*Purpose: Treg sequential migration from graft to draining lymph nodes (dLN) via afferent lymphatics is required for Treg suppressive function. Treg lymphotoxin alpha beta (LTαβ) stimulates LT beta-receptor (LTβR) on lymphatic endothelial cells (LEC) for migration. We tested the hypothesis that Treg engagement of LEC LTβR is required for allograft protection by using three genetically distinct mouse models.

*Methods: A conditional knockout (KO) of LTβR was made by crossing LTβR^fl/fl with Prox1-Cre-ER^T2, to generate KO^fl mice in which LTβR is deleted in LEC after tamoxifen treatment. Treg engagement of LTβR was tested in germline deficient LTβR^-/- and LTα^-/- mice, and regulatable KO^fl mice. Treg function was analyzed by flow cytometry and histology in the islet transplant model.

*Results: In KO^fl mice, tamoxifen treatment reduced LTβR expression only in LEC, while fibroblastic reticular cells and blood vessel endothelial cells (BEV) maintained expression. LTβR deletion in LEC reduced expression of CCL21, non-canonical NFκB kinase (NIK), chemotactic lipid sphingosine-1-phosphate (S1P), and amount of Foxp3+ Tregs in LN T cell zones. LTβR deletion did not affect normal immune system homeostasis; and overall architecture and composition of stromal cells, leukocytes, and innate lymphoid cells in primary and secondary lymphoid organs were maintained. In an in vivo model of tissue to dLN lymphatic migration, Tregs migrated less well in the KO^fl mice, while Treg entry from blood vessel (lined with BEV) into LN was not inhibited. In the islet allograft model, when Tregs were transferred locally with islets, allograft survival was reduced from 25d to 13d in KO^fl recipients (p<.03), to 15d in germline LTβR^-/- recipients (p<.03), and to 13d in wild type mice receiving LTα^-/- Treg (p<.03). Treg migrated less well from islet allografts to the dLN in the KO^fl mice and in mice receiving LTα^-/- Treg. Non-migrating Tregs became Foxp3^loCD25^lo exTreg and correlated with inhibition of LT signaling.

*Conclusions: Treg LTαβ and LEC LTβR interactions, characterized in three different genetic mouse models, demonstrate that these interactions are required for the proper execution of in vivo Treg suppressive function. Lymphotoxin regulates Treg accumulation in the tissues and migration to the LN and determines Treg stability for islet allograft protection.

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