By
From the * Department of Genetics, Beckman Center B007, Stanford University School of Medicine,
Stanford, California 94305; and the Immunex Research and Development Corporation,
Seattle, Washington 98101
Apoptosis of peripheral blood T cells has been suggested to play an important role in the pathogenesis of human immunodeficiency virus (HIV) infection. Spontaneous, Fas (CD95)-induced
and activation-induced T cell apoptosis have all been described in peripheral blood mononuclear cell cultures of HIV-infected individuals. We have previously shown that activation-induced T cell apoptosis is Fas independent in peripheral blood T cells from HIV+ individuals.
In this study, we extend and confirm these observations by using an inhibitor of interleukin-1
converting enzyme (ICE) homologues. We show that z-VAD-fmk, a tripeptide inhibitor of ICE homologues, can inhibit Fas-induced apoptosis of peripheral blood CD4+ and CD8+ T
cells from asymptomatic HIV+ individuals. z-VAD-fmk also inhibited activation (anti-CD3)- induced CD4+ and CD8+ T cell apoptosis (AICD) in some but not all asymptomatic HIV+ individuals. Apoptosis was measured by multiparameter flow cytometry. The z-VAD-fmk inhibitor also enhanced survival of T cells in anti-Fas or anti-CD3 antibody-treated cultures and inhibited DNA fragmentation. AICD that could be inhibited by z-VAD-fmk was Fas independent and could be inhibited with a blocking monoclonal antibody to tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL), a recently described member of the TNF/nerve growth
factor ligand family. The above findings show that Fas-induced T cell apoptosis is ICE dependent in HIV infection. AICD can be blocked by ICE inhibitors in some patients, and this
AICD is 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
roles in the pathogenesis of HIV infection.
Several studies have shown that spontaneous, Fas- and
activation-induced T cell apoptosis occurs in PBMCs
and purified T cells from HIV-infected individuals (1).
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
mediated by Fas-Fas ligand (FasL)1 interactions (7). 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 (12), to costimulate proliferation and cytokine production of T cells
from healthy individuals (14), and to be involved in cytotoxic T lymphocyte-mediated killing (15, 16). We and
others have recently showed that peripheral blood CD4+
and CD8+ T cells from 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, similar to Fas, has 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 on the role of Fas in T cell apoptosis of
HIV disease raised the question of whether Fas-FasL interactions are involved in the activation-induced T cell apoptosis observed in HIV infection. Using reagents that block
either Fas antigen or FasL, we recently showed that this activation-induced T cell apoptosis is Fas/FasL independent
(19). In the present study, we confirm and extend these observations by using z-VAD-fmk, a tripeptide inhibitor of
interleukin-1 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 used 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, 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 (PharMingen,
San Diego, CA). ICE inhibitors were commercially purchased
Cbz-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk; Enzyme Systems Products, Dublin, CA), Cbz-Asp-Glu-Val-Asp-fluoromethyl ketone (z-DEVD-fmk; Enzyme Systems Products),
Cbz-Tyr-Val-Ala-Asp-chloromethyl ketone (z-YVAD-cmk;
Bachem, King of Prussia, PA), and Cbz-Phe-Ala-fluoro methyl
ketone (z-FA-fmk; Enzyme Systems Products). The IL-1 Anti-Fas and Anti-CD3-induced Apoptosis and Inhibition Experiments.
PBMCs from asymptomatic HIV+ individuals were isolated
by density centrifugation on Ficoll-Hypaque. Freshly prepared
PBMCs were cultured for 24 h at 106 cells/ml in RPMI-1640
plus 5% FCS in 24-well plates coated with or without monoclonal
anti-Fas antibody CH-11 or anti-CD3 antibody OKT3. Cultures
were performed at 37°C in a 5% CO2 humid atmosphere. Plates
were coated with 500 µl/well of 5 µg/ml of antibodies in PBS,
pH 7.4, for 2 h at 37°C. Plates were then briefly washed twice
with 500 µl/well RPMI-1640 plus 5% FCS and then washed one more time as above for 30 min at 37°C. In the inhibition experiments, PBMCs were preincubated with ICE inhibitors for 3 h at
37°C in a 5% CO2 humid atmosphere before being transferred to
antibody-coated plates. Apoptosis inhibition experiments using
the Jurkat T cell line were performed by preincubating with ICE
inhibitors for 3 h and then stimulating with 200 ng/ml CH-11 in
solution. The effect of the ICE inhibitors on IL-1 Apoptosis Measurement and DNA Fragmentation.
