*Purpose: Immune suppressive therapy can be complicated by malignancy. Regulatory T cells (Treg) control immune tolerance for graft survival but cause to immune suppression in the tumor microenvironment. Treg express high levels of lymphotoxin (LT), while most tumor cells express high levels of the LT beta-receptor (LTβR), which signals through classical p65 and non-classical (NIK) NFκB pathways. We tested the hypothesis that direct Treg-cancer cell interactions stimulate LTβR signaling, differentially engage receptor associated NFκB signaling pathways, and influence the growth and metastasis of cancer cells.

*Methods: The murine melanoma cell line B16F10-eGFP and murine dermal lymphatic endothelial cell (LEC) were used in biochemical, phenotypic, and functional analyses of LTβR signaling and migration assays. Murine CD4 T cell or Treg 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: Treg express high levels of LTα1β2, while LTβR is highly expressed on B16-F10 cancer cells and LEC. LTβR classical NFκB signaling is constitutively activated in B16-F10, leading to higher expression of VCAM-1, CCR10, and CCL2 which were suppressed by LTβR-NFκB-p65 blocking peptide. LTβR non-classical NFκB-NIK signaling is also constitutively activated, leading to expression of CCL21, CCL27, and CXCL12 which were suppressed by LTβR-NFκB-NIK blocking peptide. Cancer cells chemotactically migrate across LEC, and migration is prevented by inhibiting cancer cell NFκB-NIK signaling. Treg LTα1β2 can further stimulate cancer cell LTβR to promote cancer cell migration. Treg regulated cancer cell migration is LT dependent, so that LTα^-/- Treg failed to stimulate cancer cell migration. Treg LTα1β2 also stimulated LTβR on LEC, which conditioned the LEC to further promote cancer cell TEM, by down regulating the cell junction molecule VE-cadherin. 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 both cancer cells and LEC to promote cancer cell transendothelial migration. LTβR-NFκB-signaling blocking peptides suppress cancer migration and chemokines to block cancer metastatic invasion. Thus, our study provides a rational strategy of modulating Treg activities to prevent tumor spread in transplant recipients.

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