Elucidating the Mechanisms of SA-4-1BBL Immunoprevention Through Advanced Informatics Approaches

Mice were treated subcutaneously with SA-4-1BBL or an agonistic antibody and injection site (IS) tissue and draining lymph nodes (LN) were analyzed for differentially expressed genes (DEGs). Our findings reveal that SA-4-1BBL induces a compartmentalized and temporally dynamic immune program, characterized by early effector activation at IS and sustained immune regulation in draining LN. K-means clustering of 4,564 DEGs identified 8 functionally distinct clusters, with IS-enriched clusters (1 and 4) upregulating effector genes such as Cd28, Klra1, Cd4, Cd40, and Cd40l, supporting CD4+ T and NK cell activation.

In contrast, LN-dominant clusters (2 and 5) were enriched for regulatory genes (Tnfaip3, Irf5, Col1a2), implicating roles in tissue remodeling and long-term immune memory. Compared to agonistic antibody, SA-4-1BBL elicited a more selective and durable activation of adaptive immune pathways, including TCR signaling, Th1/Th2 differentiation, and NK cytotoxicity, while avoiding systemic inflammation. Agonistic antibody, in contrast, activated broader innate inflammatory programs, including Toll-like receptor and neurodegeneration-linked pathways.

IMPRes analysis revealed that SA-4-1BBL engages sequential immune-regulatory circuits centered on Stat1, Cd247, and Ifng, and modulates the CD151–TGF-β axis, offering novel mechanistic insight into its immune-balancing effects. These findings define a compartmentalized immune architecture that underlies SA-4-1BBL’s ability to provide safe, long-lasting cancer immunoprevention. Our study highlights the value of applying comprehensive informatics techniques in decoding the spatial and functional complexity of tissue-specific immune programming.