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Department of Cellular and Developmental Biology, "La Sapienza" University, Rome, Italy;
Department of Environmental Sciences, Università della Tuscia, Viterbo, Italy; and
Center of Evolutionary Genetics, Consiglio Nazionale delle Ricerche, Rome, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The same signals promoting, in T cells, clonal expansion and differentiation into effector and memory cells are also responsible for their death. This evidence prompted analysis of the pathways that allow T cells to choose one of these two opposing functional effects (13). Particular emphasis was given to the strength of the interaction between APCs and T cells, to costimulation, and to the quality and concentration of the Ag. Within this context, recent studies have evidenced that the duration of TCR signaling is one of the most crucial parameters determining whether naive or effector T cells will be activated or deleted (14). Moreover, other studies have suggested that AICD can favor the elimination of T cells specific for persistent and disseminated Ags, such as self Ags (15, 16). Since all these data were obtained in murine systems, it has not yet been defined whether the fate of human lymphocytes is regulated by the same parameters. In fact, analysis of human T cell susceptibility to apoptosis gave contradictory results. TCR-activated lymphocytes freshly isolated from peripheral blood have been described as resistant to AICD (17, 18). On the contrary, the same T cells develop an apoptosis-sensitive phenotype either upon prolonged culture in IL-2, or after repeated Ag stimulation (19, 20, 21, 22). A general susceptibility of human T cells to AICD is difficult to accept, in that it could mean that memory T cells cannot mount an efficient immune response. As a consequence, it is possible that the human experimental systems that often rely on unphysiological polyclonal activators did not resemble the Ag-specific murine systems. In fact, TCR activation is very often analyzed in humans by using either mitogens or agonistic mAbs. Mitogens do not mimic TCR serial triggering, such as anti-CD3 Abs, because of their low off-rate (23).
Thus, to clarify the mechanisms that regulate, in human memory T cells activated by an Ag, the ability to take a decision between a suicidal or a proliferative response, we chose an experimental system that closely mimics physiological T cell activation such as human T cell lines and clones specific for viral peptides or alloantigens. Using these cells activated by different APC, we observed that memory T cells, when properly activated by an Ag, developed an AICD-resistant phenotype. In particular, we demonstrated that the resistant phenotype is directly dependent on TCR recognition of a specific Ag bound to MHC molecules on the surface of the APC. This interaction is sufficient to block the CD95 signal transduction pathways in that it results in transcriptional activation of FLIP, which inhibits the activation of FLICE and the processing of death substrates.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synthetic peptides corresponding to residues 100–115 and 307–319 of influenza hemagglutinin (HA) were used in this study (Neosystem Laboratoire, Paris, France). The anti-human Fas mAb CH11 was purchased from Upstate Biotechnology (Lake Placid, NY); the UB2 and the APO-1 were from Kamiya Biomedical (Seattle, WA). The rabbit polyclonal Ab anti-poly(ADP)-ribose polymerase (PARP) and the rIL-2 were from Boehringer Mannheim (Mannheim, Germany); the goat Ab anti-FLICE p20 (anti-Mch5) and the HRP-conjugated anti-goat IgG were from Santa Cruz Biotechnology (Santa Cruz, CA). The HRP-conjugated anti-mouse Ig and the anti-rabbit Ig were purchased from Amersham Life Science (Buckinghamshire, U.K.). The monoclonal anti-CD3 OKT3 was kindly offered by Dr. C. T. Baldari (Department of Evolutionary Biology, University of Siena, Siena, Italy). ICE-like protease inhibitor zVAD-fmk was from Bachem Peptide and Biochemical (Bubbendorf, Switzerland). Metabolic inhibitors tyrphostin (Life Technologies, Gaithersburg, MD), genistein (Life Technologies), cyclosporin A (CsA; Sigma, St. Louis, MO), and cycloheximide (Sigma) were also used in this study. PMA was purchased from Sigma, and ionomycin (I) from Calbiochem (La Jolla, CA). All of the other chemicals used were of analytical grade and were purchased from Sigma or Merck (Darmstadt, Germany).
