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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2875-2885
Tumor B Cells From Non-Hodgkin's Lymphoma Are Resistant to CD95
(Fas/Apo-1)-Mediated Apoptosis
By
Joël Plumas,
Marie-Christine Jacob,
Laurence Chaperot,
Jean-Paul Molens,
Jean-Jacques Sotto, and
Jean-Claude Bensa
From the Immunology Department, ETS Isère-Savoie and Research
Group on Lymphoma, Unité UPRES 2021, Grenoble, France.
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ABSTRACT |
Apoptosis mediated by the CD95 (Fas/Apo-1) molecule plays a crucial
role in the regulation of the B-cell immune response. In this study, we
examined the function of the CD95 antigen in B-cell-derived
non-Hodgkin's lymphoma (NHL), a malignant disease of mature B cells.
Membrane CD95 molecules were found to be constitutively expressed in a
large number of NHL, including mantle cell (MCL, n = 10), lymphocytic
(LCL, n = 10), follicular (FL, n = 11), and diffuse large cell
lymphoma (DLCL, n = 9) with, however, different levels of intensity.
Indeed, the levels of CD95 were low in MCL and LCL as compared with FL
and DLCL. However, regardless of the intensity of expression, CD95
triggering with anti-CD95 monoclonal antibody (MoAb) did not induce
apoptosis of lymphoma B cells, while these cells underwent apoptosis
after irradiation or staurosporine treatment. Further experiments were
then performed to address whether apoptosis could be restored by B-cell
activation via CD40 cross-linking. We showed that CD40 engagement in
the presence of interleukin (IL)-4 was more effective than CD40
engagement alone in upregulating the CD95 antigen and induced
CD95-mediated cell death in nontumoral B cells. Concerning
malignant B cells, CD40 ligation in the presence of IL-4 strongly
increased CD95 expression, but did not markedly increase CD95-induced
apoptosis. Furthermore, using cytotoxic T cells, we showed that CD95L
was also ineffective in inducing apoptosis in lymphoma B cells, whereas these cells were killed by the perforin pathway. Our findings suggest
that the CD95-mediated cell death pathway is altered in malignant cells
from the NHL we tested. This could be a mechanism allowing lymphoma B
cells to escape from immune regulation.
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INTRODUCTION |
CD95 (FAS/APO-1) BELONGS to the tumor
necrosis factor (TNF) receptor superfamily1,2 and mediates
apoptosis after cross-linking with CD95 ligand (CD95L)3 or
specific antibodies.4,5 The homeostasis of the immune
response is highly regulated by such interactions. Indeed,
CD95-mediated apoptosis plays a crucial role in the activation-induced
cell death of T lymphocytes6,7 and in T-cell-mediated
cytotoxicity.8,9 Concerning the human B-cell immune
response, the role of CD95 is underlined by the development of an
autoimmune lymphoproliferative syndrome in children who have defective
CD95-mediated apoptosis.10,11 Moreover, recent work has
demonstrated the key role of in vivo CD95 ligation in the expansion of
antigen-reactive B cells and elimination of tolerant B
cells.12 The dual reactivity following CD95 engagement is
regulated by signals from both the B-cell antigen receptor (BCR) and
CD40. Like CD95L, CD40 ligand (CD40L) is a member of the TNF
superfamily and is expressed by activated CD4+ T
cells.13,14 It promotes the growth of B cells by ligation with CD40. CD40 cross-linking also upregulates CD95 expression on B
cells and induces susceptibility to CD95-mediated apoptosis of tolerant
B cells12 or of B cells in the absence of BCR
stimulation.15-17 In contrast, CD40-activated B cells
become resistant to CD95-based apoptosis if the BCR is
engaged18,19 and even proliferate in vivo.12
Malignant non-Hodgkin's lymphomas (NHL) are derived from a clonal
expansion of B cells arrested at different stages of
differentiation.20 Thus, lymphoma cells are the neoplastic
counterparts of naive, activated, or memory normal B cells that each
express a unique BCR. The nature of the antigen recognized by tumoral
BCR is generally unknown or thought to be an
autoantigen.21,22 Malignant B cells also share, with normal
B cells, an antigen presenting function that allows them to generate
antitumor cytotoxic lymphocytes.23 Moreover, CD40 is
functional on tumor B cells because its ligation can induce resistance
to spontaneous apoptosis.24 CD95 expression has been
detected in NHL by immunohistochemical analysis,25-27 however, its role in the induction of apoptosis or proliferation has
not been fully investigated. In human lymphoma B-cell lines, some
reports show that CD95-ligation induces apoptosis.4,28,29 In chronic B-lymphocytic leukemia, malignant cells activated by Staphylococcus aureus Cowan I plus interleukin (IL)-2 undergo apoptosis
after CD95 engagement except in one case, in which tumor cells
proliferated.30
We investigated whether CD95 could be involved in NHL malignant B-cell
development and in their susceptibility to T-cell-mediated cytotoxicity. We show here that all isolated tumor cells express CD95,
but at various levels, and are resistant to apoptosis mediated by CD95
cross-linking either by specific monoclonal antibody (MoAb) or by CD95L
expressed on cytotoxic T cells. CD40 activation upregulates CD95
expression on malignant B cells, but poorly restores responsiveness to
CD95-mediated apoptosis.
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MATERIALS AND METHODS |
Cell lines, medium, and cytokines.
The mouse fibroblastic L cells stably transfected with the human CD40
Ligand (CD40Lig-L cells)15 was kindly provived by Dr J. Banchereau (Schering-Plough, Dardilly, France). CD40Lig-L cells, Jurkat T cells, and Epstein-Barr virus (EBV)-immortalized B-lymphoblastoid cell lines (BLCL) were grown at 37°C in 5%
CO2 in air, in RPMI 1640 containing 1 mmol/L sodium
pyruvate, 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 µg/mL
streptomycin, and nonessential amino acids (complete medium)
supplemented with 10% fetal calf serum.
Purified human rIL-4 was purchased from R&D Systems (Oxon, UK) and
human rIL-2 was provided by Roussel Uclaf (Romainville, France).
Preparation and CD40-activation of lymphoma and nontumoral B cells.
Lymphoma B cells were obtained from lymph nodes or spleens from 40 NHL
patients, including 10 lymphocytic (LCL), 10 mantle cell (MCL), 11 follicular (FL), and nine diffuse large cell (DLCL) NHL according to
the Revised European-American Lymphoma (REAL) classification.20 Nontumoral cells were obtained from lymph nodes from patients with benign hyperplasia or from spleens from cadavers. Biopsies were gently dissociated with a scalpel in RPMI 1640 and filtered through a 100-µm cell strainer to remove aggregates. The
lymphoma samples were included in this study when the malignant clone
represented more than 98%, evaluated by the positivity with anti-
or - chain MoAb, of all B cells present. The malignant or nontumoral
B lymphocytes were separated using a standard rosetting technique using
2-aminoethyl-isothiouronium bromide (AET)-sensitized sheep red blood
cells. A flow cytometric analysis with CD3/CD19 antibodies was
performed to evaluate the purity of the suspension, which was at least
97%. CD19+ B cells were then cryopreserved until
phenotypic or functional assays were performed. The viability of cells
used in all experiments was at least 90%.
