Antigen properties shape organization of FcεRI aggregates to tune mast cell signaling.

Fc receptors containing immunoreceptor tyrosine-based activation motifs (ITAMs) are critical components of the innate immune system that bridge adaptive antibody recognition to cellular effector responses. In allergic responses, the high-affinity IgE receptor, FcεRI, is activated when multivalent antigens crosslink receptor-bound IgE, yet the molecular mechanisms linking antigen structure to signaling output remain incompletely understood. Here, we compare two antigens presenting identical IgE-binding haptens but differing in geometry: the high-valency, heterogeneous DNP-BSA and the defined trivalent antigen DF3. We find that these ligands elicit distinct patterns of degranulation and FcεRI γ-chain phosphorylation, correlating with differences in the recruitment of the inhibitory lipid phosphatase SHIP1. Monte Carlo simulations predicted that each antigen generates receptor aggregates with distinct size, complexity, and inter-receptor spacing. Using direct stochastic optical reconstruction microscopy (dSTORM) and Bayesian Grouping of Localizations (BaGoL) analysis, we directly visualized the nanoscale aggregate geometry and found that DF3 induced smaller, more linear aggregates with tighter receptor spacing than DNP-BSA. Together, our results show that antigen properties, including size, valency, and epitope spacing, modulate FcεRI aggregate architecture and tune the balance of positive and negative signaling to ultimately shape mast cell outcomes.

Statement of significance: Allergic immune responses are initiated when multivalent antigens aggregate IgE-bound FcεRI on mast cells, yet the relationship between antigen structure and signaling strength remains unclear. This study combines biochemical assays, Monte Carlo modeling, and super-resolution imaging to show that allergen properties, specifically valency and nanoscale epitope spacing, govern the geometry of FcεRI aggregates and the balance of activating and inhibitory signals. These findings establish a direct mechanistic link between antigen structure and immune receptor output, providing new insight into how physical antigen features encode mast cell responses.