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CHEMOKINES
From the National Institute of Health and Medical
Research, University of Méditerranée and Department of
Hematopathology, Institut Paoli-Calmettes, Marseille, France.
Clonal expansion of activated T cells is controlled by homeostatic
mechanisms leading to cell death of a large proportion of the cells.
The CD3/TcR pathway induces cell death, mostly when triggered
in the absence of costimulatory signal. The unique T cell-specific
chemokine of the C class, lymphotactin (Lptn), has recently been shown
to inhibit the production of Th1-type lymphokines in human
CD4+ T cells. The present study shows the ability of Lptn
to costimulate the death of CD4+ T lymphocytes triggered
through CD3/TCR. The Lptn-mediated increased cell death exhibited
characteristic features of apoptosis, as mainly determined by DNA
fragmentation and exposure of an apoptotic-specific mitochondrial
antigen. This apoptosis was dependent on Fas/FasL signaling, was not
rescued by addition of interleukin 2, and proceeded with a predominant
processing of both initiator procaspase-9 and effector procaspase-7.
These caspase activities were further evidenced by specific cleavage of
poly(ADP-ribose) polymerase (PARP) and CD3/TCR Chemokines play a crucial role in immune and
inflammatory reactions by causing both the recruitment and activation
of leukocytes.1,2 With the exception of lymphotactin
(Lptn), most chemokines share a conserved 4-cysteine motif in their
N-terminus. Based on the arrangement of this motif and on both genetic
and functional criteria, chemokines are subdivided into 4 subfamilies,
designated as CXC (or In physiologic situations, peripheral T cells respond to CD3/TCR
complex triggering by proliferating, producing cytokines and
receptors, undergoing death, or becoming anergic. A variety of factors,
including the type of antigen, the magnitude of TCR stimulation, the
coordinate engagement of costimulatory molecules such as CD28, or the
production of T-cell growth factors such as interleukin 2 (IL-2), are
known to influence the destiny of T cells. IL-2 or CD28 signals
cooperate with TCR engagement to induce optimal T-cell clonal
expansion, production of cytokines,25,26 including CC
chemokines,27 and expression of cytokine receptors such as
IL-2 receptor (IL-2R).28,29 The lack of these
costimulatory signals during TCR triggering results in anergic or
apoptotic response of T cells.
Apoptosis is an active form of cell death that is crucial to maintain
self-tolerance of the peripheral immune system, by down-regulating the
cellular expansion of activated T cells.30,31 Among
molecular mechanisms involved in this process, the Fas/Fas ligand
(FasL) system is implicated as a major mediator.32 FasL
can be cleaved from the cell surface via a metalloproteinase, releasing
a soluble form.33,34 This latter form (sFasL) has been
shown not only to be less active than the membrane-bound form (mFasL),
regarding their proapoptotic activity,35 but also to
inhibit the activity of mFasL.36 The apoptotic program
includes characteristic morphologic and biochemical changes. Caspases
(cysteine aspartate-specific proteases) are critical mediators of
apoptosis characterized by their ability to cleave their substrates
after aspartic residues.37 Caspase activity results in the
cleavage of several cellular substrates38 such as the
poly(ADP-ribose) polymerase (PARP),39-41 the DNA
fragmentation factor 45 (DFF45),42,43 the CD3/TCR
Our recent work provided evidence that Lptn was optimally produced in
response to TCR engagement alone, in the absence of CD28 costimulation,
in human CD4+ T cells.46 This expression was
accompanied by an inhibitory effect of Lptn on CD4+ T cell
proliferation through the inhibition of Th1-type lymphokine production.24 In the work reported herein, we describe the
enhancement of TCR-induced apoptosis of CD4+ T cells by
Lptn, which might also explain the reported inhibition of
proliferation.24 This increase in apoptosis occurred
through Fas/FasL interaction as supported by complete inhibition in the presence of antagonist anti-Fas monoclonal antibody (mAb), by up-regulation of mFasL, and down-regulation of sFasL expression, both
induced by Lptn. This Lptn-mediated apoptosis was mostly dependent on
Fas/FasL signaling, was not completely rescued by addition of IL-2, and
proceeded with a strong processing of the initiator procaspase-9 and
the effector procaspase-7. These Lptn-induced caspase activities
resulted in specific cleavage of PARP, CD3/TCR CD4+ T-cell purification and activation
Proliferation assays
