CD40 Ligand Protects from TRAIL-Induced Apoptosis in
Follicular Lymphomas through NF-?B Activation and
Up-Regulation of c-FLIP and Bcl-xL
Marion Travert,* Patricia Ame-Thomas,*†Ce ´line Pangault,*†Alexandre Morizot,§
Olivier Micheau,§Gilbert Semana,*¶Thierry Lamy,*‡Thierry Fest,*†Karin Tarte,*†
and Thierry Guillaudeux2*
The TNF family member TRAIL is emerging as a promising cytotoxic molecule for antitumor therapy. However, its mechanism
of action and the possible modulation of its effect by the microenvironment in follicular lymphomas (FL) remain unknown. We
show here that TRAIL is cytotoxic only against FL B cells and not against normal B cells, and that DR4 is the main receptor
involved in the initiation of the apoptotic cascade. However, the engagement of CD40 by its ligand, mainly expressed on a specific
germinal center CD4?T cell subpopulation, counteracts TRAIL-induced apoptosis in FL B cells. CD40 induces a rapid RNA and
protein up-regulation of c-FLIP and Bcl-xL. The induction of these antiapoptotic molecules as well as the inhibition of TRAIL-
induced apoptosis by CD40 is partially abolished when NF-?B activity is inhibited by a selective inhibitor, BAY 117085. Thus, the
antiapoptotic signaling of CD40, which interferes with TRAIL-induced apoptosis in FL B cells, involves NF-?B-mediated induc-
tion of c-FLIP and Bcl-xLwhich can respectively interfere with caspase 8 activation or mitochondrial-mediated apoptosis. These
findings suggest that a cotreatment with TRAIL and an inhibitor of NF-?B signaling or a blocking anti-CD40 Ab could be of great
interest in FL therapy. The Journal of Immunology, 2008, 181: 1001–1011.
patients having FL will experience recurrent relapses, leading to
death (1–3). The mechanisms relevant for FL B cell prolonged
survival remain unclear. The anti-apoptotic Bcl-2 protein is over-
expressed in most FL and results from a t(14;18) chromosomal
translocation presents in 85% of the cases. However, this phenom-
enon cannot explain by itself the selective advantage given to tu-
mor cells (4). Recent microarray-based gene expression profiling
and immunohistochemical analyses have revealed that clinico-bi-
ologic outcome in FL patients is primarily predicted by specific
ollicular lymphomas (FL)3are indolent non-Hodgkin lym-
phomas and have a relatively good prognosis with a me-
dian survival as long as 10 years. However, the majority of
molecular features of nonmalignant cells instead of tumor cells
themselves (5, 6). The influence of the cellular microenvironment
on the prognosis of FL probably reflects the participation of both
immune and stromal cells in the biology and pathogenesis of this
tumor (5, 7). This microenvironmental dependency is supported by
the fact that FL B cells are very difficult to grow in vitro in the
absence of stromal cells and without stimulation of the CD40 re-
ceptor, a crucial event in the interactions between B and T cells
(8–10). CD40, a 48-kDa TNF superfamily transmembrane recep-
tor, was first identified and functionally characterized on B lym-
phocytes (11, 12) and is involved in activation and survival of
normal and malignant B cells, such as FL (8, 10). During the
germinal center (GC) reaction, CD40 strongly contributes to B
cell proliferation and differentiation, to somatic hypermutation
and isotype switching, and to memory B cell genesis (11, 13,
14). During these processes, B cells with low-affinity Ag re-
ceptors are eliminated by apoptosis to generate a B cell reper-
toire with appropriate Ag specificities. Studies on GC B cells of
human and mice origins lacking the functional CD95 receptor
have demonstrated that this death receptor, also a member of
the TNFR superfamily, is directly involved in the clonal selec-
tion of GC B cells (15, 16). Within GC, CD40 is expressed on
normal B lymphocytes and interacts as a trimer with CD40
ligand (L) expressed predominantly on CD4?activated T cells
(17). CD40 activation exerts a complex modulation of B cell
apoptosis: CD40 promotes GC B cell survival by protecting
them against CD95-induced apoptosis (18–20), but also induces
CD95 expression, thereby rendering the cells sensitive to
CD95L or CD95 agonists. Cross-linking of CD40 on tonsillar B
cells and B cell lines activates nuclear factor NF-?B/Rel tran-
scription factors (21–25). This activation is required for pro-
tection against CD95-mediated apoptosis, by up-regulating cel-
lular inhibitors of apoptosis, c-FLIP, Bcl-xLand Bfl-1/A1, or
Gadd45b (20, 26, 27). It has been demonstrated that c-FLIP
*Institut National de la Sante ´ et de la Recherche Me ´dicale, Unite ´ 917 MICA, Faculte ´
de Me ´decine, Universite ´ Rennes 1, Institut Fe ´de ´ratif de Recherche 140 Ge ´ne ´tique
Fonctionnelle Agronomie et Sante ´, Rennes, France;†De ´partement He ´matologie-Im-
munologie et The ´rapie Cellulaire and‡Service d’He ´matologie Clinique, Centre Hos-
pitalo-Universitaire Pontchaillou, Rennes, France;§Institut National de la Sante ´ et de
la Recherche Me ´dicale, Unite ´ 866, Universite ´ de Bourgogne, Dijon, France; and
¶Etablissement Franc ¸ais du Sang-Bretagne, Rennes, France
Received for publication July 11, 2007. Accepted for publication May 13, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by grants from the Re ´gion Bretagne, the Association pour
le De ´veloppement de l’He ´mato-Oncologie, and the Universite ´ de Rennes 1. M.T. was
supported by the Ligue Re ´gionale Contre le Cancer and the Socie ´te ´ Francaise
d’He ´matologie. O.M. is supported by research grants from the Institut National du
Cancer (PL098), Agence Nationale de la Recherche (ANR-06-JCJC-0103), and the
European community (ApopTrain Marie Curie RTN). A.M. is a recipient of a fel-
lowship from the French Ministry of Research and Education.
