*Purpose: Regulatory T cell (Treg) suppressor function and allograft protection depend on maintenance of transcription factor Foxp3 expression and lymphatic migration, respectively. Treg lymphotoxin (LT) αβ signals LTβ receptor (LTβR) on lymphatic endothelial cells (LEC). We tested the hypothesis that LTαβ-LTβR signaling specifically regulates Treg stability, migration, and suppressor function.

*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 and flow cytometry.

*Results: Treg maturation and execution of suppressor function depends on sequential migration from the target tissue to the draining lymph nodes (dLN). Treg LTα1β2 stimulates LTβR on LEC during afferent lymphatic migration. 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. In a transwell based Treg-LEC co-culture assay, the non-migrating Tregs became exTregs and were accompanied by increased methylation at the Foxp3 locus and decreased suppressor function. An LTβR decoy peptide blocking classical, but not non-classical LTβR NFκB signaling, prevented conversion to exTregs. Since LEC LTβR stimulates the NFκB classical pathway to increase IL-6 secretion, both neutralization of IL-6 with antibody or decoy peptide blockade of classical LTβR NFκB inhibited IL-6 and prevented conversion of Treg to exTreg. Role of classical LTβR NFκB was confirmed with human Tregs migrating across human LEC.

*Conclusions: Treg LTαb and LEC LTβR interactions are important for Treg migration, stability, and suppressor function. Disruption of the interactions results in poor migration, increased retention in the 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 IL-6 or by blocking classical LTβR NFκB.

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