*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 tested the hypothesis that LTβR drives LN structure and function to enhance Treg migration and suppressive function in allograft survival.

*Methods: LTβR^fl/fl mice were crossed with Prox1-Cre-ER^T2 mice to generate Prox1-Cre-ER^T2+/-LTβR^fl/fl(KO) mice, in which LTβR is conditionally depleted in LEC by tamoxifen treatment. The effects of LTβR depletion on Treg lymphatic migration and LN structures were analyzed by flow cytometry, immunohistochemistry, and in vivo migration assays.

*Results: In Cre-Lox mice, LTβR expression by LEC was markedly reduced by 10 days after tamoxifen treatment, while blood vessels and fibroblastic reticular cells (FRC) maintained expression. Depletion of LTβR in the LN led to a marked reduction in Foxp3+ Treg in T cell zones, and decreased CXCL12 and CCL21 expression, chemokines important for T cell migration to LV and LN. Treg from KO mice expressed lower amounts of T-bet mRNA, a regulator of Treg migration. Indeed, Treg migrated poorly from tissues to LNs in KO mice in vivo, while entry from blood into LN was not altered. LTβR depletion reduced LEC expression of non-canonical NFκB kinase (NIK) and the inflammatory and chemotactic lipid sphingosine-1-phosphate (S1P), while expression of VCAM-1, ICAM-1, and CCL19 were not affected. Many other aspects of the LN remained normal: overall architecture, cortical and medullary organization, B and T cell zone segregation, dendritic cell distribution, and structure of the FRC stromal network; and LTβR depletion did not affect composition of stromal cells, leukocytes, or innate lymphoid cells in primary or secondary lymphoid organs, suggesting normal immune system homeostasis. In mice transplanted with islets mixed with nTreg, allograft survival was reduced from 23 to 14 days (p<0.03) in the KO.

*Conclusions: LTβR depletion from LEC induced specific immune defects, including a significant reduction in the expression of CCL21, S1P, and NIK, which are important in Treg migration. It led to poor Treg migration and reduced accumulation of Foxp3+ Treg in the LN. Reduced LN Treg correlated with worse islet allograft survival. This conditional depletion models identifies LTβR as a key regulatory of Treg migration and function.

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