Second-generation Langerhans cells originating from epidermal precursors are essential for CD8⁺ T cell priming

In vivo studies questioned the ability of Langerhans cells (LCs) to mediate CD8⁺ T cell priming. To address this issue, we used intradermal immunization with plasmid DNA, a system in which activation of CD8⁺ T cells depends on delayed kinetics of Ag presentation. We found that dendritic cells (DCs) located in the skin at the time of immunization have limited ability to activate CD8⁺ T cells. This activity was mediated by a second generation of DCs that differentiated in the skin several days after immunization, as well as by lymph node-resident DCs. Intriguingly, CD8 ⁺ T cell responses were not affected following treatment with clodronate liposomes, immunization of CCR2^-/- mice, or local neutralization of CCL20. This suggests that local, rather than blood-derived, DC precursors mediate CD8⁺ T cell priming. Analysis of DC differentiation in the immunized skin revealed a gradual increase in the number of CD11c⁺ cells, which reached their maximum 2 wk after immunization. A similar differentiation kinetics was observed for LCs, with the majority of differentiating LCs proliferating in situ from epidermal precursors. By using B6/Langerin-diphtheria toxin receptor chimeric mice and LC ablation, we demonstrated that epidermal LCs were crucial for the elicitation of CD8 ⁺ T cell responses in vivo. Furthermore, LCs isolated from lymph nodes 2 wk after immunization contained the immunization plasmid and directly activated Ag-specific CD8⁺ T cells ex vivo. Thus, these results indicate that second-generation Ag-expressing LCs differentiating from epidermal precursors directly prime CD8⁺ T cells and are essential for optimal cellular immune responses following immunization with plasmid DNA.