T cell lines and clones
The alloreactive T cell line PALP was generated using DR1 homozygous PBMC as a stimulator (24). T cell clones HC3 and HC6, restricted by DRB1*0101, specific for HA100–115 and HA307–319 peptides, respectively, and the G12, DR1-specific alloreactive T cell clone have been previously described (25). The line and clones were maintained in culture by weekly stimulation with DR1-expressing PBMC, prepulsed or not with the specific peptides and rIL-2 (Boehringer Mannheim). All T cells used in these experiments were CD4+ and CD45 RO+. Moreover, CD4 phenotypes of HC3 and HC6, and G12 were Th0 and Th1, respectively.
Preparation of APC
DR1-expressing L cells (5–3.1) were generated and cultured as described (26). Monocytes and dendritic cells (DC) were purified from the freshly isolated PBMC of a DR1+ individual. The former were isolated by overnight adherence, and the latter were obtained from CD13+ cells, as described previously (27). Briefly, multistep Percoll gradient (Pharmacia Fine Chemicals, Uppsala, Sweden)-purified PBMC were cultured for 5 days in RPMI/10% FCS supplemented with 25 ng/ml of granulocyte-macrophage CSF and 1000 U/ml IL-4 (Genzyme, Cambridge, MA), and treated with 200 ng/ml of LPS. DC were removed with 0.5 M EDTA 24 h later. A DR1-homozigous EBV-B cell line was also used. Monocytes, DC, and EBV-B were pulsed with the specific Ags and then incubated with T cells.
T cell activation and analysis of apoptosis
The T cell line and clones were activated by different APCs. Briefly, T cells (5 x 105/ml) were cultured in the presence of mitomycin C (Sigma)-treated DR1-expressing L cell transfectants (5–3.1), or professional APC in 48-well plates, in a total volume of 500 µl. For HA-specific clones, the APCs were prepulsed with 10 µg/ml of peptide. For PMA/I stimulation, the cells were cultured in the presence of 0.05 µM PMA and 0.5 µM ionomycin. Apoptosis was evaluated on T cells treated with anti-Fas or rFasL and activated or not with APC or PMA + I. For anti-CD3-mediated apoptosis, CD4+ T lymphocytes were cultured in OKT3-coated wells. Apoptosis was measured using a Becton Dickinson (Mountain View, CA) FACStar flow cytometer after 18 h of culture, as described previously (28). Briefly, cells were centrifuged at 1400 rpm for 6 min and washed once with 1 ml PBS. Pellets were then carefully resuspended in PBS/0.1% Triton X-100 containing 50 µg/ml propidium iodide (PI; Sigma). The percentage of specific apoptosis was calculated as follows: percentage of specific apoptosis = (percentage of PI+ cells - percentage of spontaneous PI+ untreated cells)/(100 - percentage of spontaneous PI+ untreated cells) x 100.
PCR amplification
The gene expression was determined by RT-PCR (29). The PCR mixture contained 50 mM KCl, 10 mM Tris-HCl, 2.5 mM MgCl2, 0.2 mM dNTPs, and 0.2 µM 5' and 3' oligonucleotide primers, and 2.5 U Taq polymerase (Perkin-Elmer, Cetus, Norwalk, CT) was amplified in 0.5 ml Gene Amp tubes in a final volume of 50 µl. PCR reactions were amplified by 35 cycles at 94°C for 1 min, 60°C for 30 s, and 72°C for 30 s. PCR was conducted in the automated DNA Thermal Cycler GeneAmp PCR System 2400 (Perkin-Elmer, Cetus). The primer sequences were the following: ß-actin, 5'-GTGGGGCGCCCCAGGCACCA and ß-actin, 3'-CTCCTTAATGTCACGCACGATTTC; CD95, 5'-ATGCTGGGCATCTGGACCCT and CD95, 3'-GCCATGTCCTTCATCACACAA; Bcl-2, 5'-GGAAAGGCTCGAAATACAAGC and Bcl-2, 3'-ATTGTTCCTCCCTCCCACCC; Bcl-xL, 5'-ATTGGTGAGTCGGATCGCAGC, and Bcl-xL, 3'-AGAGAAGGGGGTGGGAGGGTA; FasL, 5'-CAAGTCCAACTCAAGGTCCATGCC and FasL, 3'-CAGAGAGAGCTCAGATACGTTGAC; FLIP, 5'-GGGAGAAGTAAAGAACAAAG and FLIP, 3'-CGTAGGCACAATCACAGCAT. PCR products were size fractionated by agarose electrophoresis and normalized according to the amount of ß-actin detected in the same mRNA sample.