CD40-activation of nontumoral and lymphoma B cells was performed using
CD40Lig-L cells. B cells were seeded in 24-well flat-bottom plates at 5 × 105 per well in the presence of 5 × 104 irradiated (7,500 rad) CD40Lig-L cells in complete
medium supplemented with 15% human A serum in the presence or absence
of 10 ng/mL IL-4 for 3 days at 37°C in 5% CO2 in air.
CD95 expression analysis.
CD95 expression on cells was determined by indirect fluorescence.
Briefly, 5 × 105 cells were incubated for 20 minutes
at 4°C with anti-CD95 MoAb CH-11 (Immunotech, Marseille, France)
and washed twice with Hanks' Balanced Salt Solution (HBSS)
supplemented with 2% newborn calf serum. They were then incubated with
0.2 mL phycoerythrin-conjugated goat antimouse IgM (Immunotech) for 20 minutes at 4°C and again washed twice.
Immunostained cells were analyzed by flow cytometry on a
FACScan (Becton Dickinson, Pont de Claix, France).
Nonspecific staining was determined using an IgM control MoAb. Both
percentages of positive cells and mean fluorescence intensities (MFI)
were recorded.
Measurement of apoptosis by 51Cr-release assays.
Cells (1 × 106 ) were labeled with 100 µCi
51Cr-sodium chromate for 1 hour at 37°C in 5%
CO2 in air, then washed three times and resuspended at
final concentration 1 × 105 cells/mL in complete
medium supplemented with 15% serum A. Each V-shaped well of 96-well
microtiter plates received 100 µL of cells (104) and 100 µL of anti-CD95 MoAb CH-11 or isotype control IgM MoAb at final
concentration indicated within the text. After 4 hours or 18 hours at
37°C, microplates were centrifuged, and 100 µL aliquots of
supernatants were assayed for radioactivity. The percentage of
apoptotic cells, evaluated by the percentage of
51Cr-release, was calculated according to the following
formula: % 51Cr-release = 100 × (ER  SR)/(MR SR), where ER, SR, MR represent experimental, spontaneous, and
maximum 51Cr-release, respectively. All expressed values
are derived from averaged quadruplicate determinations.
Measurement of apoptosis by annexin V/propidium iodide
(PI) double staining.
Annexin V binds to phosphatidylserine and allows the detection of the
loss of cell phospholipid asymmetry, an event that appears during the
early phases of apoptosis. The Apoptest containing annexin
V-fluorescein isothiocyanate (FITC) was purchased from Nexins Research
B.V. (Maastricht, The Netherlands). As described,31 cells
(5 × 105) were washed with ice-cold
phosphate-buffered saline (PBS) and were incubated for 10 minutes in
the dark in 500 µL of binding buffer containing annexin V-FITC
solution and 10 µg/mL propidium iodide (PI). Without washing, cells
were then analyzed on a FACScan. Early apoptotic cells were only
stained by annexin V-FITC, whereas late apoptotic or necrotic cells
were double-stained by annexin V-FITC and PI. The specific apoptosis,
consisting of the percentage of viable cells undergoing apoptosis by
the effect of the inducer, was calculated according to the following
formula: 100 × ([D assay D control]/100 D
control) where D represents the percentage of cells dying during the
culture through apoptosis or necrosis. For induction of apoptosis,
cells were incubated in complete medium supplemented with 15% human
serum in the presence of 1 µg of anti-CD95 MoAb CH-11 or isotype
control IgM MoAb. Irradiation exposure (3,000 rad) and staurosporin
treatment was also performed. Apoptosis was evaluated after 4 hours, 18 hours, or both at 37°C.
Cytotoxicity assay.
The cytotoxicity of allogeneic T cells against tumor targets was
measured in standard 4-hour 51Cr-release assays as
previously described.23 Briefly, 104
51Cr-labeled target cells were mixed with the effector cells at different E/T ratios (25/1 to 0.01/1). After a 4-hour incubation at
37°C in 5% CO2 in air, the radioactivity in the
supernatants was counted. The percentage of specific lysis was
calculated according to the following formula: % lysis = 100 × (ER SR)/(MR SR), where ER, SR, MR represent
experimental, spontaneous, and maximum 51Cr-release,
respectively. All expressed values are derived from averaged
quadruplicate determinations.
Effector allogeneic T cells were obtained from unidirectional mixed
lymphocyte reaction (MLR) performed by mixing HLA
mismatched purified CD3+ T lymphocytes and irradiated
(3,000 rad) lymphoma B cells, restimulated once with lymphoma cells and
IL-2. The effector cells were CD3+/T-cell receptor
(TCR)  + (99%), CD4+ and CD8+.
Before cytotoxicity assays, effector cells were preincubated for 2 hours with a mixture of phorbol myristy acetate (PMA; Sigma, St Louis,
MO; final concentration, 5 ng/mL) and the Ca2+ ionophore
ionomycin (Sigma; final concentration, 1.5 µg/mL). The cytotoxicity
tests were performed with Jurkat cells or lymphoma B cells as target
cells in complete medium supplemented with 15% human A serum in the
presence or absence of 3 mmol/L EGTA/4 mmol/L Mg2+.
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RESULTS |
CD95 expression on lymphoma B cells is heterogenous.
Membrane CD95 expression was analyzed by flow cytometry on purified
malignant B cells from 40 NHL patients, including 10 LCL, 10 MCL, 11 FL, and nine DLCL NHL. Nontumoral B cells (n = 9), EBV-immortalized (n = 5), and Jurkat T cells were also included in this study as controls.
We used the anti-CD95 MoAb CH-11 for these assays because it allows a
better detection of CD95 molecule than other clones (data not shown).
However, because MoAb CH-11 can induce cell death when used in a
soluble form,5 all experiments were performed on ice. The
absence of induction of apoptosis in this protocol was verified on
Jurkat T cells.
All the studied cells expressed CD95 and this expression was unimodal
(Fig 1). However, because the intensity of
this expression was very heterogeneous, the MFI was a more
discriminating parameter between NHL than the percentages of positive
cells (Fig 2). Our results indicate clearly
that the levels of the CD95 molecule were relatively homogeneous on
nontumoral B cells and within each group of the REAL classification
except DLCL. Lymphoma cells from LCL and MCL expressed low levels of
the CD95 molecule (mean MFI, 18 and 15, respectively), as compared with
FL (mean MFI, 57). The level of CD95 on nontumoral B cells was
relatively low (mean MFI, 36) whereas in BLCL, it was higher (mean MFI,
186).
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| Fig 1.
