(A) Ccr7 mRNA expression. Plasmacytoid DCs (CD11c^med, MHC-II^med, PDCA-1⁺, CD3^–, CD19^-), LN-resident DCs (CD11c⁺, MHC-II^med, CD3^–, CD19^–), and mDCs (CD11c⁺, MHC-II^hi, CD3^–, CD19^–) from WT and Nr4a3^–/– mice were sorted by FACS. Data represent pools of 5 mice per group. Results represent 1 of 2 independent experiments. (B) Histogram of CCR7 surface expression on mDCs. (C) MFI of CCR7 on mDCs (n = 6 mice per group). Results represent 1 of 3 independent experiments. (D) CD11c^YFP WT or CD11c^YFP Nr4a3^–/– mice were treated with R848 for 2 hours in vivo. Confocal whole-mount images of mesenteric lymphatic vessels from R848-treated CD11c^YFP WT or CD11c^YFP Nr4a3^–/– mice were analyzed using Imaris software. Lymphatics (blue) were isosurfaced, and distance transformation was applied to create a new channel that encoded the 3D distance from the lymphatic vessels. CD11c^YFP cells (green) were isosurfaced and classified as being associated with lymphatics (red) when the distance from the lymphatics was less than 15 μm. To normalize between the different data sets, the number of cells associated with lymphatics was divided by the volume of lymphatic vessels (expressed in μm³ and divided by 100,000). (E) Numbers of YFP⁺ cells associated with lymphatic vessels (red). (F) Numbers of YFP⁺ cells away from lymphatic vessels (green). (G) Day-7 BMDCs were stimulated with 0.5 μg/ml LPS for 36 hours. CD11c⁺MHC-II⁺⁺ cells were gated for CCR7 expression. (H) MFI of CCR7 expression on CD11c⁺MHC-II⁺⁺ cells was analyzed by flow cytometry. Representative histogram (top) and dot plot of individual mouse samples (bottom). Max, maximum. (I) Expression of Foxo1 mRNA and FOXO1, p-AKT, AKT, and β-actin proteins from CD11c⁺ BMDCs, stimulated with or without LPS. Results from G and I represent 1 of 3 to 4 independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, 2-tailed Student’s t test.