Background: The selective recruitment of regulatory T cells (Treg) or blockade of memory/effector T cell migration to the graft and draining lymph nodes (dLN) are crucial for graft survival. We previously demonstrated that Treg migrate to dLN to prolong graft survival, and Treg preferentially use LTαβ to engage LTβR on LEC to migrate from the graft to dLN. We hypothesized that LTαβ -LTβR mediated migration differentially engages classical NFκB and non-classical NIK signaling. Methods: Murine primary LEC and the SVEC4-10 line were used in biochemical, phenotypic, and functional analyses of LTβR signaling. Murine naïve, activated, and regulatory CD4 T cells were isolated for migration across LEC in vitro and in vivo to assess the role of LTβR signaling. Results: LEC expressed high levels of LTβR and had low level constitutive activation of LTβR, as shown by constant binding of TNF receptor-associated factor 3 (TRAF3) to the LTβR signaling complex. Stimulation of LTβR on LEC resulted in full receptor activation, with TRAF2 and TRAF3 binding to the receptor complex; and preferentially induced non-classical NFkB activation of the NIK pathway, shown by NIK accumulation, p100 processing to p52, and absence of rapid IKKα/β phosphorylation and IκBα degradation. LTβR-induced NIK accumulation was further enhanced by inhibiting cIAP, an enzyme that degrades NIK. LTβR stimulation resulted in the early up-regulation of the inflammatory molecules VCAM-1 and CCL2, mediated by classical NFκB signaling; followed by a later increase in homeostatic lymphocyte homing chemokines CCL19 and CCL21, mediated by non-classical NIK signaling. Interruption of NIK pathway by blocking TRAF3, but not TRAF2, binding to LTβR prevented T cell migration across LEC in vitro and in vivo. In contrast, blocking the classical LTβR-TRAF2/3-NFkB pathway promoted T cell transmigration by increasing CCL21 and CXCL12 secretion and decreasing VCAM-1 expression in LEC. Conclusions: LTβR is highly expressed on LEC and is constitutively activated. LTβR preferentially engages non-classical NIK signaling over the classical NFκB pathway. LTβR-TRAF3-NIK signaling regulates homeostatic chemokine production and migration of T cells. Blockade of NIK signaling alters T cell afferent lymphatic migration. These novel pathways regulate lymphatic function and the ability of effector and suppressor T cells to respond to inflammation. The molecular entities uncovered here define new targets for immune modulation in immunity and tolerance.

CITATION INFORMATION: Piao W, Brinkman C, Bromberg J. Lymphotoxin (LT) β Receptor-Mediated Non-Classical NFκB Signaling in Lymphatic Endothelial Eells (LEC) is Required for Homeostatic T Cell Migration. Am J Transplant. 2017;17 (suppl 3).

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