CD95 molecule is expressed on freshly isolated lymphoma B
cells. Flow cytometric analyses were performed on Jurkat cells (A), nontumoral B cells (B; Donor NT-1), and lymphoma B cells (C through F).
Lymphoma B cells were obtained from LCL-1 (C), MCL-5 (D), FL-6 (E), and
DLCL-4 (F) patients. Cells were stained with anti-CD95 MoAb CH-11
(dashed line) or with IgM control MoAb (solid line) followed by
PE-conjugated antimouse IgM. Percentages of positive cells and MFI
values (in brackets) are indicated for each cell population.
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| Fig 2.
The levels of CD95 expression vary according to NHL
types. CD95 expression has been analyzed by flow cytometry on
nontumoral B cells (n = 9), BLCL (n = 5), LCL (n = 10), MCL (n
= 10), FL (n = 11), and DLCL (n = 9). Cells were stained with
anti-CD95 MoAb CH-11 or with IgM control MoAb followed by PE-conjugated antimouse IgM. The MFI value was calculated by subtracting the IgM
control MFI value from the anti-CD95 MoAb MFI value. Bars indicate the
mean of MFI values within each entity.
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Resistance of lymphoma B cells to CD95-based apoptosis mediated by
anti-CD95 MoAb CH-11.
CD95 engagement induces apoptosis of activated, but not resting, normal
B cells.15-17 To determine the function of CD95 molecule on
B cells from NHL, we first studied the effect of anti-CD95 MoAb CH-11
on cell viability using a 4-hour 51Cr-release assay. This
method allows the detection of late stages of apoptosis characterized
by loss of membrane integrity, as already shown in other studies on
CD95-based lymphocyte-mediated cytotoxicity.7,9 As shown in
Fig 3, a 4-hour CD95 ligation by MoAb
induces apoptosis of Jurkat T cells in a dose-dependent manner.
Likewise, EBV-immortalized B lymphoblastoid cell lines were also
susceptible to the CD95-mediated death signal (data not shown).
However, CD95-ligation failed to induce apoptosis in three lymphoma
cell samples (Fig 3). All of the malignant B-cell suspensions, which
have been tested (two LCL, four MCL, two FL, and two DLCL), and four
nontumoral B-cell populations did not undergo apoptosis when the
membrane CD95 molecules were cross-linked by CH-11 MoAb, even at
increased incubation times (18 hours).
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| Fig 3.
Lymphoma B cells are resistant to CD95-mediated apoptosis
in an assay detecting late stages of apoptosis. Jurkat cells ( ) and
three lymphoma cell populations isolated from LCL-4 ( ), MCL-6 ( ),
and DLCL-1 ( ) patients were labeled with [51Cr]-sodium
chromate and then incubated with increased concentrations of anti-CD95
MoAb CH-11 for 4 hours. The apoptosis intensity was evaluated by the
percentage of specific 51Cr-release.
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We next used annexin V staining, as it is more suitable than the 4-hour
51Cr-release assay for detection of early stages of
apoptosis.31 Annexin V binding is combined with PI staining
to distinguish between early and late apoptotic cells. This test has
been used in several reports to measure apoptosis, in particular in
populations of germinal center B-lymphocytes.32 As a
control, Jurkat T cells that expressed high levels of CD95 molecule
(Fig 1A) were treated with anti-CD95 MoAb CH-11 or IgM control MoAb for
4 hours. As shown in Fig 4A, 38% of Jurkat
cells underwent apoptosis and were mostly in early apoptosis, as
indicated by annexin V staining. Representative dot-plots of annexin V
versus PI fluorescence obtained with lymphoma B cells from MCL (Fig
4C), FL (Fig 4D), DLCL (Fig 4E), and nontumoral (Fig 4B) B cells show
that no CD95-based apoptosis was detected after an 18-hour incubation
period. CD95 ligation never induced apoptosis either in malignant cell
populations tested (four LCL, six MCL, five FL, and four DLCL) or in
three nontumoral B-cell populations (Table
1).
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| Fig 4.
Lymphoma B cells are resistant to CD95-mediated apoptosis
in an assay detecting early stages of apoptosis. Flow cytometric analysis of Jurkat cells (A), nontumoral B cells (B; Donor NT-1), and
lymphoma B cells (C through E). Lymphoma B cells were obtained from
MCL-4 (C), FL-3 (D), DLCL-3 (E). Cells were incubated with 1 µg/mL
anti-CD95 MoAb CH-11 or with IgM control MoAb for 4 hours (A) or 18 hours (B through E) and then double-stained wirth annexin V-FITC/PI and
analyzed by flow cytometry. Numbers within dot plots represent the
percentages of cells in early apoptosis (lower right, annexin
V+/PI-) and in late apoptosis or in necrosis (upper
right, annexin V±/PI+). The specific apoptosis, that is
the percentage of viable cells undergoing apoptosis with the inducing
agent, was calculated according to the following formula:
((DCH-11 DIgM)/100 DIgM) ×100, where D represents the percentage of cells
dying during the culture by apoptosis or necrosis.
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Table 1.
Percentage of Apoptosis Induced by CD95 Cross-Linking,
Irradiation, and Straurosporine Treatment of Lymphoma and
Nontumoral B Cells
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To verify that lymphoma cells did not present major abnormalities in
intracellular mechanisms of apoptosis, we treated these malignant cells
with other apopotic inducers (Fig 5, Table
1). Apoptosis of lymphoma B cells can be induced in 4 hours and is greater after 18 hours of staurosporine treatment (Fig 5B). Irradiation exposure also induced apoptosis, but this was less marked (Fig 5A). It
is noticable that lymphoma cells become necrotic following apoptosis
during 18 hours of culture (Fig 5A and B). Most of the lymphoma cells
tested (16 patients) underwent apoptosis after irradiation exposure or
with staurosporine treatment (Table 1).
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| Fig 5.
Lymphoma B cells undergo apoptosis after irradiation or
staurosporine treatment. Lymphoma cells (FL-2 patient) were cultured for 4 hours or 18 hours (A) without treatment or after irradiation (3,000 rads), (B) with 5 µmol/L staurosporine or dimethyl sulphoxide (DMSO) as a control. Cells were stained with annexin
V-FITC/PI and then analyzed by flow cytometry. Numbers within dot plots represent the percentages of cells in early apoptosis (lower right, annexin V+/PI-) and in late apoptosis or in necrosis
(upper right, annexin V±/PI+). Percentages next to the
dot plots indicate the specific apoptosis.
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CD40 engagement greatly increases CD95 expression on lymphoma cells,
but poorly induces sensitivity to CD95-based apoptosis.