2Address correspondence and reprint requests to Dr. Thierry Guillaudeux, Institut
National de la Sante ´ et de la Recherche Me ´dicale, Unite ´ 917 MICA, Faculte ´ de
Me ´decine, Universite ´ de Rennes 1, 2, Avenue du Pr Le ´on Bernard, 35043 Rennes,
France. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: FL, follicular lymphoma; GC, germinal center; L,
ligand; FLICA, fluorochrome inhibitor of caspases.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
proteins serve as major antiapoptotic molecules during CD95-
mediated cell death (28, 29).
Proximal signaling events engaged by DR4 and DR5, the two
death receptors of TRAIL, are very similar to CD95 (30, 31).
TRAIL receptors’ ligation induces activation of caspases 8 and 10,
which in turn can induce the cleavage of Bid for initiation of ap-
optosis via the intrinsic pathway. However, some differences be-
tween CD95 and TRAIL receptor-mediated apoptosis signaling are
suggested by the finding that TRAIL, in contrast to CD95L, is
cytotoxic against many tumor cells but not against most normal
cells (32–34) and that some CD95-resistant cell lines still exhibit
sensitivity to TRAIL-mediated apoptosis (35). Moreover, ongoing
preclinical and clinical trials on different tumor models are con-
firming the potent antitumor activity of TRAIL in vivo (36). Since
FL have a slow growth profile, they may be more vulnerable to
apoptotic stimuli than to cytotoxic agents targeting dividing cells.
Then the use of new therapeutic molecules with proapoptotic func-
tion, like TRAIL, must be envisaged in FL therapies. Moreover,
studies on TRAIL-deficient mice suggest a role of TRAIL as a
tumor suppressor, where TRAIL deficiency predisposed mice to a
greater number of tumors, including B cell lymphomas, making
this ligand a new promising molecule in FL treatment (37). How-
ever, the cytotoxic response of neoplastic cells to proapoptotic
members of the TNF superfamily can be down-regulated by the
NF-?B/Rel nuclear activity (38–41). In consequence, the aim of
this work was to investigate TRAIL-mediated apoptosis in primary
FL B cells and different B cell lines from a GC origin in a context
of a strong CD40L/CD40 costimulation to mimic one of the most
important signals present in the GC microenvironment. We show
with clear evidence that CD40 signaling protects tumor B cells
from TRAIL-induced apoptosis and this is associated with a rapid
NF-?B activation, which in turn up-regulates c-FLIP and Bcl-xL.
Selective inhibition of NF-?B with appropriated drugs restores
TRAIL-induced apoptosis in B cell lymphomas.
Materials and Methods
Human tonsils were collected from children undergoing routine tonsillectomy
and primary lymph nodes were obtained from FL patients collected at diag-
nosis. Legal approval was obtained for this study from the institutional review
board of the University Hospital of Rennes. Informed consents were provided
into pieces and flushed through a 21-gauge needle. Cell suspensions were
cultured in RPMI 1640 (Invitrogen) supplemented with 10% FCS (Biowest)
and penicillin/streptomycin. The B cell population was further analyzed by
flow cytometry on CD19?CD20?cells. More than 95% of FL B cells ex-
pressed the appropriate ? or ? L chain according to the tumor monoclonal Ig
ptosis in primary follicular lymphoma
B cells with or without a CD40L
stimulation. Lymph nodes obtained
from 11 FL patients and tonsils col-
lected from 7 children undergoing
routine tonsillectomy were treated or
not with 100 ng/ml CD40L and/or
500 ng/ml TRAIL for 24 h, and apo-
ptosis was further estimated by flow
cytometry on CD19?CD20?B lym-
phocytes with an active caspase 3 as-
say. A, B cell histograms representa-
tive of one of the 11 FL patients and
one of the 7 human tonsils treated or
not with 500 ng/ml TRAIL for 24 h
are presented. The percentage of ac-
tive caspase 3-positive B cells after
CD40L, TRAIL, or CD40L plus
TRAIL treatments of the 11 lymph
nodes of FL patients (B) or the 7 chil-
dren tonsils (C) was compared with
untreated samples. Mean ? SD; ???,
p ? 0.001. Asterisks express statistics
relative to the control except when
referenced with bars.
1002TRAIL-INDUCED APOPTOSIS IN B CELL LYMPHOMA
isotype. The Burkitt lymphoma cell line BL2 was provided by J. Wiels (IGR)
and the FL-transformed B cell lines VAL, RL, and SUDHL4 were a generous
gift from C. Bastard (Centre Becquerel, Rouen, France).
Chemicals and Abs
The human recombinant soluble killer TRAIL was from Alexis Biochemi-
cals. The human recombinant soluble CD40L trimer was a generous gift
from Amgen. I?B-? phosphorylation inhibitor BAY 117085 was from Cal-
biochem. The Bcl-xLinhibitor BH3I-1?? was from Alexis Biochemicals.
FLIP protein synthesis inhibitor anisomycin was purchased from
Sigma-Aldrich. FITC-conjugated anti-CD19 and anti-CD20 mAbs were
obtained from Beckman Coulter, PE-conjugated anti-active caspase 3
apoptosis kit was purchased from BD Biosciences and FITC-conjugated
anti-annexin-V was from Roche. The anti-I?B-? Ab was obtained from
Calbiochem and the anti-Bad, anti-Bak, anti-Bax, anti-Bid, anti-Bcl-2,
anti-FADD, anti-poly(ADP-ribose) polymerase, anti-receptor interact-
ing protein, and anti-X-linked inhibitor of apoptosis protein were pur-
chased from BD Biosciences. The anti-Bcl-xLand anti-PUMA were
from Cell Signaling, the anti-FLIP NF6, anti-NOXA, anti-DR4, anti-
DR5, anti-DcR1, and anti-DcR2 Abs from Alexis Biochemicals, the
anti-Calpain from Chemicon, and the anti-?-actin from Sigma-Aldrich.
The peroxidase-conjugated goat anti-mouse or anti-rabbit Abs were ob-
tained from Bio-Rad.
