Background: Treg migration to the graft and draining lymph nodes is crucial for graft survival. Little is known about the molecular cues that regulate the entry of leukocytes across lymphatic vessels. Treg express high amounts of lymphotoxin (LT), while lymphatic endothelial cells (LEC) express high levels of the LT receptor (LTβR). LTβR signals through classical and non-classical (NIK) NFκB pathways. We hypothesized that Treg-LEC interactions stimulate LTβR signaling, differentially engage receptor associated NFκB signaling pathways, and influence the migration and immunity of other T cells.

Methods: Murine primary LEC and LEC lines were used in biochemical, phenotypic, and functional analyses of LTβR signaling. Murine naïve, activated, and regulatory CD4 T cells were isolated and migrated across LEC in vitro and in vivo. LTβR signaling was assessed with western blots, gene activation assays, and specific pharmacologic and genetic blockade.

Results: LTβR is highly expressed on LEC and lymphatic vessels. LTβR activation rapidly induces classical NFκB signaling with upregulation of the inflammatory molecules VCAM-1 and CCL2. However, LTβR preferentially engages the non-classical NIK pathway to drive homeostatic chemokines CCL21 and CXCL12 production. NIK, the hallmark of non-classical NFκB signaling, is constitutively active in resting LEC. LTβR activation further enhances NIK activity. Treg, bearing high levels of the LT ligand, are specifically poised to activate the LTβR and NIK signaling. A decoy receptor peptide that specifically blocks NIK signaling enhances VCAM-1 and integrin β4 clustering on the LEC cell surface, causing firm adhesion of T cells, thereby preventing in vitro and in vivo T cell migration.In contrast, blocking classical NFκB signaling promotes T cell migration by increasing LEC expression of CCL21 and CXCL12.

Conclusions: The LTβR on LEC is constitutively activate and engages the non-classical NIK pathway to sustain homeostatic immune cell migration. LTβR signaling preferentially engages non-classical NIK signaling over the classical NFκB pathway, and is preferentially engaged by Treg to influence homeostatic chemokine production and immune cell migration. Blocking NIK signaling inhibits leukocyte lymphatic migration, while blocking the classical arm promotes migration. The interplay of the two pathways regulates lymphatic function and the ability of effector and suppressor cells to respond to inflammation. The molecular entities uncovered here define new targets for immune modulation in immunity and tolerance.

CITATION INFORMATION: Piao W., Xiong Y., Li L., Saxena V., Bromberg J. Treg Regulate Distinct Lymphatic NFκB Signaling Pathways to Alter Migration and Immunity Am J Transplant. 2017;17 (suppl 3).

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