Cell viability and hypodiploid assessment The CD4+ T cells were activated in 24-well plates for the indicated days, as described above. Trypan blue exclusion and propidium iodide (PI) staining were assessed by light microscopy and flow cytometry, respectively. For PI assays, the cells were stained for 30 minutes at 4°C with a fluorescein isothiocyanate ( FITC)-conjugated anti-CD4 mAb, or a nonbinding matched isotype control mAb (Immunotech), washed twice in phosphate-buffered saline (PBS), and then fixed in PBS plus ethanol 70% (1 vol/5 vol) at 20°C. After
washing, they were resuspended in PI/RNAse A (10µg/250µg/mL) buffer
and incubated 30 minutes at 37°C. Samples were analyzed using a
Becton Dickinson (Mountain View, CA) FACScan cytometer. Ten thousand events were acquired and dead cells were excluded using forward- and
side-scatter gating. Percent of cells was determined in the different
phases of cell cycle, as previously described.47
Immunofluorescence staining and flow cytometric analysis (fluorescence-activated cell sorter) For estimation of surface expression of Fas, cells were stained with FITC-conjugated anti-Fas mAb (clone UB2) or a nonbinding matched isotype control mAb (both from Immunotech).The expression of 7A6 antigen, a mitochondrial membrane protein that is exposed on cells undergoing apoptosis, was analyzed by staining with a PE-coupled Apo2.7 mAb (clone 2.7A6A3, Immunotech), after activation of the cells followed by digitonin permeabilization, as recommended by the manufacturer. Fluorescence-activated cell sorter (FACS) analyses were performed as described above. In some experiments, prior to activation, cells were either left untreated or pretreated at 37°C for 15 minutes with pertussis toxin (PT), an inhibitor of G protein-coupled receptor signal transduction. Enzyme-linked immunosorbent assay detection of mFasL and sFasL Quantitative determination of human FasL protein in both lysates and supernatants from activated CD4+ T cells was performed with a commercial "sandwich" enzyme immunoassay using mouse mAbs (Oncogene Research Products, France Biochem), according to the manufacturer's instructions. Lysates and supernatants were prepared or collected, respectively, from the activated cells harvested at different times after activation between 3 and 120 hours, and were then frozen at80°C until analysis. Serial dilutions of the supernatants
and lysates were performed to ensure measuring in the linear range, and
the sensitivity of the enzyme-linked immunosorbent assay (ELISA) assay
was 0.02 ng/mL.
DNA fragmentation analysis DNA fragmentation was detected by the TUNEL method using an in situ cell death detection kit (Boehringer Mannheim, Roche Diagnostics, Meylan, France) according to the manufacturers' instructions. Briefly, CD4+ T cells were stimulated for overnight to 4 days, as described above. They were then daily harvested and washed twice in PBS. After centrifugation, cell pellets were resuspended in 200 µL PBS containing 4% paraformaldehyde for 1 hour at room temperature, and washed twice again. Permeabilization was conducted using 100 µL 0.1% Triton X-100 and 0.1% sodium citrate in PBS. After washing, cells were resuspended in TUNEL reaction mixture containing FITC-dUTP and terminal-deoxy-nucleotidyl-transferase (TdT). Control cells were incubated in TUNEL reaction mixture containing FITC-dUTP without TdT. Incubation was performed for 1 hour at 37°C before washing cells twice. Fluorescein labels incorporated in DNA strand breaks were detected by flow cytometry, as described above.Western blots The CD4+ T cells (2 × 106/point) were harvested at different times between 3 and 120 hours after stimulation and washed twice in PBS. Pellets were boiled 5 minutes in lysis buffer (10 mM Tris-HCl, pH 7.4, containing 1% sodium dodecyl sulfate [SDS] and 1 mM sodium vanadate), and then frozen at20°C. Protein lysates
were separated by appropriate percentages of SDS-polyacrylamide gel
electrophoresis (PAGE) and transferred to Immobilon-P membranes
(Millipore, Bedford, MA) as recommended by the manufacturer. Membranes
were probed with the following antibodies directed against caspase-3
(MBL, France Biochem, Nagoya, Japan), caspase-4 (Immunotech),
caspases-7, -8, -9 (MBL), DFF45 (MBL), (TCR)  -chain (6B10.2;
Santa Cruz Biotechnology, Santa Cruz, CA), PARP (Chemicon
International, Euromedex, Souffleweyersheim, France), that were
used according to the manufacturer's instructions. The protein bands
were detected by enhanced chemiluminescence (ECL, Amersham Life
Science,) at different times. To ensure that the expression of
the different proteins could be compared between the samples, blots
were reprobed with a polyclonal antibody directed against the p85
subunit of phosphatidyl-inositol-3-OH kinase (UBI, Euromedex), which
displays a uniform level of expression in T lymphocytes (not shown).