Apoptosis was
determined by staining cells with 1 µg/ml Hoechst 33342 (HO342; Molecular Probes, Eugene, OR) for 7 min on ice as
previously described (4). Cells were costained with anti-CD8-PE (Becton Dickinson, Mountain View, CA), anti-CD4-FITC
(PharMingen), and anti-CD5-Cy5 (PharMingen) or anti-CD3-
Cy5 (PharMingen) for 15 min on ice. Anti-CD3-Cy5 was used
for staining in the anti-Fas-treated cultures, whereas anti-CD5-
Cy5 was used in the anti-CD3-treated cultures. After washes and
fixing in paraformaldehyde, 30,000 cells were analyzed by flow
cytometry using a Facstar® (Becton Dickinson) and Desk software
(20). Apoptosis was determined by measuring HO342 staining
cells gated on forward and side scatter for lymphocytes. Absolute
numbers of live CD4+ and CD8+ T cell in culture were determined by measuring the percent of live nonapoptotic cells by
flow cytometry and counting the total number of PBMCs in culture (Coulter Counter model ZM; Coulter Corp., Hialeah, FL).
converting 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 infection and may play
different roles in the pathogenesis of HIV disease.
ELISA was a gift from Dr. J.S. Kenney (Syntex, Mountain View,
CA). RPMI-1640 (GIBCO BRL, Gaithersburg, MD) and FCS
(GIBCO BRL) contained <0.03 EU/ml and 0.3 EU/ml, respectively.
production
was examined by preincubating PBMCs from HIV
individuals
with inhibitors for 3 h, and then stimulating for 24 h with 1 µg/
ml LPS. Supernatants were then assayed in a specific IL-1
ELISA.
Statistical Analysis. Data were analyzed by Wilcoxon's signed-rank test for paired data, Mann-Whitney U test, and analysis of variance using JMP Statistics Guide (SAS Institute, Inc., Cary, NC).
Preincubation of cultures for 3 h with z-VAD-fmk tripeptide at 50 µM inhibited 72% of anti-Fas-induced peripheral blood T cell apoptosis in asymptomatic HIV-infected individuals
measured at 24 h by multiparameter flow cytometry (n = 11;
Fig. 1 A). z-VAD-fmk (50 µM) inhibited 24-h Fas-induced
apoptosis of both CD4+ and CD8+ T cell (67 and 78%, respectively; Fig. 1 B). z-VAD also increased the survival of
live cells in anti-Fas-treated cultures and inhibited DNA
fragmentation (Fig. 1, C and D). At 10 µM, z-VAD-fmk
inhibited 32% of anti-Fas-induced apoptosis of CD3+ T
cells (data not shown). Spontaneous 24-h CD4+ and CD8+
T cell apoptosis could not be inhibited by 50 µM z-VAD-fmk. The control peptide z-FA-fmk (50 µM) had no effect
on either Fas-induced or spontaneous T cell apoptosis. The
z-YVAD-cmk and z-DEVD-fmk tetrapeptides at 50 µM
did not inhibit anti-Fas-induced T cell apoptosis of HIV+
PBMCs (Fig 1, A-C) and concentrations as high as 200 µM still failed to inhibit apoptosis (data not shown).
Although anti-Fas-induced apoptosis of the Jurkat T cell
line could also be inhibited with z-VAD-fmk down to
concentrations of 10 µM (86% inhibition), higher concentrations of z-YVAD-cmk and z-DEVD-fmk were required
to inhibit this apoptosis (29 and 41% inhibition, respectively,
at 100 µM) (Fig. 2 A). Both z-VAD-fmk and z-YVAD-cmk at 10 µM inhibited 77 and 73% of IL-1 production
by LPS-stimulated PBMCs (Fig 2 B). z-FA-fmk had no effect on either Fas-induced apoptosis of the Jurkat T cell line
or on IL-1
production by LPS-stimulated PBMCs.
ICE Inhibitors and Anti-CD3-induced Apoptosis.
