*Purpose: To promote transplant tolerance, T regulatory cells (Treg) must enter the afferent lymphatic vasculature by migrating across lymphatic endothelial cells (LECs), and then home to graft draining lymph nodes (LNs). Treg engage LECs using unique molecular pairings that facilitate migration, including lymphotoxin alpha beta (LTαβ): lymphotoxin beta receptor (LTβR) and PD-1:PD-L1 at the Treg:LEC interface. We hypothesized that Treg interactions with LEC at the membrane interface regulate the expression and distribution of molecules required for effective migration and induction of immune tolerance.

*Methods: Mouse primary LEC were grown to confluence, seeded onto transwell inserts, and used to assess molecular interactions required for effective migration. Purified wild type (WT) and LTα knock out (KO) CD4+ T cells and Treg were separately purified by flow sorting. Treg and CD4+ T cells were transmigrated across LEC-transwell inserts towards CCL19 over 3 hours. At the completion of migration, membranes with LEC layers were stained for LTβR, PD-1, and PD-L1 and receptor distribution measured and quantified by confocal microscopy.

*Results: There were no significant differences in expression of LTβR, PD-1, or PD-L1 on the apical or basal surface of LEC that were not exposed to Treg or CD4+ populations. However, VCAM-1 expression on the basal surface of LEC layers was significantly higher at baseline in comparison to the apical surface. Migration of WT Treg induced a specific increase in the expression of LTβR, PD-1, and PD-L1 at the Treg:LEC interface. Similarly, migration of WT Treg induced site-specific increases in VCAM-1 expression at the interface. These changes in expression did not occur following transmigration of LTα KO Treg, or CD4+ non-Treg cells.

*Conclusions: Sequential Treg migration from blood to allografts to draining LNs is a requirement for optimal transplant tolerance. Treg engage LEC using novel molecular interactions including LTβR: LTαβ and PD-1:PD-L1 to enter the afferent lymphatic vasculature. Engagement of Treg to the LEC surface leads to upregulation of LTβR, PD-1, PD-L1, and VCAM-1 at the Treg:LEC interface. Migration of Treg across LEC did not globally change expression of these molecules on either the apical or basal surface indicating that this process is highly specific at the interface junction. These results provide new insights into the molecular regulation of migration and Treg function.

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