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Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2360-2368
Induction of Fas (Apo-1, CD95)-Mediated Apoptosis of Activated
Lymphocytes by Polyclonal Antithymocyte Globulins
By
Laurent Genestier,
Sylvie Fournel,
Monique Flacher,
Olga Assossou,
Jean-Pierre Revillard, and
Nathalie Bonnefoy-Berard
From the Laboratory of Immunology, INSERM, Hôpital E. Herriot,
Lyon, France.
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ABSTRACT |
Polyclonal horse antilymphocyte and rabbit antithymocyte globulins
(ATGs) are currently used in severe aplastic anemia and for the
treatment of organ allograft acute rejection and graft-versus-host disease. ATG treatment induces a major depletion of peripheral blood
lymphocytes, which contributes to its overall immunosuppressive effects. Several mechanisms that may account for lymphocyte lysis were
investigated in vitro. At high concentrations (.1 to 1 mg/mL) ATGs
activate the human classic complement pathway and induce lysis of both
resting and phytohemagglutinin (PHA)-activated peripheral blood
mononuclear cells. At low, submitogenic, concentration ATGs induce
antibody-dependent cell cytotoxicity of PHA-activated cells, but not
resting cells. They also trigger surface Fas (Apo-1, CD95) expression
in naive T cells and Fas-ligand gene and protein expression in both
naive and primed T cells, resulting in Fas/Fas-L interaction-mediated cell death. ATG-induced apoptosis and Fas-L expression were not observed with an ATG preparation lacking CD2 and CD3 antibodies. Susceptibility to ATG-induced apoptosis was restricted to activated cells, dependent on IL-2, and prevented by Cyclosporin A, FK506, and
rapamycin. The data suggest that low doses of ATGs could
be clinically evaluated in treatments aiming at the selective deletion of in vivo activated T cells in order to avoid massive lymphocyte depletion and subsequent immunodeficiency.
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INTRODUCTION |
THE POLYCLONAL antilymphocyte or
antithymocyte globulins (ATG)* are potent
immunosuppressive agents used in organ transplantation since the late
1960s. They have proved effective either as rescue treatment of first
rejection episodes and graft-versus-host reaction or as prophylactic
treatment of rejection.1 As an alternative to polyclonal
ATGs, monoclonal antibody (MoAb) OKT3 has been extensively used in
organ transplantation.2,3 However, in clinical studies, polyclonal ATGs compare favorably to OKT3 both for prophylactic use or
in rescue therapy.4 The precise mechanism of action of ATGs
is undefined, but the profound lymphocytopenia observed throughout the
treatment period mainly contributes to the immunosuppressive effect.
Various mechanisms have been proposed to explain lymphocyte depletion,
including complement-mediated cytolysis or clearance of lymphocytes by
opsonization and phagocytosis by macrophages.5 ATGs are a
mixture of multiple antibodies to various lymphocyte surface
antigens.6-8 It was recently reported that antibodies specific for HLA class I molecules,9-11 and antibodies to
CD2,12,13 CD30,14 CD45,15 and
CTLA-416 could induce apoptosis of T cells, whereas
anti-HLA class II and anti-HLA class I antibodies can also trigger
apoptosis of activated B cells.17 Antibodies to CD2, CD3,
CD45, and HLA molecules were identified in ATGs; it may therefore be
hypothesized that their binding either to resting or to activated T
cells, or both, may trigger a signal of programmed cell death.
Furthermore, ATGs contain antibodies to CD2 and CD3, which account for
their mitogenic properties.7 Repeated activation of mature
T cells through CD2 or CD3 results in apoptosis of activated T
cells.18 The major pathway of this activation-induced cell
death (AICD) uses the interaction between Fas (Apo-1, CD95) expressed
by activated T and B cells and Fas-ligand (Fas-L, CD95-L) produced by a
subset of activated T cells.19-21 The present study was
designed to investigate in vitro the different mechanisms whereby ATGs
can induce peripheral lymphocyte depletion. To this end, we measured
the capacity of ATGs bound to peripheral blood lymphocytes (PBL) to
bind human C1q and to induce complement-dependent lysis. We determined
their activity in antibody-dependent cell-mediated cytotoxicity (ADCC)
and their capacity to induce Fas and Fas-L expression. In all those
assays, we compared the sensitivity of naive versus mitogen-activated
PBL to ATG-induced lysis, in order to identify those mechanisms that
could display some specificity toward preactivated PBL. The dose
responses were analyzed according to serum concentrations achieved
during treatments. Finally, we evaluated the effect of
immunosuppressive drugs that interfere with the interleukin-2 (IL-2)
pathway (Cyclosporin A, [CsA], FK506, rapamycin) on the development
of the sensitivity to ATG-induced lysis.
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MATERIALS AND METHODS |
Antibodies and reagents.