Previously, we have shown that AICD of peripheral blood T
cells in HIV-infected individuals is Fas/FasL independent
(19). To extend these studies, we examined the effect of
the z-VAD-fmk tripeptide ICE inhibitor on anti-CD3-
induced 24-h AICD in HIV. CD5+ T cell apoptosis for
anti-CD3 alone, anti-CD3 plus z-VAD-fmk, and anti-CD3 plus z-FA-fmk was 32.3 ± 1.8, 22.9 ± 1.9, and 29.7 ± 2.4%, respectively (n = 25; Fig. 3 A); spontaneous
CD5+ T cell apoptosis was 14.1 ± 1.8%. Inhibition of
AICD by z-VAD-fmk in asymptomatic HIV+ patients correlated with the level of AICD (P <0.01; Spearman's Rho), with greater inhibition occurring in patients with
higher levels of AICD (Fig. 3 B). Inhibition of AICD by
z-VAD-fmk ranged from 0 to 87.2% (Fig. 3 B). In those
patients in which z-VAD-fmk inhibited AICD, both
CD4+ and CD8+ T cell apoptosis was inhibited (data not
shown). When AICD was inhibited, z-VAD-fmk could inhibit DNA fragmentation and enhance survival of anti-CD3 treatment cultures (data not shown). In all the above
experiments, the anti-FasL antibody failed to block both
ICE-dependent and independent AICD (Fig 4, A and B;
data not shown) confirming our previous findings with
HIV+ PBMCs (19). Addition of 100 U/ml of recombinant
IL-2 to these cultures also failed to inhibit 24-h AICD (data
not shown).
TRAIL Mediates ICE-dependent AICD in HIV-infected Individuals.
In patients with AICD that could be inhibited
by z-VAD-fmk, all of CD5+ T cell apoptosis could be inhibited by a neutralizing monoclonal antibody to TRAIL
(Fig. 4, A and B). Anti-CD3-induced 24-h apoptosis of
CD5+ T cells was 15.9 ± 2.7, 26.3 ± 3.1, 14.1 ± 1.8, 23.6 ±
3.6, 25.9 ± 3.1% for untreated, anti-CD3 antibody-treated,
anti-CD3 antibody plus anti-TRAIL-treated, anti-CD3
antibody plus anti-FasL-treated, and anti-CD3 antibody plus
isotype control-treated cultures, respectively. Anti-TRAIL
inhibited 24-h AICD in both CD4+ and CD8+ T cells.
Anti-TRAIL enhanced survival and inhibited DNA fragmentation in these anti-CD3-treated cultures (data not
shown). The blocking anti-FasL monoclonal antibody and
the isotype control antibody had no effect on T cell AICD
(Fig. 4, A and B). In patients in which z-VAD-fmk had little or no effect on AICD (<15% inhibition) neither anti-TRAIL nor anti-FasL antibody had any effect on T cell
AICD. However, the anti-FasL blocking antibody could
block anti-CD3-induced AICD of a T cell line generated
with PHA/IL-2 from a healthy HIV individual (Fig. 4 C).
Neither anti-TRAIL nor anti-FasL antibody had any effect
on spontaneous T cell apoptosis in HIV+ PBMC cultures
(data not shown).
T cell apoptosis may play an important role in the pathogenesis of HIV disease. We and others have previously shown that peripheral blood T cells from HIV-infected individuals are especially susceptible to Fas-induced apoptosis (4, 5). On the other hand, we have shown that activation-induced peripheral blood T cell apoptosis (anti-CD3 stimulation) in HIV+ individuals, is Fas independent (19). To elucidate further T cell apoptosis in HIV infection in the present study, we examined whether inhibiting ICE family homologues can inhibit Fas- and activation-induced T cell apoptosis.
Fas-induced apoptosis involves the activation of members of both the ICE and CPP32 subfamilies of cysteine
proteases (21). The tripeptide z-VAD-fmk is an irreversible inhibitor of ICE protease and is expected to inhibit
other members of the ICE subfamily of cysteine proteases
(24). z-VAD-fmk can inhibit apoptosis induced by Fas
(25), overexpression of REAPER (26) and FLICE/MACH (27), and a variety of other stimuli (28, 29). We found that
z-VAD-fmk inhibits Fas-induced peripheral blood CD4+
and CD8+ T cell apoptosis in asymptomatic HIV+ individuals, thus showing that members of the ICE subfamily of proteases are involved in this apoptosis. Tetrapeptide inhibitors of ICE (z-YVAD-cmk) or CPP32 (z-DEVD-fmk)
could not inhibit Fas-induced T cell apoptosis in PBMC
cultures. Previously, z-YVAD-cmk was shown to inhibit
Fas-induced apoptosis of purified CD4+ and CD8+ T cells
from HIV+ individuals with CD4+ T cell apoptosis being
inhibited more efficiently than CD8+ T cells (30). In our
study, the tripeptide z-VAD-fmk inhibitor was equally efficient in inhibiting Fas-induced CD4+ and CD8+ T cell apoptosis. The lack of effect of tetrapeptides in our hands is
possibly due to differences in cellular uptake of these inhibitors and this may explain why IL-1 production by
monocytes was inhibited by z-YVAD-fmk, whereas T cell
apoptosis was not.
