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How vaccination organizes effective in vivo cross-priming remains poorly defined. We show that adjuvanted vaccination engages an inducible cross-presenting program centered on murine inflammatory cDC2s (inf-cDC2s) and human DC3s derived from the human immune system mouse model, associated with effective CD8⁺ T cell cross-priming. These results extend the cellular basis of cross-presentation beyond a fixed lineage hierarchy through the specialization of responsive cross-presenting identity across species under adjuvanted vaccination. Mechanistically, vaccine-induced cross-presentation was linked to impaired late-stage autophagic flux, characterized by reduced autophagosome-lysosome fusion, in line with prolonged intracellular antigen availability and sustained cross-presentation.

Targeted genetic manipulation of DCs to enhance tumor infiltration and localized IL-12 secretion is an attractive therapeutic strategy for cancer immunotherapy. Using genome-wide CRISPR/Cas9 screening, we identified aryl hydrocarbon receptor (AhR) as the key negative regulator of IL-12 production in DCs. Early-stage Ahr-knockout TIDCs upregulated mitochondrial biogenesis gene expression and increased mtROS production while preserving mitochondrial structure, mass, and membrane potential. Mechanistically, AhR inhibition enriched mitochondria-ER contacts (MERCs), inducing mito-calcium influx and reprogramming pyruvate metabolism to drive mtROS production and IL-12 synthesis. We emphasize the importance of mtROS and mitochondria-ER dynamics as potential targets for enhancing DC function in cancer immunotherapy.