The intensity of bands was quantified using a laser densitometer
(Ultroscan LKB, Pharmacia). The level of expression of a
specific band was given as the ratio (optical density of the
band/optical density of p85) and normalized results were expressed in
arbitrary units.
Caspase-9 colorimetric assay The enzymatic activity of caspase-9 was determined in CD4+ T cells activated for the indicated days via CD3, singly or in combination with Lptn, or its neutralizing Ab, using a commercial kit (R&D Systems Europe, Abingdon, England). The assays were performed according to the manufacturer's protocol.
Lptn inhibits and induces both cell growth and death of human CD4+ T cells, respectively We previously demonstrated that Lptn inhibited the production of Th1-type lymphokines (IL-2, interferon- [IFN-]) by
CD3/TCR-activated CD4+ T cells, as well as their
proliferation.24 To determine whether this reduced
proliferation was due to decreased cell division or increased cell
death (or both), viability and cell cycle analysis for determination of
DNA content were assessed by trypan blue exclusion (Figure
1A) and PI staining (Figure 1B,C),
respectively. We observed a progressive increase in the number of
living lymphocytes on CD3 stimulation alone, which was abrogated by
Lptn (Figure 1A). A similar increase on CD3 stimulation, in the
presence of either anti-Lptn Ab (Figure 1A) or the CC chemokine RANTES
(not shown), indicated both specificity and selectivity of the Lptn inhibitory effect. Under identical conditions, PI staining revealed that the presence of Lptn not only induced a reduction in the percent
of cells at the G2/M interface (Figure 1B) but also
increased the percent of subdiploid cells (Figure 1B,C). The
accumulation of subdiploid cells was abrogated by the addition of
anti-Lptn Ab (Figure 1B,C). Thus, the inhibition of CD4+
T-cell proliferation induced by Lptn could result from both arrest in
cell growth and increase in cell death.
Lptn-mediated increase in CD4+ T-cell death occurs through an apoptotic-like mechanism In a attempt to determine whether an apoptotic-like mechanism could characterize the Lptn-mediated increase in CD4+ T-cell death, both DNA fragmentation and detection of the 7A6 mitochondrial protein at the cell surface were used. DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase-mediated nick-end labeling assay (TUNEL). Positive controls were systematically included in each activation condition by treatment of the cells with DNase I. The flow cytometric profiles presented in Figure 2A show an enhanced TUNEL staining, when Lptn was present together with CD3/TCR stimulation. The observed increase in the percent of apoptotic cells and mean fluorescence intensity (MFI) peaked at day 2 (not shown). Contrasting with the effect of Lptn, RANTES did not noticeably affect the intensity of TUNEL staining, indicating the selectivity of Lptn (Figure 2B).
