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Originally published online as doi:10.1189/jlb.0108043 on May 15, 2008

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(Journal of Leukocyte Biology. 2008;84:488-498.)
© 2008 by Society for Leukocyte Biology

Cell cycle regulation by FasL and Apo2L/TRAIL in human T-cell blasts. Implications for autoimmune lymphoproliferative syndromes

Alberto Bosque*,1, Juan I. Aguiló*, Manuel del Rey{dagger}, Estela Paz-Artal{dagger}, Luis M. Allende{dagger}, Javier Naval* and Alberto Anel*,2

* Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain; and
{dagger} Servicio de Inmunología, Hospital 12 de Octubre, Madrid, Spain

2 Correspondence: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza E-50009, Spain. E-mail: anel{at}posta.unizar.es


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ABSTRACT
 
The Fas-FasL pathway plays an important role in the homeostasis of mature lymphocytes, with defects causing autoimmune lymphoproliferative syndromes (ALPS). Human T-cell blasts are not sensitive to FasL or Apo2L/TRAIL-induced apoptosis unless they get reactivated, but either of those ligands inhibits their growth in the absence of cell death induction due to a cell cycle arrest in S-G2/M. In the present work, we have studied the mechanism(s) by which FasL or Apo2L/TRAIL regulate T-cell blast cell cycle in healthy donors and in two types of ALPS patients. Our data indicate that in human CD8+ T-cell blasts, Fas ligation, and especially Apo2L/TRAIL induce the p53-dependent decrease in cyclin-B1 levels. However, the induction of the negative cell cycle regulator p21WAF1 by FasL or Apo2L/TRAIL in either CD4+ or CD8+ T-cell blasts seems to be the main regulatory mechanism. This mechanism is dependent on caspase activation and on H2O2 generation. The increase in p21 levels by FasL or Apo2L/TRAIL is concomitant with p53 increases only in CD8+ T-cell blasts, with p21 levels maintained high for longer times than p53 levels. In CD4+ T-cell blasts p21 levels are controlled through a transient and p53-independent mechanism. The present results suggest that the etiology of ALP syndromes could be related not only to defects in apoptosis induction, but also in cell cycle regulation.

Key Words: lymphocyte proliferation • lymphocyte homeostasis • autoimmunity • death receptors


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INTRODUCTION
 
The Fas-FasL pathway plays an important role in the homeostasis of mature lymphocytes by limiting lymphocyte accumulation and minimizing reactions against self-antigens. This was clearly shown in the lymphoproliferative syndromes observed in lpr (lymphoproliferation) or gld (generalized lymphoproliferative disease) mice [1 , 2 ], which carry autosomal recessive mutations in the Fas gene or in the gene encoding FasL, respectively. Defects of the Fas pathway were identified in human patients in the following years, and the diseases associated were termed autoimmune lymphoproliferative syndromes (ALPS) [3 , 4 ]. ALPS associate lymphoproliferative manifestations, such as lymphadenopathies and hepatosplenomegaly with a specific immunological disorder consisting of hypergammaglobulinemia G sometimes associated with hyper IgA and the presence of an expanded population of TcR+CD4–CD8– double-negative T lymphocytes. Autoimmune manifestations are observed in most cases. ALPS (OMIM 601859) are classified according to the underlying genetic defect [5 , 6 ]. In ALPS type 0, a homozygous Fas mutation usually causes a complete deficiency of the Fas protein and a severe form of the disease. ALPS type I is characterized by heterozygous Fas mutation (ALPS type Ia) and more rarely, by heterozygous (ALPS-Ib [7 , 8 ];) or homozygous (ALPS-Ic [9 ];) mutation in the gene for FasL. ALPS type II is characterized by resistance to Fas-mediated apoptosis due to mutations in caspase 10 (ALPS type II). In ALPS type III, the genetic defect is unknown, although some of these patients could carry a recently described mutation in N-Ras [10 ], named ALPS type IV provisionally by the authors.

Our group reported for the first time the possible implication of APO2 ligand/TNF-related apoptosis inducing ligand (Apo2L/TRAIL), another member of the TNF/FasL family, and its receptors DR4 and DR5, in the regulation of the activation of human T cells [11 ]. This was confirmed later on in Apo2L/TRAIL knockout mice, which are more sensitive to the induction of experimental autoimmune diseases [12 ]. However, no reports exist on diseases due to defects in the Apo2L/TRAIL – DR4/DR5 system.

Human T-cell blasts are not sensitive to FasL or Apo2L/TRAIL-induced apoptosis unless they get reactivated [13 14 15 ]. However, our previous studies demonstrated that, in the presence of IL-2, either FasL or Apo2L/TRAIL is able to inhibit the growth of human T-cell blasts in the absence of overt cell death induction [15 ]. This effect was due to a cell cycle arrest in S-G2/M, affecting to either CD4+ or CD8+ T cells, and with a certain specificity of Apo2L/TRAIL for the regulation of CD8+ T-cell blast growth [16 ].

In the present work, we have studied the possible mechanism(s) by which FasL or Apo2L/TRAIL regulate T-cell blast cell cycle, analyzing their effect on the known regulators of the S-G2/M checkpoint, such as p53, p21WAF-1 and cyclin B1 [17 , 18 ]. The results obtained with T-cell blasts from healthy donors and from two different types of ALPS patients [9 , 19 ], which exhibit also different degrees of lymphoproliferation, suggest that the etiology of these syndromes could be not only related to defects in apoptosis induction, but also in cell cycle regulation.


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PATIENTS, MATERIALS, AND METHODS
 
The protocols of these studies involving cell from ALPS patients were approved by the Institutional Review Board of Hospital 12 de Octubre (Madrid, Spain). Informed consent was provided according to the Declaration of Helsinki.