Tonsils, FL lymph nodes, and B cell lines were cultured alone or with
CD40L (100 ng/ml), or TRAIL (100 or 500 ng/ml), or cotreated with
CD40L and TRAIL for 3 or 24 h. For B cell lines, apoptosis was analyzed
using a PE-conjugated anti-active caspase 3 apoptosis kit according to the
manufacturer’s instructions or a FITC-conjugated anti-annexin-V Ab. For
primary tonsils and FL samples, active caspase 3 was analyzed on selec-
tively gated CD19?CD20?B cells. For NF-?B inhibition, B cell lines and
primary tumor B cells were pretreated for 1 h with 75 nM or 1 ?M BAY
117085, respectively, and then stimulated with 100 or 500 ng/ml TRAIL
for 24 h. Active caspase 3-positive cells were quantified. Labelings were
analyzed using a FACSCalibur and CellQuest Pro software (BD
A fluorochrome inhibitor of caspases (FLICA) apoptosis detection kit
was used to revealed active caspases. Once inside the cell, FLICA inhib-
itors bind covalently to active caspases; these inhibitors are cell permeable
and noncytotoxic. The green fluorescent signal represents the amount of
active caspases present in the cell at the time the reagent was added. After
stimulation, the cell suspension was incubated with caspase 3–7, 6, 8. or 9
FLICA (AbD Serotec) for 1 h at 37°C/5% CO2. After two washes, cells
were directly analyzed by flow cytometry.
TRAIL-receptor labeling and neutralization
B cell lines were labeled with mouse mAb directed against DR4, DR5,
DcR1, DcR2, and IgG1 isotype matched as negative control. FITC-conju-
gated goat anti-mouse IgG1 secondary Ab was used. For TRAIL receptor
inhibition, cells were pretreated for 45 min with preservative-free anti-DR4
and/or anti-DR5-neutralizing Abs and then treated with 100 ng/ml TRAIL
for 24 h. FITC-annexin-V-positive cells were detected by flow cytometry.
ptosis in GC-derived B lymphoma
cell lines with or without a CD40L
stimulation. A, 16 ? 104
SUDHL4, RL, and VAL cells/well
were treated or not with 100 ng/ml
CD40L and/or 100 ng/ml TRAIL for
24 h and active caspase 3-positive
cells were estimated by flow cytom-
etry. Mean ? SD, n ? 4; ?, p ? 0.05;
??, p ? 0.01; and ???, p ? 0.001.
Asterisks express statistics relative to
the control except when referenced
with bars. B, TRAIL receptor surface
expressions were evaluated in GC-de-
rived B lymphoma cell lines by flow
DcR1, and DcR2 Abs. Isotypic con-
trols are depicted in dashed line and
specific labeling in bold line. C, Neu-
tralization of DR4 and/or DR5 was
performed on BL2 using 0.01–10
TRAIL-mediated apoptosis was eval-
uated by flow cytometry and ex-
pressed as a percentage of control val-
ues (TRAIL-induced apoptosis in
absence of Ab). Mean ? SD, n ? 3;
??, p ? 0.01 and ???, p ? 0.001. D,
16 ? 104BL2 cells/well were treated
or not with 100 ng/ml CD40L and/or
100 ng/ml TRAIL for 3 and 24 h, and
annexin V-positive cells were further
estimated by flow cytometry. Mean ?
SD; ?, p ? 0.05; ??, p ? 0.01; and
???, p ? 0.001. E, BL2 cells were
treated or not with 100 ng/ml CD40L
and/or 100 ng/ml TRAIL for 0–24 h
and the percentage of active caspase 3
cells was further estimated by flow
cytometry (mean ? SD, n ? 3).
1003The Journal of Immunology
BL2 cells (32 ? 103/well) were treated or not with CD40L, TRAIL, or a
combination of both in 96-well plates. After 24 h of culture, cells were
pulsed with 1 ?Ci/well tritiated thymidine ([3H]TdR; Amersham Bio-
sciences) for the last 12 h of culture, harvested, and counted on a liquid
scintillation analyzer. For cell cycle analysis, 2.4 ? 106BL2 cells were
treated or not with CD40L, TRAIL, or a combination of both in 75-cm2
flasks. After 24 h of culture, cells were collected and fixed overnight with
70% ethanol, then RNase A was added for 5 min. Finally, propidium iodide
was admixtured to the cells before flow cytometry analysis. Cell cycle
distribution was determined using ModFit software (Verity Software
Western blot analysis
After treatments, cells were lysed in radioimmunoprecipitation assay buffer
(50 mM Tris-HCl (pH 7.4), 1% Nonidet-P 40, 150 mM NaCl, 0,25% so-
dium deoxycholate, 1 mM EDTA, 1 mM paramethylsulfonide, 1 ?g/ml
pepstatin, leupeptin. and aprotinin) at 4°C. After 14,000 ? g centrifugation
for 30 min, the protein concentration was determined in the supernatant by
bicinchoninic acid assay. Samples were boiled for 5 min in Laemmli buffer
(62.5 mM Tris-HCl (pH 6.8), 2% SDS, 25% glycerol, and 0.01% brom-
phenol blue) containing 4.8% of 2-ME. Equal amounts of protein (30 ?g)
were loaded on 12–15% SDS-polyacrylamide gels and transferred to a
polyvinylidene difluoride membrane (Millipore). Membranes were blocked
with 5% nonfat dry milk in PBS-Tween 20 (0.1%, v/v) for 1 h and incu-
bated for 1 h at room temperature with the different Abs. Membranes were
then washed twice with PBS/Tween20 and incubated for 1 h with HRP-
conjugated goat anti-mouse or anti-rabbit Abs. Immunoreactive proteins
were visualized by a chemiluminescence protocol (ECL plus; Amersham
NF-?B activity measurement
NF-?B activation was measured with a TransAM NF-?B family kit (Active
Motif). This ELISA is based on measurements of p50-, p65-, c-Rel-, p52-,
and RelB-binding activities to specific consensus DNA sequences. Nuclear
extracts were purified according to the manufacturer’s instructions after 1
or 6 h of stimulation with CD40L (100 ng/ml), TRAIL (100 ng/ml), or
both. Five micrograms of nuclear extracts were added per ELISA well,
incubated with anti-p50, anti-p65, anti-c-Rel, anti-p52 or anti-RelB pri-
mary Abs for 1 h, washed, and then incubated with the secondary perox-
idase-conjugated Ab for 1 h. After three washes, the developing solution
was added for 10 min and absorbance was read at 450 nm.
