We have previously shown that activation-induced T cell apoptosis is Fas indie in peripheral blood T cells from HIV+ individuals. asymptomatic HIV+ individuals. z-VAD-fmk also inhibited activation (anti-CD3)C induced CD4+ and CD8+ T cell apoptosis (AICD) in some but not all asymptomatic HIV+ individuals. Apoptosis was measured by multiparameter circulation cytometry. The z-VAD-fmk inhibitor also enhanced survival of T cells in anti-Fas or anti-CD3 antibody-treated cultures and DO-264 inhibited DNA fragmentation. AICD that could be inhibited by z-VAD-fmk was Fas impartial and could be inhibited with a blocking monoclonal antibody to tumor necrosis factorCrelated apoptosis-inducing ligand (TRAIL), a recently explained member of the TNF/nerve growth factor ligand family. The above findings show that Fas-induced T cell apoptosis is usually ICE dependent in HIV contamination. AICD can be blocked by ICE inhibitors in some patients, and this AICD DO-264 is usually mediated by TRAIL. These results show that TRAIL can be a mediator of AICD in T cells. These different mechanisms of peripheral blood T cell apoptosis may play different functions in the pathogenesis of HIV contamination. Several studies have shown that spontaneous, Fas- and activation-induced T cell apoptosis occurs in PBMCs and purified T cells from HIV-infected individuals (1C5). This apoptosis has been proposed as an important mechanism in the pathogenesis of HIV disease involved in both the functional defects and depletion of CD4+ T cells (6). Previously, a number of investigators have shown that activation-induced cell death in human T lymphocytes is usually mediated by FasCFas ligand (FasL)1 interactions (7C10). Signaling through Fas, a member of the TNF/nerve growth factor (NGF) receptor superfamily (11), has been shown to induce apoptosis of T cell clones and lines (12C14), to costimulate proliferation and cytokine production of T cells from healthy individuals (14), and to be involved in cytotoxic T lymphocyteCmediated killing (15, 16). We as well as others have recently showed that peripheral blood CD4+ and CD8+ T cells from DO-264 HIV-infected individuals are especially susceptible to Fas-induced apoptosis and that this apoptosis correlates with disease progression and severity (4, 5). TNF-related apoptosis-inducing ligand (TRAIL)/Apo-2L (17, 18) has been recently cloned and been shown to be a member of the TNF/NGF family of ligands. Although TRAIL, much like Fas, has DO-264 been shown to induce apoptosis in a number of cell lines, it does not induce apoptosis in normal peripheral blood T and B cells. Thus, the biological function of TRAIL has yet to be determined. Our initial study around the role of Fas in T cell apoptosis of HIV disease raised the question of whether FasCFasL interactions are involved in the activation-induced T cell apoptosis observed in HIV contamination. Using reagents that block either Fas antigen or FasL, we recently showed that this activation-induced T cell apoptosis is usually Fas/FasL impartial (19). In the present study, we confirm and lengthen these observations by using z-VAD-fmk, a tripeptide inhibitor of interleukin-1 transforming enzyme (ICE) protease homologues. We show that although Fas induced apoptosis of peripheral blood T cells can be abrogated by z-VAD-fmk in all asymptomatic HIV+ patients, activation-induced CD4+ and CD8+ T cell apoptosis (AICD) of T cells can be inhibited in some but not all patients. We report here that TRAIL can mediate AICD of T cells. AICD of peripheral blood T cells from HIV-infected individuals that could be effectively inhibited by z-VAD-fmk could also be blocked by a neutralizing monoclonal antibody to TRAIL, but not to FasL. Our findings show that multiple mechanisms of T cell apoptosis are operative in HIV contamination and may play different functions in the pathogenesis of HIV disease. Materials and Methods Samples and Materials. Heparinized blood samples were obtained after informed consent of asymptomatic HIV+ individuals. Mouse monoclonal IgM antibody to Fas antigen (CD95) CH-11 (Immunotech, Westbrook, ME) was utilized for Fas-induced apoptosis experiments. For AICD experiments, the anti-CD3 monoclonal antibody OKT3 was used. Blocking mouse monoclonal antibody to FasL (NOK1; IgG1 isotype) was a gift by Dr. H. Yagita (Juntendo University or college, Tokyo, Japan). For TRAIL blocking, the neutralizing monoclonal antibody M180 was used (IgG1 isotype; Immunex, Seattle, WA). The monoclonal anti-TNP antibody 107.3 was used as an IgG1 isotype control (= 11; Fig. ?Fig.11 and 0.01; Wilcoxon’s signed-rank test for paired data; mean and standard errors shown). ( 0.05; Wilcoxon’s signed-rank test for paired data). (= 25; Fig. ?Fig.33 0.01; Spearman’s Rho), with greater inhibition occurring in patients with higher levels of AICD (Fig. ?(Fig.33 and = 25; horizontal bar depicts imply; 0.01 by Wilcoxon’s signed-rank test for paired data). ( 0.01; Spearman’s Rho). Open in a separate window Open in a separate window Open in a separate window Physique 4 TRAIL, KIR2DL5B antibody but not FasL, mediates.