In our studies, activation (anti-CD3)-induced CD4+
and CD8+ T cell apoptosis could be inhibited in some patients but not all, thus subdividing the HIV+ patients into
two groups with ICE-dependent and ICE-independent AICD, respectively. Spontaneous T cell apoptosis could
not be inhibited by z-VAD-fmk. The ICE-dependent peripheral blood T cell AICD in HIV+ individuals was mediated by TRAIL, as a blocking anti-TRAIL monoclonal antibody inhibited this apoptosis. TRAIL, a member of the
TNF/NGF ligand family, has been shown to induce apoptosis of transformed cell lines (17, 18) and TRAIL-induced
apoptosis requires ICE-like cysteine proteases (31). Our
findings show an involvement of TRAIL in AICD of peripheral blood T cells from HIV+ individuals. An involvement of TRAIL in T cell AICD has not been previously
reported. Recently, cytotoxic activity in long-term cytotoxic assays has been demonstrated by T cells from perforin/FasL-deficient mice (32). Only a fraction of this perforin and FasL-independent cytotoxicity can be attributed
to TNF- (32), suggesting that other members of the
TNF/NGF ligand family such as TRAIL may be involved
in long-term cytotoxicity. In our study, z-VAD-fmk and anti-TRAIL inhibited T cell AICD in some patients, and
this inhibition correlated with the level of AICD, whereas
in other patients, they failed to significantly inhibit AICD.
Whether this reflects different stages of disease or levels of
peripheral blood T cell activation in these asymptomatic
patients is not known at the moment. Patients that exhibited z-VAD-fmk-inhibitable/TRAIL-mediated T cell
AICD also had lower peripheral blood CD8+ T cell absolute counts (data not shown); although this was not statistically significant, it may indicate that CD8+ T cell subsets
are involved in this apoptosis. CD8+CD28
T cells increase in peripheral blood of HIV+ patients when CD8+ T
cells decrease (33, 34), raising the possibility that such terminally differentiated cytotoxic T cells may be involved in the TRAIL-mediated AICD we observed (35, 36). Another issue raised by our findings is whether there is increased expression of TRAIL receptors during HIV infection. Recently, three TRAIL receptors have been cloned
(DR4, TR2, and TR3; references 37), two of which
can mediate apoptosis (37, 38). It will be important to determine which one receptor or combination of receptors is involved in AICD during HIV infection. Future studies
should address these questions.
In some HIV+ individuals, z-VAD-fmk had little or no effect on AICD of peripheral blood T cells. It is not clear at the moment whether this failure to inhibit AICD is due to limitations in our experimental approach or whether this AICD is truly ICE independent. If ICE independent, this AICD may be mediated by other members of the ICE family of cysteine proteases, such as members of the CPP32 subfamily that are not inactivated by z-VAD-fmk (25). Apoptosis of T cells that cannot be inhibited by z-VAD-fmk but can be blocked by inhibitors of CPP32 have been previously reported (40). We have previously described a similar ICE-independent T cell apoptosis in HIV-1 Tat protein-enhanced AICD (41). This raises the possibility that the ICE-independent peripheral blood T cell AICD we found in HIV+ individuals may be due to Tat protein- induced apoptosis (42). IL-2 withdrawal from the murine CTLL cell line can also induce apoptosis, which cannot be inhibited by z-VAD-fmk but is abrogated by CPP32 inhibitors (40, 43), thus raising the possibility that the ICE-independent AICD in HIV+ PBMCs may be due to insufficient IL-2 production in these cultures. However, this was shown not to be the case, because recombinant IL-2 added to these cultures failed to inhibit T cell apoptosis. Another possible explanation for the ICE-independent AICD we describe is that it may be due to a bystander killing induced by cytotoxic CD8+ T cells. Such cytotoxicity cannot be inhibited by z-VAD-fmk, because granzyme B cannot be directly inhibited by z-VAD-fmk and during the protease activation cascade granzyme B circumvents the activation of ICE by directly activating CPP32 and Mch3 (40, 44- 47). In our study, spontaneous T cell apoptosis of HIV+ PBMC cultures could not be inhibited by z-VAD-fmk; whether this is the result of T cells having received the lethal hit in vivo or this apoptosis belonging to ICE-independent AICD is not clear at the moment.