RNA was extracted using a RNeasy kit (Qiagen) and cDNA was generated
using Superscript II reverse transcriptase (Invitrogen). For quantitative RT-
PCR, we used assay-on-demand primers and probes, and the TaqMan Uni-
versal Master Mix from Applied Biosystems. Gene expression was mea-
sured using the Applied Biosystems Prism 7900 Sequence Detection
System. 18S was determined as the appropriate internal standard gene. For
each sample, the cycle threshold value for the gene of interest was deter-
mined, normalized to its respective value for 18S, and compared with the
value obtained for unstimulated cells.
Retrovirus production and cell transduction
The bicistronic retroviral pMIG vector containing an internal ribosome
entry site upstream of the enhanced GFP gene (42) was used to introduce
c-FLIPLcDNA from a pcDNA3 plasmid using BglII-SalI and BglII-XhoI,
mal growth and cell cycle perturbed
after a TRAIL treatment. A and B,
BL2 (2.4 ? 106cells/flask) cells were
treated or not with 100 ng/ml CD40L
with or without 100 ng/ml TRAIL in
75-cm2flask. After 24 h of culture,
cells were collected and fixed by eth-
anol 70% overnight, then RNase A
and propidium iodide was added. The
cell cycle was analyzed by flow cy-
tometry. A is representative of one of
the four independent experiments
summarized in B (mean ? SD, n ? 4;
??, p ? 0.01; ???, p ? 0.001). C,
32 ? 103BL2 and SUDHL4 cells/
well were treated or not with 100
ng/ml CD40L with or without 100
ng/ml TRAIL in 96-well plates. After
24 h of culture, cells were pulsed with
1 ?Ci/well [3H]TdR for the last 12 h
of culture, harvested, and counted
on a liquid scintillation analyzer
(mean ? SD, n ? 6; ???, p ? 0.001).
CD40L restore a nor-
1004 TRAIL-INDUCED APOPTOSIS IN B CELL LYMPHOMA
respectively. The pMIG vector encoding Bcl-xLwas purchased from Ad-
dgene (43). Retroviruses were produced using a Retro-X Universal Pack-
aging System (BD Clontech). Briefly, GP2-293 cells were transfected us-
ing a standard calcium phosphate technique with 10 ?g of pMIG (mock,
encoding FLIP-L or Bcl-xL) and 5 ?g of pVSVG. Twelve hours after
transfection, medium was removed to stimulate cells overnight with 10 ?M
sodium butyrate. Stimulated cells were then washed twice with PBS to
remove sodium butyrate and refed with fresh medium. Viral supernatants
were collected 24–48 h after, to infect SUDHL4 cells, in the presence of
8 ?g/ml polybrene for 12 h. Infection was repeated twice, then mock-
transfected cells and cells expressing c-FLIP-L or Bcl-xLwere sorted by
flow cytometry based on GFP expression.
Statistical analyses were performed with the Student t test using GraphPad
Prism software. The significance is shown as follows: ?, p ? 0.05; ??, p ?
0.01; and ???, p ? 0.001.
Effect of CD40 triggering on TRAIL-induced apoptosis in
primary FL B cells
To address whether the proapoptotic TNF superfamily member
TRAIL could be a promising therapeutic molecule in the treatment
of FL, we first estimated its potency to induce apoptosis on pri-
mary FL B cells (Fig. 1). After a 24-h treatment with 500 ng/ml
TRAIL on a total cell population extracted from lymph nodes re-
covered from patients with FL at diagnosis, we estimated by flow
cytometry the percentage of active caspase 3-positive cells on
CD19?CD20?B lymphocytes. We observed in all 11 patients
tested that TRAIL significantly induces B cell death with a 30%
increase of active caspase 3-positive primary FL B cells according
to the control (Fig. 1, A and B). It is worth notice that on average
20% of active caspase 3-positive nontreated cells were detected
reflecting spontaneous apoptosis after 24 h of culture as already
described, and thus indirectly confirms the role played by the
lymph node microenvironment in FL B cell survival (44, 45). To
evaluate a possible side effect of TRAIL, if used as an anticancer
drug, we also tested its cytotoxicity on normal B cells from human
tonsils.In this case, TRAIL
CD19?CD20?gated B lymphocytes (Fig. 1, A and C). Toxicity
was also evaluated on the CD19?CD20?non-B cell compartment.
These cells exhibited no cell death in malignant and nonmalignant
samples (data not shown). Dose-response experiments have been
performed on total cell populations extracted from lymph nodes
and a higher concentration of TRAIL (500 ng/ml) was needed to
exhibitedno toxicity on
optotic signaling pathway by CD40L
in TRAIL-treated BL2 and SUDHL4
cells. A and C, BL2 and SUDHL4
cells were stimulated or not with 100
ng/ml CD40L and/or 100 ng/ml
TRAIL for 3 or 24 h. Cell lysates
were analyzed after stimulation by
immunoblotting. ?-Actin was used
as a loading control. B, 16 ? 104BL2
cells were stimulated or not with 100
ng/ml CD40L and/or 100 ng/ml
TRAIL for 3 and 24 h and active
FLICA detection kit by flow cytom-
etry (mean ? SD, n ? 4; ??, p ?