Rabbit ATG, batch no. 95-07, and horse antilymphocyte globulins,
batches no. 1141 and no. 5, were provided by Dr J. Carcagne (Pasteur
Merieux serums & vaccins, Lyon, France). Characteristics of each batch
have been previously reported.7 F(ab )2
fragments of ATG no. 95-07 were prepared by pepsin digestion and
purified by exclusion chromatography on protein A, following standard
procedures. Normal rabbit IgG (Zymed, San Francisco, CA) and horse
anti-rabies globulins purified according to the same procedure used for
ATGs (Pasteur Merieux serums & vaccins) were used as controls. The anti-CD52 MoAb CAMPATH-1M (IgM) was a gift from Prof H. Waldmann (Sir
Dunn School of Pathology, University of Oxford, Oxford, UK). The three
anti-Fas MoAbs were used in this study, UB2 for cytofluorometry assays;
CH11 (IgM), ZB4 (IgG1), and phycoerythrine streptavidin were obtained
from Immunotech (Marseille, France). Fluorescein-isothiocyanate (FITC)-conjugated CD25 and CD69 MoAbs were obtained from Becton Dickinson (Mountain View, CA) and two biotinylated anti-Fas-L one from
Pharmingen (San Diego, CA) and the other from Alexis Corporation (Coger
S.A., Paris, France). CD3 MoAb OKT3 was from Cilag Laboratories
(Levallois-Perret, France).
The lectin phytohemagglutinin (PHA), phorbol myristate acetate (PMA),
ionomycin, and cycloheximide (CHX) were obtained from Sigma Chemical
Co. (St Louis, MO). Rapamycin (RPM) and FK506 were gifts from Dr A. Altmann (La Jolla Institute for Allergy and Immunology, San Diego, CA),
and CsA was kindly supplied by Sandoz (Novartis, Paris, France). Human
IL-2 and rIFN- were kindly provided by Dr J. Banchereau
(Schering-Plough, Dardilly, France).
Cell preparation.
Peripheral blood was collected from healthy donors in the presence of
sodium citrate. After the addition of a calcium chloride solution,
blood was defibrinated by gentle rotation of the flask; mononuclear
cells were then isolated by centrifugation on a layer of Histopaque
(Sigma). Cells were washed three times in Hank's balanced salt
solution (HBSS) before culture. Those cell suspensions referred to as
PBL were shown to contain 3.8% ± 0.4% monocytes, as defined by
expression of CD14. For complement-mediated lysis and ADCC experiments,
peripheral blood mononuclear cells (PBMC) were obtained by
centrifugation of heparinized blood on a layer of Histopaque.
Culture medium and cell proliferation.
PBL were resuspended in RPMI 1640 (Sigma) supplemented with 10% fetal
calf serum (FCS), 2 mmol/L L-glutamine, and antibiotics (penicillin 100 U/mL, streptomycin 100 µg/mL). For the proliferation assay, cells (106/mL) were incubated in 96-well microplates
(Costar, Cambridge, MA) in the presence of PHA (5 µg/mL) or with ATGs
at the indicated concentrations. Cultures were maintained in a humid
atmosphere at 37°C containing 5% CO2 for the indicate
time.
Immunofluorescence assays.
Cells were washed with isotonic NaCl/Pi buffer containing 1% bovine
serum albumin (BSA) and 0.2% NaN3 (phosphate-buffered saline [PBS]/BSA/azide). Cells (5 × 105) were
incubated with 10 µL labeled MoAbs for 30 minutes at 4°C. Then,
after two washes in PBS/BSA/azide buffer, cells were fixed with 1%
formaldehyde in PBS/BSA/azide buffer and analyzed by flow cytometry
with a FACScan (Becton Dickinson, Pont de Claix, France). For
intracellular analysis of Fas-L expression, cells were fixed with
freshly prepared 2% paraformaldehyde in PBS and permeabilized by
saponin (0.33%) (Sigma).
Measurement of apoptosis.
After 3 days of culture, unstimulated or PHA-activated PBL were
harvested. Dead cells were removed by centrifugation on a layer of
Histopaque (Sigma), and viable cells were washed in HBSS. Viable cells
(106/mL) were incubated in 96-well microplates in the
presence of ATG or CH11 MoAb. After incubation, cell death was
evaluated by three different techniques. Measurement of mitochondrial
transmembrane potential by flow cytometry after
3,3-dihexyloxacarbocyanine (DiOC6) staining22
and detection of phosphatidylserine expression by flow cytometry after
addition of FITC-conjugated annexin V23 were performed on
the same suspensions at the indicated time. Nuclear apoptosis was
assessed by fluorescence microscopy after staining with Hoechst 33342 (Sigma) at 10 µg/mL, following previously described
methods.24 Nuclear fragmentation or marked condensation of
the chromatin with reduction of nuclear size, or both, were considered
typical features of apoptotic cells. On the basis of these
measurements, results were expressed either as percentage of apoptotic
cells or as percentage of specific apoptosis according to the
formula
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RNA isolation, reverse transcription, PCR amplification of
Fas-ligand mRNA, and quantification.
