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 highly selective enrichment of cDC1 or scalable production at high purity. Here, we established a novel in vitro culture system for selective generation of CD103 cDC1 from mouse bone marrow using defined media conditions together with recombinant FLT3L, GM-CSF, and Kit ligand (KitL), termed iDC1. iDC1 cultures enabled scalable generation of an estimated 1.5 x 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. Phenotypic and transcriptional analyses demonstrated that iDC1 closely align with the CD103 cDC1 lineage while remaining clearly distinct from macrophage populations. Functionally, iDC1 responded robustly to innate stimulation, produced interleukin-12 and inflammatory chemokines, and efficiently cross-presented cell-associated antigen to CD8 T cells. Mechanistically, KitL and GM-CSF regulated distinct stages of cDC1 generation, whereas proteomic, phospho-proteomic, and functional analyses demonstrated that GM-CSF suppresses apoptosis and oxidative stress while promoting cDC1 proliferation. iDC1 generation was dependent on the +32 kb Irf8 enhancer required for bona fide cDC1 development, and STAT5- and BRD4-associated regulatory programs were identified as important regulators of efficient iDC1 generation. Together, these findings establish iDC1 cultures as a scalable platform for studying cDC1 biology and developing cDC1-based immunotherapeutic strategies.
Sharma, S., Flynn, F., Capaldo, B., Holewinski, R., Chen, Q., Meerzaman, D., Andresson, T., Mayer, C. T.
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