Activation-Induced Death by Apoptosis in CD4 T Cells from HIV-Infected Asymptomatic Individuals

In immature thymocytes, T-cell receptor (TCR) mobilization leads to an active T-ceil suicide process, apoptosis, involved in the selection of the T-cell repertoire. We have proposed that inappropriate induction of such a cell death program in the mature CD4+ T-cell population could account for both early qualitative and late quantitative CD4+ T lymphocyte defects of human immunodeficiency virus (HIV)-infected individuals. Here, we report that the selective failure of CD4+ T cells from 38 clinically asymptomatic HIV-infected individuals to proliferate in vitro to major histocompatibility complex class II (MHC-II)-dependent TCR mobilization and to pokeweed mitogen is due to an active CD4+ T-cell death process, with biochemical and ultrastructural features of apoptosis, including DNA fragmentation in multiples of 200 base pairs, and chromatin condensation. Activation-induced cell death was not detected in T cells from any of 20 controls, and occurred in purified CD4+ T cells from HIV-infected asymptomatic individuals. Activationinduced CD4+ T-cell death was prevented by cycloheximide, cyclosporin A, and a CD28 monoclonal antibody. CD28 monoclonal antibody not only prevented apoptosis but also restored T-cell proliferation to stimuli, including pokeweed mitogen, superantigens, and the tetanus and influenza recall antigens. These findings provide new insights into the pathogenesis of AIDS and may represent a basis for the design of specific therapeutic strategies. Groux, Torpier, Monte, Mouton, Capron, Ameisen 3 H IV-infected individuals present early CD4 + T-cell functional defects (l-7) that precede the quantitative reduction in this cell population that leads to AIDS. These functional defects are detected while less than l/l ,000 TH cells are infected (8-IO), and are characterized by a selective loss of ability to proliferate in vitro to self MHC-II-restricted recall antigens and to pokeweed mitogen (PWM) (l-7). CD4+ T-cell dysfunction and depletion have been attributed to a wide range of distinct mechanisms. In particular, the early qualitative defects have been related to T-cell suppression (7,11), clonal anergia (7), autoimmune responses (12), inhibitory effects of HIV proteins (13,14), or selective HIV-mediated destruction of memory T cells, leading to the presence of only naive CD4+ T cells (15,16). We have proposed the hypothesis (17) that a single mechanism, the inappropriate re-emergence of a T-cell death program in response to activation could account for both early qualitative and late quantitative CD4+ T-cell defects from HIV-infected individuals. Programmed cell death, or activation-induced cell death, or apoptosis, is an active cell suicide mechanism of widespread biological importance (18) that constitutes the physiological response of normal immature thymocytes to activation (18-23); this process is involved in the negative selection of the T-cell repertoire, the deletion of autoreactive T-cell clones, and the establishment of self-tolerance (24). This cell suicide mechanism occurs in the absence of bystander-cell destruction, requires cell activation, Groux, Torpier, Month, Mouton, Capron, Ameisen 4 initiation of protein synthesis, and involves the activation of an endogenous endonuclease that results in a characteristic regular fragmentation of the entire cellular DNA into multiples of an oligonucleosome unit of 200 base pairs (18-28). In immature thymocytes, apoptosis is not an obligatory response to TCR stimulation, but is the consequence of incomplete signal transduction related to the nature of the antigen presenting cell and to the absence of certain co-signals (22,23,29,30). A major question in T-cell biology is thus whether TCR mobilization may also lead in certain circumstances to the re-emergence of a functional cell death program in mature T cells. We have investigated whether in vitro activation of T cells from clinically asymptomatic HIV1 -infected individuals including individuals with normal CD4+ T-cell counts and from controls with polyclonal activators and self-MHC-II-dependent recall antigens may lead to T-cell death. Since memory T cells specific for a given recall antigens are rare, and might be depleted in HIV-infected individuals, we also investigated the response to the self-MHC-II-dependent staphylococcal enterotoxin B superantigens (SEB) (31). These superantigens bind to MHC-II molecules and interact with defined Vp TCR molecules expressed by up to 30% of normal human T cells, inducing proliferation in normal mature CD4 + T cells (31) and apoptosis in immature thymocytes (21). Groux, Torpier, Mont4 Mouton, Capron, Ameisen 