iCD103-DCs are not specialized to induce tolerance. (A) DC maturation marker expression among CD11c⁺ GM-DCs or CD11c⁺B220^– FL-cDCs and CD11c⁺B220^– iCD103-cDCs in the presence or absence of overnight CpG stimulation. Unstimulated (solid black lines), CpG-stimulated (solid red lines), and isotype control stainings (gray overlay) are shown. (B-C) CellViolet-labeled CD4⁺Rag1^−/− × OTII T cells were co-cultured with GM-DCs (upper panel), FL-DCs (middle panel), or iCD103-DCs (lower panel) in the presence of OVA_323-339 peptide, IL-2, and combinations of TGFβ and RA. After 5 days, cells were analyzed by flow cytometry. (B) Plots display CellViolet dilution vs Foxp3 expression among CD4⁺ cells. (C) Quantification of proliferated Foxp3⁺ cells in different culture conditions. Mean + SD is shown. **P < .01, ***P < .001; NS, not significant; 2-way analysis of variance with Bonferroni’s post-test. (D) Relative Aldh1a2 expression among GM-DCs, FL-DCs, and iCD103-DCs is shown normalized to Hprt and GM-DCs. Results show 2 independent cDNA preparations. (E-F) Naïve WT mice were injected intravenously (IV) with equal numbers of OVA_257-264-pulsed FACS-sorted CD11c⁺ GM-DCs or CD103^hiCD11c⁺B220^– iCD103-DCs. Mice were infected 8 days later IV with 5 × 10³ colony-forming units (CFU) Lm-OVA. Seven days post infection, splenocytes were restimulated with OVA_257-264 to determine the antigen-specific CD8⁺ T-cell response. (E) Representative plots display CD62L vs interferon (IFN)-γ expression among restimulated CD8⁺ T cells. (F) Quantification of IFN-γ–secreting CD8⁺ T cells. Results are representative of 2 to 5 (A), 4 (B), or 2 (D-F) independent experiments. Data were combined from 4 independent experiments with individual culture preparations (C) or 2 independent experiments with 4 to 5 mice per experimental group (F). *P < .05, **P < .005; NS, not significant; 2-tailed Mann-Whitney U test.