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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4771-4777
Dysregulation of CD95/CD95 Ligand-Apoptotic Pathway in
CD3+ Large Granular Lymphocyte Leukemia
By
Thierry Lamy,
Jin Hong Liu,
Terry H. Landowski,
William S. Dalton, and
Thomas P. Loughran Jr
From the H. Lee Moffitt Cancer Center and Research Institute, the
Veterans's Administration Hospital, and the Departments of Medicine,
Microbiology, Immunology, and Pharmacology, University of South Florida
Medical School, Tampa, FL.
 |
ABSTRACT |
CD95 (Fas)-induced apoptosis plays a critical role in the
elimination of activated lymphocytes and induction of peripheral tolerance. Defects in CD95/CD95L (Fas-Ligand)-apoptotic pathway have been recognized in autoimmune lymphoproliferative diseases (ALPS)
and lpr or gld mice and attributed to CD95 and CD95L
gene mutations, respectively. Large granular lymphocyte (LGL) leukemia is a chronic disease characterized by a proliferation of
antigen-activated cytotoxic T lymphocytes. Autoimmune features such as
hypergammaglobulinemia, rheumatoid factor, and circulating immune
complexes are common features in LGL leukemia and ALPS. Therefore, we
hypothesize that expansion of leukemic LGL may be secondary to a
defective CD95 apoptotic pathway. In this study, we investigated
expression of CD95 and CD95L in 11 patients with CD3+ LGL
leukemia and explored the apoptotic response to agonistic CD95
monoclonal antibody (MoAb). We found that leukemic LGL from each
patient expressed constitutively high levels of CD95/CD95L, similar to
those seen in normal activated T cells. However, cells from 9 of these
11 patients were totally resistant to anti-CD95-induced apoptosis.
Similarly, cells were resistant to anti-CD3-MoAb-triggered cell death.
Lack of anti-CD95-induced apoptosis was not due to mutations in the
CD95 antigen. Leukemic LGL were not intrinsically resistant to
CD95-dependent death, because LGL from all but 1 patient underwent
apoptosis after phytohemagglutinin/interleukin-2 activation. The
patient whose leukemic LGL were intrinsically resistant to CD95 had an
aggressive form of LGL leukemia that was resistant to combination
chemotherapy. These findings that leukemic LGL are resistant to
CD95-dependent apoptosis despite expressing high levels of CD95 are
similar to observations made in CD95L transgenic mice. These data
suggest that LGL leukemia may be a useful model of dysregulated
apoptosis causing human malignancy and autoimmune disease.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
LARGE GRANULAR lymphocyte (LGL)
leukemia can be classified into CD3+ (T cell) and
CD3 (natural killer [NK] cell) type depending on
the cell lineage of the leukemic cells.1 Autoimmune
manifestations are a prominent and characteristic feature of T-LGL
leukemia. Serologic abnormalities are frequent, including
autoantibodies such as rheumatoid factor and antinuclear antibody, as
well as high levels of circulating immune complexes and polyclonal
hypergammaglobulinemia.1-5 Autoimmune disease, particularly
rheumatoid arthritis, also occurs frequently in LGL leukemia. Increased
numbers of LGL could be explained either by stimulation of
proliferation or by inhibition of apoptosis. Circulating leukemic LGL
are in the Go/G1 phase of the cell cycle6; therefore, we
hypothesize that extended cell survival may be secondary to defects in
apoptosis.