Patients
We characterized previously two different ALPS patients: 1) Patient 1: patient with a new homozygous mutation in the FasL gene (A247E), placed in the extracellular domain of the protein, presumably in the region of interaction with the Fas receptor, which resulted in loss of function. This patient could be considered as the first reported case of ALPS-Ic [9 ]; and 2) Patients 2 and 3: a child and his father with a new heterozygous mutation in the Fas death domain (DD), that are included in the ALPS-Ia group of patients [19 ].

T-cell blast generation
Human peripheral blood lymphocytes (PBL) were obtained from blood of healthy donors or the ALPS patients described by Ficoll-Paque density centrifugation and elimination of adherent cells, and day-6 T-cell blasts were obtained from them by PHA stimulation in the presence of 30 IU/ml of IL-2, as described previously [11 , 15 ].

Analysis of proliferation
The degree of proliferation of T lymphocytes during the process of T-cell blast generation was estimated by labeling with carboxyfluorescein succinimidyl ester (CFSE), as detailed previously [16 , 20 ]. Briefly, fresh PBL were washed twice in PBS and adjusted to 2 x 106/ml in PBS with 1 µM CFSE. Cells were incubated 3 min at room temperature, and the reaction was quenched by adding 200 µl of FBS per ml. Then, cells were incubated 1 min at room temperature and washed twice with PBS; T-cell blasts were generated from these labeled PBL, as indicated above. CFSE staining was determined by flow cytometry in day-6 T-cell blasts, and the number of cycles was calculated from these data.

Treatment of T-cell blasts with increasing doses of IL-2; effects of blocking anti-FasL or anti-Apo2L/TRAIL mAbs
In some experiments, day 6 T-cell blasts obtained from healthy donors were incubated during an additional 48-h period with increasing doses of IL-2 (30 or 300 IU/ml), in the presence of 1 µg/ml of the anti-FasL blocking mAb NOK-1 (PharMingen, Barcelona, Spain) or of 100 ng/ml of the anti-Apo2L/TRAIL-blocking mAb 5C2 (kindly provided by Dr. Avi Ashkenazi, Genentech, South San Francisco, CA). At the end of the incubations, cell extracts were obtained, and immunoblots were performed as indicated below. In the same experiments, cell growth was estimated by counting Trypan blue-negative cells, and results were expressed as N(t)/N(0) (number of viable cells at a given time(t)/number of viable cells at time 0).

Treatment with inhibitors
In some experiments, day 6 T-cell blasts were treated during additional 48 h with the following inhibitors in the presence of 30 UI/ml of IL-2: reduced glutathione (GSH, 5 mM; Sigma, Barcelona, Spain), which supports glutathione peroxidase activities to eliminate cellular H2O2; Ac-YVAD-CHO (YVAD, 300 µM; Bachem, Weil am Rhein, Germany), which inhibits caspase-1 and is used as a negative control of CHO group for caspase inhibitors; Ac-DEVD-CHO (DEVD; 300 µM; Bachem, Weil am Rhein, Germany), which is a specific inhibitor of caspase-3 activity; and L-buthionine-S,R-sulfoximine (BSO; 400 µM; Sigma, Barcelona, Spain), which is a specific inhibitor of Glu cysteine ligase and thus of GSH synthesis [21 ]. In other experiments, day 6 T-cell blasts were treated during 8 h with increasing doses of exogenous H2O2, from 40 nM to 1 µM, in the presence of 30 IU/ml of IL-2. After the treatments, cell extracts were obtained, and immunoblot analysis was performed as indicated below.

Treatment of CD4+ or CD8+ T-cell blasts with agonistic anti-Fas mAb or with recombinant Apo2L/TRAIL
After T-cell blast generation, CD4+ and CD8+ T cells were isolated by negative selection using magnetic cell sorting (MACS; Miltenyi, Bergisch Gladbach, Germany), following the manufacturer's protocol [22 ]. The purity of isolated CD4+ or CD8+ T cells was 90 ± 5%; impurities were never due to T cells of the opposite subset. Isolated CD4+ and CD8+ T-cell blasts were treated during additional periods of time (between 4 h and 72 h) with 100 ng/ml of the agonistic anti-Fas mAb CH11 (Upstate Biotechnology, Lake Placid, NY, USA) or with 500 ng/ml of recombinant Apo2L/TRAIL (rApo2L), in the presence of 30 IU/ml of IL-2. The rApo2L preparation used in this study corresponds to Apo2L.0, in which the monomer form predominates [23 ] and which, in contrast to previous preparations, does not contain a polyhistidine tag promoting aggregation [24 ]. rApo2L was kindly provided by Dr. Avi Ashkenazi (Genentech, South San Francisco, CA, USA). After the treatments, cell extracts were obtained, and immunoblot analysis was performed as indicated below.

Immunoblot analysis
5 x 106 cells were lysed at 4°C in 100 µl of a buffer containing 2% SDS, 20 mM Tris/HCl pH 6.8 and EDTA 1 mM and boiled for 15 min. 1 x 106 lysed cells were analyzed by SDS-PAGE on 12% gels and separated proteins transferred to nitrocellulose membranes. Membranes were then blotted with specific anti-p53 (0,166 µg/ml; Immunotech, Marseille, France), anti-p21 (1 µg/ml, sc-397; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Cyclin B1 (2 µg/ml, sc-245; Santa Cruz Biotechnology) or with anti-β-actin (0.32 µg/ml, clone AC-15; Sigma;) mouse mAb, in the conditions described previously [15 ]. The expression level of the different proteins analyzed was quantified with a densitometer (Bio-Rad, Barcelona, Spain) and normalized to the same amount of β-actin.

