In panels A and B, the results of one out of three representative experiments are shown. In another set of experiments, EA.hy926 cells were activated with TNF- and incubated with a MAb against CD54, this was followed CHIR-99021 trihydrochloride by the addition of SpA-SEA-D227A and SEA-primed T cells. strong cytotoxicity against IFN– and TNF–activated EA.hy926 which had been preincubated with SEA. Blocking of CD54 completely abrogated the T-cell attack. SEA-D227A, which has a mutated class II binding site, did not promote any cytotoxicity. A strong lysis was observed when a fusion protein consisting of protein A and SEA-D227A was added together with T cells to TNF–induced EA.hy926 and HUVEC precoated with monoclonal antibodies (MAb) directed against HLA class I, CD54, or CD106 molecules. Finally, an scFv antibody fragment reactive with an unknown EC antigen was fused with SEA-D227A. Both EA.hy926 and HMVEC were efficiently lysed by scFv-SEA-D227A-triggered cytotoxic T cells. CHIR-99021 trihydrochloride Taken together, superantigen-activated T-cell-dependent EC killing was induced when EC expressed an inflammatory phenotype. Moreover, specific MAb targeting of the superantigen to surface antigens induced EC lysis. Our data suggest that directed T-cell-mediated lysis of unwanted proliferating EC, such as those in the tumor microvasculature, can be clinically useful. Endothelial cells (EC) collection the blood vessels and form a barrier between blood components and the tissues; they also play a crucial role in inflammatory responses, immune reactions, and vascular hemostasis (24). The cytokines interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-) are secreted by leucocytes in response to numerous microorganisms during the early phase of an inflammatory response. This results in the activation of EC and production of autacoids, including prostanoids, platelet-activating factor, and nitric oxide. Activated EC display an increased cell surface expression of adhesion molecules, such as E-selectin (CD62E), ICAM-1 (CD54), PECAM-1 (CD31), and VCAM-1 (CD106), which facilitate the extravasation of leukocytes from your microvasculature to inflammatory sites in the peripheral tissues (14,21). Increased concentrations of gamma interferon (IFN-) are also detected during the later stages of an inflammatory response and may result in the induction of HLA class II surface expression, upregulation of HLA class I density, and enhanced peptide transport capacity in EC (6,23). These phenotypic changes allow EC to serve as antigen-presenting cells (APC) and suggest that EC plays an active role during several phases of an immune response. Certain strains ofStaphylococcus aureusproduce immunostimulatory exotoxins, such as harmful shock syndrome (TSS) toxin 1, staphylococcal enterotoxin A (SEA), SEB, and SEC, all of which are associated with food poisoning and TSS (for a review, see research31). These exotoxins have been denominated superantigens (SAg) due to their ability to Arnt activate a high frequency of T lymphocytes. SAg bind as unprocessed proteins to HLA class II molecules on APC and oligoclonally activate T cells expressing particular T-cell receptor V chains (25). In vivo exposure to excessive amounts of SAg results in a strong cytokine production, including IL-2, TNF-, and IFN-, which are associated with a harmful shock-like syndrome (15,27,34). Interestingly, SAg binds to not only professional APC but also to other HLA class II-bearing cells, such as activated human umbilical vein EC CHIR-99021 trihydrochloride (HUVEC) (37). It has been exhibited that bacterial SAg efficiently bind HLA class II-positive, activated EC and subsequently trigger human T cells to proliferate and CHIR-99021 trihydrochloride produce cytokines (2,17). SAg- and EC-induced T-cell activation appears to CHIR-99021 trihydrochloride be strongly inhibited by monoclonal antibodies (MAb) to CD2, CD11a, CD28, ICAM-1, and VCAM-1, suggesting that multiple adhesion pathways contribute to ECT-cell interactions (17). In the present study, we show that this SAg SEA was able to induce T-cell-directed cytotoxicity against activated HLA class II-positive EC (SAg-dependent cellular cytotoxicity [SDCC]). SEA-directed cytotoxic T lymphocytes (CTL) efficiently lysed established HLA class II-positive EC lines as well as main HUVEC and human microvascular endothelial cells (HMVEC). In addition to the SDCC against EC, we demonstrate that attenuated and mutated SEA proteins that fail to bind HLA class II proteins, can be linked to EC-reactive MAb, and target CTL to lyse EC. An scFv-SEA chimeric protein, which is usually selectively reactive to activated EC, may have a therapeutic potential for inhibition of pathological vascular growth, such as neoangiogenic processes in solid tumors. == MATERIALS AND METHODS == == Cells and reagents. == The EA.hy926 cell line was obtained from F. Lupu (Thrombosis Research Institute, London, United Kingdom) (11). The immortalized cell collection was managed in RPMI 1640 (Gibco-BRL, Paisley, United.