Leukemic LGL show many characteristics of antigen-activated T
cells.6 The physiological deletion of antigen-activated T cells occurs through apoptosis, mediated through CD95 antigen (Fas).7,8 CD95 is a transmembrane protein, belonging to the tumor necrosis receptor family.9 Ligation of CD95 by CD95
Ligand (FasL) or by anti-CD95 monoclonal antibody (MoAb) induces
apoptosis of target cells bearing CD95.10-14
CD95/CD95L-triggered apoptosis is involved in control of the immune
response, induction of peripheral tolerance, and killing of
viral-infected or malignant cells.8,15,16
A defect in CD95-dependent apoptosis is the underlying pathogenetic
mechanism in animal models of lymphoproliferative disorders associated
with autoimmune manifestations. Lpr/Lpr mice have mutations in
CD95, whereas gld/gld mice have mutations in
CD95L.17,18 Both animal models are characterized by
hypergammaglobulinemia, rheumatoid factor, and circulating immune
complexes, features similar to those observed in LGL
leukemia.19,20 Dysregulation of CD95/CD95L is also seen in
CD95L transgenic mice.21 In this model, high levels of
CD95L result in the selection of a novel population of activated T
cells that express high levels of CD95, but that are resistant to
CD95-mediated apoptosis.
In this study, we examined expression of CD95 and CD95L by leukemic LGL
and evaluated whether leukemic LGL were susceptible to CD95 and T-cell
receptor (TCR)-induced apoptosis. We found that leukemic LGL expressed
high levels of CD95 and CD95L, similar to levels seen on normal
activated T cells. Despite high CD95 expression, leukemic LGL were
resistant to CD95 or TCR-triggered cell death.
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PATIENTS AND METHODS |
Patients.
All patients met clinical criteria of T-LGL leukemia, with LGL counts
ranging from 600 to 27,000/µL (normal, 223 ± 99/µL) and
evidence of clonal TCR gene rearrangement.1 Clinical and laboratory features of these patients are shown in
Table 1. Nine patients had chronic disease
not requiring treatment. Patient no. 5 was receiving methotrexate for
neutropenia and rheumatoid arthritis; on therapy, the neutrophil count
increased into the normal range. Patient no. 10 had an aggressive form
of LGL leukemia, refractory to methotrexate. He presented with massive,
painful hepatomegaly with increased circulating LGL (16 × 109/L) and thrombocytopenia (20 to 30 × 109 platelets/L). This patient did not have   T-cell
lymphoma, because the leukemic cells were   + and
as determined by MoAb staining. Four cycles
of CHOP produced no response.
CD95 and CD95L expression.
Fresh peripheral blood mononuclear cells (PBMC) were obtained from the
11 LGL leukemia patients and from the buffy coat of healthy donors.
PBMC were isolated by Ficoll-Hypaque (Pharmacia Biotech, Uppsala,
Sweden) gradient centrifugation and then analyzed by flow cytometry
(FacScan; Becton Dickinson, Mountain View, CA).
Analysis of CD95 surface expression was performed using phycoerythrin
(PE)-conjugated UB2 MoAb (Kamiya Biomedical Corp, Tukwila, WA). To
measure CD95 expression on LGL leukemic cells, the cells were incubated
with UB2 MoAb and anti-CD57-fluorescein isothiocyanate (FITC) MoAb
(Immunotech, Marseille, France). Negative isotype control MoAbs were
IgG1-PE (Immunotech) and IgM-FITC (Dako, Copenhagen, Denmark). PBMC
from healthy donors and leukemic LGL were cultured in RPMI medium
(Fischer Scientific, Pittsburgh, PA) supplemented with 10% fetal calf
serum and activated for 2 days with 1 µg/mL phytohemagglutinin (PHA;
Sigma, St Louis, MO) and for 10 additional days with recombinant
interleukin-2 (IL-2; 100 U/mL; Chiron, Emeryville, CA). CD95 expression
was determined before and after activation. For determination of CD95
expression on normal CD57+ T cells or NK cells, three-color
staining was performed using MoAbs: CD95-PE UB2, CD57-FITC, and CD3-Cy5
(Immunotech). CD3+/CD57+ and
CD3/CD57+ cells were gated for
determination of CD95 expression.
