In Vivo CRISPR screen of dendritic cells identifies Traf2 as a critical regulator of anti-tumor immunity via modulation of non-canonical NF-kB pathway

For their ability to sample and present tumor antigens and prime cognate adaptive immune responses, the critical role of dendritic cells (DCs) in potentiating anti-tumor immune has been increasingly recognized. Yet despite their instrumental function in empowering tumoricidal adaptive immune responses, they have also been found in several cancers to foster tolerogenic immunity towards cancer antigens-underscoring the need to better understand the regulatory mechanisms that enable DCs to orchestrate a more tumoricidal response rather than a tolerogenic one. To investigate such mechanisms, we performed pathway analysis on transcriptional signatures of mature DCs found in several tumor types in both mice and humans, which identified non-canonical NF-kB (ncNF-kB) signaling as a potential regulatory pathway. Through an in vivo ProCode-based CRISPR knockout (KO) screen targeting ncNF-kB signaling molecules in DCs in Kras G12D Tp53 -/- (KP) tumors in mice, we discovered Traf2, a negative regulator of ncNF-KB pathway, as a key modulator capable of enhancing the expression of immunostimulatory molecules, particularly IL-12. Both CRISPR-mediated Traf2 knockout and DC-specific deletion using Zbtb46-Cre demonstrated that KO of Traf2 in DCs lead to elevated IL-12 production and increased frequencies of CCR7⁺CD40⁺ mature DCs upon dead-cell uptake. In congruence with their immunogenic phenotype, Traf2-KO DCs demonstrated a heightened capacity to activate OT-1 T cells. Reflective of enhanced DC immunogenicity in vitro, mice with DC-specific Traf2 knockout, when challenged with orthotopic lung tumors, showed increased IL-12 production in DCs and enhanced T cell activation-all of which led to a significantly reduced tumor burden compared to their wildtype controls. Collectively, these findings position DC-specific Traf2 inhibition as a promising strategy to reprogram dendritic cells toward potent tumoricidal immunity and enhance the efficacy of cancer immunotherapy.