Introduction: Lymphotoxin (LT) directs lymphoid organ structure, and LTβ receptor (LTβR) is involved in leukocyte migration into lymph nodes (LN) and thymocyte migration. We previously showed LT was required for murine Treg to prolong islet allograft survival, and for Treg migration from tissues into afferent lymphatics by regulating interactions with lymphatic endothelial cells (LEC). We hypothesized that human Treg would use LT to regulate their interaction and migration through human LEC.

Methods: Murine Treg were generated from Foxp3GFP mice, and flow-sorted for CD25 and Foxp3GFP. Naïve human T cells were flow-sorted from peripheral blood, and effectors and induced Treg (iTreg) were stimulated with anti-CD3, IL-2 and antigen presenting cells (APCs). iTreg received TGFβ1 and rapamycin. Thymus-derived tTreg were isolated from umbilical cord blood and expanded with anti-CD3, IL-2 and APCs. T cells were used in transmigration assays across primary mouse and human skin LEC and the murine LEC line SVEC4-10. For footpad assays tTreg were labeled with CFSE, injected into footpads, and analyzed in draining popliteal LN 12h later. LTβR fusion protein (LTβRIg) was used to block Treg LTαβ.

Results: Consistent with our previous results using mouse T cells and LEC, human Treg but not non-Treg CD4 T cell migration across human skin LEC was inhibited by LT blockade. We extended our mechanistic understanding of the action of LT. Murine Treg migration across SVEC4-10 and primary mouse LEC layers was specifically inhibited by blocking LTαβ-LTβR interactions, blocking VCAM-1, or inhibiting the non-canonical NFκB pathway. Migration of other T cell subsets was not inhibited by any of these agents. Inhibitory effects of these treatments were not additive, suggesting they were part of the same pathway. Inhibitory effects were further enhanced by simulating lymphatic fluid flow across the LEC. Finally, LT and VCAM-1 blockade also inhibited Treg but not non-Treg CD4+ T cell migration in vivo from footpad to draining LN. .

Conclusions: Both human and murine Treg but not non-Treg rely on cell surface LTαβ to stimulate LTβR on lymphatic endothelium for lymphatic migration. This mechanism involves VCAM-1 and the non-canonical NFκB pathway and operates under both static conditions and fluid flow. As lymphatic flow is modulated during inflammation this may also result in different sensitivity to this pathway depending upon the phase of the inflammatory response.

CITATION INFORMATION: Brinkman C, Hippen K, Blazar B, Bromberg J. Human and Mouse Treg Use Lymphotoxin for Migration Across Lymphatic Endothelium. Am J Transplant. 2016;16 (suppl 3).

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