*Purpose: Regulatory T cell (Treg) migration to afferent lymphatics and lymph nodes (LN), and suppressive function, require Treg lymphotoxin alpha (LTα) to stimulate LT beta-receptor (LTβR) in lymphatic endothelial cells (LEC). We tested the hypothesis that Treg engagement of LTβR is required for graft protection, using three genetically distinct mouse models.

*Methods: Treg engagement of LTβR was tested in germline LTβR^-/- and LTα^-/- mice. A conditional knockout of LTβR was made by crossing LTβR^fl/fl with Prox1-Cre-ER^T2, to generate Prox1-Cre-ER^T2+/-LTβR^fl/fl (KO^fl) mice in which LTβR is depleted in LEC by tamoxifen treatment. Treg function was analyzed by flow cytometry and histology in the islet transplant model.

*Results: In the islet allograft model, when Treg were transferred locally with the islets, allograft survival was reduced from 25d to 13d in KO^fl recipient mice (p<.03), to 15d in germline LTβR^-/- (p<.03) recipient mice, and to 15d in wild type mice receiving LTα^-/- Treg (p<.03). In an in vivo model of tissue to draining LN (dLN) lymphatic migration, Treg migrated less well in the KO^fl mice, while Treg entry from blood into LN was not inhibited by lymphatic LTβR depletion. Treg migrated less well from islet allografts to the dLN in the KO^fl mice and in mice receiving LTα^-/- Treg. In KO^fl mice, tamoxifen treatment reduced LTβR expression by LEC only, while blood vessel endothelial cells and fibroblastic reticular cells maintained expression. LTβR depletion reduced LEC expression of the chemotactic lipid sphingosine-1-phosphate and non-canonical NFκB kinase. Depletion of LTβR in the LEC of the LN also led to a marked reduction in the accumulation of Foxp3+ Treg in T cell zones and reduced CCL21 expression, a chemokine important for T cell migration to lymphatic vessels and LN. LTβR depletion did not affect overall LN architecture or composition of stromal cells, leukocytes, or innate lymphoid cells in primary or secondary lymphoid organs, suggesting normal immune system homeostasis. In vitro stimulation showed that Treg LTα directly regulated LEC CCL21 expression and secretion by stimulating LTβR.

*Conclusions: Using three different genetic mouse models, Treg-LTα-LTβR-LEC interactions were characterized. Disruption of these interactions inhibits Treg accumulation and migration to LN, execution of Treg suppressive function, and conferred significant disadvantage for islet allograft protection.

« Back to 2020 American Transplant Congress