*Purpose: Lymphotoxin (LT) α1β2 high expressing Tregs are essential for immune tolerance although inhibiting antitumor immunity. Lymphatic endothelial cells (LEC) express the LT beta-receptor (LTβR), which is ligated by LTα1β2 and signals through classical RelA/p50 and non-classical RelB/p52 NFκB pathways to regulate Treg migration. Most tumor cells also express LTβR. There are conflicting reports on the role of LTβR signaling in tumor growth, with both positive and negative effects noted. We hypothesize that LTα1β2 on Tregs directly binds both tumor cells and LEC, stimulates LTβR NFκB signaling in both, and influences tumor migration and metastasis.

*Methods: The B16F10-Flu/GFP melanoma and dermal LEC were used in biochemical, phenotypic, and functional analyses of LTβR signaling and migration assays. Mouse CD4 T cells or Tregs were isolated from wild type (WT) or LTα^-/- Foxp3-GFP Tg mice. LTβR signaling was evaluated with highly specific LTβR-NFκB blocking peptides. WT or LTβR^-/- C57/BL6J mice were used for melanoma growth and mortality assays.

*Results: LTβR was highly expressed on B16-F10 melanoma and many other cancer cells. LTβR classical NFκB signaling was constitutively activated in B16-F10 and was inhibited by both LTβR-specific classical (nciLT) and non-classical (nciLT) NFκB blocking peptides. Non-classical NFκB signaling was also constitutively active, was enhanced by LTβR agonistic mAb, and was blocked by nciLT. LTα1β2 high expressing WT but not LTα^-/- Treg stimulated RelB/p52 NFκB signaling in B16F10. Blocking NFκB signaling in B16F10 with nciLT or ciLT inhibited B16F10 chemotactic transendothelial migration (TEM) across LEC. Tregs also stimulated LTβR^high LECs, causing down regulation of VE-cadherin and increasing chemokine expression to promote melanoma TEM. Host LTβR-deficiency did not affect melanoma growth but reduced mortality, suggesting a defect in metastasis. Both ciLT and nciLT further enhanced survival of melanoma-bearing mice.

*Conclusions: Treg LTα1β2 stimulates both tumor cells and LEC to enhance tumor migration. Treg ligates LTβR on B16F10 to enhance non-classical RelB/p52 NFκB signaling to promote metastatic migration. Treg also ligates LTβR on LEC and licenses the LEC to promote B16F10 TEM. Blocking both arms of LTβR-NFκB-signaling suppresses B16F10 melanoma lymphatic TEM and enhances survival of melanoma-bearing mice. Our observations provide a rational strategy to modulate Treg activities to prevent tumor spread in transplant recipients.

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