Introduction: Lymphotoxin (LT) regulates the organization, development, and maintenance of lymphoid organs. Lymphotoxin beta receptor (LTΒR) signaling has been implicated in T cell migration in the thymus and dendritic cell positioning in the germinal center. However, the role of LT in T cell migration to lymph node (LN) via high endothelial venules (HEV) or lymphatics has not been explored. In this study we tested the hypothesis that LT- LTΒR interactions are important for T cell migration to LN.

Methods: T cell subsets were generated using Foxp3GFP reporter mice, and flow purified based upon CD44, CD25, and Foxp3GFP expression. qRT-PCR quantitated gene expression. T cells were used in transmigration and time-lapse assays of movement on lymphatic endothelial SVEC4-10 cells or blood endothelial MS-1 cells. For in vivo migration assays, T cell subsets were labeled with tracking dyes, injected via the foot pad or intravenously, and analyzed at various times.

Results: Treg had higher levels of LTΑ mRNA than naÏve or effector CD4 T cells. In addition, natural Treg (nTreg) and azacytidine-induced nTreg-like cells (Aza Treg) expressed more LTΑ than TGFΒ induced Treg (iTreg). nTreg expressed more LTΒ mRNA than the other cell types. Compared to other members of LT molecular family, only naÏve CD4 T cells expressed substantial levels of LIGHT mRNA. Transmigration assays showed that blockade of the LTΒR pathway resulted in reduced nTreg and iTreg transmigration across the lymphatic endothelial cell line, but not the blood endothelial cell line; while transmigration of naÏve and effector CD4 T cells was not inhibited. Time lapse imaging showed that initial Treg contact with lymphatic endothelium but not blood vascular endothelium was also altered by blockade of the LTΒR pathway. Finally, blockade of the LTΒR pathway in vivo resulted in reduced Treg infiltration of LN via afferent lymphatics but not via blood, while again naÏve and effector T cell entry into LN was not reduced.

Conclusions: Treg, but not naÏve or effector T cells, rely upon stimulation of the LTΒR on lymphatic endothelium for migration to LN via afferent lymph. This interaction is not required for LN entry via HEV. This reveals a previously undescribed role for the LTΒR pathway in regulating T cell entry into LN. Since nTreg are known to be specialized to engage the afferent lymph pathway to enter LN and be fully suppressive, modulating this pathway may allow targeted disruption or enhancement of Treg migration to LN, thus either suppressing or enhancing effector T cell responses.

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