The present report extends and confirms our previous studies (19) in which anti-CD3-induced AICD in HIV+ PBMCs was shown to be Fas independent. AICD of purified CD8+ T cells in HIV infection has previously been shown to be Fas independent, although purified CD4+ T cell AICD was shown to be Fas mediated (30). This apparent discrepancy may be the result of using purified versus mixed cell populations. We believe our studies using PBMCs may be more relevant to what may be happening in vivo since they address the interactions of different cellular subsets. Purified CD4+ or CD8+ T cells may reflect a more artificial system, because T cells are restricted to interacting exclusively to cells of the same subpopulation. On the other hand, different modes of T cell stimulation may result in different mechanisms of AICD; this has been reported for superantigen-induced apoptosis, which has been shown to require Fas-FasL interactions, whereas anti-CD3-induced T cell death can proceed in the absence of Fas (48). In our experiments, we chose to examine AICD induced by immobilized anti-CD3 antibody, since this would reflect antigen stimulation of T cells more closely. Recently, FasL upregulation on monocytes was shown to be deficient during HIV infection (49). If also true for T cells, this FasL deficiency may explain the paradox of having Fas/FasL-independent T cell AICD in HIV+ individuals whose peripheral blood T cells are sensitive to Fas killing.
Our findings show that in HIV+ individuals Fas-induced apoptosis of peripheral blood CD4+ and CD8+ T cells requires the activation of cysteine proteases of the ICE subfamily. Activation-induced T cell apoptosis in some patients could be inhibited by z-VAD-fmk. This AICD in HIV+ individuals was mediated by TRAIL, showing a role for TRAIL in T cell AICD. Our findings indicate that multiple mechanisms of T cell apoptosis are operative in HIV infection and, thus, possibly contributing differently to disease pathogenesis.
Address correspondence to Peter D. Katsikis, Department of Genetics, Beckman Center B007, Stanford University School of Medicine, Stanford, CA 94305. Phone: 415-723-5054; FAX: 415-725-8564; E-mail: katsikis{at}auhs.edu
Received for publication 6 June 1997 and in revised form 1 August 1997.
M.E. Garcia-Ojeda was supported by a National Science Foundation fellowship. J.F. Torres-Roca was supported by Stanford Immunology Training grant AI-07290. This work was supported by National Institutes of Health grants CA 42509, LM 04836, and AI 31770, and a grant from the Unicorn Foundation.The authors would like to thank Drs. T. Merigan and M. Winters for use of facilities at the Center for AIDS Research (Stanford Medical School, Stanford, CA).
1. | Meyaard, L., S.A. Otto, R.R. Jonker, M.J. Mijnster, R.P. Keet, and F. Miedema. 1992. Programmed death of T cells in HIV-1 infection. Science (Wash. DC). 257: 217-219 [Medline]. |
2. | Oyaizu, N., T.W. McCloskey, M. Coronesi, N. Chirmule, V.S. Kalyanaraman, and S. Pahwa. 1993. Accelerated apoptosis in peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus type-1 infected patients and in CD4 cross-linked PBMCs from normal individuals. Blood. 82: 3392-3400 [Abstract]. |
3. | Groux, H., G. Torpier, D. Monte, Y. Mouton, A. Capron, and J.C. Ameisen. 1992. Activation-induced death by apoptosis in CD4+ T cells from human immunodeficiency virus-infected asymptomatic individuals. J. Exp. Med. 175: 331-340 [Abstract]. |