To confirm that cell death induced by Lptn was due to apoptosis, we
performed immunodetection of 7A6 mitochondrial protein, which is
specifically expressed on apoptotic cells.48 In
correlation with the TUNEL assay results, the 7A6 expression increased
on CD3 stimulation in the presence of Lptn, as indicated by the
percentage of positive cells (Table 1),
and more variably by MFI (not shown). This increase was totally
abrogated by the addition of anti-Lptn Ab or PT (Table 1), supporting
specificity and coupling to G
Lptn-induced increase in CD4+ T-cell apoptosis is mostly Fas dependent Fas signaling is the best known inducer of apoptosis in CD4+ T cells.49,50 To examine whether this signaling could contribute to the Lptn-induced cell death, we first examined the effects of agonistic (7C11) and antagonist (ZB4) anti-Fas mAbs on CD4+ T-cell proliferation. For this purpose, T cells were preincubated with the antibodies and thymidine uptake was then measured, as described.24 As shown in Figure 3A, a slight reduction in the CD3-mediated proliferation was induced by the agonistic 7C11 mAb, whereas no effect of the antagonist ZB4 mAb was detected. In contrast, this latter antibody completely reversed to the control level (CD3 stimulation alone) the Lptn-mediated inhibition of proliferation, whereas the agonistic anti-Fas mAb had no significant effect (Figure 3A).
To further confirm the involvement of the Fas/FasL system, we examined
the expression of both the soluble and membrane-bound forms of FasL in
the same ELISA assays, the results of which are presented in Table
2. In absence of Lptn, the amount of
sFasL progressively accumulated to reach approximately 10 ng/mL between days 3 and 5, whereas in presence of Lptn, a very constant lower level
(around 2 ng/mL) persisted during the same time (Table 2). This 5-fold
reduction in the amount of sFasL in supernatants of the Lptn-treated
cells was probably not due to the decrease in cell number induced by
Lptn, because this reduced amount of sFasL was detected before the
decrease in cell number. In sharp contrast with this, a constant low
level (1-2 ng/mL) of mFasL expression was measured in the absence of
Lptn, as opposed to a 3-fold higher level in the presence of Lptn
(Table 2). This increase in mFasL expression peaked at day 2, concomitant with the peak of Lptn-induced cell death. The expression of
Fas that progressively increased on CD3 stimulation was additionally
up-regulated by Lptn, at early times after activation (not shown). The
inefficiency of Lptn to enhance cell death, on CD3 stimulation in the
presence of IL-2, was associated with an opposite pattern of mFasL
expression, because this expression was down-regulated by the presence
of Lptn (not shown).
Finally, to more directly investigate the implication of Fas signaling in the Lptn-induced apoptosis, we examined the effect of the antagonist anti-Fas ZB4 mAb on DNA fragmentation using the TUNEL assay. As shown in Figure 3B, preincubation of the cells with ZB4 mAb did not affect the intensity of TUNEL staining on CD3 stimulation alone, whereas it reduced staining intensity in the presence of Lptn, nearly to the levels detected in the absence of Lptn. A comparable inhibitory effect of ZB4 mAb was observed on the Lptn-induced increase in 7A6 antigen expression (data not shown). Collectively, these results supported a role of the Fas/FasL pathway in Lptn-mediated apoptosis. Selective activation of both initiator and effector caspases in response to Lptn-induced apoptosis Because caspase activation is a common final step required for many apoptotic stimuli, we examined the effect of a pan-caspase inhibitor, z-VAD-fmk, on DNA fragmentation in CD3-activated T cells in the presence or absence of Lptn. As shown in Figure 4A, z-VAD-fmk completely inhibited the Lptn-mediated increase in TUNEL staining, when compared with TUNEL positivity obtained by CD3 stimulation alone. In addition, the reduced thymidine uptake in Lptn-treated cells was also overcome by this inhibitor, whereas that of untreated cells was not affected (not shown). Because z-VAD-fmk has been shown to titrate a large number of caspases, we further investigated the contribution of specific initiator and effector caspases in Lptn-mediated apoptosis. The processing of these 2 sets of caspases was analyzed by Western blots, performed