Statistical analysis
Mean values were compared using the two-tailed Student’s t test for independent means. Differences were not regarded as significant if P > 0.05.


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RESULTS
 
T cells from an ALPS-Ic patient but not from ALPS-Ia patients show increased lymphoproliferation in vitro
CFSE labeling gets "diluted" to half of its value each time that cells divide, so this method is very useful to estimate the number of cell divisions in a given population [16 , 20 ]. After 6 days in culture, CFSE labeling was analyzed by flow cytometry comparatively in T-cell blasts from healthy donors and in T-cell blasts generated from the ALPS patients analyzed (Fig. 1 ). As shown in Fig. 1A , top, the degree of proliferation of T-cell blasts from the ALPS-Ic patient, with an homozygous mutation in the extracellular domain of FasL (patient 1 [9 ]), was clearly higher than that of T cells from a healthy donor, dividing a mean value of 6 times (Fig. 1B , solid circle). In the experiment shown, the percentage of T cells that divided 5 times or more in the healthy donor was of 31% (25.4±16% as a mean of eight healthy donors), while this population was a major one in T cells from patient 1, arriving to 72.7%. In a previous study, it was observed that the in vitro proliferation rate of T-cell blasts from the ALPS-Ia patient 3, with a heterozygous mutation in the Fas death domain, was also slightly higher than that of the control T cells used in that experiment ([19 ]; compare also solid squares in Fig. 1B ). However, in the new experiment shown in Fig. 1A , bottom, this higher degree of proliferation was not confirmed (compare also solid triangles in Fig. 1B ). The representation of all of the values obtained using T-cell blasts from eight different healthy donors, shown in Fig. 1B , indicates that only the in vitro proliferation rate of T cells from patient 1 (mean divisions, 6) is higher than the mean proliferation obtained in healthy donors (mean divisions, 2.82±0.98), while the pooled values obtained for patients 2 and 3, son and father with the same heterozygous mutation in the Fas death domain (mean divisions 3.62±0.90), are in fact within the range of variation of normal T cells.


Figure 1
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  		Figure 1. T lymphocytes from an ALPS-Ic patient show higher lymphoproliferation in vitro than T lymphocytes from healthy controls or from ALPS-Ia patients. Fresh PBL were isolated from healthy controls or from the patients indicated, labeled with CFSE, day 6 T-cell blasts generated, as indicated in Patents, Materials, and Methods, and then analyzed by flow cytometry. (A) CFSE labeling of day 6 T-cell blasts from two healthy donors (black lines) or either from the ALPS-Ic patient 1 (red line, top) or from the ALPS-Ia patient 3 (green line, bottom) is shown. (B) The mean number of divisions in each case was calculated from the CFSE staining data at day 6. Data are the individual results obtained in T-cell blasts from eight different healthy donors (Control), in one experiment using T cells from patient 1 (Pat. 1; ALPS-Ic) or in two different experiments using T cells from patient 3 (solid triangle and square) and in one experiment using T cells from patient 2 (open triangle; Pat. 2 and 3; ALPS-Ia). Paired control/patient 1 data corresponding to Fig. 1A , top, are shown as solid circles; paired control/patient 3 data corresponding to Fig. 1A , bottom, are shown as solid triangles; paired control/patient 3 data appearing in a previous publication [19 ] are shown as solid squares.

The initial cell populations in each case were different, since an atypical double-negative population (DN) is present in PBL from ALPS patients (25% in patient 1; 12% in patient 2; 5% in patient 3) and also the CD4/CD8 ratio is inverted with respect to normal values (1.4±0.3 as a mean in the eight healthy donors used; 0.64 in patient 1; 0.81 in patient 2; and 0.57 in patient 3 [9 , 19 ]). During the blast generation process, the CD4/CD8 ratio is reduced in T cells from healthy donors due to the higher proliferation of CD8+ compared with CD4+ T cells (0.8±0.15 as a mean in the eight healthy donors used). Regarding ALPS patients, during T-cell blast generation, the DN population was lost in all three patients, in agreement with their reported anergic state [25 ], and the CD4/CD8 ratio did not change so much (0.38 in patient 1; 0.81 in patient 2; and 0.44 in patient 3). Hence, at the blast stage, the point at which experiments were performed, the cell populations in the different samples were not so different, with a higher proportion of CD8+ over CD4+ T cells. In addition, the cell populations present in T-cell blasts from patient 1 or from patient 3 were equivalent, and the increased proliferation was only observed for patient 1.

Results shown in Fig. 1 indicate that FasL is clearly involved in controlling the proliferation rate of human T-cell blasts in vitro. More vigorous antigen-specific proliferation was observed previously in gld mice, especially in CD4+ T cells [26 , 27 ], but no analysis of cell-cycle regulators was performed in those studies.

The process described in human T cells seems death-domain independent, because the proliferation rate of the ALPS-Ia patients studied, in which signal transduction through the Fas death domain is abolished, is within the normal range. However, the ALPS-Ia patients studied do exhibit in vivo lymphoproliferation, and this could be related to the increased proliferation of memory T cells that survive activation-induced cell death (AICD), as demonstrated in our previous study [19 ].