4. | Katsikis, P.D., E.S. Wunderlich, C.A. Smith, L.A. Herzenberg, and L.A. Herzenberg. 1995. Fas antigen stimulation induces marked apoptosis of T lymphocytes in human immunodeficiency virus-infected individuals. J. Exp. Med. 181: 2029-2036 [Abstract]. |
5. | Estaquier, J., T. Idziorek, W. Zou, D. Emilie, C. Farber, J. Bourez, and J. Amiesen. 1995. T helper type 1/T helper type 2 cytokines and T cell death: preventive effect of interleukin 12 on activation-induced and CD95 (Fas/APO-1)-mediated apoptosis of CD4+ T cells from human immunodeficiency virus-infected persons. J. Exp. Med. 182: 1759-1767 [Abstract]. |
6. | Fauci, A.S.. 1993. Multifactorial nature of human immunodeficiency virus disease: implications for therapy. Science (Wash. DC). 262: 1011-1018 [Medline]. |
7. | Alderson, M.R., T.W. Tough, T. Davis-Smith, S. Braddy, B. Falk, K.A. Schooley, R.G. Goodwin, C. . Smith, F. Ramsdell, and D.H. Lynch. 1995. Fas ligand mediates activation-induced cell death in human T lymphocytes. J. Exp. Med. 181:71-77. |
8. | Brunner, T., R.J. Mogil, D. LaFace, N.J. Yoo, A. Mahboubi, F. Echeverri, S.J. Martin, W.R. Force, D.H. Lynch, C.F. Ware, et al . 1995. Cell-autonomous Fas (CD95)/Fas-ligand interaction mediates activation-induced apoptosis in T-cell hybridomas. Nature (Lond.). 373: 441-444 [Medline]. |
9. | Dhein, J., H. Walczak, C. Baumler, K.M. Debatin, and P.H. Krammer. 1995. Autocrine T-cell suicide mediated by APO-1/(Fas/CD95). Nature (Lond.). 373: 438-441 [Medline]. |
10. | Ju, S.T., D.J. Panka, H. Cui, R. Ettinger, M. el-Khatib, D.H. Sherr, B.Z. Stanger, and A. Marshak-Rothstein. 1995. Fas(CD95)/FasL interactions required for programmed cell death after T-cell activation. Nature (Lond.). 373: 444-448 [Medline]. |
11. | Smith, C.A., T. Farrah, and R.G. Goodwin. 1994. The TNF receptor superfamily of cellular and viral proteins: activation, costimulation, and death. Cell. 76: 959-962 [Medline]. |
12. | Yonehara, S., A. Ishii, and M. Yonehara. 1989. A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor. J. Exp. Med. 169: 1747-1756 [Abstract]. |
13. | Trauth, B.C., C. Klas, A.M. Peters, S. Matzku, P. Moller, W. Falk, K.M. Debatin, and P.H. Krammer. 1989. Monoclonal antibody-mediated tumor regression by induction of apoptosis. Science (Wash. DC). 245: 301-305 [Medline]. |
14. | Alderson, M.R., R.J. Armitage, E. Maraskovsky, T.W. Tough, E. Roux, K. Schooley, F. Ramsdell, and D.H. Lynch. 1993. Fas transduces activation signals in normal human T lymphocytes. J. Exp. Med. 178: 2231-2235 [Abstract]. |
15. | Kagi, D., F. Vignaux, B. Ledermann, K. Burki, V. Depraetere, S. Nagata, H. Hengartner, and P. Golstein. 1994. Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity. Science (Wash. DC). 265: 528-530 [Medline]. |
16. | Lowin, B., M. Hahne, C. Mattmann, and J. Tschopp. 1994. Cytolytic T-cell cytotoxicity is mediated through perforin and Fas lytic pathways. Nature (Lond.). 370: 650-652 [Medline]. |
17. | Wiley, S.R., K. Schooley, P.J. Smolak, W.S. Din, C.P. Huang, J.K. Nicholl, G.R. Sutherland, T.D. Smith, C. Rauch, C.A. Smith, et al . 1995. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity. 3: 673-682 [Medline]. |
18. |
Pitti, R.M.,
S.A. Marsters,
S. Ruppert,
C.J. Donahue,
A. Moore, and
A. Ashkenazi.
1996.
Induction of apoptosis by
Apo-2 ligand, a new member of the tumor necrosis factor cytokine family.
