*Purpose: Regulatory T cells (Treg) must migrate from tissues via lymphatic vessels (LV) to the draining lymph node (dLN) to induce tolerance. During migration, Treg lymphotoxin alpha (LTα) stimulates lymphotoxin beta-receptor (LTβR) signaling on lymphatic endothelial cells (LEC) lining the LV and dLN. We hypothesized that LEC LTβR regulates Treg migration and suppression by modulating LN structure and function.

*Methods: LTβR^fl/fl mice were crossed with Prox1-Cre-ER^T2 mice to generate knock out (KO) mice, which lack LTβR expression in LEC after tamoxifen treatment. Littermate Prox1-Cre-ER^T2-/-LTβR^fl/fl wild type (WT) were used as controls. Using flow cytometry, immunohistochemistry and in vivo and transwell based in vitro migration assays, the effects of LTβR depletion on Treg lymphatic migration and LN structures were analyzed.

*Results: In KO 10 days after tamoxifen treatment, LTβR expression was markedly reduced specifically on LEC, yet maintained in fibroblastic reticular cells and blood vessel endothelial cells. This selective inhibition resulted in poor Treg migration in KO mice from tissues to draining LN. These effects were confirmed in an islet allograft model, where Treg transferred into the islet graft migrated poorly to the draining LN in KO mice, resulting in reduced allograft survival from 25d to 13d (p<.03). Migrating Treg maintained high Foxp3 expression, while non-migrating Treg lost Foxp3 and CD25 expression to become exTreg. LTβR depletion did not affect LN architecture, but reduced accumulation of Foxp3+ cells in LN cortical ridge, the LN microdomain important for Treg induction. LTβR depletion modulated expression of selected chemokines important for T cell migration. It decreased expression of CXCL12 and CCL21, while expression of VCAM-1, ICAM-1, and CCL19 remained similar to WT. LTβR depletion also reduced expression of non-canonical NFκB kinase (NIK) and the chemotactic lipid sphingosine-1-phosphate (S1P) in LEC. In a transwell based Treg-LEC co-culture assay, non-migrating Treg became exTreg due to both non-canonical NIK and canonical NFκB LTβR signaling in LEC.

*Conclusions: LTβR depletion from LEC inhibited Treg migration from tissues to LN and reduced accumulation of Foxp3+ Treg in the LN. Non-migrating Treg became exTreg via NFκB LTβR signaling in LEC and correlated with poor islet allograft survival. LTβR is identified as a key regulator of Treg migration and subsequently suppressive function for ensuring graft survival.

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