Figure 1.
PD-L1 is expressed on isolated LCs and in LCs in situ of ACD patients.
A. Left side: Immunofluorescence microscopy analysis of the expression of langerin and PD-L1 on the surface of human MoLCs and LCs derived from blood monocytes and epidermal tissue of healthy donors, respectively. Right side: Expression of langerin and CD1a in MoLCs (upper chart) and LCs (lower chart) detected by flow cytometry. Bar = 10 µm. B. Expression of HLA-DR and PD-L1 in skin sections of ACD donors before and 72 hours after challenge with 5% NiSO4, as detected by confocal laser scanning microscopy. Representative images of two stack series in the epidermal layer are shown. Bar = 10 µm.
Figure 2.
Intraepidermal PD-L1 increases in ACD patients after challenge with nickel.
A. PD-L1 and nuclei are visualized by Alexa Fluor 488 (green) and 7-AAD (red) immunofluorescence in sections from lesions of ACD patients after being exposed to 5% NiSO4, for 0 hours and 72 hours. Bar = 50 µm. B. Biopsies taken from inflamed skin tissue areas of ACD donors. The sections were incubated with anti-PD-L1, stained with EnVision+ System-HRP AEC, and counterstained with hematoxylin. Representative light microscopy images covering the profile from the upper stratum corneum down to dermal rete pags are depicted. Bar = 50 µm.
Figure 3.
PD-L1 and HLA-DR are upregulated in MoLCs after stimulation with allergens.
MoLCs were exposed to graded doses of DNCB, nickel or SDS for 24 hours and stained by anti-PD-L1 and anti-HLA-DR, respectively. FACS analyses were gated on viable cells. A. Protein expression is shown as dark gray graphs in overlay histograms. Cells stimulated under the same conditions were stained with isotype-controls and are represented by light gray. B. Bars indicate fold increase of mean fluorescence intensities (MFI) compared to untreated control. Results shown are representative for four independent experiments with different donors.
Figure 4.
Blockage of PD-L1 enhances cytokine release from stimulated Th cells.
A. FACS analyses of CD86 or PD-L1 on MoLCs, dark gray, or isotype-controls, light gray. Numbers depicted in the histograms show mean fluorescence intensities. MoLCs were stimulated by LPS or PGN for 24 hours. Histograms shown are from the same donor representative for results obtained with three different donors. B – E. Secretion of cytokines by CD4+T cells in coculture with MoLCs. MoLCs were stimulated by LPS or PGN (as in A.), and incubated with IgG1, anti-PD-L1, or anti-CD86. Secretion of IL-17, IL-22, TNF-α, and IFN-γ in pg/ml was detected via ELISA 7 days after starting the coculture. Data shown are from 4 different donors, indicated by the following symbols: ▴, ▪, •, ♦. Wo, without stimulus.
Figure 5.
Nickel induces release of IL-22 and TNF-α after blockage of PD-L1.
A., B. Secretion of cytokines by CD4+T cells while being in coculture with MoLCs. MoLCs were either stimulated by nickel for 24 hours (+) or not (−), and incubated with anti-PD-L1 (+) or not (−). Protein secretion was detected via ELISA in the supernatants 7 days after starting the coculture of MoLCs with allogeneic CD4+T cells. Secretion levels (in pg/ml) are shown for IL-22 and TNF-α. Data shown are from 4 different donors and visualized by scatter plots; donors 1–4 are indicated by the following symbols: ▴, ▪, •, ♦.
Figure 6.
PGN and nickel induce IFN-γ and IL-17 in CCR6+ and CCR6+/CCR4+ cells.
T memory cells were incubated with MoLCs pretreated with anti-PD-L1 (+) or not (−). A. Expression of CCR6 and CCR4 in CD4+T cells enriched from the blood of healthy donors by depletion of non-CD4 and naïve (CD45RA+)T cells. T memory subpopulations were gated as CCR6– (P5), CCR6+/CCR4– (P6), or CCR6+/CCR4+ (P7) cells. B. Release of IFN-γ and IL-17 by sorted subpopulations. MoLCs were stimulated by nickel or PGN for 24 hours and incubated or not with anti-PD-L1. Secretion levels (in pg/ml) are shown for IFN-γ and IL-17 7 days after starting the coculture. Data reported as mean +/− SD of triplicates are representative for 3 different donors. Wo, without stimulus.