T-cell blasts from an ALPS-Ic patient but not from ALPS-Ia patients show reduced levels of p53 and p21WAF-1
Our previous studies demonstrated that, in the presence of IL-2, either FasL or Apo2L/TRAIL inhibit the growth of human T-cell blasts in the absence of overt cell death induction, due to a cell cycle arrest in S-G2/M [15 , 16 ]. The main regulator of the G2/M transition is the protein p21WAF-1, which binds to cyclin B1 and inhibits CDK1 activity. The induction of p21WAF-1 expression results in cell cycle arrest in the G2/M and G1/S transitions [17 ]. On the other hand, it has been demonstrated that p21WAF-1 is important in the regulation of T-cell proliferation, at least in the secondary response of memory T cells and that p21WAF-1 knockout mice have a tendency to develop autoimmune disease in greater or lesser extent, depending on their genetic background [28 , 29 ]. p21 induction can be mediated through p53-dependent and -independent mechanisms [18 ]. In addition, the late-onset degradation of cyclin-B1 through a p53-dependent, p21WAF-1-independent mechanism has been also described [30 , 31 ].

As a consequence, protein levels of p53 and p21WAF-1 were analyzed on T cells from healthy donors and from the ALPS patients described during the process of blastic transformation. As shown in the representative experiment of Fig. 2A , levels of p21 and of p53 increased after blastic transformation in healthy donors and in the ALPS patients, indicating that the proliferation of T cells is a tightly regulated process. However, although the levels of these proteins were the same in T-cell blasts from the healthy donors or from the ALPS-Ia patient 2, they were significantly reduced in T-cell blasts from the ALPS-Ic patient 1. The quantification of the percentage of p53/β-actin and p21/β-actin ratios in T-cell blasts from the patients with respect to those obtained in T-cell blasts from healthy donors in the three experiments performed is shown in Fig. 2B , showing consistent reductions of 39 ± 0.2% and 38 ± 1.4% in the p21/β-actin or p53/β-actin ratios in T-cell blasts from patient 1 (P<10–6). This result correlates with the increased in vitro lymphoproliferation observed in T-cell blasts from this patient in comparison with the ALPS-Ia patients and with the normal controls shown in Fig. 1 .


Figure 2
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  		Figure 2. p53 and p21 levels in T lymphocytes from healthy donors and from ALPS-Ic and ALPS-Ia patients. (A) Anti-p53, p21 or β-actin immunoblots were performed on extracts from fresh PBL (day 0) or day 6 T-cell blasts (day 6), obtained from healthy donors (Control) or from the ALPS-Ic patient 1 or the ALPS-Ia patient 2, as indicated. Two different anti-p21 immunoblots are shown. The positions of p53, p21, or β-actin are indicated on the left of the corresponding blots. The extracts used correspond to 1 x 106 cells, and expression levels of the proteins analyzed were quantified in a densitometer and normalized to the same amount of β-actin. Results are representative of three different experiments. (B) The results obtained for the p53/β-actin and p21/β-actin ratios in cells from the patients with respect to those obtained in cells from healthy donors are shown. Results are expressed as a percentage of the control and are the mean ± SD of the three different experiments indicated above. ***, P < 10–6.

Blocking of death ligands in T-cell blasts from healthy donors reduced the expression levels of p21WAF-1
In human T-cell blasts, the plasma membrane expression of FasL or Apo2L/TRAIL is extremely low, and the proteins are stored in cytoplasmic multivesicular bodies, being secreted in their bioactive form associated with the internal exosomes upon an additional activation [32 , 33 ]. In a previous work, it was shown that incubation of human T-cell blasts with increasing amounts of IL-2 induced a reduction of their proliferation rate and that specific blocking of FasL prevented this reduction. This regulatory effect was due to the secretion of bioactive FasL associated with exosomes, which is increased by high doses of IL-2 [34 ]. We extend here this study to Apo2L/TRAIL. Day 6 T-cell blasts from healthy donors were cultured for an additional 48-h period in the presence of increasing doses of IL-2 (30 or 300 IU/ml) and in the presence or absence of the blocking anti-FasL mAb NOK-1 and/or the blocking anti-Apo2L/TRAIL mAb 5C2. In these experiments, the higher proliferation rate was obtained with 30 IU/ml of IL-2, since the N(t)/N(0) value obtained was around 1.5 as a mean at 48 h, indicating that around 50% of cell growth was observed. However, growth in the presence of 300 IU/ml was always lower (no more than 20% of growth in 48 h). Culture in the presence of the anti-FasL and/or anti-Apo2L/TRAIL-blocking antibodies increased cell growth in the presence of 30 IU/ml, while the anti-FasL mAb restored growth to exactly the same level in the cultures made in the presence of 300 IU/ml, with a partial effect of the anti-Apo2L/TRAIL mAb (Fig. 3A ). Hence, IL-2 favors T-cell blast growth control by mechanisms dependent on FasL and/or Apo2L/TRAIL.


Figure 3
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  		Figure 3. Effect of FasL and Apo2L/TRAIL-blocking antibodies on the growth of T-cell blasts from healthy donors. Relationship with p21 and p53 levels. (A) Day 6 T-cell blasts were cultured for an additional period of 48 h in the presence of 30 or 300 IU/ml of IL-2, as indicated, in the presence or absence (solid bars) of either 1 µg/ml of the anti-FasL-blocking mAb NOK-1 (open bars), 100 ng/ml of the anti-Apo2L/TRAIL blocking mAb 5C2 (gray bars) or a combination of both (hatched bars) and cell growth estimated by counting Trypan blue-negative cells. Results are expressed as the ratio between the number of viable cells at 48 h (N(t)) and the number of viable cells at time 0 (N(0)). Results are the mean ± SD of experiments performed with three different donors. *, P < 0.05; **, P < 0.03. Statistical significance in each sample was calculated with respect to results obtained in control cells (solid bar). (B) After the incubations described, cell extracts were obtained and anti-p53, anti-p21, or anti-β-actin immunoblots performed. The extracts used correspond to 1 x 106 cells, and the expression level of p21 and p53 were quantified in a densitometer and normalized to the same amount of β-actin. The immunoblot shown is representative of experiments performed with T-cell blasts from the same three healthy donors used in A. (C) The results obtained for the p21/β-actin and p53/β-actin ratios after the different treatments with respect to those obtained in untreated cells are shown. Results are expressed as a percentage of the control and are the mean ± SD of the three different experiments indicated above. *, P < 0.05.

