NK1R-signaled BMDCs are required to home in sDLNs to promote type 1 immunity. (A) Flow cytometric analysis of the expression of homotypic adhesion molecules and the chemokine receptor CCR7 by untreated-DCs, NK1R-DCs, Ag-DCs, and Ag-NK1R-DCs. Mean ± 1 SD of MFI of 3 independent experiments are illustrated. (B) Microscopic analysis (H&E, PAS) of sDLN, 24 hours after subcutaneous immunization with WT or CCR7^KO BMDCs untreated (DC), NK1R-DCs, or Ag-NK1R-DCs. Paracortical areas (dotted lines) are defined by the presence of PAS⁺ high endothelial veins (arrowheads, inset). H&E and PAS, ×200. Inset, ×500. (C) Identification by fluorescence microscopy of CFSE⁺ NK1R-DCs 24 hours after subcutaneous injection and homed in the subcapsular and paracortical areas of sDLN (dotted lines). Nuclei were stained blue with DAPI, ×200. (D) Colocalization of CFSE⁺ Ag-NK1R-DCs (injected subcutaneously, 24 hours prior), and endogenous CD11c⁺CD11b⁺, CD11c⁺CD11b⁻, and CD11c⁺Ly6C⁺ DCs in sDLNs (top and bottom left panels) and CD3⁺ T cells (bottom right panel). Fluorescence microscopy, ×100 and ×200. (E) Comparative analysis of CTL function in WT mice skin-vaccinated with WT or CCR7^KO, NK1R-DCs, Ag-DCs, or Ag-NK1R-DCs. The Ag-specific CTL activity was measured 7 days after immunization by in vivo killing assays. Means ± 1 SD from 5 mice per group are shown. CFSE, carboxyfluorescein diacetate succinimidyl ester; DAPI, 4,6 diamidino-2-phenylindole.