In contrast to DC-stimulated CD8+ T cells, we detected a high percentage of CD80 expression on proliferating iLEC-primed CD8+ T cells (Fig. exogenous Ags, in turn causing dysfunctional activation of CD8+ T cells under homeostatic conditions. Thus, we suggest that steady-state lymphatic drainage may contribute to peripheral tolerance by delivering self-Ags to lymph nodeCresident leukocytes, as well as by providing constant exposure of draining peripheral Ags to LECs, which maintain tolerogenic cross-presentation of such Ags. Introduction The lymphatic system transports interstitial fluid, Ags, solutes, and immune cells from your periphery and earnings them to the blood circulation after surveillance Lin28-let-7a antagonist 1 through lymph nodes (LNs), thereby initiating adaptive immune responses (1C3). In addition to effector immune responses, LNs are important sites for the maintenance of peripheral tolerance. LN stromal cells, which include lymphatic endothelial cells (LECs) and blood endothelial cells (BECs), as well as fibroblastic reticular cells (FRCs) in the T cell zone, are thought to contribute to tolerance induction of autoreactive T cells that escape central memory (4), as well as regulate the contraction of inflammatory responses (5). Indeed, the lymphatic endothelium is usually emerging as an important player in shaping immunity and tolerance (1C3, 6C10). For example, LECs were shown to suppress maturation of dendritic cells (DCs) (1, 4, 11) and their subsequent priming of CD8+ T Lin28-let-7a antagonist 1 cells in a contact-dependent manner (4, 5, 9). In addition, LECs, as well as FRCs, can directly prime CD8+ T cells (5); they express components of the Ag-presentation machinery, including MHC class I and II molecules (6C9, 12), and were shown to directly contribute to peripheral tolerance by expression and presentation of endogenous peripheral tissue Ags (PTAs), leading to compromised CD8+ T cell activation (6C9). They are also sensitive to pathogen-associated molecular patterns via the expression of various users of the TLR family (8, 11). Together, these studies established LECs as contributors to the maintenance of peripheral tolerance to endogenously expressed self-Ags. However, little is known about whether LECs as APCs have the ability to capture and process exogenous Ags for CD8+ T cell deletion. Although so-called professional APCs, such as CD8a+ DCs, can process exogenous Ags for cross-presentation to CD8+ T cells, some nonhematopoietic cell types also were shown to be capable of cross-presentation (13). For example, liver sinusoidal endothelial cells (LSECs) are thought to capture and cross-present circulating Ag to CD8+ T cells, leading to CD8+ T cell deletion and the establishment of a tolerogenic environment (14). This is especially important in the liver, where LSECs are among the first cells to encounter the large diversity of foreign Ags from food, as well as TLR agonists from commensal sources (15). Similarly, LECs are the first cells to contact extracellular Ags that arise in the periphery and drain into lymphatic vessels after, for example, tissue damage, inflammation, or contamination. We recently showed that a foreign Ag (OVA) expressed by an orthotopically implanted tumor could be cross-presented by tumor-associated LECs that, when isolated, could drive dysfunctional activation of cognate CD8+ T cells and promote tumor progression (16). Because tumors use physiological mechanisms to promote tolerance for their survival (17), we hypothesized that a comparable mechanism of Ag cross-presentation by LECs may exist under steady-state conditions to promote tolerance against self-Ags. In this article, we demonstrate that, under homeostatic conditions, LECs constitutively uptake and cross-present exogenous Ags to CD8+ T cells. We further show that LEC-activated T cells are more rapidly apoptotic, upregulate so-called exhaustion markers (PD-1, CTLA-4, and CD80), secrete less IFN- and IL-2, and express lower levels of the activation markers CD25, CD44, and CD69 compared with T cells activated by mature DCs. Together, these data suggest that LECs help to maintain CD8+ T cell tolerance to exogenous Ags that are encountered in lymph under steady-state conditions, which may be important for preventing autoimmune reactions against self-Ags after contamination or injury. Materials and Methods Reagents Lin28-let-7a antagonist 1 Elf1 All chemicals were from Sigma-Aldrich (Buchs, Switzerland), unless otherwise noted. The mature MHC class I epitope, OVA256C264 (SIINFEKL) peptide, was from GenScript (Piscataway, NJ). Endotoxin-free OVA was from Hyglos (Bernried am Starnberger Observe, Germany). Lin28-let-7a antagonist 1 Abs.