Fig. 6.

The pDCs primed with CM or IFNβ are more likely to produce IFNα. a Schematic overview of the priming experiment. The pDCs were stimulated for 2 h with conditioned medium (CM), 0.5 µg/mL CpG-C, or control medium. Cells were washed, coated with cytokine capture reagent, encapsulated in droplets, and stimulated individually with 0.01 µg/mL IL-3 or 50 µg/mL CpG-C for 12 h. CM was generated from bulk pDC cultures stimulated with 5 µg/mL CpG-C at a density of 25,000 cells/well. Cytokine-secreting cells were detected using flow cytometry. b The fraction of IFNα-secreting cells is plotted against different treatment conditions. c Co-expression of CCR7, CD40, CD86, and TNFα by single IFNα⁺ and IFNα⁻ pDCs was analyzed. Shown is the relative contribution of each functional response pattern to the total pDC population. d The pDCs were primed with different concentrations of CM and stimulated with 50 µg/mL CpG-C. Shown is the fraction of IFNα-secreting cells plotted against CM concentration; n > = 3. Dots indicate mean and whiskers indicate SEM. e The pDCs were treated as described above, primed with 10% CM, and stimulated with different concentrations of CpG-C. Shown is the fraction of IFNα-secreting cells plotted against CpG-C concentration; n = 3. f The pDCs were primed with 10% CM, control medium, or 500 U/mL IFNβ, and stimulated with 50 µg/mL CpG-C. The fraction of IFNα-secreting cells is plotted against different treatment conditions; n > = 3. g Schematic model illustrating stochastic IFNα-production by pDCs. Few pDCs produce IFNα constitutively without stimulation by TLR ligands, here resembled by differentiation from a freshly isolated pDC (pDC₀) to an IFNα-secreting pDC (pDC₁). Literature indicates that this is not IRF7 dependent but NF-κB and AP-1²⁷. Upon TLR9 triggering the IRF7-dependent pathway is activated which also allows differentiation to IFNα-secreting pDC at a much higher rate. Despite involvement of the IRF7 pathway, still only very few pDCs produce IFNα. Paracrine signaling via the type I IFN receptor can increase this rate, probably via the upregulation of IRF7 expression, leading to a large fraction of cells expressing IFNα. After producing IFNα, pDCs become refractory to re-stimulation (pDC₂) and eventually die (Φ). b, f Welch-corrected two-sample t-test; *p < 0.05, **p < 0.01