at different times on CD3 stimulation in the presence or absence of Lptn. With respect to the initiator caspases, a clear processing of procaspase-9 was observed in Lptn-treated cells as opposed to untreated cells (Figure 4B). This processing peaked at day 2 (Figure 4B,C) and returned to the control level (Lptn-untreated cells) at day 3 (Figure 4C). With regard to the expression of procaspase-8, a transient increase was observed at day 2 in both activation conditions (not shown). The procaspase-8 levels seemed, however, to be steadily lower (from 0.5-5 days) in the presence of Lptn (Figure 4B and not shown). As an internal positive control, a clear processing of the procaspase-8 was detected in the presence of Lptn, on CD3 and CD28 costimulation (Figure 4B). Because no cleaved fragment of caspase-9 could be detected even after long exposures of the blots, the activity of this caspase was investigated at different times on CD3 stimulation in the presence or absence of Lptn, using LEHD, a synthetic tetrapeptide substrate in colorimetric assays, as described in "Materials and methods." As seen in Figure 4D, a 6-fold increase in caspase-9 activity, peaking at day 2, was measured in Lptn-treated cells, as compared with either untreated cells or Lptn-treated cells in conjunction with the anti-Lptn Ab. Consequently, the kinetics of increase in caspase-9 activity correlated closely with the kinetics of both decrease in caspase-9 proform expression and increase in DNA fragmentation, observed in response to Lptn.
We next considered whether some effector caspases could also be
specifically processed by Lptn under identical conditions. By Western
blotting, a marked decrease in levels of the caspase-7 proenzyme was
detected from day 1 on CD3 stimulation, in the presence but not in the
absence of Lptn (Figure 5A). This
decrease peaked from day 2 to at least day 3 (Figure 5B, and not shown
for day 4). As an internal control, no significant variation in
caspase-7 proenzyme expression was detected on CD3 and CD28
costimulation, irrespective of the presence or absence of Lptn (Figure
5A and not shown). The processing of caspase-7 appeared to be selective because caspase-3 (not shown) and caspase-4 (Figure 5A) proenzymes remained at constant levels in the presence of Lptn. For caspase-3, a
9-fold increased expression was observed at day 2 on CD3 stimulation alone (not shown). With regard to procaspase-4, expression profiles were similar in the presence or absence of Lptn (Figure 5A and not
shown) following CD3 stimulation, but not on CD3 and CD28 costimulation
(3-fold increase in absence of Lptn) (Figure 5A).
Specific cleavage of caspase substrates in response to Lptn-induced apoptosis Because the effector caspase-7 was specifically processed after CD3 triggering in the presence of Lptn, we next examined whether caspase substrates such as PARP, CD3/TCR-chain, or DFF45, which have been shown as targets for caspase-7,40,41,43,44 could be involved in our Lptn-dependent apoptotic process. As shown in Figure
6A, on CD3 stimulation, the PARP (p116)
protein appeared to be more efficiently cleaved in Lptn-treated cells
than in untreated cells, mainly as the result of a stronger reduction
in expression of the p116 precursor than of an increase in expression
of the p85 cleaved product. Such an effect peaked around day 3 (Figure 6A). As an additional substrate undergoing reduction in its expression during apoptosis,44 the CD3/TCR -chain also underwent a
transient loss of expression on CD3 stimulation, in the presence of
Lptn, with a peak around day 3, in contrast with a progressive increase in expression in the absence of Lptn (Figure 6A,B). Finally, because the cleavage of DFF45, an inhibitor of caspase-activated
deoxyribonuclease (CAD)42,43 is known to play a critical
role in apoptotic DNA fragmentation, we analyzed DFF45 expression
profile in the cell extracts used for expression analyses of PARP, CD3
-chain, and caspases. Results shown in Figure 6A showed that DFF45
did not appear to be cleaved in the presence of Lptn, a result in
agreement with the absence of detection of caspase-3 processing in
these conditions (not shown). Altogether, these results indicate
that the apoptogenic effect of Lptn on CD3-activated
CD4+ T cells was accompanied by a selective processing of
both caspases and substrates.