Modulation of the ap-
1005The Journal of Immunology
induce strong FL B cell apoptosis as compared with the B cell lines
(100 ng/ml) used in the next section (data not shown). This was
due to the admixture of TRAIL-sensitive and -resistant cells ob-
tained after tissue extraction, as the CD19?CD20?B lymphocyte
fraction represented ?50% of the total cell population. These re-
sults confirmed the specific antitumoral activity of TRAIL. Then,
we asked whether the strong survival signal provided by CD40 on
B cells could interfere with a TRAIL treatment. To address this
question, we treated normal and neoplastic B cells with 100 ng/ml
human recombinant CD40L, alone or in cotreatment with 500
ng/ml TRAIL for 24 h. We observed that CD40L alone protected
both normal and FL-derived primary B cells from spontaneous
apoptosis, as already addressed but also efficiently protected tumor
cells from TRAIL-induced apoptosis when used in cotreatment
(Fig. 1, B and C). These results indicate that CD40 triggering, a
strong signal in GC B cell differentiation, contributes to GC B cell
survival but will also interfere with TRAIL-induced apoptosis
Effect of CD40 triggering on TRAIL-induced apoptosis in
GC-derived B lymphoma cell lines
To characterize the molecular mechanisms which sustain CD40
protection against TRAIL-induced apoptosis, we tested different
cell lines derived from GC lymphomas. We retained four of them,
BL2, SUDHL4, VAL, and RL, and we analyzed for their sensi-
tivity to TRAIL with or without a CD40L cotreatment (Fig. 2A).
We consistently observed that the BL2 and SUDHL4 cell lines
were highly sensitive to 100 ng/ml TRAIL with ?85% of active
caspase 3-positive cells, when RL was an intermediate responder
and VAL was much less sensitive to the same dose of the recom-
binant protein after a 24-h treatment. Dose-response experiments
showed that BL2 and SUDHL4 were sensitive to a lower dose of
TRAIL (10 ng/ml) with 60% of active caspase 3-positive cells
after a 24-h treatment (data not shown). We analyzed surface ex-
pression of DR4, DR5, DcR1, and DcR2 on the four different cell
lines to appreciate whether the differences in TRAIL sensitivity
were linked to receptor expression levels. As demonstrated in Fig.
2B, the level of cell surface expression of DR4 and DR5 was
similar in the four different cell lines. The only differences we
observed were for the decoy receptors, with DcR2 almost absent
on BL2 but significantly expressed on VAL and RL. However,
DcR2 was also highly expressed on the very sensitive SUDHL4
cell line. VAL was the only cell line expressing a significant
amount of DcR1 on its cell surface. Using specific neutralizing
Abs directed against DR4 and DR5, we analyzed the respective
involvement of each death receptor in TRAIL-induced apoptosis in
BL2 (Fig. 2C). We showed that most of the death signal is engaged
through DR4 in this cell line since the anti-DR4 Ab inhibits
TRAIL-induced apoptosis in a dose-dependent manner. High con-
centrations of this Ab (10 ?g/ml) reduced by 65–70% TRAIL-
induced apoptosis, whereas at this concentration, the anti-DR5 Ab
had no significant effect.
When cotreated with CD40L, BL2 and SUDHL4 showed a clear
protection from TRAIL-induced apoptosis. This effect was obvi-
ous on BL2 and SUDHL4 but could also be significantly noticed
on RL, which exhibited a moderate sensitivity to TRAIL (Fig. 2A).
After annexin-V labeling and using an active caspase 3 assay, we
showed that CD40L protection against TRAIL-induced apoptosis
was noticeable but weak after 3 h and was clear after 6–24 h of
cotreatment, reaching a protection effect of at least 40% (Fig. 2, D
and E). Since BL2 and SUDHL4 exhibited the same pattern of
response to TRAIL and CD40 signaling as primary FL cells, we
then focused our investigations on these two cell lines.
Effect of CD40 ligation on cell growth and cell cycle regulation
on TRAIL-stimulated GC-derived B lymphoma cell lines
To fully appreciate the effect of TRAIL and CD40L on cell cycle
regulation, we analyzed BL2 and SUDHL4 cell growth and BL2
cell cycle after a 24-h treatment with 100 ng/ml TRAIL and/or 100
ng/ml CD40L (Fig. 3). After treating BL2 separately with TRAIL,
we observed that this stimulation was associated with a strong
increase of the sub-G1cell population, an increase of the G0-G1
subpopulation, and a decrease of the S and G2-M phases reflecting
apoptosis (sub-G1phase) and a blockade of the cell cycle (Fig. 3,
A and B). In agreement, a strong decrease of cell growth using
thymidine incorporation was obtained (Fig. 3C). BL2 treated with
CD40L exhibited a slight increase of thymidine incorporation as
compared with the control but with a similar repartition of cells in
each phase of the cell cycle compared with the control. This dif-
ference in thymidine incorporation observed between CD40L-
treated and nontreated cells was due to a slight reduction of spon-
taneous apoptosis detected in the sub-G1subpopulation (Fig. 3B).
When the cells were cotreated with TRAIL and CD40L, the
sub-G1cell population was considerably reduced as compare with
TRAIL alone. We also noticed that cotreated BL2 cells continued
to proliferate with an increase of thymidine incorporation and a
recovery of the cell cycle. These last results that we also confirmed
on the SUDHL4 cell line (Fig. 3C), associated with the data on
apoptosis (Fig. 2), indicate that CD40 signaling not only protects
BL2 and SUDHL4 cells were stimulated or not with 100 ng/ml CD40L
and/or 100 ng/ml TRAIL for 15 min. Cell lysates were analyzed by immuno-
blotting for I?B?. ?-Actin was used as a loading control. B and C, 100 ? 106
BL2 cells were serum starved for 15 h and stimulated or not with 100 ng/ml
CD40L and/or 100 ng/ml TRAIL for 1 h (B) or 6 h (C). Nuclear extracts were
analyzed by ELISA as described in Materials and Methods. NFYA ELISA
was used as a loading control (mean ? SD, n ? 3).
CD40L activates the NF-?B1 and NF-?B2 pathways. A,
1006TRAIL-INDUCED APOPTOSIS IN B CELL LYMPHOMA
lymphoma B cells from TRAIL-induced apoptosis but also re-
stores the proliferation process of these cells.
Modulation of the apoptotic signaling pathway by CD40L in
TRAIL-treated BL2 and SUDHL4 cells
Since CD40L protects GC-derived B cell lymphomas from apo-
ptosis and restores their progression into the cell cycle, we next
analyzed the apoptotic signaling pathway engaged after CD40L
and TRAIL costimulation on the BL2 and SUDHL4 cell lines.