However, no net reduction was observed in p21 levels at 30 IU/ml of IL-2 (P>0.05). The effects on cell growth were accompanied by net reductions in p21 levels at the high IL-2 concentration and especially when both blocking antibodies were used in combination, as shown in the representative immunoblot analysis of Fig. 3B and in the quantifications shown in Fig. 3C : a 52 ± 5% of reduction as a mean in the p21/β-actin ratio (P<0.05). However, there was no effect on p53 levels (Fig. 3B and 3C , bottom; P>0.05), suggesting the predominance of a p21-dependent, p53-independent mechanism, at least when a mixed T-cell blast population is used.

FasL and Apo2L/TRAIL regulate cell cycle progression in human T-cell blasts by caspase activation and H2O2 generation
The "classical" signal transduction pathway activated by the ligation of death receptors, such as Fas/CD95, DR4, or DR5, implicates the intracellular cascade of caspase activation [35 , 36 ]. Recently, it has been demonstrated that at least Fas ligation in human T-cell blasts is also associated with the caspase-independent production of a moderate level of reactive oxygen species, specifically hydrogen peroxide, through the activation of NADPH oxidases [34 , 37 ]. Hence, caspase inhibitors and exogenous GSH, which supports GSH peroxidase activity, the main H2O2-depleting enzyme [38 ], were used to inhibit each of these pathways, respectively. As shown in the representative experiment of Fig. 4A , the caspase-3 inhibitor Ac-DEVD-CHO did not affect p53 levels more than the irrelevant caspase-1 inhibitor Ac-YVAD-CHO at the low IL-2 dose of 30 IU/ml. At the higher IL-2 dose, Ac-DEVD-CHO induced a reduction in p53 levels when compared with cells treated with Ac-YVAD-CHO. This reduction was of 38 ± 4% as a mean of the two experiments performed (Fig. 4B , left), but it did not yield a significant result. On the other hand, GSH did not affect p53 levels more than the irrelevant inhibitor Ac-YVAD-CHO and did not increase the effect of Ac-DEVD-CHO when used in combination (Fig. 4A and 4B) .


Figure 4
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  		Figure 4. Hydrogen peroxide and caspases are involved in the control of p53 and p21 expression in human T-cell blasts. (A) Day 6 T-cell blasts were cultured with 30 or 300 IU/ml of IL-2 for an additional period of 48 h in the absence (–) or presence of either 5 mM of reduced L-glutathione (GSH), 300 µM of Ac-DEVD-CHO (DEVD), or of the irrelevant Ac-YVAD-CHO (YVAD), or a combination of GSH and DEVD, as indicated. Cell extracts were obtained, and anti-p53, anti-p21, or anti-β-actin immunoblots were performed. The extracts used correspond to 1 x 106 cells, and the expression level of p21 and p53 were quantified in a densitometer and normalized to the same amount of β-actin. Results are representative of experiments performed with T-cell blasts from two different healthy donors. (B) The results obtained for the p21/β-actin and p53/β-actin ratios after the different treatments with respect to those obtained in untreated cells are shown. Results are expressed as a percentage of the control and are the mean ± SD of the two different experiments indicated above. *, P < 0.05; **, P < 0.03. (C) Cell growth was estimated in the same experiments by counting Trypan blue-negative cells. Results are expressed as the ratio between the number of viable cells at 48 h (N(t)) and the number of viable cells at time 0 (N(0)) and are the mean ± SD of experiments performed with the same two healthy donors. *, P < 0.05; **, P < 0.03. Statistical significance in each sample was calculated with respect to results obtained in control cells (solid bar).

In the same representative experiment, these inhibitors had significant effects on p21 levels, as shown in Fig. 4A and 4B , indicating again a predominance of p21-mediated mechanisms. At the low IL-2 dose of 30 IU/ml, only the combination of GSH+DEVD resulted in a significant reduction of p21 levels (60±4% of reduction as a mean, P<0.05; Fig. 4B , right). When the high IL-2 dose of 300 IU/ml was used, both GSH and Ac-DEVD-CHO did reduce p21 levels to extremely low levels (around 80% of reduction in the p21/β-actin ratio, P<0.03; Fig. 4B , right). Cell growth was estimated in parallel in these experiments, and results are shown in Fig. 4C . Either GSH or DEVD increased cell proliferation in the presence of the high IL-2 concentration from 45% to around 70%, but only the combination of both agents resulted in a statistically significant increase (90% of proliferation; P<0.05). The increase in proliferation observed using GSH+DEVD approached that observed by using the blocking anti-FasL mAb NOK-1 (110% of proliferation; P<0.03). The specificity of the caspase inhibitors used has been demonstrated previously [39 ] and was corroborated in this study by immunoblot analysis of inhibition of caspase processing (data not shown).