Total cellular RNA was isolated from 5 × 106 cells,
following the method of Chomczynski and Sacchi.25 Reverse
transcription of 1 µg RNA was performed using the first-stand cDNA
synthesis kit (Pharmacia Biotech, Orsay, France) in a total reaction
volume of 15 µL. After 90 minutes at 37°C, the reaction was
terminated by heating for 4 minutes at 95°C. PCR was performed in
mixtures containing 1 µL cDNA derived from 10 ng total RNA, primers
(100 ng of each; Eurogentech, Seraing, Belgium), 2.5 µL 10 × PCR
buffer (Promega, Charbonnieres, France) containing 1.5 mmol/L
MgCl2, 0.05 mmol/L of each dNTP, and 0.5 U of Taq
polymerase (Promega). Primers for Fas-L and Actin included Fas-L sense
primer 5CCATTT-AAC-AGG-CAA-GTC-CAA-CTC-3, Fas-L anti-sense primer
5CAA-CAT-TCT-CGG-TGC-CTG-TAA-C-3, actin sense primer
5GGG-TCA-GAA-GGA-TTC-CTA-TG 3, and actin anti-sense primer
5GGTCTCAAACATGSATCTGGG-3. These primers were designed to discriminate
between the amplification of cDNA (low size PCR products) and
contaminating genomic cDNA (high size PCR products). For each amplicon,
23 to 35 amplification cycles (1 minute at 94°C, 1 minute at 58°C,
and 1 minute at 72°C) were performed with the PCR system 9600 (Perkin
Elmer, Montigny-le-Bretonneux, France). Semiquantitative evaluation of
amplification products was performed as described by Morgan et
al.26 Briefly, each PCR product (15 µL) was
electrophoresed on agarose gel (2%) stained with ethidium bromide and
photographed using polaroid type 665 positive/negative film. The
specificity of PCR reaction was confirmed by the expected size of the
amplification products. The PCR signal intensities were quantitated by
scanning the negative film using a Desktop Scanning Densitometer
(PDI/Pharmacia Biotech, Saint-Quentin-Yvelines, France) and by
evaluating the integrated trace optical density (OD) for each band
using Quantity One Software (PDI/Pharmacia Biotech). The point for
samples comparison in the exponential amplification range was selected
by inspection from semi-logarithmic plots of OD versus cycle numbers.
To correct for variations in the amount of input cDNA, results were
expressed as the ratio Fas-L OD/actin OD at the point previously
determined.
Complement-mediated lysis.
Resting or PHA-activated PBMC were labeled with
Na251 CrO4 for 2 hours at room
temperature and washed twice. They were resuspended in medium at 2 × 106 cells/mL, and 100 µL of the suspension was added to
round-bottomed microtiter plates containing 50 µL of an appropriate
dilution of the antibody. After incubation for 10 minutes at room
temperature, 50 µL of 40% fresh or heat-inactivated (56°C, 30 minutes) autologous serum (obtained from defibrinated blood) was added.
The cell suspensions were incubated at 37°C for 30 minutes, then
centrifuged at 100g for 2 minutes, and 100 µL of the
supernatant was collected for measurement of released radioactivity.
Controls without antibody were used to measure the spontaneous
radioactivity release. The percentage of specific 51Cr
release was calculated using the
formula
C1q binding.
A total of 20 µL of ATGs or control Ig in PBS/BSA/azide was added to
PBMC pellets (4 × 105) and incubated at 37°C for 30 minutes. After two washes in PBS, samples were separated in two and
incubated at room temperature for 30 minutes in the presence of 50 µL
of autologous serum or heat-inactivated (56°C, 30 minutes) serum as a
control. After two washes, cells were incubated with 10 µL of
polyclonal goat anti-C1q FITC antibody (1/50 Cappel, Durham, NC) at
4°C for 30 minutes. After two washes, cells were fixed
with 1% formaldehyde in PBS/BSA/azide buffer and analysis performed on
a FACScan flow cytometer.
Antibody-dependent cell cytotoxicity.
Resting and PHA-activated PBMC were labeled with
Na251 CrO4 for 2 hours at room
temperature and washed twice. They were resuspended in medium at 1 × 106 cells/mL, and 50 µL of the suspension was added to
round-bottomed microtiter plates containing 50 µL of an appropriate
dilution of the antibody. After incubation for 10 minutes at room
temperature, 100 µL of effector cells (25 × 106
cells/mL) was added. The cell suspensions were incubated at 37°C for
6 hours, then centrifuged at 100g for 2 minutes and 100 µL of
the supernatant collected for measurement of released radioactivity as
for complement-mediated lysis.
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RESULTS |
ATGs induce apoptosis of activated lymphoblasts.
