Project Summary/Abstract Foxp3+ regulatory CD4+ T cells (Treg) induce and maintain tolerance. Treg also limit the tempo and strength of normal immune responses to environmental antigens and pathogens. How Treg are regulated to manifest these functions is incompletely understood. Treg co-opt helper T cell (Th) transcription factors to express similar selected receptors and migrate to Th resident sites to suppress immunity. Since Treg and Th responses are not precisely aligned, these observations do not fully define mechanisms of how Treg are regulated, nor identify how their function is channeled to be in the “right place at the right time”. The mechanisms of how Treg functions are geographically and kinetically distinct from conventional CD4+ T cells (Tconv) remain elusive. We demonstrated that specific trafficking is required for Treg to suppress allograft rejection: Treg sequentially migrate from blood through microvascular endothelium into tissue, and then from tissue through afferent lymphatics to the draining lymph nodes (dLN). If this sequence is interrupted, particularly lymphatic migration, Treg fail to be activated and suppress Tconv and dendritic cells (DC) in tissue and LN. Indeed, Treg lose CD25 and Foxp3, becoming exTreg. Thus, a major requirement for normal Treg function, and a step not required for Tconv, is afferent lymphatic migration. Our overall hypothesis is that the Treg-lymphatic interaction is an important regulatory nidus for suppression and tolerance, and our goal is to define the key events and structures that regulate this interaction. Our publications demonstrate novel attributes of Treg migration. Thymus derived (nTreg) and peripherally induced Treg (iTreg) express high cell surface lymphotoxin (LT). Treg use LT to migrate across afferent lymphatic endothelial cells (LEC). Treg LT binds the LEC LT-receptor (LTR) to stimulate non-canonical NFB-inducing kinase (NIK). NIK signaling facilitates Treg transendothelial migration (TEM) by inducing LEC responses, including increased CCL21 expression, VCAM-1 rearrangement, and basal lamellipodia that engage Treg. Our new studies show that the Treg-LEC interaction: 1.) uses LT-LTR to condition LEC for permissiveness for gating the TEM of other leukocytes (i.e. T, B, DC, M); 2.) determines if exTreg are induced; and 3.) relies on PD-1(Treg)/PD-L1(LEC) interactions for Treg TEM. Our data demonstrate novel Treg-LEC interactions that serve essential roles for Treg suppression. Since Treg must migrate through tissues and interact with afferent lymphatics, and since Treg uniquely deploy diverse interactions with LEC that regulate migration and suppression, these observations lead us to the following specific hypotheses: 1.) Treg-LEC LT-LTR interactions regulate the permissiveness of LEC for gating TEM of many other leukocyte subsets and acts as a gatekeeper for immunity, suppression, and resolution of inflammation; 2.) Treg-LEC interactions determine if Treg mai...