*Purpose: Regulatory T cell (Treg) suppressor function and allograft protection depend on maintenance of transcription factor Foxp3 expression and lymphatic migration. Lymphatic endothelial cell (LEC) lymphotoxin (LT) β receptor (LTβR) binds with Treg LTα1β2 to stimulate LEC responses that permit Treg migration. We tested the hypothesis that Treg LTαβ engagement of LEC LTβR regulates additional LEC functions that determine Treg stability.

*Methods: Treg stability, migration and function were analyzed in vitro in a transwell based LEC migration assay and in vivo in islet allograft and footpad migration models. Mice with deletion of LTα^-/-, LTβR^-/-, and Prox1-Cre-ER^T2+/-LTβR^fl/fl (KO^fl) in which LTβR is deleted in LEC by tamoxifen treatment, were used. Tregs were analyzed by immunofluorescence microscopy, ELISA and flow cytometry.

*Results: In a transwell based Treg-LEC co-culture assay, where Tregs transmigrate from the basal to the apical surface of LEC, the non-migrating Treg became Foxp3^loCD25^lo exTreg. ExTreg conversion was accompanied by methylation at the Foxp3 locus and decreased suppressor function. ELISA demonstrated preferential secretion of IL-6 by LEC on the basal (abluminal) surface. An LTβR decoy peptide blocking classical, but not non-classical, LTβR NFκB signaling prevented IL-6 secretion and conversion of Tregs to exTregs. Likewise, neutralization of IL-6 with antibody also prevented exTreg conversion. These findings were confirmed with human Tregs migrating across human LEC. The actions of decoy peptides and anti-IL-6 were confirmed in vivo in the footpad migration assay. Disruption of Treg-LEC interactions resulted in impaired islet allograft survival from 25d to 13d in KO^fl mice (p<.03), to 15d in LTβR^-/- mice (p<.03), and to 13d in mice receiving LTα^-/- Treg (p<.03). Conditional or germline deletion of LTβR on LEC, or deletion of LTα on Treg, led to Treg retention in the graft and poor migration from tissues to the dLN. Non-migrating Tregs were converted to Foxp3^loCD25^lo exTreg.

*Conclusions: Treg LTαβ and LEC LTβR interactions are important for Treg migration, stability, and suppressor function. Disruption of the interaction results in poor migration, increased retention in graft, loss of Foxp3 expression, and thereby the ability to protect the allograft. The exTreg have the potential to become T effector cells and cause allograft rejection. ExTreg conversion can be prevented therapeutically by inhibiting LEC IL-6 or LTβR NFκB.

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