Knowing that ATGs could induce apoptosis of B-cell lines and to a
lesser extent, T-cell lines,27 we examined whether such mechanism could also take part in the elimination of peripheral T
lymphocytes. Three-day PHA-activated PBL, as well as nonactivated PBL,
were treated with ATG no. 95-07, F(ab)2 fragments of ATG, anti-Fas MoAb CH11 as positive control, and normal rabbit IgG as
negative control. Apoptosis was evaluated by DiOC6[3] and
annexin V staining (Fig 1) and by
fluorescence microscopy after staining with Hoechst 33342 (Fig
2). The results showed that ATG no. 95-07 at nonmitogenic concentrations (10 µg/mL), their F(ab)2
fragments, and the anti-Fas MoAb CH11 induced apoptosis of 30% to 40%
of PHA-activated PBL, whereas resting PBL were not sensitive (Figs 1
and 2). Similar results were observed with ATG no. 1141 obtained from
horse (data not shown). Interestingly ATG no. 5 containing CD18, CD11a,
anti- 2m, and anti-HLA DR antibodies, but no CD3, CD2, and CD5
specificities, and which is not mitogenic at concentrations ranging
from 1 to 1,000 µg/mL, did not induce apoptosis at 10 and 100 µg/mL
(Fig 2; data not shown). Normal rabbit did not induce cell death of
resting or activated PBL (Fig 2). Similar experiments were repeated
with PBL activated by a 3-day culture period with PMA (10 ng/mL) plus
ionomycin (500 ng/mL), PMA (10 ng/mL) plus OKT3 (100 ng/mL), or a
mitogenic concentration of ATG no. 95-07 or no. 1141 (100 µg/mL).
Whatever the activator used, the addition of ATGs (10 µg/mL) or
F(ab)2 fragments thereof resulted in specific apoptosis
ranging from 20% to 50% (data not shown).
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| Fig 1.
Effect of ATGs on mitochondrial transmembrane potential
and on phosphatidylserine expression. PBL were activated for 3 days in
presence of PHA (5 µg/mL). After removal of dead cells, medium alone,
ATG no. 95-07 (10 µg/mL), or CH11 anti-Fas MoAb (1 µg/mL) was
added. After 12 hours,   m modifications were evaluated by staining
with DiOC6 (3). The expression of phosphatidylserine at the
surface membrane was evaluated after 15 hours by measuring annexin-V
binding. The percentage of cells with decreased mitochondrial potential
membrane or increased expression of phosphatidylserine are indicated
for each histogram. Results from one typical experiment among four
showing similar percentages.
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| Fig 2.
ATGs induce apoptosis of activated T lymphocytes. PBL
were cultured in presence of medium alone or PHA (5 µg/mL) for 3 days. Dead cells were removed and viable cells were treated for 20 hours with ATG no. 95-07, F(ab)2 fragments of ATG no.
95-07, ATG no. 5 or normal rabbit IgG at 10 µg/mL or with the agonist
anti-Fas MoAb CH11 at 1 µg/mL. Protection by the antagonist anti-Fas
MoAb, was tested by pre-incubating PBL or PHA-activated cells for 1 hour with ZB4 MoAb at 2 µg/mL. The percentage of apoptotic cells was
determined by fluorescent microscopy after staining with Hoechst 33342. Results are expressed as mean ± SEM of five different experiments or
as mean of two experiments for ATG no. 5.
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ATG-induced apoptosis is fully inhibited by an antagonist anti-Fas
antibody.
The apoptotic activity of ATGs was effective only on activated T cells,
which express Fas and which are sensitive to Fas-mediated apoptosis28; we therefore studied whether ATG-induced
apoptosis was dependent on Fas/Fas-L interaction. To this end,
PHA-activated PBL were incubated for 1 hour with the antagonist
anti-Fas MoAb ZB4, which blocks the interaction between Fas and Fas-L,
before addition of ATG no. 95-07, ATG F(ab)2 fragments or
CH11 MoAb. As shown in Fig 2, ATG-induced apoptosis was completely
blocked by ZB4, indicating that ATG-induced apoptosis of activated T
cells required Fas/Fas-L interaction. This idea was re-enforced by the
observation that simultaneous addition of ATG no. 95-07 (10 µg/mL)
and CH11 resulted in the same percentage of apoptotic cells as with
each antibody tested alone (data not shown). This result suggests that the same subset of activated T cells is the target of ATGs and anti-Fas
antibodies. Furthermore, it shows that ATGs do not contain anti-Fas
blocking antibodies, at least in sufficient amount to be detected in
this assay.
ATGs induce Fas and Fas-L expression.
In an effort to obtain further evidence for a possible role of
Fas/Fas-L interaction in ATG-induced apoptosis, we examined whether
ATGs would induce Fas-L expression in both resting and activated-PBL.