Detection of CD95L expression required intracellular staining as
previously described.22,23 PBMC were washed twice with phosphate-buffered saline (PBS) and fixed for 10 minutes at 4°C with 0.5% paraformaldehyde-PBS. After centrifugation, PBMC were resuspended in 0.1% Triton X-100-PBS for 3 minutes. PBMC were then
washed and incubated with 2 µg anti-CD95L C-20 MoAb (Santa Cruz
Biotechnology, Santa Cruz, CA) for 30 minutes at 4°C and then
rinsed three times in PBS containing 1% bovine serum albumin and 0.1%
sodium azide. Nonspecific binding sites were blocked for 30 minutes
with 20% normal swine serum. The cells were washed again and incubated
with an FITC-conjugated swine antirabbit Ig MoAb (Dako) for 30 minutes
at 4°C. Normal rabbit Ig (Dako) diluted to the same protein
concentration as the primary antibody was used as a negative control.
After washing, the cells were analyzed using flow
cytometry.24,25
Apoptosis assay.
For the apoptosis assays, unactivated or activated PBMC were
transferred to a 96-well culture plate at a concentration of 5 × 105/mL. The cells were then incubated with 1 µg/mL of anti-CD95 MoAb (CH 11; Kamiya Biomedical Corp) or 10 µg/mL
of anti-CD3 MoAb (BC3; kindly provided by C. Anasetti, FHCRC, Seattle,
WA) for 24 or 48 hours. The same conditions were used to induce cell
death induced by 50 µmol/L of C2-ceramide (Sigma). Determination of
apoptosis was performed by staining with 7-amino-actinomycin D (7-AAD;
Calbiochem, San Diego, CA) and propidium iodide (PI; Molecular Probes,
Inc, Eugene, OR), as described.24,25 For 7-AAD
staining, cells were incubated with 7-AAD at a concentration of 20 µg/mL for 30 minutes at 4°C in the dark. The cells were then
resuspended in PBS and analyzed using flow cytometry. For PI staining,
cells were incubated with 50 µg/mL of PI for 30 minutes at room
temperature, after fixation overnight with 70% ethanol. To ensure that
apoptotic signal was related specifically to anti-CD95, the cells were
preincubated with 500 ng/mL CD95 blocking ZB4 MoAb (Kamiya Biomedical
Corp) for 60 minutes before treatment with the apoptosis-inducing MoAb CH11. T-cell leukemia CEM cell line was used as a positive control. CD95- or TCR-specific apoptosis was determined as follows: (% of
apoptotic cells in the assay well  % of apoptotic cells in the
control well)/(100 % of apoptotic cells in the control well) × 100.
Cell sorting.
In experiments examining apoptosis of activated LGL, flow cytometry was
used to isolate the leukemic CD57+ cells. Cells (12 to 15 × 106) were washed in PBS, resuspended in 300 µL of
PBS, and incubated for 30 minutes at 4°C with CD57 FITC MoAb.
CD57+ cells were then sorted on a FACStar (Becton
Dickinson) cell sorter and directed to apoptosis assay as described
above. The purity of enriched CD57+ cells was 93% to 96%.
Western blotting.
Cells were lysed in a buffer composed of 1% Nonidet P-40, 10 mmol/L
Tris (pH 7.4), 0.1 mmol/L phenylmethylsulfonyl fluoride, EDTA, 10 mmol/L iodometacine, 1 µg/mL leupeptin, 1 µg/mL apoprotin, 0.4 mmol/L Na orthovanadate, and antipain for 45 minutes at 4°C. Equal
amounts of protein were loaded on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to
nitrocellulose membrane. After blocking with PBS buffer containing 5%
milk and 0.1% tween, the membranes were probed with N-20 anti-CD95L
(Santa Cruz Biotechnology) for 2 hours at room temperature followed by horseradish peroxidase (HRP)-conjugated secondary antibody. Membranes were washed and developed using a chemoluminescent detection system (ECL; Amersham Life Science, Arlington Heights, IL).
Analysis of CD95 gene.
