REA141), anti-ILT4 (clone: REA184), anti-PDL-1 (clone: REA1197), and anti-FasL (clone: NOK-1) (all from Miltenyi Biotec).
Techniques: Cell Culture, Expressing, Flow Cytometry, Staining