iCD103-DCs upregulate CCR7, efficiently enter lymph nodes, and induce protective T-cell immunity. (A) GM-DCs and iCD103-DCs were kept untreated (unstim) or were stimulated for 16 hours with CpG, heat-killed Listeria monocytogenes (HK-Lm), or heat-killed M bovis (HK-BCG). CCR7 expression is shown for live-gated CD11c⁺B220^–CD103^hi iCD103-cDCs and CD11c⁺ GM-DCs (black lines). Overlays indicate control stainings (gray lines). (B-C) GM-DCs were labeled with CFSE and mixed with CellViolet-labeled iCD103-DCs (GM^CFSE/iCD103^CV) or vice versa (GM^CV/iCD103^CFSE). (B) Flow cytometric analysis of DC mixtures before injection (upper panel) and recovered DCs from popliteal lymph nodes (popLN) 48 hours after subcutaneous injection in combination with CpG. (C) Migration ratio calculated as the frequency of labeled iCD103-DCs recovered from popLN (2 per mouse) after 48 hours over the initial frequency in the injected DC mixtures. A ratio of 1 indicates equivalent migration capacity of GM-DCs and iCD103-DCs. ***P = .001; 1-sample Student t test compared with value of 1.0. (D) iCD103-DCs and GM-DCs were stimulated and FACS-sorted as CD11c⁺B220^–CD103^hi or CD11c⁺ cells. DCs were then pulsed with OVA_257-264 and OVA_323-339 peptides, washed, and injected subcutaneously. Pulsed splenocytes served as a control. After 7 days, mice were bled and leukocytes were restimulated with OVA_257-264 in the presence of Brefeldin A followed by flow cytometric analysis. The frequencies of CD62L^–IFN-γ⁺ cells among CD8⁺ T cells are quantified. (E) Vaccinated mice were challenged on day 8 with a lethal dose of Lm-OVA (1 × 10⁶ CFU) IV. CFUs in the spleen were determined 3 days later. (F-G) iCD103-DCs were stimulated with M bovis BCG and transferred subcutaneously into mice. Identically treated samples without DCs (No DCs) were injected as a control. Seven days later, splenocytes were restimulated with Ag85B_240-254 and analyzed by flow cytometry. (F) Representative plots show CD62L vs IFN-γ expression among CD4⁺ live-gated cells. (G) Quantification of Ag85B_240-254-specific IFN-γ–producing cells. Results are representative of or quantify 2 to 3 independent experiments (A), 2 independent experiments with 2 to 3 (B-C,F-G), 2 independent experiments with 4 to 5 (D), or 2 independent experiments with 1 to 4 (E) mice per group. **P < .01, **P < .001; NS, not significant; 2-tailed Mann-Whitney U test.