Sufficient material was available for mutational analyses of the CD95
coding sequence in 5 patients. These analyses were performed by reverse
transcription-polymerase chain reaction (RT-PCR)
single-stranded conformation polymorphism (SSCP) followed by cDNA
sequencing. Total RNA was extracted from PBMC of LGL leukemia patients
or normal subjects and transcribed to cDNA. The open reading frame of
the CD95 cDNA was amplified with three overlapping sets of primers and
analyzed by SSCP under previously established
conditions.26,27 The cytoplasmic region, which encompasses
the signal transducing death domain, was further examined for mutations
by denaturing gradient gel electrophoresis (DGGE). The cDNAs showing
mobility shifts were extracted from the gel and reamplified using the
same primer set. PCR products were subcloned into the TA cloning
vector pCR2.1 (Invitrogen, La Jolla, CA), and multiple clones
were selected for bidirectional sequencing (ALF, Piscataway, NJ).
| |
RESULTS |
CD95/CD95L expression.
PBMC from all patients expressed constitutively high levels of surface
CD95 and CD95L protein (Table 2). The mean
percentage of CD95+ LGL cells was 88% ± 7%, similar
to that of activated T cells (94% ± 3%) and much higher than that
in normal PBMC, in which CD95 is expressed at a relatively low level
(35% ± 11%). Almost 80% of leukemic CD57+ cells
coexpressed CD95 (Fig 1), whereas in normal
PBMC, only a minority of CD57+ cells expressed CD95 (32% ± 12%). Because normal CD57+ cells may be either
CD3 or CD3+, we performed three-color
analysis of normal PBMC to further delineate CD95 expression. We found
that CD95 was expressed on 32% ± 10% of normal CD3+,
CD57+ cells (n = 10). Therefore, there was a much higher
frequency of CD95 expression on leukemic LGL compared with their normal CD3+, CD57+ counterparts. It is of interest
that the percentage of normal CD57+ cells expressing CD95
after activation was similar to that seen constitutively in leukemic
LGL. Figure 2 shows the results of flow
cytometry detection of CD95L. As previously described, CD95L was
expressed on normal PBMC only after activation. In contrast, we found
constitutive expression of CD95L on PBMC from all LGL leukemia
patients. The level of expression of CD95L on leukemic LGL appeared
higher than that observed on activated normal T cells. The mean
fluorescence intensity of CD95L was 3.9 (range, 1.7 to 7) in leukemic
LGL, as compared with 1.5 (range, 1.43 to 1.6) in activated PBMC.
Western blot analysis of whole cell lysates confirmed the elevated
levels of CD95L in leukemic LGL compared with normal activated PBMC
(not shown).
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| Fig 1.
Representative flow cytometry results showing
coexpression of CD95 on CD57+ leukemic LGL from patient
no. 9. Ninety-one percent and 41% of the cells express CD95 (graph on
the left) and CD57 (graph on the center), respectively. In dual
fluorescence, 82% of the CD57+ cells coexpress CD95
(graph on the right).
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| Fig 2.
Determination of CD95L expression on normal PBMC (before
and after PHA/IL-2 activation) and in 2 cases of freshly isolated LGL
leukemia (cases no. 4 and 11). The cells were fixed and permeabilized
and then stained with anti-CD95L MoAb (C-20) followed by secondary
antirabbit-FITC MoAb. The shaded area represents the level of
fluorescence obtained with an isotype control MoAb, the unshaded area
represents the level with CD95L MoAb.
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Results of apoptosis assay (Table
3).
Freshly isolated PBMC from 10 of 11 patients showed no apoptosis after
24 hours of exposure to anti-CD95 MoAb (Fig
3). After prolonged incubation with anti-CD95 MoAb for 48 hours, cells
still remained resistant, except for patient no. 3. In this patient, 31% of the cells were apoptotic. Similar results were seen after 48 hours of anti-CD3 MoAb activation. Absence of apoptosis was observed in
9 of 10 patients, with only patient no. 3 showing slight susceptibility
with 28% apoptotic cells (not shown). Ceramide-induced cell death was
detected in all 8 cases studied and was comparable to normal PBMC (47% ± 13% v 42% ± 11%, respectively; not shown).
