*Purpose: Regulatory T cell (Treg) lymphatic migration and maintenance of transcription factor Foxp3 expression are required for suppressor function and allograft protection. Treg stimulate lymphotoxin beta receptor (LTβR) on lymphatic endothelial cells (LEC). We tested the hypothesis that Treg-LEC engagement is necessary for Treg migration, sustaining Foxp3 expression, and suppressor function.

*Methods: Treg stability, migration and function were analyzed in vivo in an islet allograft model, and in vitro in a transwell based LEC migration assay. Mice with deletion of lymphotoxin alpha (LTα^-/-), LTβR^-/-, and Prox1-Cre-ER^T2+/-LTβR^fl/fl (KO^fl) in which LTβR is depleted in LEC by tamoxifen treatment, were used.

*Results: Treg differentiation and execution of suppressor function required sequential migration from the target tissue to the draining LN. Treg use LTα1β2 to stimulate LTβR on LEC for migration to LN via afferent lymphatics. Disruption of LTα1β2-LTβR interactions between Treg and LEC 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 depletion 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 lost Foxp3 and CD25 expression to become exTreg. In a transwell based Treg-LEC co-culture assay, the non-migrating Treg became exTreg and lost Foxp3, CD25, CD39, GITR, and LTαβ expression while maintaining CD49b, CD73, and CTLA4 expression. Conversion to exTreg was accompanied by methylation at the Foxp3 locus and decreased suppressor function. Since LTβR stimulates the NFκB classical pathway to secrete IL-6, blockade of NFκB or neutralization of IL-6 prevented exTreg conversion. CD39 catalyzes adenosine production, and supplementation with adenosine also prevented exTreg conversion in an adenosine receptor 2A (A2AR) dependent manner.

*Conclusions: Treg-LEC LTα-LTβR interactions are important for Treg migration, stability and suppressor function. Disruption of the interactions 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 IL-6 or LTβR NFκB, or by stimulating A2AR.

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