To this end, PBL were first cultured in presence of a mitogenic
concentration of ATG no. 95-07 (100 µg/mL) or PHA or medium alone for
3 days. After elimination of dead cells, preactivated PBL were then
incubated for 6 hours with medium alone, ATG no. 95-07 at nonmitogenic
(10 µg/mL) and mitogenic (100 µg/mL) concentrations or PHA, and
induction of Fas-L mRNA was analyzed by RT-PCR. ATG no. 95-07 at either
10 or 100 µg/mL induced Fas-L mRNA expression by nonactivated and by
preactivated-PBL (Fig 3). Similar
experiments performed with freshly isolated PBL showed that ATG no.
95-07 (10 and 100 µg/mL), but not control rabbit IgG, strongly
induced Fas-L mRNA expression (Fig 3).
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| Fig 3.
Expression of Fas-L mRNA induced by ATGs. (Left) PBL were
cultured in presence of medium alone, ATG no. 95-07 (100 µg/mL) or
PHA (5 µg/mL) for 3 days. Dead cells were removed, and viable cells
were stimulated with normal rabbit IgG at 100 µg/mL, ATG no. 95-07 at
10 µg/mL and 100 µg/mL or PHA at 5 µg/mL for 6 hours. (Right)
Freshly isolated PBL were stimulated with normal rabbit IgG at 100 µg/mL or ATG no. 95-07 at 10 µg/mL and 100 µg/mL for 6 hours.
mRNA of each sample was amplified by RT-PCR as described in Materials
and Methods with primers specific for actin or Fas-L. The number of
amplification cycles selected within the exponential phase of PCR was
29 for actin and 32 for Fas-L. The PCR products were separated on 2%
agarose gel and the PCR signal intensities were quantified by scanning
the negative film. Results are expressed as the ratio of absorbance of
Fas-L/absorbance of actin (mean ± SEM of three experiments).
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In parallel, surface expression of Fas and Fas-L molecules, but CD25
and CD69 activation markers as well, was analyzed by flow cytometry on
PBL cultured in the presence of ATG no. 95-07 at 10 and 100 µg/mL for
1 to 3 days. At mitogenic concentrations (100 µg/mL), ATG no. 95-07 induced CD69, CD25, Fas, and Fas-L expression (Fig
4). Surface expression of Fas, CD69, and
CD25 reached a maximum at day 2, and that of Fas-L at day 1. At
nonmitogenic concentrations (ie, 10 µg/mL), ATG no. 95-07 still
induced expression of CD69, Fas, and Fas-L, but not that of the CD25
molecule, suggesting that, at low concentrations, ATGs
drive lymphocytes into the G1 phase of the
cell cycle but did not allow them to progress to S phase because of the
absence of CD25 expression. Interestingly ATG no. 5 at 100 µg/mL did
not induce CD69 Fas and Fas-L expression (Fig 4), nor did it trigger
apoptosis (Fig 2). Finally, these experiments were completed by
intracellular staining of Fas-L in paraformaldehyde-fixed and
saponin-permeabilized cells. The results indicate that ATG no. 95-07 (10 and 100 µg/mL) increased intracellular Fas-L in both resting and
preactivated PBL, with a maximum on days 1 to 2 (Fig 4; data not
shown). Histograms of fluorescence (Fig 4) show that a small subset of
PBL is positive before activation, whereas after stimulation by ATG,
most of the lymphocyte population becomes Fas-L positive.
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| Fig 4.
Effect of ATGs on CD69, CD25, Fas, and Fas-L surface
expression. PBL were cultured in presence of medium alone or ATG no. 95-07 and ATG no. 5 at 10 µg/mL and 100 µg/mL for 3 days. At days 0, 1, 2, and 3, surface expression of CD69, CD25, Fas, and Fas-L was
determined by cytofluorometry. In parallel, incorporation of
[3H]TdR uptake during the last 8 hours of culture was
measured (med 367 ± 41 cpm, ATG no. 5 10 µg/mL 391 ± 23 cpm,
ATG no. 5 100 µg/mL 252 ± 26 cpm, ATG no. 95-07 10 µg/mL
532 ± 53 cpm, and ATG no. 95-07 100 µg/mL 11,500 ± 103 cpm).
Histograms of Fas-L expression at day 1 are shown. Representative of
four experiments with ATG no. 95-07 and of two with ATG no. 5.
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Interference with the IL-2 pathway reduces ATGs-induced apoptosis.
Knowing that IL-2 is required for acquisition of susceptibility to
Fas-mediated apoptosis,29,30 we analyzed the effect of
immunosuppressive agents that interfere with the IL-2 pathway on
ATG-induced cell death. PBL were cultured with PHA in the presence of
CsA or FK506, which block IL-2 expression at a transcriptional level,
or with RPM, which blocks IL-2 signaling. After 3 days, cells were
treated with ATGs or F(ab)2 fragments. The presence of
CsA, FK506, or RPM, during T-cell activation, markedly decreased apoptosis mediated by ATG no. 95-07 or their F(ab)2
fragments (Fig 5). In keeping with these
results, we observed that addition of rIL-2 during the last 24 hours of
cell culture, to PBL activated by PHA in the presence of CsA restored
the sensitivity to ATG and F(ab)2-induced apoptosis (Fig
5B). Conversely, the addition of interferon- (IFN-) restored
T-cell proliferation,29 but not the sensitivity to
ATG-induced apoptosis. In agreement with previous
reports,29,30 similar effects were observed as regards sensitivity to Fas-mediated apoptosis (Fig 5B).
