Fig. 6 | Nature Communications

Fig. 6

From: Single-cell analysis reveals that stochasticity and paracrine signaling control interferon-alpha production by plasmacytoid dendritic cells

Fig. 6The alternative text for this image may have been generated using AI.

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

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