Human mast cell subtype plasticity and lineage stability shaped by microenvironmental signals

Abstract

Background and Objectives: Mast cells exist as two functionally distinct subtypes, MCT and MCTC, whose identity is shaped by microenvironmental signals during tissue maturation. TGF-β has been identified as an instructive signal for MCT differentiation, and prior work has demonstrated that MCTC can acquire aspects of MCT identity upon TGF-β exposure. Whether mature MCT identity is actively maintained by ongoing TGF-β signaling, the transcriptional and functional extent of plasticity in both subtypes, and the contribution of SMAD3 to these responses remain uncharacterized. Methods: MCT and MCTC were differentiated from peripheral blood CD34+ progenitors in vitro. To examine plasticity in mature cells, TGF-β was withdrawn from MCT and added to MCTC for one week prior to analysis. To interrogate canonical signaling, the SMAD3-specific inhibitor SIS3 was applied concurrently with TGF-β. Transcriptional remodeling was assessed by bulk RNA sequencing, functional plasticity by IgE-mediated cysteinyl leukotriene and prostaglandin D2 measurement, and surface phenotypic remodeling by flow cytometric profiling. Results: TGF-β withdrawal from MCT and addition to MCTC produced asymmetric transcriptional responses, with 713 and 1,799 differentially expressed genes, respectively, spanning granule component, eicosanoid biosynthesis, and receptor gene programs. TGF-β bidirectionally regulated IgE-mediated eicosanoid production across both subtypes. Certain features of MCT identity, including low MRGPRX2 surface expression, were resistant to TGF-β withdrawal, whilst others, including CD33 and eicosanoid biosynthetic capacity, required continuous TGF-β signaling for their maintenance. In MCTC, TGF-β addition induced broad transcriptional and functional remodeling alongside suppression of MCTC-enriched surface markers. SMAD3 inhibition selectively attenuated TGF-β-driven cysteinyl leukotriene production in MCT and integrin α2B expression in both subtypes, demonstrating context-dependent and mediator-specific SMAD3 contributions. Conclusion: Human mast cell subtype identity comprises both developmentally consolidated programs that are resistant to acute microenvironmental change and actively maintained programs requiring continuous TGF-β signaling. MCT and MCTC differ fundamentally in the architecture of this relationship, with MCTC exhibiting broader susceptibility to TGF-β-driven remodeling. These findings reframe mast cell subtype identity as a dynamically maintained state shaped by microenvironmental signals.