These results suggest that caspase activation and H2O2 generation are implicated in the regulation of p21 expression, with more clear results at high IL-2 concentrations. To confirm these data, especially regarding H2O2 generation, we also used L-buthionine-S,R-sulfoximine (BSO), a specific inhibitor of glutathione synthesis [21 ] and exogenous GSH in similar experiments. In the representative experiment shown in Fig. 5A , top, BSO increased p21 levels over that observed in control T-cell blasts, while GSH reduced again the level of this protein. The increase induced by BSO or the decrease induced by GSH were significant, as shown in the quantification of Fig. 5A , middle: 21 ± 1% of increase and 37.5 ± 10% of decrease respectively, as a mean of the three experiments performed (P<0.03). The increase in p21 levels induced by this dose of BSO was associated with an almost complete inhibition of cell growth in the absence of cell death. However, GSH had no significant effect on cell growth in these experiments (Fig. 5A , bottom). GSH and BSO had an important impact in the blast generation process, as shown in Fig. 5B . While GSH increased the proliferation rate of activated T cells, increasing the percentage of cells that divided 1, 2, 3, and 4 times and the mean number of divisions by 30%, BSO retarded proliferation without inducing cell death, increasing the percentage of cells that did not divide from 50% in the control to 76%, and decreasing the mean number of division by 65%. The effects of GSH or of BSO on the levels of H2O2 in human T-cell blasts was determined by DCF-DA fluorescence and flow cytometry in previous studies [34 ] and corroborated also in the experiments performed in this study (data not shown).


Figure 5
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  		Figure 5. Regulation of p21 expression by H2O2 in human T-cell blasts from healthy donors and from the ALPS-Ic patient. (A) Day 6 T-cell blasts were cultured for an additional period of 48 h with 30 IU/ml of IL-2 in the absence (C) or in the presence of either 400 µM BSO or 5 mM GSH, as indicated. Extracts from day 6 T-cell blasts before the treatments were also analyzed for comparison. After the treatments, extracts were obtained and anti-p21 or β-actin immunoblots performed (top). The extracts used correspond to 1 x 106 cells, and the expression level of p21 was quantified in a densitometer and normalized to the same amount of β-actin. The immunoblot shown is representative of experiments performed with T-cell blasts from three different healthy donors. The results obtained for the p21/β-actin ratio after the different treatments with respect to those obtained in untreated cells are shown (middle). Results are expressed as a percentage of the control and are the mean ± SD of the three different experiments indicated. **, P < 0.03. Cell growth was estimated in parallel by counting Trypan blue-negative cells (bottom). Results are expressed as the ratio between the number of viable cells at 48 h (N(t)) and the number of viable cells at time 0 (N(o)). Results are the mean ± SD of experiments performed with the same three donors. **, P < 0.03. Statistical significance in each sample was calculated with respect to results obtained in control cells (solid bar). (B) Fresh PBL were isolated from a healthy donor and labeled with CFSE; day 4 T-cell blasts were generated in the presence or absence (Control, solid histograms) of 5 mM GSH (green histogram) or 400 µM BSO (red histogram) and then analyzed by flow cytometry. Numbers in the histograms indicate the mean number of divisions calculated from the CFSE labeling data in each case. (C) Day 6 T-cell blasts from the ALPS-Ic patient 1 were cultured with 30 IU/ml of IL-2 for 8 h in the absence (–) or in the presence of 1 µM H2O2. Cells were collected, and immunoblots against p21 and β-actin were performed.

We reasoned that the same effect of BSO on p21 could be imitated by adding controlled amounts of H2O2. We performed a dose-response of H2O2 on human T-cell blasts and observed that an increase on p21 levels could be already observed at 40 nM and that it did not induce cell death in 8-h experiments until doses of 1 µM were reached, but that at higher doses, cell death was evident (data not shown). Then, we treated T-cell blasts from the ALPS-Ic patient with 1 µM H2O2 for 8 h and observed a net increase in their low p21 levels (Fig. 5B) . These results suggest for the first time that controlled pro-oxidant treatments could be beneficial for lymphoproliferation control in these patients.

Studies in separated CD4+ and CD8+ populations of human T-cell blasts
To study the changes in the expression of cell cycle regulators, separated CD4+ and CD8+ populations of human T-cell blasts were treated with the agonistic anti-Fas mAb CH11 or with recombinant Apo2L/TRAIL (rApo2L) in the presence of exogenous IL-2 for several time periods, from 4 to 72 h, as indicated in our previous studies [15 , 16 , 34 , 40 ]. After these incubation periods, the expression of p21, p53, or cyclin B1 was analyzed by immunoblot and quantified as a percentage of the expression of each protein in untreated cells at time zero, as shown in Fig. 6 .


Figure 6
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  		Figure 6. Effect of the agonistic anti-Fas mAb CH11 and of recombinant Apo2L/TRAIL (rApo2L) on p21, p53, and cyclin B1 levels in CD4+ or CD8+ T-cell blasts. (A–D) Day 6 CD4+ or CD8+ T-cell blasts were purified by MACS, as indicated in the experimental procedures. After isolation, cells were either lysed (C) or cultured with 30 IU/ml of IL-2 for different time periods (4, 24, 48, or 72 h, as indicated) in the absence or in the presence of either 100 ng/ml of the cytotoxic anti-Fas mAb CH11 or 500 ng/ml of rApo2L, as indicated. After the incubations, cells were collected, and immunoblots against p21 (A for CD4+; B for CD8+), p53, cyclin-B1 (C for CD4+; D for CD8+) and β-actin (A–D) were performed. Expression levels were quantified in a densitometer and normalized to the same amount of β-actin. The immunoblots shown are representative of experiments performed with T-cell blasts from two different healthy donors. The results obtained for the p21/β-actin ratios (A and B) or for the p53/β-actin and cyclin-B1/β-actin ratios (C and D) after the different treatments with respect to those obtained in untreated cells at time zero are shown. Results are expressed as a percentage of the control and are the mean ± SD of the two different experiments indicated. *, P < 0.05; **, P < 0.03. (E) Cell growth was estimated in parallel by counting Trypan blue-negative cells. Results are expressed as the ratio between the number of viable cells at the different times indicated (N(t)) and the number of viable cells at time 0 (N(0)). Results correspond to the mean ± SD of the two experiments performed. *, P < 0.05; **, P < 0.03. Statistical significance in each sample was calculated with respect to results obtained in control cells at each time point (open circles).

