Background:Selective recruitment of regulatory T cells (Treg) to draining lymph nodes is crucial for graft survival, and LTαβ-LTβR signaling is required to direct Treg into afferent lymphatics. Activated LTβR induces both classical NFκB and alternative NIK pathway dependent inflammatory and migration responses, but blocking the entire LTαβ-LTβR axis does not effectively regulate immunity and inflammation. We selectively targeted each arm of the NFκB pathway to reveal molecular mechanisms of T cell migration, and aimed to develop therapeutic peptides to promote graft tolerance. Methods: Two LTβR-derived cell-permeable decoy peptides, LTA and LTB, comprised of the N-terminal cell-penetrating sequence of the Drosophila antennapedia peptide were designed and evaluated. LTA was constructed to block only the alternative NIK pathway, while LTB blocked only the classical NF-κB pathway. Murine lymphatic endothelial cells (LEC) were used in biochemical and functional analyses. Naïve, activated, and regulatory CD4 T cells were isolated for migration across LEC in vitro and in vivo to assess the role of LTbR signaling. Results: LTA sequestered TNF receptor-associated factor 3 (TRAF3) but not TRAF2 from the LTbR complex, while LTB prevented both TRAF3 and TRAF2 binding to the receptor complex in LTβR-activated LEC. LTA inhibited LTβR/NIK-mediated p100 processing to p52, and suppressed expression of the homeostatic chemokines CXCL12, CCL19, and CCL21 in LEC. In contrast, LTB inhibited NFκB-mediated IκBα phosphorylation and degradation, and inhibited expression of the inflammatory molecules CCL2 and the VCAM-1. The peptide-mediated effects were highly specific, as TNFR1-mediated NFκB signaling was unaltered by LTA or LTB. LTA prevented CD4 T cell transmigration across the LEC, while suppressing CCL21 and CXCL12 secretion and β-catenin expression, and transiently promoting VCAM-1 expression in LEC. In contrast, LTB increased CCL21 and CXCL12 secretion in LEC, and promoted T cell transmigration. Conclusions: Selective targeting of LTβR-mediated non-classical NIK signaling in LEC efficiently prevented T cell migration across LEC, suggesting the essential role of LTβR-TRAF3-NIK-p100/p52 pathway in LEC during T cell migration. LTA is a potent LTβR-TRAF3-NIK signal blocking agent and migration inhibitor. LTB specifically targets LTβR-mediated TRAF2-classical NFκB signaling, and promotes Treg transmigration across LEC. These peptides may act as novel therapeutic interventions to promote allograft survival.

CITATION INFORMATION: Piao W, Xiong Y, Brinkman C, Bromberg J. Regulation of T Cell Migration by Specifically Targeting Lymphotoxin β Receptor (LTβR)-Mediated Non-Canonical NFkB Signaling. Am J Transplant. 2017;17 (suppl 3).

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