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| Fig 5.
(A) Effect of immunosuppressive agents on ATG-mediated
apoptosis. PBL were cultured for 3 days with PHA (5 µg/mL) and CsA (250 ng/mL), FK506 (10 nmol/L) or RPM (60 nmol/L) were added at the
onset of the culture. Apoptosis was determined by fluorescence microscopy after staining with Hoechst 33342, 20 hours after treatment with ATG no. 95-07 or their F(ab)2 fragments at 10 µg/mL. (B) Effect of addition of exogenous IL-2 or IFN-. PBL were
cultured for 3 days with PHA (5 µg/mL); medium alone (gray bars) or
CsA (250 ng/mL) (black bars) were added at the onset of the culture. Recombinant IL-2 (25 U/mL) or rIFN- (500 U/mL) was added during the
last 24 hours of activation. Apoptosis was determined by fluorescence microscopy after staining with Hoechst 33342, 20 hours after treatment with ATG no. 95-07, their F(ab)2 fragments at 10 µg/mL
or the CH11 (1 µg/mL) MoAb. (Results are expressed as mean ± SEM
of three different experiments).
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Furthermore, CsA and FK506 were described as strongly inhibiting Fas-L
expression in murine T-cell hybridomas.31 Thus, we have
tested whether incubation of 3-day PHA-activated PBL with CsA, just
before ATG treatment would interfere with ATG-induced apoptosis. A
3-hour preincubation of PHA-blasts with CsA or CHX inhibited
ATG-induced cell death but did not interfere with apoptosis induced by
the anti-Fas MoAb (Fig 6). These data
suggest that immunosuppressive agents that interfere with the IL-2
pathway can prevent ATG-induced apoptosis by inhibiting either Fas-L
synthesis or the acquisition of sensitivity to Fas-L-mediated cell
death by activated T cells.
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| Fig 6.
ATG-induced apoptosis is inhibited by CsA and requires
protein synthesis. PBL were incubated for 3 days in the presence of PHA
(5 µg/mL). Dead cells were removed and viable cells were incubated for 3 hours with CsA (250 ng/mL) or CHX (0.5 µg/mL) before treatment with ATG no. 95-07 (10 µg/mL) or CH11 (1 µg/mL). Apoptosis was determined by fluorescence microscopy after staining with Hoechst 33342. Results are expressed as mean ± SD of three different
experiments.
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ATGs induce complement-mediated cytolysis at supramitogenic
concentrations.
Binding of human C1q was measured by incubation of PBL in the presence
of ATGs and fresh human serum, followed by flow cytometry assessment of
the amount of bound C1q per cell. Heat-inactivated human serum was used
as control. Maximal binding was achieved at 1 mg/mL. At lower ATG
concentrations, only rabbit, but not equine, ATG bound C1q (Fig
7). C1q binding was comparable between resting PBL and preactivated cells.
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| Fig 7.
C1q binding to PBL or PHA-blasts sensitized with ATGs.
PBL or PHA blasts were labeled with increasing amount of rabbit ATG (no. 95-07) or horse ATG (no. 1141) and then with autologous serum (solid line) or heat-inactivated serum as control (dashed line). C1q
binding was detected by using FITC-goat anti-C1q polyclonal antibody
and cell analyzed by flow cytometry as described in Materials and
Methods. Representative of three independent experiments.
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The ability of ATGs to induce resting or PHA-activated PBMC lysis was
evaluated in the presence of an exogenous source of human complement.
Minimal cytolysis was observed at 10 µg/mL with equine ATG, whereas
maximal cytolysis was only achieved at very high concentrations (1 mg/mL) of ATGs. As a positive control of complement-mediated cytolysis,
we used the CAMPATH-1M MoAb, which, in agreement with a previous
report,32 induced about 80% lysis at 10 µg/mL. Of note,
no difference was observed, whether ATGs were obtained from horse (no.
1141) or rabbit (no. 95-07), and whether resting or PHA-activated PBMC
were used as target cells in the complement-dependent lysis assay (Fig
8).
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| Fig 8.
Complement-mediated lysis of PBMC versus PHA-Blasts. PBMC
or 3-day PHA-activated PBMC were labeled with 51Cr and
incubated with rabbit ATG (no. 95-07) ( ), horse ATG (no. 1141)
( ), control horse ( ) or rabbit IgG ( ) or the anti-CD52 MoAb
CAMPATH-1M (IgM) ( ) at the indicated concentrations, for 30 minutes
at 37°C, in the presence of 10% autologous serum. Results are
expressed as specific release as defined in Materials and Methods
(mean ± SEM of three different experiments).
