Targeting DNGR-1 with Fangchinoline Elevates Dendritic Cell Antigen Cross-Presentation-Mediated Antitumor Immunity in Melanoma.

Cross-presentation of exogenous antigens by dendritic cells (DCs) is essential for initiating CD8+ T cell-mediated anti-tumor immunity. DNGR-1 (CLEC9A), a C-type lectin receptor on conventional type 1 DCs (cDC1s), facilitates uptake and cytosolic delivery of dead cell-associated antigens. However, no small-molecule modulators of DNGR-1 have been reported. Here, we identify fangchinoline-a natural bisbenzylisoquinoline alkaloid-as a small-molecule modulator that enhances antigen cross-presentation in a DNGR-1-dependent manner. Functional screening shows fangchinoline promotes MHC-I-restricted cross-presentation and CD8+ T cell priming. It binds a DNGR-1 pocket (Trp209, Glu225, Arg226), confirmed by surface plasmon resonance, thermal shift, probe labeling, and mutagenesis. DNGR-1 engagement activates the Syk-Nox2 pathway, increasing phagosomal reactive oxygen species and membrane permeabilization to enable cytosolic antigen access. Fangchinoline exerts subset-dependent effects: in GM-CSF/IL-4-derived DCs and endogenous cDC1-associated settings in vivo, it enhances cross-presentation-associated function, whereas in Flt3L-induced DCs with high basal DNGR-1 expression it promotes DNGR-1-dependent cell death. In melanoma models, fangchinoline synergizes with DC transfer and PD-1 blockade to promote CD8+ T cell-mediated tumor control. It enhances antigen-specific CD8+ T cell activation in human DC-T cell co-cultures. Together, these findings establish fangchinoline as a DNGR-1-targeting modulator for context-dependent enhancement of anti-tumor immunity.