Western blot analyses were performed for most of the molecules of
the apoptotic cascade (Fig. 4, A and C) and FLICA was used to
evaluate caspase activation after 3 and 24 h of stimulation (Fig.
4B). We first noticed that caspase 8 cleavage induced by TRAIL
was slightly decreased by a CD40L cotreatment after 3 h. This
difference was significant after 24 h, with a reduction of active
caspase 8 of ?40–50%. Associated with this reduction of active
caspase 8, we observed an up-regulation of c-FLIP after cotreat-
ment, also detected with CD40L alone (Fig. 4, A and C). This
up-regulation was faint at 3 h but very clear after 24 h. As a
consequence, Bid truncation detected after TRAIL treatment was
almost completely inhibited when CD40L was added to the culture
for 24 h. We also observed that receptor interacting protein was
cleaved after a 24-h treatment with TRAIL as already described in
other cell types (46). This cleavage was inhibited after a costimu-
lation with CD40L. Among the other components of the apoptotic
signaling pathway, the proapoptotic member Bax was up-regulated
after 24 h of TRAIL treatment and a second band with a lower
molecular mass of p18 appeared, corresponding to the cleavage of
the p21 Bax protein. This smaller fragment, previously described
by Wood et al. (47) and Cao et al. (48) as being more efficient in
cell death, is generated in the late phase of apoptosis as a cleavage
product of calpain (49, 50). We indeed confirmed calpain activa-
tion with the appearance of proteolysis products associated with its
activation after 24 h of stimulation. CD40L cotreatment modulated
calpain activation and cleavage of Bax p21 into p18. These events
participated in the reduction of TRAIL-induced apoptosis by
CD40L. Similarly, CD40L prevented Bad cleavage detected under
TRAIL stimulation as already observed in other models (51). The
up-regulation of the BH3-only member NOXA after a 24-h treat-
ment with TRAIL was also partially inhibited by a cotreatment
with CD40L. We did not detect any changes after the different
stimulations for the other proapoptotic members Puma and Bak, as
for the anti-apoptotic Bcl-2 protein. Conversely, the anti-apoptotic
protein Bcl-xLwas induced after 24 h of treatment with CD40L,
cleaved with TRAIL as described by Mu ¨lher et al. (52), and pro-
tected from cleavage and still up-regulated after a cotreatment.
signaling induced by CD40L restores
TRAIL-induced apoptosis. A, 16 ?
104BL2 or SUDHL4 cells were stim-
ulated or not with 75 nM BAY
117085 for 1 h and then stimulated or
not with 100 ng/ml CD40L and/or
100 ng/ml TRAIL for 24 h, and active
caspase 3 was estimated by flow cy-
tometry (mean ? SD, n ? 4; ?, p ?
0.05). B, 1 ? 106primary FL B cells
obtained from six patients were pre-
treated or not with 1 ?M BAY
117085 for 1 h and then stimulated or
not with 100 ng/ml CD40L and/or
500 ng/ml TRAIL for 24 h. Apoptosis
was further estimated by flow cytom-
etry on CD19?CD20?gated B lym-
phocytes with an active caspase 3 as-
say. Active caspase 3 (ratio/control)
was determined as followed: percent-
age of active caspase 3 of treated FL
B cells/percentage of active caspase 3
of nontreated FL B cells of the same
individual (mean ? SD, n ? 6; ?, p ?
0.05 and ??, p ? 0.01). C, BL2 and
SUDHL4 cells were stimulated or not
with 75 nM BAY 117085 for 1 h and
then stimulated or not with 100 ng/ml
CD40L and/or 100 ng/ml TRAIL for
24 h. Cell lysates were analyzed by
immunoblotting for c-FLIP, Bcl-xL,
and ?-actin. D, Real-time PCR quan-
titation of c-FLIP and Bcl-xLexpres-
sion was evaluated on BL2 and
SUDHL4 cells stimulated or not with
75 nM BAY 117085 for 1 h and then
stimulated or not with 100 ng/ml
CD40L and/or 100 ng/ml TRAIL for
5 h. Each sample was normalized to
18S. Results are representative of one
of four independent experiments.
Inhibition of NF-?B
1007The Journal of Immunology
This molecule with c-FLIP is probably another major player in the
protection mediated by CD40L against TRAIL-induced apoptosis.
As a consequence of the upstream modulation provided by the
CD40L signaling on TRAIL-induced cell death, we observed a
significant inhibition of caspase 9, 6, 7, and 3 cleavages at 24 h.
Caspase activation induced by TRAIL was reduced by 40–50%
after a CD40L cotreatment. This corroborates the 40% protection
from apoptosis observed after a cotreatment and previously de-
scribed in Fig. 2. Finally, CD40L also modulated some inhibitors
of apoptosis, in particular X-linked inhibitor of apoptosis protein
which is protected from degradation after TRAIL stimulation. As
a final consequence of these modulations in the apoptotic pathway,
CD40L reduced poly(ADP-ribose) polymerase cleavage, ulti-
mately protecting the cells from DNA degradation.
NF-?B activation by CD40L is responsible for its protective
effect against TRAIL-induced apoptosis
We observed that c-FLIP and Bcl-xLwere both up-regulated after
24 h of CD40L treatment in BL2 and SUDHL4. These genes are
known to be NF-?B target genes, the main signaling pathway en-
gaged after CD40L stimulation. We then asked whether the pro-
tection mediated by CD40L was directly linked to its ability to
induce NF-?B. NF-?B/Rel transcription factors are dimers of pro-
teins (p50/p105 or NF?B1, p52/p100 or NF?B2, p65 or RelA,
c-Rel and RelB) that have ?300-aa Rel regions. The NF-?B/Rel
complexes are either found in cell nuclei or retained in the cyto-
plasm by inhibitors of the I?B (?–?) family; these latter are pro-
teolyzed on cell stimulation by a number of agents, allowing NF-
?B/Rel dimers to reach the nucleus and control the expression of
a wide range of genes. We analyzed NF-?B activation by Western
blot in BL2 and SUDHL4 with an anti-I?B? Ab and by ELISA to
evaluate the level of the different NF-?B subunits, p50, p65, c-Rel,
p52, RelB, into the nucleus of the BL2 cells (Fig. 5). When the two
cell lines were treated with CD40L with or without TRAIL, I?B?