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| Fig 3.
Freshly isolated LGL are resistant to CD95 and
anti-CD3-mediated apoptosis. CEM-sensitive cell line was used as a
positive control for CD95-induced apoptosis (top panel). The cells were
incubated with media alone (histograms on the left) or with anti-CD95
(CH11, 1 µg/mL) for 24 and 48 hours, as shown. The cells were then
stained with 7-AAD and analyzed by flow cytometry. The LGL leukemic
cells are analyzed in the bottom panel. The percentage of apoptotic
cells is indicated in each histogram. Although leukemic LGL were
resistant to both anti-CD95- and anti-CD3-induced apoptosis (BC3, 10 µg/mL), they remained susceptible to ceramide.
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After activation with PHA and IL-2, PBMC from 10 of 11 patients showed
susceptibility to anti-CD95, with a mean percentage of apoptotic cells
of 50% (range, 30% to 76%). Likewise, exposure to anti-CD3 MoAb
induced an apoptotic response in 10 of 11 patients, with a mean
percentage of apoptotic cells of 45% (range, 29% to 63%). The
apoptotic effect of anti-CD95 MoAb was specifically inhibited by ZB4
MoAb (Fig 4). To ensure that apoptosis was
occurring in LGL leukemic cells rather than in normal cells expanded
after 10 days of IL-2 activation, CD57+ cells from 3 patients were sorted after activation and exposed to anti-CD95 and
anti-CD3 MoAb. Purity after cell sorting was greater than 93%
CD57+ cells in each case.
Figure 5 shows that 76% of the
CD57+ cells underwent apoptosis after anti-CD95 MoAb in
patient no. 4. Similar results were seen after activation with anti-CD3
MoAb (not shown). The same data were obtained with the
CD57+-sorted cells from patients no. 5 and 7.
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| Fig 4.
Protection of CD95-induced apoptosis by CD95 blocking
MoAb ZB 4. The activated PBMC of patient no. 11 were preincubated with
ZB 4 (500 ng/mL for 60 minutes) and then exposed to
anti-CD95 for 48 hours. The cells were incubated with 50 µg/mL of PI
for 30 minutes at room temperature, after fixation overnight with 70%
ethanol, and the DNA content was analyzed using flow cytometry.
Histograms on the left, center, and right represent the results after
incubation with serum alone, anti-CD95, and ZB4 + anti-CD95,
respectively. The percentage of apoptotic cells is indicated on the
left side of each panel.
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| Fig 5.
Flow cytometry results showing that leukemic LGL are
susceptible to anti-CD95-induced apoptosis after activation. The PBMC
of patient no. 4 were cultured initially with PHA (1 µg/mL) for 2 days and then with IL-2 (100 U/mL) for 10 more days. The cells were
then stained with CD57+ and sorted on FacStar. The
purified CD57+ cells (94%) were then incubated with
anti-CD95 MoAb (CH11, 1 µg/mL) for 48 hours and stained with 7-AAD
before analysis using flow cytometry. The graph on the left represents
the control (media alone); the graph on the right represents the cells
incubated with CH11. The percentage of apoptotic cells is shown on the
upper-right quadrant.
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Analysis of CD95 gene mutation.