J. Biol. Chem.
271:
12687-12690
|
19. | Katsikis, P.D., M.E. Garcia-Ojeda, E.S. Wunderlich, C.A. Smith, H. Yagita, K. Okumura, N. Kayagaki, M. Alderson, L.A. Herzenberg, and L.A. Herzenberg. 1996. Activation- induced peripheral blood T cell apoptosis is Fas independent in HIV-infected individuals. Int. Immunol. 8: 1311-1317 [Abstract]. |
20. | Moore, W.A., and R.A. Kautz. 1986. Data analysis in flow cytometry. In Handbook of Experimental Immunology. Blackwell Scientific Publications, Oxford. 30.1-30.11. |
21. | Enari, M., H. Hug, and S. Nagata. 1995. Involvement of an ICE-like protease in Fas-mediated apoptosis. Nature (Lond.). 375: 78-81 [Medline]. |
22. | Los, M., M. Van de Craen, L.C. Penning, H. Schenk, M. Westendorp, P.A. Baeuerle, W. Droge, P.H. Krammer, W. Fiers, and K. Schulze-Osthoff. 1995. Requirement of an ICE/CED-3 protease for Fas/APO-1-mediated apoptosis. Nature (Lond.). 375: 81-83 [Medline]. |
23. | Enari, M., R.V. Talanian, W.W. Wong, and S. Nagata. 1996. Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis. Nature (Lond.). 380: 723-726 [Medline]. |
24. | Dolle, R.E., D. Hoyer, C.V. Prasad, S.J. Schmidt, C.T. Helaszek, R.E. Miller, and M.A. Ator. 1994. P1 aspartate-based peptide alpha-((2,6-dichlorobenzoyl)oxy)methyl ketones. J. Med. Chem. 37: 563-564 [Medline]. |
25. | Slee, E.A., H. Zhu, S.C. Chow, M. MacFarlane, D.W. Nicholson, and G.M. Cohen. 1996. Benzyloxycarbonyl- Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK) inhibits apoptosis by blocking the processing of CPP32. Biochem. J. 315: 21-24 [Medline]. |
26. | Pronk, G.J., K. Ramer, P. Amiri, and L.T. Williams. 1996. Requirement of an ICE-like protease for induction of apoptosis and ceramide generation by REAPER. Science (Wash. DC). 271: 808-810 [Abstract]. |
27. | Muzio, M., A.M. Chinnaiyan, F.C. Kischkel, K. O'Rourke, A. Shevchenko, J. Ni, C. Scaffidi, J.D. Bretz, M. Zhang, R. Gentz, et al . 1996. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/ APO-1) death-inducing signaling complex. Cell. 85: 817-827 [Medline]. |
28. | Zhu, H., H.O. Fearnhead, and G.M. Cohen. 1995. An ICE-like protease is a common mediator of apoptosis induced by diverse stimuli in human monocytic THP.1 cells. FEBS Lett. 374: 303-308 [Medline]. |
29. |
Fearnhead, H.O.,
D. Dinsdale, and
G.M. Cohen.
1995.
An
interleukin-1 ![]() |
30. |
Estaquier, J.,
M. Tanaka,
T. Suda,
S. Nagata,
P. Golstein, and
J.C. Ameisen.
1996.
Fas-mediated apoptosis of CD4+ and
CD8+ T cells from human immunodeficiency virus-infected
persons: differential in vitro preventive effect of cytokines
and protease antagonists.
Blood.
87:
4959-4966
|
31. | Marsters, S.A., R.M. Pitti, C.J. Donahue, S. Ruppert, K.D. Bauer, and A. Ashkenazi. 1996. Activation of apoptosis by Apo-2 ligand is independent of FADD but blocked by CrmA. Curr. Biol. 6: 750-752 [Medline]. |
32. | Braun, M.Y., B. Lowin, L. French, H. Acha-Orbea, and J. Tschopp. 1996. Cytotoxic T cells deficient in both functional fas ligand and perforin show residual cytolytic activity yet lose their capacity to induce lethal acute graft-versus-host disease. J. Exp. Med. 183: 657-661 [Abstract]. |
33. | Caruso, A., A. Cantalamessa, S. Licenziati, L. Peroni, E. Prati, F. Martinelli, A.D. Canaris, S. Folghera, R. Gorla, A. Balsari, et al . 1994. Expression of CD28 on CD8+ and CD4+ lymphocytes during HIV infection. Scand. J. Immunol. 40: 485-490 [Medline]. |
34. | Choremi-Papadopoulou, H., V. Viglis, P. Gargalianos, T. Kordossis, A. Iniotaki-Theodoraki, and J. Kosmidis. 1994. Downregulation of CD28 surface antigen on CD4+ and CD8+ T lymphocytes during HIV-1 infection. J. Acquir. Immune Defic. Syndr. 7: 245-253 [Medline]. |
35. |
Effros, R.B.,
R. Allsopp,
C.P. Chiu,
M.A. Hausner,
K. Hirji,
L. Wang,
C.B. Harley,
B. Villeponteau,
M.D. West, and
J.V. Giorgi.