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ATGs induce antibody-dependent cell cytotoxicity at low
concentrations.
ATGs no. 95-07 and no. 1141 were tested for their ability to induce
ADCC of both resting and PHA-activated PBMC. We observed that this
effect was concentration dependent, with a maximal cytotoxicity at 1 µg/mL of ATG no. 95-07 and effective only when PHA-activated PBMC
were used as target cells (Fig 9). As
expected, the ADCC phenomenon was not observed with F(ab)2
fragments of ATG no. 95-07 and was restricted to ATG from rabbit
origin, because ATG no. 1141 did not induce cell lysis at
concentrations ranging from 0.01 to 100 µg/mL.
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| Fig 9.
Antibody-dependent cell cytotoxicity of PBMC versus
PHA-blasts. PBMC or 3-day PHA-activated PBMC were labeled with
51Cr and incubated with rabbit ATG (no. 95-07), horse ATG
(no. 1141), or their F(ab)2 fragments at the indicated
concentrations in presence of effector cells for 6 hours at 37°C.
Results are expressed as specific release as in Fig 8. Representative
of two independent experiments.
|
|
| |
DISCUSSION |
Both horse antilymphocyte globulins and rabbit ATGs are still used in
the treatment of severe aplastic anemia, organ allograft rejection, and
graft-versus-host disease (GVHD), but their mechanisms of action remain
largely unknown. A major common feature of ATG treatment is peripheral
lymphocyte depletion,1,4,5,33 which usually persists
throughout the administration period and slowly reverses thereafter.
Although not formally demonstrated in clinical studies, lymphocyte
depletion is likely to account for the immunosuppressive activity of
ATGs.34 The present study addressed the mechanisms of
peripheral lymphocytopenia, with special emphasis on the differential
susceptibility of preactivated T cells (PHA blasts) versus nonactivated
T cells to ATG-induced cell death. ATGs contain multiple antibody
specificities with little batch-to-batch variability despite the use of
different cell sources (thymocytes, T-cell lines, or B-cell lines) and
different immunization protocols.6-8 We therefore tested
two ATG preparations of horse anti-human lymphocyte globulins (no.
1141) and rabbit anti-thymocyte globulins (no. 95-07) currently used in
organ and bone marrow transplantation, as well as one horse ATG
preparation (no. 5) previously used in kidney transplantation (selected
because of its highly unusual lack of mitogenic activity related to the absence of demonstrable CD2 and CD3 specificities).7 Horse anti-lymphocyte globulins are administered at 10 to 15 mg/kg/d,33 and rabbit ATGs at 1.0 to 1.2 mg/kg/d, resulting
in average serum levels of 0.5 mg/mL and 80 to 200 µg/mL,
respectively.5 These dosages have been selected mostly on
empiric grounds, but individual dosage adjustment to maintain absolute
T-cell numbers of 50 to 100 cells/µL did not result in a major
decrease in daily doses.33 It is worth noting that the
10-fold dosage difference between equine and rabbit ATGs is not
paralleled by differences in either specific antibody titers (eg, CD2,
CD3, CD4, CD8)7 or in vitro functional properties such as
T-cell activation5,6,35,36 or B-cell
apoptosis.27
Complement-dependent lysis is initiated by the binding of human C1q to
ATG-coated cells. At low and intermediate ATG concentrations, C1q
binding was demonstrable with rabbit ATG on both PBL and PHA blasts but
remained borderline or not detectable with equine ATG (Fig 7). As shown
with chimeric monoclonal antibodies of different isotypes, C1q binding
may not be correlated with cell lysis.37 Therefore, we used
the highly sensitive chromium release assay to measure
complement-dependent lysis. The data (Fig 8) indicate that equine and
rabbit ATGs are equally effective on PBMC and PHA blasts, but only at
high concentrations. In keeping with our observation, complement
consumption, as measured by decreased serum CH50 activity, was recorded
in some patients during equine ATG treatment, but never with rabbit ATG
(Y. Lebranchu, personal communication, January 1997).
ADCC has been suggested as a possible mechanism of lymphocyte depletion
by ATG.1,5 NK cells present in peripheral blood are potent
effectors of Fc receptor-dependent cell lysis. Our results indicate
that only PHA blasts, but not PBMC, can be lysed through an ADCC
mechanism, suggesting that rabbit ATGs could display some selectivity
toward preactivated lymphocytes, should a similar mechanism operate in
vivo. Equine ATG on the other hand was completely ineffective in this
assay.
