Background:Lymphotoxin beta receptor (LTβR) mediated signaling in lymphatic endothelium cells (LEC) is important for migration of regulatory T cells (Treg) to afferent lymphatic vessels (LV) and lymph nodes (LN), and for Treg suppressive function for allograft survival. To study Treg-LTβR engagement, we developed and characterized an in vivo conditional, regulatable knockout of the LTβR.

Methods:LTβR^fl/fl mice were crossed with Prox1-Cre-ER^T2 transgenic mice to generate Prox1-Cre-ER^T2+/-LTβR^fl/fl mice, in which LTβR is depleted in LEC by tamoxifen treatment. The effects of LTβR depletion on Treg lymphatic migration and migration related molecules were analyzed by flow cytometry and histology.

Results: In Cre-Lox mice, LTβR expression by LEC was markedly reduced by 10 days after tamoxifen treatment, while blood vessel endothelial cells and fibroblastic reticular cells (FRC) maintained expression. LTβR depletion did not affect stromal cell or leukocyte cell counts or percentages in primary or secondary lymphoid organs, suggesting normal immune system homeostasis. Histologic analysis of LN did not show significant differences in the overall architecture, cortical or medullary organization, B and T cell zone segregation, dendritic or plasmacytoid dendritic cell distribution, or the structure of the FRC network. Depletion of LTβR did lead, however, to a marked reduction in the accumulation of Foxp3+ Treg and the expression of CCL21 in the LN. CCL21 is a ligand for CCR7, and the major chemokine important for T cell migration to LV and LN. In vitro stimulation assays showed that both LTβR and Treg directly regulated CCL21 expression and secretion by LEC, confirming that Treg-LTβR-LEC interactions are specific and physiologically important. LTβR depletion also reduced LEC expression of non-canonical NFκB kinase (NIK) and the inflammatory and chemotactic lipid sphingosine-1-phosphate (S1P). However, expression of other LEC factors important in Treg migration such as VCAM-1, ICAM-1, and CCL19 were not affected.

Conclusions: Depletion of LTβR from LEC by administration of tamoxifen did not induce global morphologic or immune abnormalities. In contrast, there was a significant reduction in the accumulation of Foxp3+ Treg in the LN, and expression of molecules that are regulated by LTβR signaling and important for Treg migration, such as CCL21, S1P, and NIK. This model allows study of acute and chronic LTβR mediated regulation of Treg migration and function in a normal environment.

CITATION INFORMATION: Saxena V., Piao W., Xiong Y., Li L., Wagner C., Bromberg J. Lymphotoxin Beta Receptor Modulation in Lymphatic Endothelial Cells Alters Regulatory T Cell Dynamics Am J Transplant. 2017;17 (suppl 3).

« Back to 2018 American Transplant Congress