Function ablating mutations in the CD95 gene have been shown to be
causative in some autoimmune diseases. Using RT-PCR SSCP and DGGE
analysis, followed by cDNA sequencing of PCR products, we examined the
CD95 coding sequence to determine if mutations might account for the
failure of LGL leukemia cells to undergo apoptosis when exposed to
anti-CD95 MoAb. No mutations were detected in the CD95 coding sequence
of the LGL leukemia patients studied. Four of the five patients
examined expressed a previously documented polymorphism at bp 836, which does not alter the amino acid sequence.
| |
DISCUSSION |
CD3+ LGL leukemia is a chronic clonal T-cell
lymphoproliferative disorder recognized as a distinct entity using
clinical, immunological, and molecular parameters.1,2,28
The clonal expansion of leukemic LGL may require a multistep
pathogenesis. Leukemic LGL have many characteristics of
antigen-activated cytotoxic T lymphocytes (CTL): (1) They express a
T-cell cytotoxic phenotype and can be activated via the CD3/CD16
pathway.6,29 (2) They constitutively express perforin and
CD95L.30,31 (3) At least in some cases, they use a
restricted V repertoire, reinforcing the hypothesis of antigenic
selection.32 Increased numbers of LGL could be explained
either by a stimulation of proliferation or inhibition of programmed
cell death. CD95/CD95L interactions play a major role in the induction
of cell death after T-cell activation.8-11 CD95 is weakly
expressed on the surface of resting T cells and is upregulated after
antigen activation.9,14 The accumulation of peripheral T
cells might result from a defect in removing antigen-activated T cells.
Therefore, expansion of LGL leukemic cells could be due to a defect in
the CD95 apoptotic pathway.
We studied the CD95/CD95L apoptotic pathway in 11 cases of T-LGL
leukemia. We found a constitutively high surface expression of CD95 and
CD95L in all 11 cases at levels similar to, if not greater than, those
seen in normal activated T cells. LGL specifically expressed CD95,
because almost 80% of CD57+ LGL coexpressed CD95. Despite
this high level of CD95 expression and evidence of a constitutively
activated T-cell phenotype, freshly isolated cells from 9 of 11 patients were completely resistant to anti-CD95-induced apoptosis. In
the 2 remaining patients (patients no. 1 and 3), CD95-induced apoptosis
was lower than levels seen in control cells, especially in patient no.
3, whose cells displayed a slight apoptotic response only after 2 days
of anti-CD95 MoAb exposure. A similar pattern of resistance to
TCR-triggered cell death was also observed. Normal T cells become
sensitive to apoptosis after activation, when expression of high level
of CD95 and CD95L is observed. Our observations that leukemic LGL is
resistant to apoptosis despite expressing high levels of both CD95 and
CD95L suggest that this apoptotic pathway is dysregulated in LGL
leukemia.
These results of CD95 resistance are similar to findings observed in
animal models of lymphoproliferation and autoimmune disease occurring
in lpr and gld mice.19,20 The pathogenesis
of CD95 resistance in these murine models is due to mutations in CD95 and CD95L, respectively.17,18 Recently, resistance to
anti-CD95-induced apoptosis has been described in a human disease,
termed autoimmune lymphoproliferative syndrome (ALPS).33-37
The clinical and biological features of ALPS are very similar to those
observed in LGL leukemia, including the following: (1) The majority of
the uniquely expanded CD4, CD8
cells in ALPS are CD57+.33 (2) The patients
present with splenomegaly, hypergammaglobulinemia, and autoimmune
hemolytic anemia, thrombocytopenia, or neutropenia. CD95 gene mutations
primarily involving the death domain have been described in these
patients. We previously reported the absence of mutations in the CD95
death domain in seven patients.31 However, some patients
with ALPS have had mutations in regions of CD95 other than the death
domain.34 For these reasons, we examined the entire CD95
antigen cDNA in the LGL leukemia patients. We found no evidence for
CD95 mutation in each of the 5 patients studied. We have also found no
evidence for CD95 mutation in 4 additional patients (unpublished
observations). Therefore, dysregulated CD95-dependent
apoptosis in LGL leukemia does not result from mutations in CD95.
