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Blood, Vol. 91 No. 8 (April 15), 1998:
pp. 2875-2885
By
From the Immunology Department, ETS Isère-Savoie and Research
Group on Lymphoma, Unité UPRES 2021, Grenoble, France.
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.
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.
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.
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- 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.
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 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 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 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.
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).
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).
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.
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.
Submitted September 16, 1997;
accepted November 25, 1997.
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.
1.
Nagata S,
Golstein P:
The Fas death factor.
Science
267:1449,
1995
2.
Gruss HJ,
Dower SK:
Tumor necrosis factor ligand superfamily: Involvement in the pathology of malignant lymphomas.
Blood
85:3378,
1995
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
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
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
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
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
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
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
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
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
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
22.
Zelenetz AD,
Chen TT,
Levy R:
Clonal expansion in follicular lymphoma occurs subsequent to antigenic selection.
J Exp Med
176:1137,
1992
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
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
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
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
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
Introduction
Abstract
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%.
SR)/(MR 
+ (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+.
) 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.
