Scalable generation of pure CD103+ cDC1 from iDC1 cultures.

Abstract

Aims: Conventional type 1 dendritic cells (cDC1) specialize in cross-presentation and interleukin-12 production and are critical for immunity against intracellular pathogens and tumors, but remain rare in vivo, limiting mechanistic and translational studies. Existing bone marrow-derived dendritic cell (BMDC) methods do not achieve selective enrichment of cDC1 or scalable production at high purity. We therefore aimed to establish a high-efficiency BMDC culture system for selective generation of CD103+ cDC1 from mouse bone marrow.

Methods: Mouse bone marrow cells were cultured in defined medium with recombinant FLT3L, low-dose GM-CSF, and Kit ligand (KitL) to generate induced cDC1 (iDC1). Phenotypic, transcriptomic, proteomic, phospho-proteomic, and functional analyses were performed and compared with established BMDC methods and ex vivo immune cell populations.

Results: iDC1 cultures enabled scalable generation of an estimated 1.5 × 10⁹ CD103⁺ cDC1 at greater than 95% purity from a single mouse, representing at least a 75-fold increase relative to previous recombinant cytokine-based methods. iDC1 closely aligned with the cDC1 lineage while distinct from macrophages. Functionally, iDC1 responded robustly to innate stimulation, secreted IL-12p40 and inflammatory chemokines, and efficiently cross-presented cell-associated antigen to CD8⁺ T cells. Mechanistically, KitL and GM-CSF cooperated during early cDC1 generation, whereas GM-CSF promoted proliferation and survival during later stages of culture. iDC1 generation depended on the +32 kb Irf8 enhancer, and STAT5- and BRD4-associated regulatory programs contributed to efficient iDC1 generation.

Conclusion: iDC1 cultures are a scalable platform for studying cDC1 biology and may facilitate development and preclinical evaluation of cDC1-based immunotherapeutic strategies.