Figure 1.
Plasmacytoid dendritic cells show weak T cell stimulatory capacity.
A: Flow cytometric analysis of human PBMC and DC. pDC are characterized as lineage− CD11c− CD303+ cells (middle panel). Dot plots (right side) exemplarily depicts isolated pDC, percentage indicates purity. Histograms show surface expression of CD40, CD80, CD86, CD58 and HLA-DR on resting and CpG/IL-3-activated pDC and cytokine cocktail activated cDC. Isotype-matched control mAb staining is shown in grey. One representative result of three independent experiments is shown. B: T cell stimulatory capacity of activated pDC and cDC. CD4+ or CD8+ Teff were stimulated with allogeneic pDC or cDC at different DC:Teff ratios. Teff proliferation was determined on day 4 of culture by incorporation of 3H-Tdr and presented as mean ± SD of triplicates. One representative experiment out of 3 is shown. C: Expansion of DC-stimulated Teff. Expansion rates of CD4+ or CD8+ Teff after stimulation with allogeneic cDC (n = 4) or pDC (n = 2), determined at day seven. The figure shows the relative fold expansion of initially stimulated Teff. Bar indicates mean values. D: Cytokine profiles of Teff seven days after stimulation with pDC or cDC. Numbers represent the percentage of cytokine-positive Teff. One typical result out of six is shown. ** p<0.01.
Figure 2.
T effector cell proliferation induced by plasmacytoid dendritic cells in presence of regulatory T cells.
A: Absence of Treg function in DC-stimulated cocultures with Teff. CD4+ (upper panel) or CD8+ (lower panel) Teff alone or in coculture with Treg (Treg:Teff 1∶1) from the same donor were stimulated with activated allogeneic pDC or cDC (left and middle panel). Polyclonal stimulation served as control for validation of Treg function (right panel). Proliferation was assessed on day 4 of culture by incorporation of 3H-Tdr and is depicted as mean ± SD of triplicates. One representative result out of 44 (pDC) respectively 9 (cDC) independent experiments is shown. B: Increased Treg frequencies in cocultures. CD4+ Teff were stimulated with activated pDC in presence of increasing Treg numbers. Mean values of three independent experiments are shown as proliferation of cocultures normalized to CD4+ Teff alone (set to 100%). C: Prestimulation of Treg. Treg were prestimulated with anti-CD3 mAb, allogeneic pDC or pDC plus anti-CD3 mAb for 20 h prior their use in suppression assays with syngenic CD4+ Teff (Teff:Treg 1∶1) or left untreated. T cell proliferation was assessed on day 4. Proliferation in cocultures was normalized to Teff proliferation in absence of Treg (set to 100%). D: Addition of exogenous IL-2 and IFN-γ to suppression assays. Cocultures (Teff:Treg 1∶1) were stimulated with allogeneic pDC (Teff/Treg:pDC 5∶1) in absence or presence of IL-2 or IFN-γ. Polyclonal stimulation served as control. Proliferation was analyzed on day 4 of culture by 3H-Tdr incorporation, presented as normalized to Teff proliferation in absence of Treg (set to 100%). Diagrams show mean values (± SD) of three independent experiments.
Figure 3.
Plasmacytoid dendritic cells are unable to induce proliferation of regulatory T cells.
A: Proliferation of DC-stimulated Treg alone or in coculture with Teff. eFluor®670-labeled Treg were stimulated alone (left panel) or in coculture with CD4+ Teff (ratio 1∶1, right panel). Allogeneic pDC or cDC (Treg/Teff:DC 5∶1) were used for stimulation, stimulation with anti-CD3 mAb plus irradiated T cell-depleted PBMC (standard culture) served as control. Proliferation was determined on day 4 of culture by flow cytometrical analysis of eFluor®670 dilution. One representative result of three independent experiments is shown. B: Treg proliferation induced by allogeneic pDC versus cDC. Treg were cocultured at a Treg:DC ratio of 5∶1 with either pDC or cDC. Treg stimulated with anti-CD3 mAb plus T cell-depleted PBMC served as control. Treg proliferation was determined by 3H-Tdr incorporation at day 4 of culture. Diagram shows Treg proliferation normalized to proliferation in standard culture (pDC: n = 43; cDC: n = 9) ** p<0.01.
Figure 4.
Neutralization of proinflammatory cytokines does not restore Treg suppression.
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 3H-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%).
Figure 5.
Plasmacytoid dendritic cells are unable to activate the suppressive function in human regulatory T cells.
A: Surface expression of CTLA-4 and GARP on resting and differently stimulated Treg. Upper panels: flow cytometric characterization of resting CD25+ Tregs stained for indicated markers. Lower panels: Treg were stimulated with anti-CD3 mAb (5 µg/ml) plus T cell-depleted PBMC or allogeneic cDC (1∶5), pDC (1∶5) or pDC plus 5 µg/ml anti-CD3 mAb for 48 h. Flow cytometric analysis of CTLA-4 (extracellular expression), GARP and Foxp3 is shown. Percentages of the indicated surface marker on Foxp3+ Treg are displayed. One representative result out of four is shown. B: Additional anti-CD3 mAb stimulation restored Treg function in cocultures with Teff and pDC. CD4+ Teff cocultured with Treg (Treg:Teff 1∶1) were stimulated with allogeneic pDC alone or in presence of additional anti-CD3 mAb or anti-CD28 mAb in different concentrations or plate bound anti-CD40L (10 µg/ml). Proliferation (mean ± SD of triplicates) was analyzed on day 4 by 3H-Tdr incorporation and normalized to untreated cocultures (set to 100%). One representative result of three is shown. C: Blockade of CTLA-4 and GARP in pDC/anti-CD3-stimulated cocultures. CD4+ Teff cocultured with Treg (Treg:Teff 1∶1), stimulated with allogeneic pDC plus anti-CD3 mAb (0.5 µg/ml). Where indicated, anti-CTLA-4 mAb or anti-GARP mAb (10 µg/ml each) were added. Proliferation (mean ± SD of triplicates) was analyzed on day 4 and normalized to untreated cocultures (set to 100%). Results are shown as mean proliferation (± SD) of three independent experiments.