*Purpose: Regulatory T cell (Treg) suppressor function for allograft survival requires maintenance of transcription factor Foxp3 and sequential migration from tissues to lymph nodes (LN). Treg function depends on their engagement with lymphotoxin beta receptor (LTβR) on lymphatic endothelial cells (LEC). We tested the hypothesis that Treg-LEC engagement is required for maintaining Foxp3 expression and Treg function.

*Methods: Mice with deletion of lymphotoxin alpha (LTα^-/-); LTβR^-/-; and Prox1-Cre-ER^T2+/-LTβR^fl/fl (KO^fl) mice, in which LTβR is depleted in LEC by tamoxifen treatment, were used. Treg stability, migration and function were analyzed in vivo in an islet allograft model, and in vitro in a transwell based LEC migration assay.

*Results: Treg use LTα1β2 to stimulate LTβR on LEC for migration to draining LN (dLN) via afferent lymphatics. Conditional or germline depletion of LTα in Treg or LTβR in LEC prevented Treg lymphatic migration from tissues to dLN. In an islet allograft model, depletion of LTβR on LEC, or deletion of LTα on Treg, led to Treg retention in the graft and poor migration to the dLN. Treg differentiation and execution of their suppressor function required sequential migration from the target tissue to the dLN. Modulation of LTβR on LEC or LTα on Treg resulted in downregulation of Foxp3 expression in Treg and generation of exTreg. Disruption of LTα1β2-LTβR interactions between Treg and LEC resulted in impaired allograft survival from 25d to 13d in KO^fl mice (p<.03), to 15d in LTβR^-/- mice (p<.03), and to 15d in mice receiving LTα^-/- Treg (p<.03). In a transwell based Treg-LEC co-culture assay, migrating Treg maintained high Foxp3 and CD25 expression. The non-migrating Treg became exTreg and lost Foxp3, CD25, CD39, GITR, and LTαβ expression while maintaining CD49b, CD73, and CTLA4 expression. Since LTβR stimulates the NFκB classical pathway to secrete IL-6, blockade of NFκB or neutralization of IL-6 reversed exTreg conversion. CD73 and CD39 catalyze adenosine production, and adenosine addition also reversed exTreg conversion.

*Conclusions: Treg-LEC LTα1β2-LTβR interactions are important for Treg migration, stability and suppressor function. Disruption of the interaction results in poor migration, increased retention in graft, loss of Foxp3 expression, and thereby their ability for allograft protection. The exTreg have the potential to become T effector cells and cause allograft rejection. ExTreg conversion can be prevented therapeutically.

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