*Purpose: Treg immune suppression which is critical for transplant graft survival may also enhance tumor growth and metastases. Treg lymphotoxin (LTα1β2) may directly engage the LT beta-receptor (LTβR), which most tumor cells express, and signal through classical p65 and non-classical (NIK) NFκB pathways. We tested the hypothesis that direct Tregs-cancer cell interactions stimulate LTβR NFκB signaling pathways and influence the migration of cancer cells.

*Methods: The murine melanoma cell line B16F10-eGFP and murine dermal lymphatic endothelial cells (LEC) were used in biochemical, phenotypic, and functional analyses of LTβR signaling and migration assays. Murine CD4 T cells or Tregs were isolated from wild type or LTα^-/- mice, and co-cultured with tumor cells or LEC. LTβR signaling was assessed with western blots and immunohistochemistry, and with highly specific LTβR-NFκB blocking peptides.

*Results: LTβR was highly expressed on B16-F10 and many other cancer cells. LTβR classical NFκB signaling was constitutively activated in B16-F10, leading to higher expression of VCAM-1, CCR5, CXCL10 and CCL2 which were enhanced by inflammatory cytokines and suppressed by LTβR-NFκB-p65 blocking peptide. LTβR non-classical NFκB-NIK signaling was also constitutively activated, leading to expression of CCL21, CCL27, and CXCL12 which were suppressed by LTβR-NFκB-NIK blocking peptide. Cancer cells chemotactically migrated across LEC, and migration was prevented by inhibiting cancer cell NFκB-NIK signaling. Treg LTα1β2 further stimulated cancer cell LTβR to enhance cancer cell migration. LTα^-/- Treg failed to stimulate cancer cell migration, so that Treg regulated cancer cell migration is LT dependent. Treg LTα1β2 also stimulated LTβR on LEC, which conditioned the LEC by down regulating the cell junction molecule VE-cadherin, and thus further promoted cancer cell migration. Co-inoculation of B16-F10 with inhibitors of NFκB signaling suppressed melanoma tumor growth and metastases in vivo.

*Conclusions: Treg LTα1β2 directly signals LTβR on cancer cells to promote cancer cell metastatic migration. Treg LTα1β2 directly signals LTβR on LEC to promote cancer cell transendothelial migration. LTβR-NFκB-signaling blocking peptides suppress cancer migration and chemokines to block cancer metastatic invasion. These observations provide a rational strategy to modulate Treg activities to prevent tumor spread in transplant recipients.

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