Introduction: Lymphotoxin (LT) coordinates the development, organization and structure of lymphoid organs, and LTβreceptor (LTβR) is involved in thymocyte migration. However, LT has no described role in T cell migration in the periphery. We previously showed that LT was required for Treg to prolong islet allograft survival, and therefore examined the role of LT in T cell lymphatic migration and interactions with lymphatic endothelial cells (LEC) in vitro and in vivo. We found that LT controls important aspects of afferent but not efferent lymphatic migration for Treg, but not conventional CD4+ T cells (Tconv).

Methods: Treg and Tconv were generated using Foxp3GFP mice, and flow-sorted via CD25 and Foxp3GFP expression, or isolated from C57BL/6 (B6) or LTα deficient mice (LTα KO) using CD4 and CD25 beads. T cells were used in transmigration assays in vitro across primary LEC from resting B6 lymph nodes (LN). For in vivo assays, T cells were labeled with tracking dyes, injected into ear pinnae, and analyzed at various times by whole mount fluorescent immunohistochemistry, or intravital imaging. LTβR fusion protein (LTβRIg) was used to block Treg LTαβ interactions with LEC LTβR.

Results: Treg migration across primary LEC was specifically inhibited by blocking LTαβ-LTβR interactions. Treg interacted with protrusions on the basal surface of LEC monolayers, with co-localization of VCAM-1 and moesin in the LEC with the Treg. This interaction was inhibited when LTαβ-LTβR interactions were blocked with LTβRIg. When injected into ear pinnae, Treg migrated in close proximity to lymphatics, and migration was inhibited for Treg but not Tconv with LTβRIg or with using LTα KO Treg. Treg-LT interactions with LTβR in ears were also specifically associated with the presence of filopodia-like protrusions from lymphatic vessels.

Conclusions: Treg rely upon cell surface LTαβ to stimulate LTβR on lymphatic endothelium for interstitial and lymphatic migration. These interactions alter the distribution of adhesive and cytoskeletal adapter proteins in LEC, and are associated with dynamic changes in their morphology. This reveals a novel role for LTαβ-LTβR in regulating Treg lymphatic migration and interaction with lymphatic endothelium. This interaction is required for Treg to move from tissues to afferent lymphatics to draining LN and be fully suppressive. Thus, targeting this pathway may allow modulation of Treg migration to LN, either suppressing or enhancing immunity or tolerance.

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