Recent reports have demonstrated that CD40 triggering on resting normal
B cells upregulates CD95 expression and induces their susceptibility to
CD95-mediated apoptosis.15-17 We first determined optimal
activation conditions to increase CD95 expression and to induce
CD95-based apoptosis responsiveness by performing 3-day cultures with
nontumoral B cells (Donor NT-1) in the presence or absence of
irradiated CD40Lig-L cells, IL-4, or both. To induce CD95-mediated
death, anti-CD95 MoAb was added on day 3 and apoptosis was evaluated 18 hours later. Results in Fig 6 represent one
of two separate experiments (Table 2). We
showed that CD40 activation upregulated CD95 expression on B cells (Fig
6C) and this phenomenon was stronger in the presence of IL-4 (Fig 6D),
whereas IL-4 alone had little effect (Fig 6B) compared with cells
cultured with medium (Fig 6A). Furthermore, CD40 cross-linking in the
presence of IL-4 restored CD95-based apoptosis more efficiently than
culture with CD40Lig-L cells alone (Fig 6C and D). Cells cultured with
medium or IL-4 did not undergo apoptosis (Fig 6A and B).
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| Fig 6.
CD40 ligation and CD40 ligation plus IL-4 upregulate CD95
expression on nontumoral B cells and induce CD95-based apoptosis. CD95
expression and apoptosis induction were measured on 3-day cultured B
cells (Donor NT-1) in mediun alone (A), or with IL-4 (B), CD40Lig-L
cells (C) and IL-4 plus CD40Lig-L cells. CD95 expression was analyzed
by flow cytometry after staining with anti-CD95 MoAb CH-11 (dashed
line) or with IgM control MoAb (solid line) followed by PE-conjugated
antimouse IgM. Numbers within histogram plots represent MFI values. For
CD95-mediated apoptosis experiments, cultured cells were incubated with
1 µg/mL anti-CD95 MoAb CH-11 or with IgM control MoAb for 18 hours
and then double-stained with annexin V-FITC/PI and analyzed by flow
cytometry. Numbers within dot plots represent the percentages of cells
in early apoptosis (lower right, annexin V+/PI-) and in
late apoptosis or in necrosis (upper right, annexin V±/PI+). Percentages next to the dot plots indicate the
specific apoptosis.
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Table 2.
Modulation of CD95 Expression and Sensitivity to
CD95-Mediated Apoptosis of Lymphoma and Nontumoral B Cells by CD40L and
IL-4
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We then examined the effect of CD40Lig-L cells/IL-4 culture on the
modulation of CD95 expression and function on lymphoma B cells. As
indicated in Table 2, CD40 ligation and IL-4 activation significantly
upregulated CD95 expression on lymphoma B cells. However, little
apoptosis occured after incubation with anti-CD95 MoAb for the last 18 hours of culture (Fig 7 and Table 2),
except for one case (MCL-6) where the specific apoptosis reached 37%. The intensity of CD95-mediated apoptosis was not significantly different when lymphoma cells were cultured with CD40Lig-L cells or
IL-4 alone, as compared with CD40Lig-L cells plus IL-4.
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| Fig 7.
CD40 ligation and CD40 ligation plus IL-4 poorly induce
sensitivity to CD95-based apoptosis. CD95 expression and apoptosis induction were measured on 3-day cultured B cells (Patient FL-3) in
mediun alone (A), or with IL-4 (B), CD40Lig-L cells (C), and IL-4 plus
CD40Lig-L cells. CD95 expression was analyzed by flow cytometry after
staining with anti-CD95 MoAb CH-11 (dashed line) or with IgM control
MoAb (solid line) followed by PE-conjugated antimouse IgM. Numbers
within histogram plots represent MFI values. For CD95-mediated
apoptosis experiments, cultured cells were incubated with 1 µg/mL
anti-CD95 MoAb CH-11 or with IgM control MoAb for 18 hours and then
double-stained with annexin V-FITC/PI and analyzed by flow cytometry.
Numbers within dot plots represent the percentages of cells in early
apoptosis (lower right, annexin V+/PI-) and in late
apoptosis or in necrosis (upper right, annexin V±/PI+).
Percentages next to the dot plots indicate the specific apoptosis.
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Resistance of lymphoma cells to CD95-induced lysis, but not
perforin-mediated cytotoxic T-cell killing.
CD95/CD95L interactions and perforin/granzyme B pathways are the two
main lytic mechanisms used by cytotoxic T cells.8,9 Thus,
we next addressed the question of the ability of the physiologic ligand
of CD95 (CD95L) expressed by cytolytic T cells to induce apoptosis of
lymphoma B cells. For this purpose, we performed cytotoxic assays with
activated T cells using a standard 4-hour 51Cr-release test
in the presence or absence of EGTA that blocks the perforin pathway
(Fig 8). Allogeneic cytolytic T cells were obtained after mixed lymphocyte reaction culture using lymphoma B cells
as stimulators and then activated by PMA/ionomycin to upregulate CD95L
expression as described elsewhere.33 Activated cells were
tested with Jurkat cells to control functional expression of CD95L. As
shown in Fig 8A, the lysis of Jurkat cells is only CD95-based, as it
was not significantly changed in the presence of EGTA. When lymphoma
cells were used as targets (Fig 8B), lysis that exceeded 45% in the
absence of EGTA was completly abrogated in its presence. These results
indicate that CD95 ligation by functional CD95L did not induce
apoptosis of lymphoma B cells, and that the perforin pathway was the
main mechanism by which malignant B cells were killed by cytotoxic T
cells.
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| Fig 8.
Resistance of lymphoma cells to CD95, but not perforin,
lytic mechanisms of cytolytic T cells. Allogeneic antilymphoma T cells generated by MLR were preincubated for 2 hours with 5 ng/mL PMA and 1.5 µg/mL ionomycin. The subsequent 4-hour cytotoxicity assay was
performed with Jurkat (A) or lymphoma B cells (B) in the presence of
medium () or EGTA-Mg2+ (). The cytotoxicity
intensity was evaluated by the percentage of 51Cr-release.
Experiments 1 and 2 were performed with lymphoma cells isolated from
LCL-3 and MCL-1 patients, respectively.
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| |
DISCUSSION |
B-cell NHL is a particular neoplastic disease because the malignant
clone develops essentially from the immune lymphoid system. In vivo,
the development of normal B-cell responses appears to be under the
control of both specific antigen recognition via the B-cell receptor
(BCR) and CD4+ T lymphocytes via CD40L and CD95L
molecules.12 Without adequate BCR engagement,
CD40-activated B cells undergo apoptosis by CD95 ligation.12,15-19 This mechanism seems to allow the
expansion of antigen-reactive B cells and elimination of tolerant B
cells.
In NHL, an alteration of the control of B-cell growth or survival leads
to cellular accumulation and tumor development. Thus, the
overexpression or altered expression of proteins involved in apoptosis
or the cell cycle are often found in NHL: Bcl-2 in follicular
lymphoma,34 Bcl-6 in diffuse large cell
lymphoma,35 cyclin D1 in mantle cell
lymphoma,36 c-myc in Burkitt's lymphoma,37 and
p53 in several types of lymphoma.38,39 However, a single abnormality is not sufficient to explain the development of
lymphoma.40 Moreover, the observation that isolated
lymphoma cells died rapidly in culture suggested that interaction with
other cells in the tumoral environment is needed for their survival.