Recently, an autoimmune lymphoproliferative disease (ALD) has been
described with generalized autoimmune manifestations but without
expansion of dual CD4/CD8-negative T cells.38
Interestingly, the CD95+ cells are resistant to
anti-CD95-induced apoptosis but do not display any CD95 gene
mutations. Ceramide is the second messenger produced by hydrolysis of
sphingomyelin after a CD95 apoptotic signal.39 In ALD
patients, ceramide induced cell death is deficient, suggesting a
downstream alteration of the apoptosis pathway.38 Ceramide-induced apoptosis was normal in LGL leukemia patients, suggesting that the cause of apoptotic resistance is different in LGL
leukemia compared with ALD patients.
The mechanism resulting in resistance to CD95-induced apoptosis in LGL
leukemia is not known. From our study we do know that resistance is not
due to lack of CD95 surface expression or mutant CD95 protein, as seen
in myeloma samples or myeloma cells lines.25,27 We also
know that there is not an intrinsic defect in apoptosis in LGL
leukemia, because leukemic cells from 10 of 11 patients underwent
apoptosis after activation with IL-2. It is conceivable that lack of
IL-2 in vivo might explain the apoptotic resistance. IL-2 predisposes
peripheral T cells to CD95 and anti-CD3/TCR-induced cell
death.14 IL-2-deficient mice
(IL-2/) develop a fatal disease with
lymphadenopathy, splenomegaly, and multiorgan T-cell
infiltration.40 Activated T cells from these
IL-2/ mice display a CD95-resistant
phenotype, although they express CD95 similarly to cells from their
IL-2+/+ littermates. Although LGL leukemic cells
constitutively express p75 IL-2 receptor, they do not produce IL-2 gene
transcripts or secrete IL-2 even after anti-CD3 MoAb
activation.41,42
Leukemic cells from 1 patient remained resistant to both CD95 and TCR
triggered cell death even after activation with IL-2. No abnormalities
were observed in CD95 gene, suggesting that the defect in apoptosis is
distal to the receptor. This patient had an aggressive clinical course,
presenting with pancytopenia and massive hepatomegaly, which was
refractory to combination chemotherapy. We recently reported that
chronic LGL leukemias express relatively high levels of multidrug
resistance gene (MDR1) and that P-glycoprotein was functionally active
in CD57+ leukemic LGL.43 It is of interest that
leukemic LGL from this patient showed a high level of P-glycoprotein
surface expression (not shown). It has been suggested that
chemotherapeutic agents may induce apoptosis through the CD95/CD95L
pathway.16 Anthracycline-resistant cell lines
are also resistant to CD95-induced apoptosis.25 Therefore, elucidation of the mechanism of resistance to CD95-mediated apoptosis in this patient may also help delineate mechanisms involved in drug
resistance.
Our data suggest that LGL leukemia can serve as a useful model of
dysregulated apoptosis causing human malignancy and autoimmune disease.
Our results showing that leukemic LGL express high levels of CD95 yet
are resistant to CD95-mediated apoptosis are similar to findings
observed in CD95L transgenic mice. In this animal model,
expression of CD95L leads to disease manifestations. High levels of soluble CD95L have been found in sera from LGL leukemia patients.44 Growth of hematopoietic colonies in vitro is
negatively regulated by activation of the CD95 pathway.45
Mature neutrophils undergo apoptotic death through CD95
triggering.46 Taken together, these data suggest that
secretion of CD95 ligand may be a mechanism leading to neutropenia in
LGL leukemia. Studies investigating this hypothesis are ongoing in our
laboratory.
| |
FOOTNOTES |
Submitted February 17, 1998;
accepted August 10, 1998.
Supported by the Veterans Administration. T.L. is a recipient of
"Association pour la Recherche Contre le Cancer" and
"Pharmacia" grants. The Flow Cytometry and Molecular Biology Core
laboratories at H. Lee Moffitt Cancer Center and Research Institute
were used in the course of this work.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Thomas P. Loughran, Jr, MD, H. Lee Moffitt
Cancer Center and Research Institute, 12902 Magnolia Dr, Tampa, FL
33612; e-mail: Loughrat{at}moffitt.usf.edu.
| |
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Abstract
Introduction