5 MATERIALS AND METHODS Study subjects. Peripheral blood was obtained from 38 HIV-infected individuals in the Service des Maladies Infectieuses, Centre Hospitalier de Tourcoing, France. They were 26 males and 12 females, all clinically asymptomatic (stage II of the Center for Disease Control (CDC) classification); 25 were CDC stage IIA (no biological abnormalities, CD4>500/mm3, mean 856); 13 were CDC stage IIB (biological abnormalities, CD4<500/mm3, mean 345). HIV infection was related to homosexuality (n=15), heterosexual contact (n=14), intravenous drug use (n=7), or blood transfusion (n=2). Controls were 20 HIV-seronegative donors from the medical staff. Cell preparations. Peripheral blood mononuclear cells (PBMC) were obtained on Ficoll-Hypaque, and cells were cultured as previously described (32). In some experiments, CD4+ or CD8+ T cells were purified by negative selection with magnetic beads coated by anti mouse IgG (Dynal, Biosys, France). Cells (50.109 were plated to plastic Petri dishes in order to harvest adherent cells by scraping. Non adherent cells were incubated with 5pg/ml of CD20, CD56, MHC-II, CD4 or CD8 monoclonal antibodies (mAb) in a volume of 5ml in RPMI for 30mn. Subsequently, excess antibody was removed by washing twice in RPMI. The cells were then resuspended in 5ml RPMI with magnetic beads (according to the manufacturer’s instructions). Groux, Torpier, Mont4 Mouton, Capron, Ameisen 6 This mixture was rotated in the cold for 30mn and the cells were passed through a magnetic field twice to remove the cells that had bound to the magnetic beads. Cells were 98% pure as assessed by cytofluorometry. In some experiments, PBMC were depleted either in CD4+ or CD8+ T cells by using the same general method, cytofluorometry analysis revealing less than 2% contaminating cells. Cell proliferation assays. Cell proliferation assays were performed in 96-well culture plates (Nunc) in a final volume of 200~~1 as previously described (33). PBMC (2.5 105/ml) were cultured in RPMI/lO% fetal calf serum. Mitogens (purchased from Sigma, France) were used respectively at the following final concentrations: phytohemagglutinin (PHA), lOpg/ml; concanavalin A (ConA), lO~g/ml; pokeweed mitogen (PWM), lO~g/ml; staphylococcal enterotoxin B superantigens (SEB), lpg/ml; the CD3 mAb was used at lpg/ml. Tetanus toxoid (TT) recall antigen (Biomerieux, France) was used at lO~g/ml; and Influenza A hemagglutinin (Infl) recall antigen (Eurobio, France) at lO~g/ml. After 3 days for mitogens or 6 days for antigens, cultures were pulsed with 1pCi of 3[H]-thymidine (5 Ci/mmol, Amersham, France) during the final 15h of incubation, and harvested. Evaluation of cell death by Trypan Blue exclusion. Cells were incubated in 96-well plates with various stimuli in the same conditions as for proliferation assays. They were harvested by pipetting and diluted I:2 with 0.1% trypan blue in PBS. The live and dead cells were counted in a hemocytometer. Groux, Tot-pier, Month, Mouton, Capron, Ameisen 7 DNA fragmentation assays. DNA fragmentation was determined according to the methods of Wyllie and Morris (28) and Newell et al. (33) with slight modifications. In brief, IO7 cells were collected by centrifugation at 2009 for IOmn, and lysed in 1 ml hypotonic lysing buffer (5mM Tris Ph 7.4, 5 mM EDTA, 0.5% Triton X 100). The lysates were centifuged at 13,OOOg for 15mn. Supernatants were deproteinized by extraction once in phenol/chloroform and twice in chloroform/isoamyl alcohol (24:l) and precipitated at -20°C in 50% isopropanol, 130 mM NaCI; after electrophoresis on 2% agarose slab gels, DNA was stained by ethidium bromide. Electronmicroscopy. Cells were fixed in 1% glutaraldehyde in O.lM sodium cacodylate buffer pH 7.4 for 2h at +4”C. Pellets were post fixed in 1% aqueous osmium tetroxide for 1 h, en-block stained in 1% aqueous uranyl acetate for 6h and embedded in araldite. Sections were stained with uranyl acetate and lead citrate before examination with a Philips EM 420 electron microscope. Monoclonal antibodies and chemicals. The monoclonal antibodies (mAb) used in this study were CD3 (X35-7, IgG2a), generous gift from Dr. Bourel (France); CD28 (9.3, IgG2a), generous gift from Oncogen Corp.; CD28 (CLB 28/l, IgGl), purchased from Jansen; CD20 (IOB20, IgM), CD56 (IOT56, IgGl), HLA-DR (IOT2a, IgG2b), CD4 (10T4, lgG2a), CD8 (IOT8, IgG2a), were all purchased from Immunotech, France; CD5 (A50, IgGl), ‘CD44 (P245, IgG2a), generous gifts from Dr. A. Bernard, France. Groux, Torpier, Monte, Mouton, Capron, Ameisen 8 Cyclosporin A was purchased from Sandoz, France; and Cycloheximide from Sigma. Statistical analysis. Statistical significance was assessed by using a Student t test.