disappeared after 15 min due to its fast phosphorylation and ubiq-
uitylation, which drive this inhibitor to its degradation by the pro-
teasome (Fig. 5A). On ELISA, p50, constitutively expressed in
BL2, p65, and c-Rel were all rapidly induced during the first hour
of a CD40L treatment, reflecting their translocation into the nu-
cleus (Fig. 5B). These three NF-?B members belong to the NF-
?B1 pathway. P52 and RelB which belong to the NF-?B2 pathway
were slightly induced after 1 h of stimulation (data not shown) and
significantly detected in the nucleus after 6 h (Fig. 5C). These data
indicate that CD40L stimulation induces very efficiently both the
classical and alternative NF-?B pathways, even in cotreatment
Inhibition of NF-?B signaling induced by CD40L restores
To confirm the role played by the NF-?B signaling pathway after
CD40L stimulation in the prevention of TRAIL-induced apoptosis
in B lymphoma cells, we performed our treatments with or without
a specific inhibitor of NF-?B signaling which prevents I?B? phos-
phorylation. Our results showed that the inhibition of TRAIL-in-
duced apoptosis by CD40L on BL2 and SUDHL4 was consider-
ably reduced when BAY 117085 was added to the culture (Fig.
6A). Similar results were obtained on primary FL B cells (Fig. 6B).
We also noticed that spontaneous apoptosis was reduced when
primary FL B cells were cultured with CD40L and was then re-
stored when BAY 117085 was added into the culture medium.
This indicates clearly that spontaneous apoptosis, observed when
tumor B cells are removed from their microenvironment, can be
partially prevented after activation of the NF-?B pathway. To con-
firm the role played by c-FLIP and Bcl-xL, the two NF-?B-targeted
genes in the modulation of TRAIL-induced apoptosis, we first per-
formed quantitative RT-PCR and Western blot analysis on BL2-
and SUDHL4-stimulated cells treated or not with BAY 117085. As
resistance after CD40L stimulation of GC-derived B cell lines. A, 16 ? 104
BL2 or SUDHL4 cells were stimulated or not with 100 ?M BH3I-1?? or 30
nM anisomycin for 1 h and then stimulated or not with 100 ng/ml CD40L
and/or 100 ng/ml TRAIL for 24 h, and active- caspase 3 was estimated by
flow cytometry (mean ? SD, n ? 4; ?, p ? 0.05). B, Not transfected, mock,
FLIPL, or Bcl-xL-transfected SUDHL4 cells were treated or not for 24 h
with 100 ng/ml TRAIL and active caspase 3 was estimated by flow cy-
tometry (mean ? SD, n ? 4; ???, p ? 0.001). C, FLIPLand Bcl-xL
overexpression in the SUDHL4-transfected cell line was estimated by
Western blot. ?-Actin was used as a loading control.
c-FLIP and Bcl-xLare the two key molecules in TRAIL
1008TRAIL-INDUCED APOPTOSIS IN B CELL LYMPHOMA
shown in Fig. 6D, the up-regulation of c-FLIP and Bcl-xLobserved
by Western blot in Fig. 4A was due to the induction of both gene
expressions detected by quantitative RT-PCR after CD40L stim-
ulation alone or in cotreatment with TRAIL. This up-regulation
was almost completely blocked after treatment with the NF-?B
inhibitor. As a consequence, the up-regulation of c-FLIP and
Bcl-xLproteins after CD40L stimulation was abrogated in the
presence of BAY 117085 (Fig. 6C). To definitely address the role
played by c-FLIP and Bcl-xLin the inhibition of TRAIL-induced
apoptosis in GC lymphoma B cells, we treated BL2 and SUDHL4
with BH3I-1?? a specific Bcl-xLinhibitor or anisomycin previously
described as a potent FLIP protein synthesis inhibitor (53). We
observed that these two chemicals fully restored TRAIL-induced
apoptosis in BL2 and SUDHL4 costimulated with CD40L (Fig.
7A). We finally generated SUDHL4-transfected cells with FLIPL
and Bcl-xLusing GFP-labeled retroviral vectors. After cell sorting
of GFP?SUDHL4 cells for purity, we verified the high protein
expression of FLIPLor Bcl-xLby Western blotting (Fig. 7C) and
then treated mock, FLIPL, or Bcl-xL-transfected cells with or with-
out TRAIL (Fig. 7B). We clearly showed that SUDHL4 when
overexpressing one of these two antiapoptotic proteins were resis-
tant to TRAIL-induced apoptosis. These results altogether place
these two molecules as the main actors of TRAIL-resistance in GC
B cell lymphomas.
Standard therapies for FL include immunotherapy (such as ritux-
imab, an anti-CD20 mAb) either alone or in combination with
chemotherapy or radiotherapy. Nevertheless, none of these thera-
pies are curative, especially in the case of multirecurrent disease.
Auto or allogenic stem cell transplantation is hampered by treat-
ment-related toxicity. Then, innovative approaches are urgently
needed. The most common cytotoxic agents used for the treatment
of advanced cancers act by inducing apoptosis of tumor cells
through activation of the caspase cascade. The understanding of
the malignant B cell mechanisms of apoptosis is therefore an im-
portant issue to the improvement of active therapies on slowly
TRAIL, a molecule of the TNF family, which selectively target
tumor cells through a strong activation of apoptosis is a very prom-
ising molecule in therapeutic approaches against B cell lympho-
mas. In our study, we demonstrate that primary FL B cells and
GC-derived B lymphoma cell lines are sensitive to TRAIL-in-
duced apoptosis. All of the 11 patients with FL at diagnosis were
consistently and similarly sensitive to TRAIL. The apoptotic sig-
naling is mediated mainly by DR4 in these cells. This indicates that
patients developing FL could either be treated by TRAIL or by an
agonistic Ab directed against DR4. These data agree with other
studies describing DR4 as the principal TRAIL receptor involved
in TRAIL-induced apoptosis in other lymphoid malignancies (54,
55). We cannot exclude that the other TRAIL receptors, in partic-
ular the two decoy receptors DcR1 and DcR2, participate in the
level of TRAIL sensitivity as it is suggested by our results on RL
and more noticeably VAL, which express high levels of these de-
coy receptors and are less sensitive to TRAIL than BL2 cells. The
SUDHL4 FL-transformed derived B cell line exhibited however
high sensitivity to TRAIL despite high expression levels of DcR2.
