*Purpose: Sequential migration of regulatory T cells (Treg) from graft to afferent lymphatics and lymph nodes (LN) is needed for Treg suppressive function. Migration requires Treg lymphotoxin alpha (LTα) to stimulate LT beta-receptor (LTβR) in lymphatic endothelial cells (LEC). We used three genetically distinct mouse models to test the hypothesis that Treg engagement of LTβR is required for allograft protection.

*Methods: Treg engagement of LTβR was tested in germline deficient LTβR^-/- and LTα^-/- mice. 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 depleted in LEC after tamoxifen treatment. Treg function was analyzed by flow cytometry and histology in the islet transplant model.

*Results: In vitro, Treg LTα stimulated LTβR and regulated expression and secretion of LEC CCL21, a chemokine important for T cell migration to lymphatic vessels and LN. In KO^fl mice, tamoxifen treatment reduced LTβR expression only in LEC, while fibroblastic reticular cells and blood vessel endothelial cells maintained expression. Depletion of LTβR in the LEC of the LN led to a marked reduction in the expression of CCL21, non-canonical NFκB kinase (NIK), chemotactic lipid sphingosine-1-phosphate (S1P) and accumulation of Foxp3+ Tregs in T cell zones. 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 model of tissue to draining LN (dLN) lymphatic migration, Tregs migrated less well in the KO^fl mice, while Treg entry from blood into LN was not inhibited. In the islet allograft model, when Tregs were transferred locally with the islets, allograft survival was reduced from 25d to 13d in KO^fl recipient mice (p<.03), to 15d in germline LTβR^-/- (p<.03) recipient mice, 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 lost Foxp3 and CD25 expression to become exTreg.

*Conclusions: Using three different genetic mouse models, Treg-LTα-LTβR-LEC interactions were characterized. Disruption of these interactions inhibits Treg accumulation and migration to LN, execution of Treg suppressive function, and conferred significant disadvantage for islet allograft protection.

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