Antigen Geometry Tunes Mast Cell Signaling Through Distinct FcϵRI Aggregation andStructural Changes

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, Fc{varepsilon}RI, 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 Fc{varepsilon}RI {gamma}-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 Fc{varepsilon}RI aggregate architecture and tune the balance of positive and negative signaling to ultimately shape mast cell outcomes. Statement of SignificanceAllergic immune responses are initiated when multivalent antigens aggregate IgE-bound Fc{varepsilon}RI 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 Fc{varepsilon}RI 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.