In agreement with the observations made when blocking endogenous FasL and/or Apo2L/TRAIL in the mixed population (see Fig. 3 ), which resulted in reductions in p21 levels, either CH11 or rApo2L induced a clear increase in p21 expression in the CD8+ subpopulation at all times assayed, arriving at a maximum 67 ± 6% increase of the p21/β-actin ratio at 24 h for both agents (P<0.05; Fig. 6B ). In the CD4+ subpopulation, a similar significant increase was observed at 4 h of treatment, but it was not observed at longer times (Fig. 6A) .

Regarding p53, we could only observe an increase with respect to the control in CD8+ T cells (Fig. 6D) . After 24 h of culture in IL-2, p53 levels somewhat decreased, but Fas ligation caused these levels to be elevated (25±8% of increase of the p53/β-actin ratio with respect to time zero), and treatment with rApo2L significantly increased them (44±4% of increase of the p53/β-actin ratio with respect to time zero; P<0.05). This increase at 24 h preceded a drastic reduction in the levels of cyclin B1 at 48 h, especially again in the case of treatment with rApo2L (43±3% and 84±2% of reduction in the cyclin B1/β-actin ratio by treatment with CH11 or rApo2L, respectively, P<0.03; Fig. 6D ). This p21-independent, p53-dependent mechanism of cell cycle arrest was described previously [30 , 31 ]. Regarding CD4+ human T cells, significant increases in p53 levels at 24 h and reductions of cyclin B1 levels at 48 h are already observed in the untreated controls cultured in the presence of IL-2, without additional effects of CH11 or rApo2L (Fig. 6C) .

Cell growth was estimated in parallel in the same experiments, with results in agreement with the previous reports on the preferential effect of rApo2L on the IL2-dependent growth of the CD8+ subset at 24 h (no effect on CD4+ T cells; 63±14% inhibition of cell growth on CD8+ T cells [16 ]), and with Fas ligation inhibiting growth almost completely in both subsets at this time point. At 48 h, either anti-Fas or rApo2L inhibited CD4+ T-cell growth around 50%, while anti-Fas reduced CD8+ T-cell growth by 38 ± 5% and rApo2L by 55 ± 5%.


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DISCUSSION
 
Several p53-dependent mechanisms have been described to explain G2/M cell cycle arrest in response to genotoxic stress [18 ]. One of them is the decrease in the levels of cyclin B1 [30 , 31 ]: a decrease in the levels of this protein results in inhibition of the cyclin B1-dependent kinase (CDK1 or Cdc2), necessary for the G2 transition to mitosis. In our case, this process has been observed especially after ligation of DR4 or DR5 only in CD8+ T-cell blasts. The observation that Apo2L/TRAIL activated this mechanism preferentially in CD8+ T-cell blasts is the first indication to explain the preferential effect of Apo2L/TRAIL on cell cycle progression of this subset of T cells [16 ], something that also has implications in the maintenance of memory CD8+ T cells [41 ].

On the other hand, one of the main negative regulators of cell cycle progression is the protein p21WAF-1. An increase in the levels of this protein inhibits the activity of the cyclin-dependent kinases and specifically that of CDK1 [42 ]. Several p53-dependent and -independent mechanisms for the induction of p21 expression have been proposed [17 , 18 ]. In our case, p21 induction is clearly observed after Fas/CD95, DR4, or DR5 ligation in human T-cell blasts in both T-cell subpopulations (Fig. 6) . In addition, p21 expression is reduced by blocking endogenous FasL and/or Apo2L/TRAIL in the mixed population (Fig. 3) and seems to constitute the main regulatory mechanism. Previous reports indicated that p21 knockout female mice on a 129/Sv X C57BL/6 mixed background developed a lupus-like disease [28 ]. However, later studies made in p21 knockout mice on the lupus-prone genetic background BSXB did not support this conclusion [43 ]. More recent studies made in p21 knockout mice on the C57BL/6 genetic background, which is not prone to autoimmunity, supported a role of p21 in the regulation of T-cell proliferation, at least in secondary responses of memory T cells, with a tendency to develop autoimmunity with age, although to a lesser extent than 129/Sv X C57BL/6 mice [29 ]. However, the connection with FasL or Apo2L/TRAIL signaling reported here was not characterized in those studies.

The data obtained in the present work would indicate that p21 induction by FasL and/or Apo2L/TRAIL follows two different pathways: a fast and transient (4 h), p53-independent mechanism that seems to predominate in the CD4+ subpopulation, and a p53-dependent mechanism, with maximal p53 and p21 induction at 24 h, but with p21 levels maintained high for longer times in CD8+ T cells. It has been reported that CD4+ murine T-cell blasts were sensitive to Fas-induced cell death, while CD8+ T-cell blasts were not [44 ]. This result was confirmed by us for human T-cell blasts, but in the absence of IL-2 during the assays [40 ]. In the presence of IL-2, however, the effect of Fas ligation is similar between both subsets, cell growth inhibition instead of cell death induction [16 ]. The results obtained in CD4+ T-cell blasts are in agreement with a previous study performed in mouse thymocytes, in which it was demonstrated that FasL induced p21 expression in a p53-independent fashion, although in that study the net result of FasL treatment was cell death induction [45 ].

In addition, Fas ligation in human T-cell blasts will result in the caspase-independent, reactive oxygen species (ROS)-dependent induction of Foxo3a expression and consequently in the expression of the proapoptotic member of the Bcl-2 family Bim. This would sensitize T-cell blasts to deletion by cytokine deprivation, one of the main mechanisms of down-regulation of T-cell responses [34 ].