Interestingly, one report has described that specific CD4+
T-cell clones induced the proliferation of follicular lymphoma cells in
vitro.41 Furthermore, lymphoma B cells can proliferate after CD40 cross-linking.24 The fact that both CD40L and
CD95L are expressed on activated CD4+ T cells led us to
postulate that lymphoma cells are responsive to CD40 activation and
resistant to CD95-mediated apoptosis, independently of BCR triggering.
Such a resistance to CD95-based apoptosis could be one mechanism
involved in the development of the malignant process in NHL, allowing
the escape of malignant B cells from immune regulation. Our
observations provide evidence that such a mechanism probably exists.
The present study describes the expression and the functionality of the
CD95 molecule in a large number of tumor cells and addresses the
question of the involvement of CD95 in NHL oncogenesis and tumor
progression.
We first showed that all tumor cells were positive for CD95 expression
with, however, a magnitude of positivity that varies according to NHL
groups. In accordance with immunohistologic analysis,25-27 the highest levels of CD95 expression were encountered in FL and DLCL,
and the lowest in LCL and MCL. These observations were in agreement
with studies reporting CD95 expression on normal B cells at different
maturation stages.19,25 Indeed, germinal center B cells
from which FL and most DLCL are probably derived, express high levels
of the CD95 molecule. In contrast, naive B cells, which are thought to
be the normal counterparts of MCL and most LCL, express low levels.
The susceptibility of normal B cells to CD95-mediated apoptosis depends
on their activation state. Interestingly, we showed that CD95-ligation
did not induce apoptosis of tumor cells isolated from from MCL and LCL
NHL that are considered to be resting cells and expressed few CD95
molecules.
Conversely, FL and DLCL cells, which are considered to be activated,
particularly DLCL, which are cycling, were also not responsive to
CD95-based apoptosis. Such an absence of concordance between CD95
expression and CD95-mediated cell death has already been described in
cases of normal B cells. Indeed, normal B lymphocytes were rendered
sensitive to CD95-mediated death after mitogenic activation by
CD40-cross-linking12,15,17 or pokeweed mitogen (PWM).30 Our results showed that CD40
engagement plus IL-4 greatly upregulated the expression of membrane
CD95 molecules on lymphoma, as well as on nontumoral B lymphocytes. In
these conditions, CD95 ligation induced a strong apoptosis in
nontumoral activated B cells, whereas little apoptosis was detected in
activated lymphoma cells. Concerning apoptosis induction in nontumoral
human B cells, our results conflict with those published in the mouse
system in which IL-4 induces CD95 resistance in CD40-stimulated primary B cells.42 Our results show that CD95 cross-linking induced more apoptosis in nontumoral B cells activated by CD40 plus IL-4 than
by CD40 alone. The resistance of CD40-activated lymphoma cells to
apoptosis was not due to IL-4, as similar results were obtained without
IL-4. Discrepancies in experimental procedures could explain these
differences.
The data presented in this study strongly suggest that the CD95 death
pathway is partially blocked in human lymphoma cells and that
activation by CD40 in the presence of IL-4 is not sufficient to restore
it. This blockade was not related to late abnormalities in
intracellular mechanisms of apoptosis because tumor B cells undergo
apoptosis in response to other inducers such as irradiation or
staurosporine treatment.
Several mechanisms could interact with the CD95 signaling pathway to
inhibit apoptosis. Overexpression of Bcl-2 in follicular lymphoma cells
and also in other tumors has been proposed as a mechanism blocking the
apoptotic process induced by the CD95 molecule.26,43 However, some studies have shown that CD95-mediated apoptosis is only
partialy inhibited by Bcl-2.44,45 Other molecules could also interfere with CD95-mediated apoptosis, such as Bcl-xL, another Bcl-2-related protein that also prevents apoptosis and seems to be
commonly expressed in lymphoma cells.46 Interestingly,
Bcl-x expression is strongly enhanced by CD40 cross-linking in murine B
cells and human tonsillar B-cell centroblasts.47,48 It
would be of interest to measure Bcl-2 and Bcl-x expression in lymphoma cells after CD40 engagement in the presence of IL-4.
CD95-induced apoptosis could also be altered by mutations in genes
coding for proteins of the CD95 death-inducing signaling complex as
described in lymphoma T cells.49 At present, only one study
has reported CD95 gene alterations in B-cell NHL, but at a relatively
low frequency.50 Interestingly, novel proteins, one called
sentrin and the others FLIPs (FLICE-inhibitory proteins), have been recently described that protect against CD95-induced cell
death.51,52 An overexpression of such proteins in lymphoma cells could participate in their resistance to CD95-mediated apoptosis.
One alternative mechanism of resistance to CD95 apoptosis in lymphoma
cells could be related to BCR signaling. Antigen receptor engagement
has been shown to protect normal B cells from CD95-mediated apoptosis.12,18,19 In NHL, the existence of an antigen
recognized by tumoral BCR has rarely been described. However, the
demonstration of somatic hypermutations with intraclonal diversity in
follicular lymphoma suggests that antigen stimulation and selection is
involved in the evolution of the malignant clone.21,22
Little is known about the intracellular signaling of BCR in lymphoma B
cells. It can also be supposed that abnormalites driving constitutive signaling by the BCR could protect activated lymphoma B cells from
CD95-induced death. Further investigations will be neccessary to
explore the precise role of tumoral BCR in the resistance of lymphoma
cells to CD95-mediated cell death.
The resistance of lymphoma cells to CD95-mediated apoptosis that we
have demonstrated could signify that, in vivo, tumor cell could escape
from the regulatory control of CD4+ lymphocytes and partly
from the antitumor cytotoxic CD8+ T cells. Indeed, the
CD95-death pathway is the major mechanism for CD4+ T cells
to mediate apoptosis and one of the two killing pathways used by
CD8+ T lymphocytes.8,9 We have previously
described that allogeneic T lymphocytes were able to lyse tumor
cells.23 Thus, we tested whether CD95 cross-linking by CD95
ligand, rather than by MoAbs, could drive apoptosis in lymphoma cells.
We have shown that neither CD4+ nor CD8+
effector cells were able to induce the death of tumor cells via a
CD95-based mechanism. Indeed, the majority of the cytotoxicity observed
against tumor cells (>45%) was strictly dependent on the perforin
pathway. These results also support the notion that CD8+
lymphocytes represent good, and perhaps the best, effectors to kill
tumor cells.
