*Purpose: Little is known about the molecular cues that regulate the entry of leukocytes across lymphatic vessels. Regulatory T cells (Treg) express high amounts of lymphotoxin (LT), while lymphatic endothelial cells (LEC) express high levels of the LT beta-receptor (LTβR), which signals through classical and non-classical (NIK) NFκB pathways. We tested the hypothesis that Treg-LEC interactions stimulate LTβR signaling, differentially engage receptor associated NFκB signaling pathways, and influence the migration and immunity of other immune cells.

*Methods: Murine primary LEC were used in biochemical, phenotypic, and functional analyses of LTβR signaling. Murine CD4 or CD8 T cells, B cells, neutrophils, macrophages, and Treg were isolated from wild type (WT) or LTα^-/- GFP-Foxp3 reporter mice, and migrated across wild type or LTβR^-/- LEC in vitro and lymphatic vessels in vivo. LTβR signaling was assessed with western blots and immunohistochemistry. BALB/c islets were transplanted to C57BL/6 recipients.

*Results: Homeostatic patrolling Treg express high levels of surface LTαβ, which is further increased by activation during inflammation. LTβR is highly expressed on LEC and lymphatic vessels, and migrating Treg engage the LTβR and rapidly activate classical NFκB pathway to induce LEC expression of the migration related molecule VCAM-1. Treg also preferentially activate non-classical NFκB-NIK signaling to induce CCL21 and CXCL12, and to down regulate the cell junction molecules VE-cadherin and occludin. The Treg-LEC interaction promotes subsequent lymphatic transendothelial migration (TEM) of other immune cells, including CD4 and CD8 T cells, B cells, neutrophils, macrophages, and dendritic cells. Treg regulated TEM is LT dependent, so that LTα^-/- Treg and LTβR^-/- LEC failed to regulate immune cell migration. WT but not LTα^-/- Treg co-transplanted with islets prolonged graft survival (median survival, 25 vs 15 days, p<.045). There were fewer T cells, B cells, and DC infiltrating the graft after co-transplantation with WT compared to LTα^-/- Treg, suggesting LT-dependent Treg mobilization of inflammatory cell egress from the inflamed graft.

*Conclusions: In addition to the role of immune suppression, Treg condition LEC for permissiveness for TEM. In inflammation, Treg are dispatched to the inflamed tissue and mobilize immune cell TEM to allow efficient resolution of inflammation and decreased local tissue destruction. The Treg-LEC crosstalk uncovers new immune functions of Treg as modulators in immunity and tolerance.

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