Transfection of COS cells and preparation of soluble human FasL
Monkey COS cells (2 x 106 cells), kindly supplied by Dr. A. M. Guerrini, (Department of Cellular and Developmental Biology, "La Sapienza" University of Rome, Rome, Italy) were transiently transfected in 100-mm petri plates with 5 µg of the mammalian expression plasmid pEX-hFL1 carrying the full-length human FasL cDNA using the DEAE-dextran method, as reported previously (30). After 72 h, the soluble FasL was concentrated from the supernatant with Centriprep columns (Amicon, Beverly, MA), and was tested in an apoptosis induction assay.
FLICE and PARP analysis by Western blotting
A total of 107 T cells, either APC activated or not, was treated with 2 µg/ml anti-APO-1 for 10 min at 37°C and then lysed in lysis buffer (30 mM Tris-HCL, pH 7.5, 150 mM NaCl, 1 mM Pefabloc-SP and small peptide inhibitors, 1% Nonidet P-40). CD95 was then precipitated for 3 h at 4°C with protein A-Sepharose (Amersham). After immunoprecipitation, the beads were washed five times with 10 vol of lysis buffer. For Western blotting, immunoprecipitates or cytosolic extracts were separated by 12.5% or 8% SDS-PAGE, transferred to Hybond nitrocellulose membrane (Amersham), blocked with 10% milk in PBS/Tween (PBS + 0.1% Tween-20) for at least 1 h, and washed and incubated with the primary Ab in blocking buffer for 16 h at 4°C. Blots were developed with HRP-conjugated secondary Ab in PBS/Tween. After washing with PBS/Tween, blots were developed with the chemoluminescence method (ECL) according to the manufacturer’s instructions (Amersham).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD4+ T cell clones (HC3, HC6, and G12) were activated
with plastic-bound anti-CD3 mAb OKT3 or with murine fibroblasts
expressing the specific Ag to evaluate the effect of TCR/CD3
cross-linking by Abs or TCR serial triggering by MHC-peptide complexes
on AICD induction in T cells repeatedly stimulated by an Ag and
expanded with IL-2. The artificial APCs were used for their lack of
costimulatory molecules, thus making it possible to dissect the role of
Ag activation and costimulation in AICD. Apoptosis was measured after
18 h of activation. We found that anti-CD3 stimulation was
able to induce a massive apoptosis of T cells, whereas APC activation
did not show any detectable apoptotic effect (Fig. 1
A). These observations were confirmed by the
results of proliferation assays showing that APC-stimulated T cells
proliferated, whereas OKT3 stimulation was unable to induce DNA
synthesis (data not shown).
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B).
These results are the first evidence that memory T cells become
resistant to AICD when they are physiologically activated by an Ag. CD95-mediated apoptosis is blocked in Ag-activated memory T cells
Depending on the activation signals, both CD95- and FasL-positive
T cells displayed a sensitive and a resistant phenotype to AICD. As a
consequence, CD95 transduction pathways in Ag-activated AICD-resistant
T cells were analyzed. The T cell clones were cultured in the presence
or absence of Ag-pulsed APC and each time one of the three agonistic
anti-Fas mAbs, APO-1, CH11, and UB2, was added simultaneously (see
Fig. 2, A–C). As shown, all of
the agonistic anti-Fas mAbs induced apoptosis in a dose-response
manner in T cells alone in culture. However, when APCs were present, a
significant decrease of apoptosis induction was observed. To confirm
these data, we treated the same T cells, activated or not by the Ag
with rFasL, and we observed the same result (Fig. 2D).