Apoptosis of T cells plays an important role in controlling immune responses and is influenced by cytokines/growth factors. This report clearly provides the first demonstration of the direct involvement of Lptn, the unique member of the C chemokine subfamily, in human CD4+ T-cell death, as an additional mechanism to explain the Lptn-induced antiproliferative effects.24 In conjunction with CD3/TCR signaling, Lptn behaves like a death costimulator for recently activated CD4+ T cells. The peak of Lptn-induced apoptosis is observed around day 2, time that is probably required for the cells to express the receptor for Lptn or to become susceptible to activation-induced cell death. The abrogation of the Lptn-mediated apoptosis by PT is in accordance with both the nature of the receptor for Lptn15 and the previously demonstrated PT sensitivity of the inhibition of Th1-type lymphokine production by Lptn.24 In contrast with Lptn, our TUNEL-based results showed no effect of the CC chemokine RANTES on CD4+ T-cell apoptosis. So far, procytotoxic or proapoptotic effects have only been reported for CXC or CC chemokines in the context of human immunodeficiency virus (HIV)-suppressing activities.21-23 Whereas the apoptotic effect of Lptn toward primary CD4+ T cells was direct, that of SDF-1 toward primary CD8+ T cells only occurred in the presence of macrophages.21 The expression of Fas and FasL, known as critical mediators of elimination of activated T cells, requires activation signals through CD3/TCR complex.50 FasL has been shown to be processed and shed from the surface of human T cells.34 Although sFasL is less active than mFasL in killing T lymphocytes, it can also inhibit the activity of mFasL.35,36 We demonstrate here that Lptn both increased the expression of mFasL and reduced that of sFasL. We hypothesize that the combination of these events might contribute to enhance the apoptotic activity of FasL signaling through its receptor and therefore might account for the increase in CD4+ T-cell death induced by Lptn. Such a conclusion is also supported by the following observations: (1) the antagonist anti-Fas mAb abrogated the Lptn-mediated apoptosis, as determined by DNA fragmentation and surface expression of the 7A6 mitochondrial antigen; (2) Lptn and agonistic anti-Fas mAbs did not display additive effects when mixed together at optimal doses; and (3) using a metalloproteinase inhibitor, we observed a correlation between the increase in 7A6 apoptotic antigen expression and the inhibition of sFasL (not shown). Various apoptotic pathways triggered by distinct stimuli include a final step of caspase activation, involving particular caspases that can be tissue specific but also stimulus specific within the same cell type. The identification of specific caspases and their substrates could therefore be useful to propose specific targets for inhibition of apoptosis.44 Initiator caspases with long prodomains like caspases-8, -9, and -10 usually cleave and activate effector caspases like caspases-3 and -7.37 In this study, we clearly demonstrated the activation of caspase-9 as a predominant initiator caspase implicated in Lptn-mediated apoptosis of CD4+ T cells. We cannot exclude, however, a minor or later contribution of caspase-8 to the apoptotic process of both Lptn-treated or untreated cells, on CD3 stimulation. Effectively, according to the study of Droin and colleagues,51 the up-regulation of procaspase-8 expression at day 2 could be associated with a subsequent phase of apoptosis. Our results may, therefore, suggest either a regulatory role of caspase-8 in a caspase-9-driven apoptosis,52 or alternatively, an involvement of caspase-8 in proliferation or other T-cell activation-related events, as already reported.53,54 Our inability to detect a processing of caspase-10 did not exclude its potential contribution to the Lptn-mediated apoptotic process, as well as for other nontested caspases or different proteases, as for instance calpain.55 We demonstrate a processing of caspase-7, but not of the caspases-3 and -4, in Lptn-mediated apoptosis. Interestingly, caspase-4 activation has been more implicated in generation of proinflammatory cytokines56 than in apoptosis.57 Our observation is thus in accordance with one of the most important features of the CD28 costimulation, namely, its ability to increase the production of such cytokines.25,26 Alam and coworkers54 showed that the activation of caspases-6, -7, and -3, was required as a downstream event for T-cell proliferation. In line with this, CD3-stimulated T cells, which exhibit a more vigorous proliferation in absence than in presence of Lptn, display a dramatic increase in caspase-3 proenzyme expression. Several proteins, like PARP and DFF45, which play a role in DNA repair
