Introduction: To induce tolerance, regulatory T cells (Treg) migrate from tissues via afferent lymphatics to draining lymph nodes (dLN). To do so, Treg surface lymphotoxin alpha/beta (LTαβ) stimulates the lymphotoxin beta-receptor (LTβR) on lymphatic endothelial cells (LEC). We hypothesized that Treg engagement of LTβR is required to maintain Foxp3 expression and Treg function for allograft protection.
Materials and Methods: Prox1-Cre-ERT2 mice crossed with LTβR^fl/fl mice generated knockout (KO^fl) mice, which lacked LTβR expression in LEC after tamoxifen treatment. Littermate wild type (WT^fl) were used as controls. Germline LTα KO and LTβR KO were also used. Using flow cytometry, immunohistochemistry and in vivo and transwell based in vitro migration assays, the effects of LTβR depletion on Treg lymphatic migration and stability were analyzed.
Results: Treg LTαβ stimulated LTβR on LEC for migration to dLN. Conditional or germline deletion of LTα in Treg or LTβR in LEC prevented Treg migration from tissues to dLN. In an islet allograft model, deletion of LTβR on LEC, or LTα on Treg, led to Treg retention in the graft and poor migration to dLN. Treg differentiation and execution of suppressor function required sequential migration from the islets to the dLN. Disruption of LTαβ-LTβR interactions resulted in impaired 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<.001). Modulation of LTβR on LEC or LTα on Treg resulted in downregulation of Foxp3 expression and generation of exTreg in vivo. In a transwell based in vitro model of Treg transendothelial migration across polarized LEC, non-migrated Treg became exTreg. Conversion to exTreg required direct interaction with the basal surface of LEC. ExTreg conversion was accompanied by hypermethylation at the Foxp3 locus. ExTreg conversion relied on canonical NFkB signaling via LTβR in LEC, causing increased IL-6 production by the basal surface of LEC, and the IL-6 then induced exTreg conversion. Furthermore, migrating Treg that resisted exTreg conversion had increased expression of the ectonucleotidase CD39. Blocking CD39 or the related ectonucleotidase CD73 inhibited migration and enhance exTreg conversion, while adenosine supplementation prevented exTreg conversion and enhanced migration.
Conclusion: 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 and correlated with poor islet allograft survival. Treg LTαβ - LEC LTβR signaling is important for Treg migration, suppressive function, and maintenance of Foxp3 stability. This interaction defines LEC expressed IL-6 as a detrimental factor, while Treg expressed ectonucleotidases as protective factors for Treg stability and function. These molecular interactions can be targeted for therapeutic manipulation of immunity and suppression.