Further evidence of the resistance to CD95-mediated apoptosis of
Ag-activated T cells derives from the experiments in which the cleavage
of the ICE cascade substrate, PARP, in whole cell lysates, was analyzed
in Western blot by using an anti-PARP-specific Ab. The ICE family
protease inhibitor z-VAD-fmk was also included. We observed that
although CD95 ligation induced PARP proteolysis from a 116-kDa form to
a cleaved 85-kDa form, the simultaneous APC activation of
anti-Fas-treated T cell clones or the presence in culture of
z-VAD-fmk leads to the complete disappearance of p85 PARP (Fig. 2E). The above experimental evidences suggest that
Ag-mediated TCR engagement protects T cells from CD95-mediated cell
death.
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, a more pronounced
protection was observed when the anti-Fas was added 4 h after
APC activation, suggesting that a more sustained TCR stimulation
increases the protective phenomenon.
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A). The
results showed that a better protective effect was reached by
increasing peptide concentration. However, fibroblast-stimulated T
cells required much higher peptide concentrations compared with EBV-B.
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B). As
expected, all APC used were effective in protecting T cell clones, and
the DC, the most efficient APC (32), almost completely rescued
CD4+ T cells from anti-Fas-mediated apoptosis. These
results evidence that TCR engagement with peptide-MHC complexes is able
to protect T cells from apoptosis, although secondary signals including
costimulation systems decrease the level of TCR occupancy required for
optimal activation. TCR signaling suppresses the activation of CD95 death pathway executioners
To investigate the reason that CD95 molecules failed to mediate
apoptotic pathways in TCR-stimulated T cells, we analyzed the CD95
signaling pathway (33) in our system. CD95 can trigger two apoptotic
pathways: the sphingomyelin-ceramide pathway and the IL-1ß-converting
enzyme (ICE)/ICE-like protease cascade (34, 35). Moreover, it
has recently been observed that ceramide generation during CD95
activation is blocked by ICE protease inhibitors: this suggests that
ceramide production is downstream of the ICE-like protease cascade
(36). We therefore concentrated our investigation on the caspase
cascade. All caspases are released as inactive proenzymes that have to
be activated by proteolytic cleavage after specific aspartate residues
(37). The first ICE-like protease involved in the CD95-mediated
signaling pathway is FLICE, which is activated after CD95 engagement by
recruitment in the CD95 death-inducing signaling complex, DISC (38, 39). The active subunits p20 and p10 of FLICE are released in the
cytosol, where in its activated form FLICE cleaves PARP (40). The
impaired recruitment of FLICE in the DISC from mitogen-activated T
cells transiently resistant to AICD has recently been described (17).
Therefore, we analyzed whether FLICE recruitment and processing were
inhibited in Ag-activated T cells. To examine the recruitment and the
activation of FLICE, we immunoprecipitated CD95 upon stimulation with
anti-APO-1 mAb from cell lysates of either activated or
nonactivated T cells. The Western blot analysis with goat
anti-FLICE p20 Ab revealed that FLICE is not CD95 associated in the
anti-Fas-treated Ag-activated T cells, but is recruited in the DISC
of CD95-induced apoptosis-susceptible cells. Therefore, the processed
form of FLICE p20 was observed only in the latter (Fig. 5).
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The evidence that Ag activation interfered with the CD95 death
pathway prompted us to investigate which step (or steps) in the TCR
pathway was responsible for this interference. The effect of a panel of
different inhibitors of early, intermediate, and late events of TCR
signal transduction on the protection from CD95-induced apoptosis (Fig. 6) was tested. Both the PTK inhibitors
tyrphostin and genistein were effective in blocking the
Ag-pulsed APC-protective effect, suggesting that the "TCR
rescue step" was downstream of PTKs. Moreover, APC-activated T cells
acquire sensitivity to CD95-mediated AICD in the presence of CsA and
cycloheximide. CsA is an antagonist of the
Ca2+/calmodulin-dependent serine/threonine protein
phosphatase calcineurin, which regulates translocation of NF-ATc
proteins to the nucleus in a Ca2+-dependent way (41, 42).
Cycloheximide acts as translation inhibitor. Taken together, these
results suggest that the TCR-dependent activation-signaling pathway
could result in the neosynthesis of some antiapoptotic proteins, and
that calcium mediators can play a role in this transcriptional
activation.