1996.
Shortened telomeres in the expanded CD28![]() |
36. |
Fiorentino, S.,
M. Dalod,
D. Olive,
J.G. Guillet, and
E. Gomard.
1996.
Predominant involvement of CD8+CD28![]() |
37. |
Pan, G.H.,
K. O'Rourke,
A.M. Chinnaiyan,
R. Gentz,
R. Ebner,
J. Ni, and
V.M. Dixit.
1997.
The receptor for the cytotoxic ligand TRAIL.
Science (Wash. DC).
276:
111-113
|
38. | Walczak, H., M.A. Degli-Esposti, R. Johnson, N. Boiani, M. Gerhart, P. Smolak, K. Schooley, M. Timour, J. Waugh, C.A. Smith, et al. 1997. Identification and characterization of a second apoptosis-mediating receptor for TRAIL. EMBO (Eur. Mol. Biol. Organ.). In press. |
39. |
Degli-Esposti, M.A.,
P. Smolak,
H. Walczak,
J. Waugh,
C.P. Huang,
R. DuBose,
R. Goodwin, and
C.A. Smith.
1997.
Cloning and characterization of TRAIL-R3, a novel member of the emerging TRAIL receptor family.
J. Exp. Med.
186:
1165-1170
|
40. |
Sarin, A.,
M.L. Wu, and
P.A. Henkart.
1996.
Different interleukin-1![]() |
41. | Katsikis, P.D., M.E. Garcia-Ojeda, L.A. Herzenberg, and L.A. Herzenberg. 1997. HIV Tat protein enhances activation but not Fas (CD95) induced peripheral blood T cell apoptosis in healthy individuals. Int. Immunol. 9: 835-841 [Abstract]. |
42. | Li, C.J., D.J. Friedman, C. Wang, V. Metelev, and A.B. Pardee. 1995. Induction of apoptosis in uninfected lymphocytes by HIV-1 Tat protein. Science (Wash. DC). 268: 429-431 [Medline]. |
43. |
Vasilakos, J.P.,
T. Ghayur,
R.T. Carroll,
D.A. Giegel,
J.M. Saunders,
L. Quintal,
K.M. Keane, and
B.D. Shivers.
1995.
IL-1![]() |
44. |
Smith, D.J.,
M.J. McGuire,
M.J. Tocci, and
D.L. Thiele.
1997.
IL-1![]() |
45. | Darmon, A.J., D.W. Nicholson, and R.C. Bleackley. 1995. Activation of the apoptotic protease CPP32 by cytotoxic T-cell-derived granzyme B. Nature (Lond.). 377: 446-448 [Medline]. |
46. |
Quan, L.T.,
M. Tewari,
K. O'Rourke,
V. Dixit,
S.J. Snipas,
G.G. Poirier,
C. Ray,
D.J. Pickup, and
G.S. Salvesen.
1996.
Proteolytic activation of the cell death protease Yama/CPP32
by granzyme B.
Proc. Natl. Acad. Sci. USA.
93:
1972-1976
|
47. | Martin, S.J., G.P. Amarante-Mendes, L. Shi, T.H. Chuang, C.A. Casiano, G.A. O'Brien, P. Fitzgerald, E.M. Tan, G.M. Bokoch, A.H. Greenberg, et al . 1996. The cytotoxic cell protease granzyme B initiates apoptosis in a cell-free system by proteolytic processing and activation of the ICE/CED-3 family protease, CPP32, via a novel two-step mechanism. EMBO (Eur. Mol. Biol. Organ.) J. 15: 2407-2416 [Abstract]. |
48. | Mogil, R.J., L. Radvanyi, R. Gonzalez-Quintial, R. Miller, G. Mills, A.N. Theofilopoulos, and D.R. Green. 1995. Fas (CD95) participates in peripheral T cell deletion and associated apoptosis in vivo. Int. Immunol. 7: 1451-1458 [Abstract]. |
49. |
Sieg, S.,
D. Smith,
Z. Yildirim, and
D. Kaplan.
1997.
Fas
ligand deficiency in HIV disease.
Proc. Natl. Acad. Sci. USA.
94:
5860-5865
|