or maintenance, are cleaved by caspases in apoptotic cells.58 Although almost all caspases can cleave PARP in
vitro, its in vivo cleavage is more likely attributed to caspases-3 and -7.39-41 The observed cleavage of PARP in CD3-activated
cells in the presence of Lptn is in accordance with the increase in
procaspase-7 processing. The effect of caspase-3 on the cleavage of
DFF45 has been shown to be more effective than that of
caspase-7.43 We also observed a simultaneous absence of
DFF45 cleavage and caspase-3 activation in response to Lptn, which is
in complete agreement with the previous proposition of caspase-3 being
the major regulator of DFF45 proteolysis.43 A reduction in
the expression of CD3/TCR Although IL-2 is a T-cell survival factor, it can also confer susceptibility to apoptosis to TCR-stimulated T cells,30 by increasing the expression of death molecules like Fas/FasL. We have previously shown that a strong IL-2 deprivation was induced by Lptn, on CD3 stimulation.24 This phenomenon, however, is probably not responsible for the increased apoptosis mediated by Lptn. This notion could be supported by the following observations: (1) the addition of IL-2, even at high doses, was unable to completely rescue the CD3-activated T cells from the Lptn-induced apoptosis, and (2) a reciprocal pattern expression of both mFas and sFasL was induced by Lptn in CD3-activated cells in the presence of IL-2. This suggests an inhibitory effect of IL-2 on the particular Fas-dependent Lptn-mediated CD4+ T-cell death pathway. The deprivation of the same lymphokines that drive T cells into cycle such as IL-2 can induce a death different from apoptosis, with no role of FasL.30 In contrast, as shown by the present work, Lptn is able to directly exert a feedback control of T-cell activation by apoptosis. By demonstrating the apoptotic function of Lptn, which was previously shown by us to inhibit proliferation of CD4+ T cells, through abrogation of Th1-type lymphokine production,24 we further emphasize the role of Lptn in the in vitro clearance of CD3-activated CD4+ T cells, by proposing Lptn as a crucial modulator of TCR signals. During thymic development, the elimination of self-reactive immature T cells is ensured through clonal deletion via apoptosis, and it would be thus interesting to analyze the potential role of Lptn in such a deletional mechanism. Finally, the induction of in vivo peripheral deletion of inappropriately activated mature CD4+ T cells by Lptn-mediated apoptotic signals could be considered as a basis for the elaboration of new therapies using selective immunosuppression.
Submitted August 21, 2000; accepted November 15, 2000.
Supported by INSERM and by grants from the Fédération Nationale des Centres de Lutte Contre le Cancer and the Association pour la Recherche sur le Cancer (ARC).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Chantal Cerdan, INSERM U119, Université Méditerranée, 27, Boulevard Leï Roure, 13009 Marseille, France; e-mail: cerdan{at}marseille.inserm.fr.
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CXCR4 and CD4 mediate a rapid CD95-independent cell death in CD4+ T cells.
Proc Natl Acad Sci U S A.
1998;95:12556 |
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Abstract
Introduction
), CC (
), C (
).
, CD3; 
, CD3 plus
Lptn; 
, CD3 plus Lptn plus anti-Lptn. (B) A typical flow cytometric
profile of PI-stained CD4+ T cells. Cells were activated
with CD3 mAb in the presence or absence of Lptn. At day 2 after
activation, the different stages of cell cycle were analyzed for nuclei
DNA content after PI uptake. The percentages of cells in the different
stages (subdiploid [< 2n DNA], G0/G1 [2n
DNA], and S+G2+M [> 2n DNA]) are indicated. In
parallel, CD4 staining ensured a 90% purity of the cultured cells (not
shown). (C) Quantitative expression of the data shown in panel A. CD4+ T cells were activated for 2 days with the indicated
stimuli. Each graph represents the mean values ± SD of 3 independent experiments.
, CD3
plus ZB4.