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Recently, several groups have reported that FLIP is a FLICE
inhibitor that has a main role in regulating sensitivity to AICD (12, 43, 44). We therefore examined whether the TCR protection from
CD95-mediated AICD was due to FLIP induction in our model.
APC-activated T cells in the presence or absence of anti-Fas
agonistic mAb APO-1 were cultured for 18 h. RNA was extracted and
assayed for FLIP mRNA expression by RT-PCR. As shown in Fig. 7A, APC activation leads to a strong
induction of FLIP expression that is completely maintained in activated
cells treated with APO-1. It is interesting to note that unstimulated T
cells, grown for 7 days in the presence of IL-2, did not show any FLIP
expression, supporting the recent evidence that IL-2 down-regulates
FLIP expression (44). To confirm that FLIP induction was responsible
for the resistance to apoptosis of T cells properly activated by the
Ag, T cells stimulated by OKT3 were analyzed for FLIP expression. T
cells stimulated by specific Ag were used as control of FLIP
expression. The results in Fig. 7B show that the activation
of T cells by unphysiological polyclonal activators results in the lack
of FLIP expression. These results demonstrate that the TCR-protective
effect on CD95-mediated AICD is due to the induction of the specific
FLICE inhibitor FLIP, and that during a correct activation process the
CD95 transduction pathway is specifically blocked also when CD95 is
triggered. To confirm this conclusion, we assayed the CD95-treated
activated T cells for FLIP mRNA expression in the presence of the same
panel of metabolic inhibitors shown above in Fig. 6. We were able to
demonstrate that both genistein and tyrphostin inhibited FLIP
mRNA. Moreover, CsA completely blocked the TCR-mediated induction of
FLIP mRNA (Fig. 7C). Taken all together, these results
evidence that the ability of TCR triggering to transform memory T cells
in AICD-resistant phenotypes is associated with the neosynthesis of
FLIP.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although Ag stimulation rapidly up-regulates both CD95 and FasL expression and their interaction triggers AICD, the presence of CD95 and FasL on the cell membrane does not characterize the T cell phenotype sensitive to AICD. In fact, activated naive T cells in mice (45) and freshly isolated lymphocytes in humans (17, 18) have been described as resistant to AICD even though they are CD95 and FasL positive. This suggested that other factors must control the susceptibility to CD95-mediated apoptosis, and particular importance has been given to CD95-mediated pathways. It is worth noting that the main difference observed between CD95-susceptible and CD95-resistant phenotypes in the same cell lines concerns the ability of a functional DISC to recruit FLICE (17). The resistance to AICD has more recently been attributed to high levels of intracellular FLIP, which, interacting with the adaptor protein FADD (Fas-associated death domain protein) and the protease FLICE, blocks CD95 signaling (12). No data are available on the ability of Ag recognition to mediate the activation of FLIP expression in human memory T cells. Our evidence that AICD-resistant phenotypes expressing CD95 and FasL are also positive for FLIP represents a novel and interesting observation. In fact, what emerges from our data is that TCR triggering results in a steady state in which the synthesis rates of apoptotic molecules (CD95 and FasL) are balanced by the synthesis rate of antiapoptotic molecules (FLIP). Therefore, our evidence that in potentiating activation signals, either increasing Ag concentration or adding costimulation, an increase of the protection from CD95-mediated apoptosis is observed, makes it possible to speculate that FLIP synthesis also may be influenced. A correlation between TCR-induced expression of molecules involved in either AICD or T cell activation has already been reported (11). In fact, it has been shown that CD45-dependent PTK events and subsequent Ras and calcineurin activation are required for optimal expression of FasL following TCR ligation. The presence of a NF-AT binding site in the promoter of FasL has also been demonstrated, reinforcing the previous observation that CsA inhibited FasL expression (46). The regulation of FLIP expression still remains unknown. However, our experiments with PTK and calcineurin inhibitors, performed to assess which biochemical signaling mechanisms the lymphocytes use to couple TCR stimulation to FLIP expression, suggest that FasL and FLIP expression may use common regulatory pathways. Furthermore, the evidence that PMA/I up-regulates FasL (47) and FLIP expression in our system supports this hypothesis.