We have demonstrated recently that DcR2-mediated TRAIL inhi-
bition occurs through a TRAIL-dependent interaction with DR5,
leading to caspase 8 inhibition within the TRAIL death-inducing
signaling complex (56). It remains unclear why SUDHL4 cells
exhibit TRAIL sensitivity since they express both agonistic recep-
tors. In this context, it is noteworthy that BL2 cells engage pri-
marily DR4 to trigger TRAIL-induced cell death. Therefore, it
remains to be determined whether the selective engagement of
DR4 can be inhibited by DcR2 (54).
In this study, we have also shown that CD40L is capable of
partially inhibiting the apoptotic effect of TRAIL on primary B cell
lymphoma and B lymphoma cell lines as well as the activation of
the cysteine protease caspases 8, 9, 6, 7, and 3. These results un-
cover a new mechanism of resistance to cytotoxic agents conferred
by adjacent nontumoral cells expressing CD40L. This is particu-
larly important in the context of FL which originates from GC,
where B cell selection and differentiation are tightly dependent on
a CD40L stimulus provided mainly by TFH cells, a specific
CXCR5highICOShighCD4?T cell subpopulation present within
GC (57). This mechanism also prevents normal tonsil B cells and
FL B cells from spontaneous apoptosis in culture and identifies this
stimulation as crucial to their prolonged survival in vitro and also
probably in vivo.
Modulation by CD40L of TRAIL-induced apoptosis in GC-de-
rived B cell lymphoma is mediated by the rapid activation of the
canonical NF-?B1 pathway. Proteins of the NF-?B2 pathway are
also activated but after a prolonged activation as already reported
in different models (58). These results are in agreement with the
inhibition of I?B? degradation by BAY 117085, the potent NF-?B
inhibitor (data not shown), which almost completely reverses the
protective properties of CD40 against TRAIL-induced apoptosis.
This clearly indicates the main role played by the NF-?B signaling
pathway in this context. We have also evaluated by Western blot
the activation of the PI3K/Akt and MAPK pathways (p44/42MAPK,
JNK, and p38) after CD40L activation and have shown no involve-
ment of these signaling pathways as opposed to previous data ob-
tained on multiple myeloma and chronic lymphocytic leukemia B
cells (59, 60). We also confirmed these results using specific in-
hibitor of the PI3K/Akt, MAPK, p38, and JNK pathways (data not
c-FLIP and Bcl-xLgenes, direct targets of NF-?B transcription
factors, are both up-regulated in our study under a sustained
CD40L stimulation. The anti-apoptotic molecule c-FLIP acts at the
initiation phase of TRAIL-induced apoptosis. Both c-FLIP iso-
forms, c-FLIPshortand c-FLIPlong(61), interfere with caspase 8
activation by inhibiting the processing of procaspase 8 at the
death-inducing signaling complex (28, 62), resulting in the block-
ade of the apoptotic cascade. CD40L-induced protection against
CD95-mediated apoptosis has also been recently described by
Eeva et al. (63) in a human FL cell line. These authors showed that
this protection was associated with a rapid up-regulation of c-FLIP
and confirmed, with our present results on FL and results on other
tumors, that this inhibitory molecule is a key player in the inhibi-
tion of cell death induced by different TNF death receptor family
members in human lymphoid malignancies including FL (64, 65).
In addition to c-FLIP, Bcl-xLis also involved in the antiapop-
totic signaling of CD40 as a direct target gene of the NF-?B tran-
scription factors. Recent data in solid tumors have shown that
Bcl-xLwas responsible for the development of acquired TRAIL
resistance (66, 67). Our results demonstrate that in GC-derived B
lymphoma cells, this antiapoptotic protein is also up-regulated un-
der a CD40L stimulation. When NF-?B activation after CD40L
stimulation is blocked by a specific inhibitor of I?B? phosphory-
lation, the functional regulation played by BAY 117085 is asso-
ciated with a blockade of c-FLIP and Bcl-xL.The key role played
by either c-FLIP or Bcl-xLin the resistance to TRAIL in B cell
lymphoma after the engagement of the NF-?B signaling pathway
was definitely proven after BL2 and SUDHL4 treatment with spe-
cific inhibitor of either Bcl-xLor c-FLIP and in SUDHL4- trans-
fected cells with one of these two antiapoptotic genes (Fig. 7).
These results have to be taken into account in B lymphoma cancer
1009The Journal of Immunology
therapy, because CD40 signaling provided by TFH cells on the
GC-derived B cells could completely abolish a beneficial antitu-
mor effect mediated by TRAIL through NF-?B activation. In this
context, BAFF, another TNF family member also involved in nor-
mal B cell survival and B cell lymphoma proliferation (68, 69),
through activation of the NF-?B pathway could also cooperate
with CD40L to prevent TRAIL-induced apoptosis in FL B cells.
Collectively, our results strongly suggest that microenvironmen-
tal signals are at least in part responsible for the modulation of FL
survival in vitro and in vivo. Blockade of such signals may facil-
itate the entry of FL cells into the death pathway and might po-
tentially provide novel approaches to alter the sensitivity of FL to
therapy. As a consequence, the use of a combination of TRAIL or
anti-DR4 agonistic Abs with pharmacological inhibitors of NF-?B
signaling or blocking anti-CD40 Abs may represent an attractive
alternative therapy for FL.
We address a special thanks to Amgen which provided us with human
CD40L. We also thank Ce ´line Monvoisin and Gersende Lacombe (IFR140
GFAS) for technical assistance.
The authors have no financial conflict of interest.
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1011The Journal of Immunology