*Purpose: Lymphotoxin beta receptor (LTβR) mediated signaling in lymphatic endothelium cells (LEC) is important for migration of regulatory T cells (Treg) to afferent lymphatic vessels (LV) and lymph nodes (LN), and for Treg suppressive function for allograft survival. We developed an in vivo conditional, regulatable knockout of the LTβR to test the hypothesis that Treg-LTβR engagement is required for allograft protection.

*Methods: LTβR^fl/fl mice were crossed with Prox1-Cre-ER^T2 transgenic mice to generate Prox1-Cre-ER^T2+/-LTβR^fl/fl (KO^fl) mice, in which LTβR is depleted in LEC by tamoxifen treatment. The effects of LTβR depletion on Treg lymphatic migration and migration related molecules were analyzed by flow cytometry and histology in an islet transplant model and in vivo migration assay.

*Results: In Cre-Lox mice, LTβR expression by LEC was markedly reduced by 10 days after tamoxifen treatment, while blood vessel endothelial cells and fibroblastic reticular cells maintained expression. LTβR depletion reduced LEC expression of non-canonical NFκB kinase (NIK) and the inflammatory and chemotactic lipid sphingosine-1-phosphate (S1P). Depletion of LTβR in the LN also led to a marked reduction in the accumulation of Foxp3+ Treg in T cell zones and CCL21 expression, a chemokine important for T cell migration to LV and LN. LTβR depletion did not affect overall LN architecture or composition of stromal cells, leukocytes, or innate lymphoid cells in primary or secondary lymphoid organs, suggesting normal immune system homeostasis. In an in vivo lymphatic migration model of tissue to draining LN (dLN), Treg migrated less well in the KO^fl mice, while Treg entry from blood into LN was not regulated by LTβR depletion. In vitro stimulation showed that both LTβR and Treg directly regulated CCL21 expression and secretion by LEC, confirming that Treg-LTβR-LEC interactions are specific and physiologically important. In the islet allograft model, Treg adoptively transferred locally with the graft migrated less well to the dLN in the KO^fl mice, and allograft survival was reduced from 23d to 14d in KO^fl mice (p<.03) and to 13d in germline LTβR^-/-(p<.03) mice.

*Conclusions: Depletion of LTβR from LEC induced specific immune defects, with a significant impact on the accumulation and migration of Foxp3 expressing Treg in the LN. It conferred significant disadvantage to Treg for islet allograft protection. This study shows that Treg-LTβR engagement is important for Treg to execute their suppressive function.

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