Another feature of apoptosis-resistant phenotype is the expression of some molecules of the Bcl-2 family, such as Bcl-2 and Bcl-xL (48). Despite the fact that these proteins have been described to inhibit the proteases involved in CD95-derived apoptotic pathways and that an increase of Bcl-xL has been correlated with T cell resistance to CD95-mediated apoptosis (17), the molecular and functional interactions between the CD95 and Bcl-2 pathways have not been clarified, even though a physical interaction has been excluded (49). However, a pronounced role of Bcl-2 and Bcl-xL has been demonstrated in another apoptotic process called passive cell death, in which T cells undergo cell death when they are deprived of activation stimuli and/or growth factors. Our evidence that Bcl-2 and Bcl-xL are expressed in both AICD-sensitive and resistant phenotypes further supports the evidence that Bcl-2 is not involved in the regulation of AICD (50). However, we cannot exclude that Bcl-2 posttranslational modifications occur and modify the role of this protein in T cell AICD (51).
If we assume that memory T cells express an AICD-resistant phenotype every time that they encounter an Ag, when do these cells express a susceptible phenotype? We can find the answer to this question by analyzing the time course of CD95, FasL, and FLIP expression. Both FasL and FLIP disappear after Ag recognition in just a few hours or days, respectively (8, 12), while CD95 expression is quite permanently expressed on memory T cells (52). This means that AICD protection is a transient phenomenon triggered to permit the activation and amplification of cells properly stimulated by an Ag. Once the Ag-mediated activation is exhausted, the CD95 pathways become operative once again, as demonstrated by the susceptibility of T cells maintained in culture with IL-2 to anti-Fas- and FasL-mediated apoptosis. This suggests that in vivo, those cells susceptible to CD95-mediated apoptosis can be lysed by FasL expressed on Ag-activated bystander T cells.
Different factors have been shown to play a role in regulating the development of AICD-sensitive phenotypes, such as the duration and the quality of antigenic signaling, and costimulation. The importance of the signals mediated by TCR engagement is underlined in the literature, and the authors stress the importance of using physiological stimuli in dealing with this issue (15, 16, 18). A hypothesis that greatly supports this theory is suggested by the results of the Lanzavecchia group. It is shown that while ligands with appropriate off-rates such as peptide-MHC complexes or bacterial superantigens on APC can serially trigger TCR, high affinity anti-CD3 Abs are incapable of serial triggering because their low off-rate does not allow multiple engagements to occur (23). Our present data on apoptosis induction by OKT3 strongly support the importance of antigenic stimulation as the major factor determining the development of an AICD-sensitive phenotype. Selective induction of apoptosis by altered TCR ligands further supports this concept. Variants of pigeon cytochrome c, able to induce FasL expression and TCR aggregation on the cell surface but unable to elicit a common pattern of tyrosine phosphorylation of the TCR-associated signal transduction chains, have been described to favor cell death (53). The importance of partial T cell signaling in apoptosis induction also emerges from our recent paper on Ag recognition displayed by human MHC class II-expressing T cells. Using this system of T:T Ag presentation, we demonstrated that T cells, despite being unable to produce IL-2, became FasL+ and susceptible to CD95-mediated apoptosis (52).
In conclusion, our data allowed us to design a scenario of the regulation of human memory T cell number, which agrees with that observed in vivo in mice (16). In particular, we have demonstrated that AICD is not operative in Ag-activated memory T cells, unless Ag concentration decreases. This means that these cells are no longer protected and may be easily eliminated by FasL+ bystander T cells. Thus, our experimental system, in which the signals that regulate CD95 transduction pathways are well defined, may represent a good model for thoroughly examining the molecules responsible for the mechanisms in memory T cells regulating the susceptibility or the resistance of AICD.
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Acknowledgments |
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Footnotes |
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2 M.M.D. and F.S. have equally contributed to this work.