We show in this report that a large number of B cells from distinct NHL
groups were intrinsically resistant to CD95-mediated apoptosis,
including DLCL or FL, which are considered to arise from activated
cells. Interestingly, CD40 engagement poorly induced CD95-mediated cell
death. So, particularly in NHL, the resistance to CD95-induced cell
death is probably an important step in tumor development or
progression, as this property allows lymphoma cells to escape from
CD4+ T-cell-mediated apoptosis and partially from
CD8+ T-cell-mediated cytotoxicity. However, the blockade
in the CD95 signaling pathway cannot be considered as the sole
mechanism involved in the tumoral development because children who have
defective CD95-mediated apoptosis develop an autoimmune syndrome as
their primary disease rather than a cancer.10,11
Furthermore, recent work53 has shown that CD95-deficient
mice (lpr) lack malignant tumors, but develop lethal B-cell
lymphoma when they are deficient in T cells, suggesting a
CD95-independent role for T cells in lymphoma development. Thus, in
NHL, resistance to CD95-based apoptosis and functional abnormalities of
T lymphocytes, such as anergy,54 could be of prime
importance in facilitating lymphoma development or progression.
| |
FOOTNOTES |
Submitted September 16, 1997;
accepted November 25, 1997.
Supported by Grant No. 1241 from the "Association pour la Recherche
sur le Cancer."
Address reprint requests to Joël Plumas, PhD,
Laboratoire d'Immunologie, Etablissement de Transfusion Sanguine de
l'Isère et de la Savoie, BP 35, F-38701 La Tronche Cedex,
France.
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.
| |
ACKNOWLEDGMENT |
We are grateful to the staff of the Blood Center Immunological
Department, the Grenoble CHU and Annecy CHR hematological departments, and to E. Keddari for their collaboration. We also thank Pierre Garrone
for his comments and helpful suggestions.
| |
REFERENCES |
1.
Nagata S,
Golstein P:
The Fas death factor.
Science
267:1449,
1995[Abstract/Free Full Text]
2.
Gruss HJ,
Dower SK:
Tumor necrosis factor ligand superfamily: Involvement in the pathology of malignant lymphomas.
Blood
85:3378,
1995[Abstract/Free Full Text]
3.
Suda T,
Takahashi T,
Golstein P,
Nagata S:
Molecular cloning and expression of the Fas ligand, a novel member of the tumor necrosis factor family.
Cell
75:1169,
1993[Medline]
[Order article via Infotrieve]
4.
Trauth BC,
Klas C,
Peters AMJ,
Matzku S,
Möller P,
Falk W,
Debatin KM,
Krammer PH:
Monoclonal antibody mediated tumor regression by induction of apoptosis.
Science
245:301,
1989[Abstract/Free Full Text]
5.
Yonehara S,
Ishii A,
Yonehara M:
A cell killing monoclonal antibody (anti-Fas) to a cell surface antigen co-downregulated with the receptor of tumor necrosis factor.
J Exp Med
169:1747,
1989[Abstract/Free Full Text]
6.
Ju ST,
Panka DJ,
Cui H,
Ettinger R,
El Khatib M,
Sherr DH,
Stanger BZ,
Marshak-Rothstein A:
Fas (CD95)/FasL interactions required for programmed cell death after T cell activation.
Nature
373:444,
1995[Medline]
[Order article via Infotrieve]
7.
Alderson MR,
Tough TW,
Davis Smith T,
Braddy S,
Falk B,
Schooley KA,
Goodwin RG,
Smith CA,
Ramsdell F,
Lynch DH:
Fas ligand mediates activation induced cell death in human T lymphocytes.
J Exp Med
181:71,
1995[Abstract/Free Full Text]
8.
Lowin B,
Hahne M,
Mattmann C,
Tschopp J:
Cytolytic T cell cytotoxicity is mediated through perforin and Fas lytic pathways.
Nature
370:650,
1994[Medline]
[Order article via Infotrieve]
9.
Kägi D,
Vignaux F,
Ledermann B,
Bürki K,
Depraetere V,
Nagata S,
Hengartner H,
Golstein P:
Fas and perforin pathways as major mechanisms of T cell mediated cytotoxicity.
Science
265:528,
1994[Abstract/Free Full Text]
10.
Rieux Laucat F,
Le Deist F,
Hivroz C,
Roberts IAG,
Debatin KM,
Fischer A,
De Villartay JP:
Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity.
Science
268:1347,
1995[Abstract/Free Full Text]
11.
Fischer GH,
Rosenberg FJ,
Straus SE,
Dale JK,
Middelton LA,
Lin AY,
Strober W,
Lenardo MJ,
Puck JM:
Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome.
Cell
81:935,
1995[Medline]
[Order article via Infotrieve]
12.
Rathmell JC,
Townsend SE,
Xu JC,
Flavell RA,
Goodnow CC:
Expansion or elimination of B cells in vivo: Dual roles for CD40 and Fas (CD95) ligands modulated by the B cell antigen receptor.
Cell
87:319,
1996[Medline]
[Order article via Infotrieve]
13.
Durie FH,
Foy TM,
Masters SR,
Laman JD,
Noelle RJ:
The role of CD40 in the regulation of humoral and cell mediated immunity.
Immunol Today
15:406,
1994[Medline]
[Order article via Infotrieve]
14.
Banchereau J,
Bazan F,
Blanchard D,
Briere F,
Galizzi JP,
Van Kooten C,
Liu YJ,
Rousset F,
Saeland S:
The CD40 antigen and its ligand.
Annu Rev Immunol
12:881,
1994[Medline]
[Order article via Infotrieve]
15.
Garrone P,
Neidhardt EM,
Garcia E,
Galibert L,
Vankooten C,
Banchereau J:
Fas ligation induces apoptosis of CD40-activated human B lymphocytes.
J Exp Med
182:1265,
1995[Abstract/Free Full Text]
16.
Lagresle C,
Bella C,
Daniel PT,
Krammer PH,
Defrance T:
Regulation of germinal center B cell differentiation.
J Immunol
154:5746,
1995[Abstract]
17.
Schattner EJ,
Elkon KB,
Yoo DH,
Tumang J,
Krammer PH,
Crow MK,
Friedman SM:
CD40 ligation induces APO-1/Fas expression on human B lymphocytes and facilitates apoptosis through the APO-1/Fas pathway.
J Exp Med
182:1557,
1995[Abstract/Free Full Text]
18.
Rothstein TL,
Wang JKM,
Panka DJ,
Foote LC,
Wang Z,
Stanger B,
Cui H,
Ju ST,
Marshak-Rothstein A:
Protection against Fas-dependent Th1-mediated apoptosis by antigen receptor engagement in B cells.
Nature
374:163,
1995[Medline]
[Order article via Infotrieve]
19.
Lagresle C,
Mondiere P,
Bella C,
Krammer PH,
Defrance T:
Concurrent engagement of CD40 and the antigen receptor protects naive and memory human B cells from APO-1/Fas mediated apoptosis.
J Exp Med
183:1377,
1996[Abstract/Free Full Text]
20.
