*Purpose: Lymphotoxin (LT) α1β2 is crucial for lymphatic organ development and orchestration of immune responses. LTα1β2 is preferentially expressed by regulatory T cells (Treg) for afferent lymphatic migration. The LT beta-receptor (LTβR) is highly expressed in lymphatic endothelial cells (LEC), and signals predominantly via non-classical NFκB (NIK) pathways. Here we show that IL-2R and CD3-mediated signaling pathways preferentially increase LTαβ expression in Treg, and test the hypothesis that this stimulates LTβR signaling to LEC to regulate the migration of leukocytes from inflamed tissues.

*Methods: Murine primary LEC were used in biochemical, phenotypic, and functional analyses of LTβR signaling. Murine naïve, activated, and regulatory CD4 T cells were isolated and migrated across LEC in vitro and lymphatic vessels in vivo. LTβR signaling was assessed with western blots, gene activation assays, and specific pharmacologic and genetic blockade.

*Results: IL-2R signaling induced stronger LTα1β2 expression on induced Treg (iTreg) and natural Treg (nTreg), compared to T cell receptor (TCR) signaling. IL-2 induced classical NFκB and mitogen-activated protein kinases (MAPKs) activation in iTreg and nTreg, but these subsets differentially utilized these signaling pathways. Blocking NFκB abolished both IL-2 and TCR driven LT expression in nTreg, while blocking c-Jun NH2-terminal kinase (JNK) and extracellular-signal-regulated kinase (ERK) inhibited LT expression to a lesser extent, indicating NFκB plays a major role in nTreg. In iTreg, blocking ERK abolished both IL-2 and TCR-mediated LT increases, while NFκB blockade had less inhibitory effect, suggesting iTreg were more affected by ERK and JNK activities (Table). Activated iTreg and nTreg had the highest levels of LTαβ, and also showed the most efficient lymphatic migration. More efficient Treg migration prolonged islet graft survival by enhancing the resolution of inflammation through mobilization of inflammatory cells out of the inflamed graft.

*Conclusions: IL-2R and TCR signal through NFκB and MAPKs pathways to promote LT expression in Treg. There are differential signaling patterns in patrolling Treg in homeostasis versus iTreg inflammation. During inflammation, the highest levels of LTαβ in activated Treg endow these cells with the ability to harness LTβR signaling on LEC, thus modulating LEC structure and function and thereby condition the local environment for inflammation resolution and immune suppression.

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