3 Address correspondence and reprint requests to Dr. Enza Piccolella, Dipartimento di Biologia Cellulare e dello Sviluppo, Università "La Sapienza," Via degli Apuli 1, 00185 Rome, Italy. E-mail address:
4 Abbreviations used in this paper: AICD, activation-induced cell death; CsA, cyclosporin A; DC, dendritic cell; DISC, death-inducing signaling complex; FasL, Fas ligand; FLICE, IL-1ß-converting enzyme-like protease; FLIP, IL-1ß-converting enzyme-like protease-like inhibitory protein; HA, hemagglutinin; HRP, horseradish peroxidase; I, ionomycin; ICE, IL-1ß-converting enzyme; PARP, poly(ADP)-ribose polymerase; PI, propidium iodide; PTK, protein tyrosine kinase.
Received for publication August 17, 1998. Accepted for publication December 23, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Lß2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a cytoplasmic regulatory molecule. Cell 86:233.[Medline]
chain residues critical for binding of the toxic shock syndrome toxin superantigen. J. Exp. Med. 176:1779.. J. Exp. Med. 179:1109.This article has been cited by other articles:
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 |
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  C. Scotta, L. Tuosto, A. M. Masci, L. Racioppi, E. Piccolella, and L. Frasca Hypervariable region 1 variant acting as TCR antagonist affects hepatitis C virus-specific CD4+ T cell repertoire by favoring CD95-mediated apoptosis J. Leukoc. Biol., August 1, 2005; 78(2): 372 - 382. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Carbonneil, H. Saidi, V. Donkova-Petrini, and L. Weiss Dendritic cells generated in the presence of interferon-{alpha} stimulate allogeneic CD4+ T-cell proliferation: modulation by autocrine IL-10, enhanced T-cell apoptosis and T regulatory type 1 cells Int. Immunol., July 1, 2004; 16(7): 1037 - 1052. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Gati, N. Guerra, C. Gaudin, S. Da Rocha, B. Escudier, Y. Lecluse, A. Bettaieb, S. Chouaib, and A. Caignard CD158 Receptor Controls Cytotoxic T-Lymphocyte Susceptibility to Tumor-Mediated Activation-Induced Cell Death by Interfering with Fas Signaling Cancer Res., November 1, 2003; 63(21): 7475 - 7482. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Strauss, I. Knape, I. Melzner, and K.-M. Debatin Constitutive Caspase Activation and Impaired Death-Inducing Signaling Complex Formation in CD95-Resistant, Long-Term Activated, Antigen-Specific T Cells J. Immunol., August 1, 2003; 171(3): 1172 - 1182. [Abstract] [Full Text] [PDF]
|
|
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M. Iwashiro, W. Jinyan, M. Toda, W. Linan, T. Kato, and K. Kuribayashi Effective anti-tumor adoptive immunotherapy: utilization of exogenous IL-2-independent cytotoxic T lymphocyte clones Int. Immunol., December 1, 2002; 14(12): 1459 - 1468. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Castilleja, D. Carter, C. L. Efferson, N. E. Ward, K. Kawano, B. Fisk, A. P. Kudelka, D. M. Gershenson, J. L. Murray, C. A. O'Brian, et al. Induction of Tumor-Reactive CTL by C-Side Chain Variants of the CTL Epitope HER-2/neu Protooncogene (369-377) Selected by Molecular Modeling of the Peptide: HLA-A2 Complex J. Immunol., October 1, 2002; 169(7): 3545 - 3554. [Abstract] [Full Text] [PDF]
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|
|
|
|
|
L. Frasca, C. Scotta, G. Lombardi, and E. Piccolella Human Anergic CD4+ T Cells Can Act as Suppressor Cells by Affecting Autologous Dendritic Cell Conditioning and Survival J. Immunol., February 1, 2002; 168(3): 1060 - 1068. [Abstract] [Full Text] [PDF]
|
|
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|
O. F. Bathe, N. Dalyot-Herman, and T. R. Malek IL-2 During In Vitro Priming Promotes Subsequent Engraftment and Successful Adoptive Tumor Immunotherapy by Persistent Memory Phenotypic CD8+ T Cells J. Immunol., October 15, 2001; 167(8): 4511 - 4517. [Abstract] [Full Text] [PDF]