Harris NL,
Jaffe ES,
Stein H,
Banks PM,
Chan JKC,
Cleary ML,
Delsol G,
Dewolfpeeters C,
Falini B,
Gatter KC,
Grogan TM,
Isaacson PG,
Knowles DM,
Mason DY,
Mullerhermelink HK,
Pileri SA,
Piris MA,
Ralfkiaer E,
Warnke RA:
A revised European-American classification of lymphoid neoplasms: A proposal from the international lymphoma study group.
Blood
84:1361,
1994[Free Full Text]
21.
Friedman DF,
Cho EA,
Goldman J,
Carmack CE,
Besa EC,
Hardy RR,
Silberstein LE:
The role of clonal selection in the pathogenesis of an autoreactive human B cell lymphoma.
J Exp Med
174:525,
1991[Abstract/Free Full Text]
22.
Zelenetz AD,
Chen TT,
Levy R:
Clonal expansion in follicular lymphoma occurs subsequent to antigenic selection.
J Exp Med
176:1137,
1992[Abstract/Free Full Text]
23.
Plumas J,
Chaperot L,
Jacob MC,
Giroux C,
Mollens JP,
Sotto JJ,
Bensa JC:
Malignant B lymphocytes from non-Hodgkin's lymphoma induce allogeneic proliferative and cytotoxic T cell responses in primary mixed lymphocyte cultures: An important role of costimulatory molecules CD80 (B7-1) and CD86 (B7-2) in the stimulating function of tumor cells.
Eur J Immunol
25:3332,
1995[Medline]
[Order article via Infotrieve]
24.
Johnson PWM,
Watt SM,
Betts DR,
Davies D,
Jordan S,
Norton AJ,
Lister TA:
Isolated follicular lymphoma cells are resistant to apoptosis and can be grown in vitro in the CD40/Stromal cell system.
Blood
82:1848,
1993[Abstract/Free Full Text]
25.
Möller P,
Henne C,
Leithäuser F,
Eichelmann A,
Schmidt A,
Brüderlein S,
Dhein J,
Krammer PH:
Coregulation of the APO-1 antigen with intercellular adhesion molecule-1 (CD54) in tonsillar B cells and coordinate expression in follicular center B cells and in follicle center and mediastinal B cell lymphomas.
Blood
81:2067,
1993[Abstract/Free Full Text]
26.
Kondo E,
Yoshino T,
Yamadori I,
Matsuo Y,
Kawasaki N,
Minowada J,
Akagi T:
Expression of Bcl-2 protein and Fas antigen in non-Hodgkin's lymphomas.
Am J Pathol
145:330,
1994[Abstract]
27.
Xerri L,
Carbuccia N,
Parc P,
Hassoun J,
Birg F:
Frequent expression of Fas/APO-1 in Hodgkin's disease and anaplastic large cell lymphomas.
Histopathology
27:235,
1995[Medline]
[Order article via Infotrieve]
28.
Falk MH,
Trauth BC,
Debatin KM,
Klas C,
Gregory CD,
Rickinson AB,
Calender A,
Lenoir GM,
Ellwart JW,
Krammer PH,
Bornkamm GW:
Expression of the APO-1 antigen in Burkitt's lymphoma cell lines correlates with a shift towards a lymphoblastoid phenotype.
Blood
79:3300,
1992[Abstract/Free Full Text]
29.
Schattner EJ,
Mascarenhas J,
Bishop J,
Yoo DH,
Chadburn A,
Crow MK,
Friedman SM:
CD4+ T cell induction of Fas mediated apoptosis in Burkitt's lymphoma B cells.
Blood
88:1375,
1996[Abstract/Free Full Text]
30.
Mapara MY,
Bargou R,
Zugck C,
Döhner H,
Ustaoglu F,
Jonker RR,
Krammer PH,
Dörken B:
APO-1 mediated apoptosis or proliferation in human chronic B lymphocytic leukemia: Correlation with Bcl-2 oncogene expression.
Eur J Immunol
23:702,
1993[Medline]
[Order article via Infotrieve]
31.
Vermes I,
Haanen C,
Steffens-Nakken H,
Reutelingsperger C:
A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V.
J Immunol Methods
184:39,
1995[Medline]
[Order article via Infotrieve]
32.
Koopman G,
Reutelingsperger CPM,
Kuijten GAM,
Keehnen RMJ,
Pals ST,
Van Oers MHJ:
Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis.
Blood
84:1415,
1994[Abstract/Free Full Text]
33.
Vignaux F,
Golstein P:
Fas based lymphocyte mediated cytotoxicity against syngeneic activated lymphocytes: A regulatory pathway?
Eur J Immunol
24:923,
1994[Medline]
[Order article via Infotrieve]
34.
Tsujimoto Y,
Bashir MM,
Givol I,
Cossman J,
Jaffe E,
Croce CM:
DNA rearrangements in human follicular lymphoma can involve the 5' or the 3' region of the Bcl-2 gene.
Proc Natl Acad Sci USA
84:1329,
1987[Abstract/Free Full Text]
35.
Offit K,
Coco FL,
Louie DC,
Parsa NZ,
Leung D,
Portlock C,
Ye BH,
Lista F,
Filippa DA,
Rosenbaum A,
Ladanyi M,
Jhanwar S,
Dalla Favera R,
Chaganti RSK:
Rearrangement of the Bcl-6 gene as a prognostic marker in diffuse large cell lymphoma.
N Engl J Med
331:74,
1994[Abstract/Free Full Text]
36.
De Boer CJ,
Schuuring E,
Dreef E,
Peters G,
Bartek J,
Kluin PM,
Van Krieken HJM:
Cyclin D1 protein analysis in the diagnosis of mantle cell lymphoma.
Blood
86:2715,
1995[Abstract/Free Full Text]
37.
Dalla Favera R,
Bregni M,
Erikson J:
Human c-myc onc gene is located on the region of chromosome 8 that is translocated in Burkitt lymphoma cells.
Proc Natl Acad Sci USA
79:7824,
1982[Abstract/Free Full Text]
38.
Gaidano G,
Ballerini P,
Gong JZ,
Inghirami G,
Neri A,
Newcomb EW,
Magrath IT,
Knowles DM,
Dalla-Favera R:
p53 mutations in human lymphoid malignancies: Association with Burkitt's lymphoma and chronic lymphocytic leukemia.
Proc Natl Acad Sci USA
88:5413,
1991[Abstract/Free Full Text]
39.
Sander CA,
Yano T,
Clark HM,
Harris C,
Longo DL,
Jaffe ES,
Raffeld M:
p53 mutation is associated with progression in follicular lymphomas.
Blood
82:1994,
1993[Abstract/Free Full Text]
40.
McDonnell TJ,
Korsmeyer SJ:
Progression from lymphoid hyperplasia to high-grade malignant lymphoma in mice transgenic for the t(14,18).
Nature
349:254,
1991[Medline]
[Order article via Infotrieve]
41.
Umetsu DT,
Esserman L,
Donlon TA,
DeKruyff RH,
Levy R:
Induction of proliferation of human follicular (B type) lymphoma cells by cognate interaction with CD4+ T cell clones.
