Regulatory T cells differentially undergo transmigration or prolonged intravascular patrolling at distinct phases of the inflammatory response.

Regulatory T cells (Tregs) play important roles in limiting inflammatory responses in the skin. During these responses, Treg abundance increases and interfering with their recruitment results in exacerbation of inflammation and increased endothelial activation. However, the mechanisms whereby Tregs enter the skin remain poorly understood. The aim of this study was to use intravital microscopy to investigate adhesion and transmigration of Tregs in the dermal microvasculature in a two-challenge model of contact sensitivity. Using intravital confocal microscopy of Foxp3-GFP mice, we visualised endogenous Tregs and assessed their interactions in the dermal microvasculature at different stages of the response. Four hours after hapten challenge, Tregs underwent adhesion with ~25% of these cells proceeding to transmigrate, a process dependent on CCR4. At 24 h, Tregs adhered but no longer underwent transmigration, instead remaining in prolonged contact with the endothelium, migrating over the endothelial surface. Fours after a second challenge, Treg transmigration was restored, although in this case transmigration was CCR4-independent. Notably, at 24 hours but not 4 hours after challenge, endothelial cells expressed MHCII. Moreover, inhibition of MHCII reduced Treg adhesion, demonstrating an unexpected role for MHCII in Treg attachment to the endothelium. Together these data show that Treg adhesion and transmigration can be driven by different molecular mechanisms at different stages of an antigen-driven inflammatory response. In addition, Tregs can undergo prolonged migration on the inflamed endothelium, potentially affording them the opportunity to modulate endothelial adhesive function. Animal work was approved by the Monash Medical Centre Animal Ethics Committee.