The "classical" signal transduction pathway activated by the ligation of death receptors, such as Fas/CD95, DR4 or DR5, implicates the intracellular cascade of caspase activation [35 , 36 ]. Recently, it has been demonstrated that at least Fas ligation in human T-cell blasts is also associated with the caspase-independent production of a moderate level of ROS, specifically hydrogen peroxide, through the activation of NADPH oxidases [34 , 37 ]. Experiments shown in Figs. 4 and 5 indicate that, especially at high IL-2 concentrations, in which growth regulation by endogenous FasL and/or Apo2L/TRAIL are more evident (see Fig. 3A ), p21 levels are maintained high through either a caspase- or a ROS-dependent mechanism. Of course, if the signal is strong enough, or if T-cell blasts are undergoing a reactivation through their antigen receptor [13 , 15 ], the caspase-dependent apoptotic cell death of these T cells will finally proceed.

The main conclusion of this study is that multiple and concomitant mechanisms are acting in the control of cell cycle progression of human T-cell blasts by FasL and Apo2L/TRAIL, suggesting a complex picture that is not completely understood. On the other hand, the biochemical mechanisms connecting the ligation of their receptors with the cell cycle control mechanisms described are not known.

The present work could have a certain clinical relevance in the understanding of the etiology of the different types of ALPS and probably of other autoimmune diseases. It is clear that the ALPS-Ic patient [9 ], or the other case reported with a heterozygous deletion in FasL (ALPS-Ib [7 ]), developed a much more severe disease than ALPS-Ia patients [6 , 19 ]. The T-cell blasts of at least the two ALPS-Ia patients used in this study, although unable to activate the Fas-induced, FADD and caspase-dependent apoptotic pathway, are still able to activate the Fas-induced, NADPH oxidase-dependent generation of discrete levels of hydrogen peroxide [34 ]. On one hand, this allows the induction of Bim expression in those ALPS-Ia patients, but not in the ALPS-Ic patient, sensitizing or not, respectively, activated T-cell blasts to death by cytokine deprivation [34 ]. On the other hand, the maintenance of this signal transduction pathway results in the induction of the same elevated levels of p21 in T-cell blasts from the ALPS-Ia patients as in T-cell blasts from healthy donors, while they are clearly reduced in T-cell blasts from the ALPS-Ic patient (Fig. 2) . This reduction in p21 levels correlates with the exacerbated lymphoproliferation observed in vitro in those T-cell blasts (Fig. 1) and can be corrected with controlled pro-oxidant treatments, which also modulate lymphocyte proliferation (Fig. 5) . Another possibility would be that in heterozygous ALPS-Ia patients in which apoptotic Fas signaling is abolished, this alternative signal transduction pathway was maintained, such as it has been described for the activation of NF-{kappa}B [46 ], while the homozygous mutation in FasL observed in the ALPS-Ic patient would completely abolish all signal transduction due to Fas ligation. However, the experiments performed with T-cell blasts from healthy donors and inhibitors indicate that an H2O2-dependent, caspase-independent pathway exists, which is able to regulate p21 levels. Hence, it could be that this signal transduction pathway, which is maintained in ALPS-Ia patients, would be enough to guarantee elevated p21 levels in T-cell blasts and to prevent, at least in vitro, the exacerbated lymphoproliferation observed in T-cell blasts from the ALPS-Ic patient. The in vivo outcome is normally more complex, and lymphoproliferation observed in ALPS-Ia patients could be not only related to defects in apoptosis induction, but also to defects in the caspase-dependent pathway described. In addition, it could be also related to the increased proliferation of T cells that survive activation-induced cell death (AICD) in these ALPS-Ia patients, as demonstrated in our previous study [19 ]. T cells surviving AICD exhibit a memory phenotype and are resistant to apoptosis induction through c-FLIPS and Bcl-xL up-regulation [15 ].

Previous studies have demonstrated the capability of FasL to reverse signaling in T cells. Although in one study, it was shown that this reverse signaling resulted in toxicity on primary CD4+ murine T cells [47 ], the final consensus in either murine or human T cells has been that this is, in fact, a costimulatory signal during the activation of naïve T cells [44 , 48 ]. However, if this were the predominant effect of FasL and not the ligation of Fas, the reported mutations in FasL observed in gld mice and in ALPS-Ib and -Ic patients would result in a proliferation defect and not in lymphoproliferation, as it is the case [7 8 9 , 49 ].

The etiology of ALPS is exclusively associated with defects in Fas-induced apoptosis. Our previous studies have demonstrated that, at least in normal human T-cell blasts, FasL and Apo2L/TRAIL are also able to regulate T lymphocyte proliferation in the absence of cell death induction [15 , 16 , 34 ]. This observation should be also taken into account to explain the etiology of the different types of ALPS, especially when the main clinical manifestation of these diseases is lymphoproliferation. These observations could be also relevant for the understanding of other autoimmune diseases, in which defective signalization through FasL or Apo2L/TRAIL or their respective receptors may be implicated.


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ACKNOWLEDGEMENTS
 
This work was supported by grants SAF2004-03058 and SAF2007-65144 from Ministerio de Educación y Ciencia/Fondo Social Europeo (Spain) to A. A., A. B., and J. I. A. were supported by a FPU and a FPI fellowships, respectively, from Ministerio de Educación y Ciencia. We gratefully acknowledge Dr. Avi Ashkenazi, Genentech, Inc. (South San Francisco, CA, USA) for rApo2L and anti-Apo2L/TRAIL mAb 5C2 and Kermit Macpherson, University of Zaragoza, for editing the article for English.


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FOOTNOTES
 
1 Current address: Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA. Back

Received January 17, 2008; revised March 31, 2008; accepted April 14, 2008.


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