The major homeostatic mechanism that prevents lymphoid tissue
hyperplasia despite repeated antigenic stimulations and T- or B-cell clonal expansion is activation-induced cell death (AICD) mediated by Fas/L-Fas interaction.38 We therefore
investigated the possible contribution of the Fas pathway in
ATG-induced lympholysis. Fas-L is constitutively expressed in a variety
of tissues, including immunologically privileged sites (eg, eye,
Sertoli cells), some tumors,39,40 and
monocytes41 and produced by a subset of T cells after
repeated activation through the TCR/CD3 or CD2 pathways, or
both.42 Knowing the T-cell mitogenic properties of
ATGs,35,36 we were not surprised to observe that
restimulation by ATGs of PBL preactivated by various mitogens,
including ATGs themselves, triggered Fas-L gene expression (Fig 3).
However, quite unexpectedly, ATGs were also found to induce Fas-L mRNA
and protein expression in nonpreactivated PBL, even at low
concentrations (10 µg/mL) sufficient to trigger CD69, but not CD25,
expression and therefore remain below the mitogenic threshold (Fig 4).
Although they express Fas receptors, these CD25 negative cells do not
respond to IL-2 and therefore cannot become sensitive to Fas-dependent
apoptosis, as discussed below. The fact that blocking Fas/Fas-L
interaction completely suppressed ATGs-induced apoptosis (Fig 2)
provides unequivocal evidence for a role of the Fas pathway in
ATG-mediated lymphocyte cell death. Target cells for Fas-L should not
only express Fas receptors that are rapidly induced upon activation, but should also become sensitive to Fas-mediated apoptosis, a property
that is strictly dependent on an IL-2 signal.29,30 Hence
pharmacological interference with the IL-2 pathway in activated T
cells, by the addition of CsA, FK506, or rapamycin, prevents Fas-positive cells from becoming sensitive to ATG- and to Fas-L- (or
agonist anti-Fas antibody)-dependent apoptosis. CsA also inhibits Fas-L expression.31,43 Therefore, concomitant
administration of ATGs with any immunosuppressive agent that
interferes with the IL-2 pathway (eg, CsA, FK506, rapamycin, CTLA-4-Ig,
or CD25 antibodies) is likely to prevent Fas-dependent ATG-induced
lymphocyte depletion. Furthermore, this mechanism of lymphocyte
apoptosis may be impaired in clinical situations associated with high
plasma levels of soluble Fas.
In conclusion, this in vitro study describes some of the mechanisms
that may account for lymphocyte depletion during ATG therapy. However,
one should keep in mind that opsonization and subsequent phagocytosis
by spleen, liver, and lung macrophages is likely to account for the
massive and rapid lymphocytopenia observed with the current protocols.
Nevertheless, other mechanisms should be considered, some of which
could represent a therapeutic objective in the design of future
protocols aimed at a more selective immunosuppression. Complement-dependent lysis does not discriminate between resting and
preactivated T cells. Because it is achieved at high ATG
concentrations, it may occur in treatment with horse ATG, but this is
less likely with rabbit ATG. In this respect, the relevance of
complement-dependent lymphocytotoxicity for the standardization of ATG
preparations is questionable. Serum ATG concentrations achieved with
current dosages are mitogenic for peripheral T cells. Hence, they could trigger Fas-L expression and induce sensitivity to Fas-L in the vast
majority of T cells, unless CsA or FK506 that block these processes is
administered concomitantly. An important finding of this study is that
some ATG at low, submitogenic concentrations may trigger Fas-L
expression, resulting in the selective death of preactivated, but not
resting, lymphocytes. An ATG preparation (no. 5) lacking mitogenic
activity, and with no demonstrable CD2 and CD3 specificities, was
devoid of this property, suggesting that "lymphocyte activating"
antibodies (eg, CD2, CD3) may be critical in achieving Fas-dependent
apoptosis. Similarly, ADCC that also occurs at low rabbit ATG
concentration selectively targets activated, but not resting, T cells.
These properties could be used in protocols aiming at the selective
elimination of in vivo activated T cells (eg, donor-specific
alloreactive T cells in organ transplantation, recipient-specific T
cells in GVHD), while sparing nonactivated T cells. Such protocols
would require much lower doses than those currently used, in order to
maintain serum ATG concentrations within a 10- to 20-µg/mL range,
instead of 100 µg/mL. Their feasibility will be evaluated
in the cynomolgus monkey and, depending on the outcome of these
experiments, clinical trials may be considered.
| |
FOOTNOTES |
Submitted July 8, 1997;
accepted November 11, 1997.
*
Within the context of this report, ATG is used to refer to
either antithymocyte or antilymphocyte globulins.
Supported by Institut National de la Santé et de la Recherche
Médicale and by Région Rhône Alpes Grant No.
H098730000.
The first two authors contributed equally to this work and therefore
share the first authorship.
Address reprint requests to Nathalie Bonnefoy-Berard, PhD,
INSERM U80, Hôpital E. Herriot, 69437 Lyon Cedex 03, France.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
Prof Y. Lebranchu (Tours) is thanked for sharing unpublished
observations.
| |
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Abstract
Introduction
Abstract