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D. Chatterjee, I. Schmitz, A. Krueger, K. Yeung, S. Kirchhoff, P. H. Krammer, M. E. Peter, J. H. Wyche, and P. Pantazis Induction of Apoptosis in 9-Nitrocamptothecin-treated DU145 Human Prostate Carcinoma Cells Correlates with de Novo Synthesis of CD95 and CD95 Ligand and Down-Regulation of c-FLIPshort Cancer Res., October 1, 2001; 61(19): 7148 - 7154. [Abstract] [Full Text] [PDF]
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 |
|
 S.-i. Fujii, K. Shimizu, T. Shimizu, and M. T. Lotze Interleukin-10 promotes the maintenance of antitumor CD8+ T-cell effector function in situ Blood, October 1, 2001; 98(7): 2143 - 2151. [Abstract] [Full Text] [PDF]
|
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N. Blank, M. Kriegel, T. Hieronymus, T. Geiler, S. Winkler, J. R. Kalden, and H.-M. Lorenz CD45 Tyrosine Phosphatase Controls Common {{gamma}}-Chain Cytokine-Mediated STAT and Extracellular Signal-Related Kinase Phosphorylation in Activated Human Lymphoblasts: Inhibition of Proliferation Without Induction of Apoptosis J. Immunol., May 15, 2001; 166(10): 6034 - 6040. [Abstract] [Full Text] [PDF]
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J. Zhang, T. Bardos, K. Mikecz, A. Finnegan, and T. T. Glant Impaired Fas Signaling Pathway Is Involved in Defective T Cell Apoptosis in Autoimmune Murine Arthritis J. Immunol., April 15, 2001; 166(8): 4981 - 4986. [Abstract] [Full Text] [PDF]
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H. Yoshikawa, Y. Nakajima, and K. Tasaka Enhanced Expression of Fas-Associated Death Domain-Like IL-1-Converting Enzyme (FLICE)-Inhibitory Protein Induces Resistance to Fas-Mediated Apoptosis in Activated Mast Cells J. Immunol., December 1, 2000; 165(11): 6262 - 6269. [Abstract] [Full Text] [PDF]
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S. Fulda, E. Meyer, and K.-M. Debatin Metabolic Inhibitors Sensitize for CD95 (APO-1/Fas)-induced Apoptosis by Down-Regulating Fas-associated Death Domain-like Interleukin 1-Converting Enzyme Inhibitory Protein Expression Cancer Res., July 1, 2000; 60(14): 3947 - 3956. [Abstract] [Full Text]
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F. Somma, L. Tuosto, M. S. G. Montani, M. M. Di Somma, E. Cundari, and E. Piccolella Engagement of CD4 Before TCR Triggering Regulates Both Bax- and Fas (CD95)-Mediated J. Immunol., May 15, 2000; 164(10): 5078 - 5087. [Abstract] [Full Text] [PDF]
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 I. Suzuki and P. J. Fink The dual functions of Fas ligand in the regulation of peripheral CD8+ and CD4+ T cells PNAS, February 15, 2000; 97(4): 1707 - 1712. [Abstract] [Full Text] [PDF]
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) or
not (
) by peptide-pulsed DR1+ murine fibroblasts was
treated with different concentrations of CH11 (A), UB2
(B), APO-1 (C) anti-Fas agonistic
mAbs, and soluble rFasL (D), for 24 h. Apoptosis
was evaluated by FACS analysis, and the values are expressed as a
percentage of specific apoptosis. The percentage of hypodiploid cells
in unstimulated cultures was less than 15%. Data are representative of
five independent experiments performed with both HC6 and G12 T cell
clones. PARP proteolysis is reported in E. Alloreactive
G12 clone was incubated with DR1-transfected murine fibroblasts (APC)
or with wild-type fibroblasts (CTR). Anti-Fas mAb CH11 (100 ng/ml) was
added to unactivated (CH11) or to APC-activated T cells (APC + CH11).
The caspase inhibitor zVAD-fmk at 40 µM was added to unactivated
CH11-treated T cells (zVAD). After 8 h of incubation, the cells
were lysed and the lysates were subjected to 8% SDS-PAGE. Western blot
analysis with rabbit anti-PARP Ab revealed the full-length PARP,
p116, and its proteolysed fragment p85. The results are representative
of four independent experiments performed with both APO-1 and CH11
mAbs.
