2C, upper panel). Membrane ruffling was dynamic and we observed new ruffles continuously forming and collapsing for at least 30 min. Interestingly,

BMMCs in contact with WT Tregs exhibited a smooth plasma membrane morphology with minimal membrane ruffling (Fig. 2C, intermediate panel), likely corresponding to the absence of MCs degranulation. On the contrary, when BMMCs were conjugated with OX40-deficient Tregs the ruffling response was not reduced (Fig. 2C, lower panel). The morphological evidence for the inhibition of the BMMC degranulation response mediated by Treg through the OX40–OX40L axis were validated by the reduced amount of released β-hexosaminidase (Fig. 2D). The same effect was also observed

using PMCs (Supporting Information Fig. S2). Together, these results provide the first morphological evidence for the find more role of the OX40–OX40L axis in the Treg-mediated inhibition of MC degranulation, but the evidence Enzalutamide concentration of conjugates between MC and OX40-deficient Tregs does not exclude the involvement of other receptor–ligand counterparts in the MC–Treg connections. During synapse formation, changes in cell shape and cytoskeleton rearrangement modulate Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels, thus contributing to sustained Ca2+ signals 22. Indeed, impaired Ca2+ signals were detected in cells whose morphology did not change during cell–cell interactions 22. We have previously demonstrated that, in a co-culture system, Tregs inhibit an intracellular ((Ca2+)i) rise in activated MCs, by preventing extracellular Ca2+ influx without modifying Ca2+ mobilization from intracellular stores 4. To evaluate whether the contact between a single Treg and an MC is sufficient to inhibit extracellular Ca2+ influx, fluorescence time-lapse microscopy experiments were conducted to monitor cytoplasmic Ca2+ in the single cells. IgE-presensitized BMMCs were loaded with the Ca2+ dye Fura2 acetoxymethyl ester (Fura2-AM)

and incubated with Tregs. The cells were allowed to establish physical connection before Ag addition. Differential interference contrast (DIC) images were used to follow MC–T cell interactions over time, and the ratio of Fura2 emission upon excitation at 340 and 380 nm was used to determine the intracellular levels of cytosolic-free this website Ca2+. Upon Ag triggering, a sustained rise in cytoplasmic Ca2+ was observed in BMMCs not interacting with Tregs (Fig. 3A), which was still elevated 5 min (86.6±3.0% of the peak value) and 10 min (86.0±6.1%) after Ag stimulation (Fig. 3B). In contrast, in BMMCs forming conjugates with Tregs, while the initial response was indistinguishable from BMMCs alone (Fig. 3A), intracellular Ca2+ decreased to 24.5±4.1% of the peak amplitude after 5 min and returned to pre-stimulation values at 10 min (1±0.55% of the peak amplitude) (Fig. 3B).