iregDC extrinsic MyD88 signaling, but not direct LCMV infection or IFNβ signaling is required for iregDC differentiation.

<p><b>A.</b> WT mice were CD8 depleted and then infected with LCMV-Cl13-GFP. Flow plots show GFP expression (i.e., LCMV infection) in splenic iregDC and stimDC (gated on CD39 and CD95) on day 9 after infection. In parallel, naïve splenic DC (N), and iregDC and stimDC were FACSorted based on GFP expression on day 9 after infection. The level of GFP+ stimDC was too small to obtain adequate numbers for RNA isolation. Bar graphs show relative expression of IL-10 RNA, LCMV-GP RNA and the MFI of PDL1 protein expression from the sorted populations. To obtain sufficient cell numbers for the RNA analyses, infected and uninfected iregDC and stimDC were sorted from a pool of 8 mice. Data show one of two experiments. <b>B.</b> Flow plots show the percent and bar graphs the number of iregDC and stimDC in the indicated mouse strain from CD11b+ DCs at day 9 after LCMV-Cl13 infection. <b>C.</b> Bar graphs indicate plasma IL-10 concentration and PDL1 expression in WT, IFNαR-/-, and MyD88-/- mice at day 9 after LCMV-Cl13 infection. <b>D.</b> Naïve WT and MyD88-/- monocytes were transferred into WT mice and analyzed on day 9 after LCMV-Cl13 infection as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005356#ppat.1005356.g004" target="_blank">Fig 4E</a>. <b>E.</b> Mixed chimera mice were generated using bone marrow from WT and MyD88-/- bone marrow. Flow plots depict WT and MyD88-/- lineage splenic CD11b+ DCs at day 9 after LCMV-Cl13 of WT. Histogram shows PDL1 expression on iregDCs from WT (white) and MyD88-/- (grey) lineage cells. Bar graphs indicate PDL1 expression and the number of iregDC and stimDC from each lineage in the chimera mice. Data are representative of 2 or more independent experiments each consisting of 3–5 mice per group. *, p<0.05.