J Immunol
144:2250,
1990
42.
Foote LC,
Howard RG,
Marshak-Rothstein A,
Rothstein TL:
IL-4 induces Fas resistance in B cells.
J Immunol
157:2749,
1996[Abstract]
43.
Yang E,
Korsmeyer SJ:
Molecular thanatopsis: A discourse on the Bcl-2 family and cell death.
Blood
88:386,
1996[Free Full Text]
44.
Itoh N,
Tsujimoto Y,
Nagata S:
Effect of Bcl-2 on Fas antigen mediated cell death.
J Immunol
151:621,
1993[Abstract]
45.
Takayama S,
Sato T,
Krajewski S,
Kochel K,
Irie S,
Millan JA,
Reed JC:
Cloning and functional analysis of BAG-1: A novel Bcl-2 binding protein with anti cell death activity.
Cell
80:279,
1995[Medline]
[Order article via Infotrieve]
46.
Xerri L,
Parc P,
Brousset P,
Schlaifer D,
Hassoun J,
Reed JC,
Krajewski S:
Predominant expression of the long isoform of Bcl-X (Bcl-xL) in human lymphomas.
Br J Haematol
92:900,
1996[Medline]
[Order article via Infotrieve]
47.
Wang Z,
Karras JG,
Howard RG,
Rothstein TL:
Induction of Bcl-x by CD40 engagement rescues sIg induced apoptosis in murine B cells.
J Immunol
155:3722,
1995[Abstract]
48.
Tuscano JM,
Druey KM,
Riva A,
Pena J,
Thompson CB,
Kehrl JH:
Bcl-x rather than Bcl-2 mediates CD40 dependent centrocyte survival in the germinal center.
Blood
88:1359,
1996[Abstract/Free Full Text]
49.
Cascino I,
Papoff G,
De Maria R,
Testi R,
Ruberti G:
Fas/APO-1 (CD95) receptor lacking the intracytoplasmic signaling domain protects tumor cells from Fas mediated apoptosis.
J Immunol
156:13,
1996[Abstract]
50.
Xerri L,
Carbuccia N,
Birg F:
Search for rearrangements and/or allelic loss of the Fas/APO-1 gene in 101 human lymphomas.
Am J Pathol
104:424,
1995
51.
Okura T,
Gong L,
Kamitani T,
Wada T,
Okura I,
Wei CF,
Chang HM,
Yeh ETH:
Protection against Fas/APO-1 and tumor necrosis factor mediated cell death by a novel protein, sentrin.
J Immunol
157:4277,
1996[Abstract]
52.
Thome M,
Schneider P,
Hofmann K,
Fickenscher H,
Meinl E,
Neipel F,
Mattmann C,
Burns K,
Bodmer JL,
Schroter M,
Scaffidi C,
Krammer PH,
Peter ME,
Tschopp J:
Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors.
Nature
386:517,
1997[Medline]
[Order article via Infotrieve]
53.
Peng SL,
Robert ME,
Hayday AC,
Craft J:
A tumor-suppressor function for Fas (CD95) revealed in T cell-deficient mice.
J Exp Med
184:1149,
1996[Abstract/Free Full Text]
54.
Kudoh S,
Wang Q,
Hidalgo OF,
Rayman P,
Tubbs RR,
Edinger MG,
Kolenko V,
Panuto J,
Bukowski R,
Finke JH:
Responses to T cell receptor CD3 and interleukin-2 receptor stimulation are altered in T cells from B cell non Hodgkin's lymphomas.
Cancer Immunol Immunother
41:175,
1995[Medline]
[Order article via Infotrieve]
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February 1, 2003;
101(3):
949 - 954.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. E. Straus, E. S. Jaffe, J. M. Puck, J. K. Dale, K. B. Elkon, A. Rosen-Wolff, A. M. J. Peters, M. C. Sneller, C. W. Hallahan, J. Wang, et al.
The development of lymphomas in families with autoimmune lymphoproliferative syndrome with germline Fas mutations and defective lymphocyte apoptosis
Blood,
July 1, 2001;
98(1):
194 - 200.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
L. Chaperot, N. Bendriss, O. Manches, R. Gressin, M. Maynadie, F. Trimoreau, H. Orfeuvre, B. Corront, J. Feuillard, J.-J. Sotto, et al.
Identification of a leukemic counterpart of the plasmacytoid dendritic cells
Blood,
May 15, 2001;
97(10):
3210 - 3217.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J P Dales, J Plumas, F Palmerini, E Devilard, T Defrance, A Lajmanovich, V Pradel, F Birg, and L Xerri
Correlation between apoptosis macroarray gene expression profiling and histopathological lymph node lesions
Mol. Pathol.,
February 1, 2001;
54(1):
17 - 23.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
T. Takakuwa, Z. Dong, H. Takayama, F. Matsuzuka, S. Nagata, and K. Aozasa
Frequent Mutations of Fas Gene in Thyroid Lymphoma
Cancer Res.,
February 1, 2001;
61(4):
1382 - 1385.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
T. Decker, F. Schneller, T. Sparwasser, T. Tretter, G. B. Lipford, H. Wagner, and C. Peschel
Immunostimulatory CpG-oligonucleotides cause proliferation, cytokine production, and an immunogenic phenotype in chronic lymphocytic leukemia B cells
Blood,
February 1, 2000;
95(3):
999 - 1006.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Knorr, C. Amrehn, H. Seeberger, A. Rosenwald, S. Stilgenbauer, G. Ott, H.-K. Muller Hermelink, and A. Greiner
Expression of Costimulatory Molecules in Low-Grade Mucosa-Associated Lymphoid Tissue-Type Lymphomas in Vivo
Am. J. Pathol.,
December 1, 1999;
155(6):
2019 - 2027.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. G. Wickremasinghe and A. V. Hoffbrand
Biochemical and Genetic Control of Apoptosis: Relevance to Normal Hematopoiesis and Hematological Malignancies
Blood,
June 1, 1999;
93(11):
3587 - 3600.
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. L. ten Berge, D. F. Dukers, J. J. Oudejans, K. Pulford, G. J. Ossenkoppele, D. de Jong, J. F.M.M. Misere, and C. J.L.M. Meijer
Adverse Effects of Activated Cytotoxic T Lymphocytes on the Clinical Outcome of Nodal Anaplastic Large Cell Lymphoma
Blood,
April 15, 1999;
93(8):
2688 - 2696.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Gronbak, P. t. Straten, E. Ralfkiaer, V. Ahrenkiel, M. K. Andersen, N. E. Hansen, J. Zeuthen, K. Hou-Jensen, and P. Guldberg
Somatic Fas Mutations in Non-Hodgkin's Lymphoma: Association With Extranodal Disease and Autoimmunity
Blood,
November 1, 1998;
92(9):
3018 - 3024.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract