DAP12 enhances CAR-NK cell function via NR4A2-mediated upregulation of 4-1BB expression

Natural killer (NK) cells rely on adaptor proteins CD3ζ/FcRγ, DAP10 and DAP12 for receptor assembly and activation signaling. Among these, DAP12 is unique in that it also participates in inhibitory receptor complexes, offering potential for therapeutic modulation of NK cell activation. However, its downstream signaling mechanisms remain poorly understood. Here, by generating chimeric antigen receptor (CAR) constructs incorporating each adaptor in NK92MI cells, we find that all adaptors support similar degranulation, but DAP12 drives the strongest cytokine secretion and upregulation of inhibitory receptors. Transcriptomic analysis reveals that DAP12 specifically induces the co-stimulatory molecule 4-1BB (TNFRSF9). Antibody blockade confirms that 4-1BB promotes cytokine secretion via interaction with 4-1BBL on target cells. Mechanistically, combining bioinformatics and agonist-based assays, we identify the transcription factor NR4A2 as a key driver of DAP12-mediated 4-1BB upregulation. Our findings uncover a previously unrecognized DAP12-NR4A2-4-1BB-cytokine axis in NK cell activation and establish DAP12 as a pivotal mediator of balanced NK responses. Together, these results provide a mechanistic rationale for 4-1BB-based CAR-NK efficacy and support the integration of DAP12 into next-generation CAR-NK platforms.