Defining the immunoregulatory molecular drivers of dendritic cells using CRISPR screens in ex-vivo modelsvivo models

Conventional dendritic cell type 1 (cDC1) plays a central and indispensable role in coordinating antitumor T-cell responses by producing cytokines such as IL-12 and expressing key regulatory molecules including PD-L1. Despite their importance in shaping antitumor immunity and influencing responses to modern immunotherapies, the molecular mechanisms controlling cDC1 activation and maturation remain poorly defined. This gap in knowledge is compounded by the extremely low abundance of cDC1 within the tumor microenvironment (TME), which limits the ability to experimentally interrogate their biology and apply genetic perturbation tools at scale. To address these challenges, we developed, optimized, and compared robust ex vivo differentiation systems capable of generating physiologically relevant cDC1-like cells from both murine and human hematopoietic stem cells (HSCs).Using a carefully designed two-stage culture platform incorporating Fms-like tyrosine kinase 3 ligand (Flt3L), DLL1-mediated Notch signaling, and granulocyte-macrophage colony-stimulating factor (GM-CSF), we efficiently produced CD103⁺ cDC1-like populations from mouse bone marrow progenitors and Clec9a⁺CD141⁺ cDC1-like cells from G-CSF-mobilized human peripheral blood CD34⁺ HSCs. These ex-vivo derived cells closely mirrored their in-vivo counterparts, exhibiting the expected surface immunophenotypes, high IL-12 secretion upon stimulation measured by ELISA, and strong functional responsiveness to TLR agonists and cell-associated antigens. Upon activation, cells consistently upregulated classical maturation markers including PD-L1, MHCII, and CD40, demonstrating their ability to undergo appropriate immune stimulation-driven maturation.To support downstream genetic studies, we optimized viral transduction strategies for primary HSCs, a major technical barrier in the field. An ecotropic retroviral system combined with retronectin substantially improved gene delivery efficiency in murine cells, while an amphotropic pseudotyped retroviral approach enhanced transduction of human CD34⁺ HSCs. These advances enable reliable introduction of CRISPR-based perturbations into precursor cells prior to differentiation.Together, this scalable, high-yield differentiation and gene-delivery platform provides a powerful foundation for conducting CRISPR-based genetic screens aimed at identifying the molecular regulators orchestrating cDC1 activation and maturation. These insights have the potential to inform the development of next-generation immunotherapies that harness or enhance dendritic cell-driven antitumor immunity.Defining the immunoregulatory molecular drivers of dendritic cells using CRISPR screens in ex-vivo models