In the latter case, the secretory mechanism involves

intragranular compartments organized as tubular vesicles or tubular networks, which bud from donor granules and relocate specific granule products in response to stimulation 24. Consequently, PMD would accomplish discharge of secretory constituents from storage granules without granule-to-granule and granule-to-plasma membrane fusion events and without direct granule opening to the cell exterior, as we have observed in our experiment. PMD has been demonstrated to occur in case of cytokine secretion 23, 24, but the molecular mechanisms underlying PMD are largely unknown. In particular, very little is known about what governs the cell decision to opt for either release of entire granules or PMD, and the precise molecular mechanisms that regulate mobilization of vesicle-associated secretory MK-2206 datasheet aliquots in a PMD manner. In light of these results, it can be speculated that the lowered availability of cytosolic Ca2+ in activated MCs interacting with Tregs could be responsible for unsuccessful exocytosis but could be enough for promoting PMD. This could explain the selective inhibitory effect of Tregs on the secretion of pre-stored and usually early released mediators and the delay of TNF-α release observed at early time point. In conclusion, this study describes the dynamic and functional profile

of MC–Treg interactions. This cross-talk is not restricted to BMMCs but is a common feature of mature MCs and human MCs. Importantly, AZD6738 in vitro Liothyronine Sodium we found that this cross-talk is regulated on a single-cell level also providing the first morphological evidence for a role of the OX40–OX40L axis in Treg inhibition of MC function. However, the dynamics of Treg–MC conjugates reflects a complex synaptic structure and a more detailed analysis is necessary to understand the molecular composition of this interaction. Moreover, the evidence of PMD in MCs interacting with Tregs underlines the necessity to understand all events and mechanisms governing differential sorting, packing and

secretion of granule-stored mediators. Our findings pave the road to identify selective secretory pathways that are still partially unknown and might regulate MC degranulation without modifying their innate immune functions. C57BL/6 mice were purchased from Harlan (Harlan Italy), C57BL/6 OX40-deficient mice were kindly provided by M. Colombo in Milan, Italy. CD4+CD25+ cells were purified using the CD25+ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. By flow cytometry analysis, cells were more than 90% Foxp3+. BMMCs were obtained by in vitro differentiation of BM cells taken from mouse femur as described 4. After 5 wk, BMMCs were monitored for c-kit and FcεRI expression by flow cytometry. Purity was usually more than 97%.