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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 5 (March 1), 1998:
pp. 1644-1652
Apoptosis of Malignant Human B Cells by Ligation of CD20 With
Monoclonal Antibodies
By
Daming Shan,
Jeffrey A. Ledbetter, and
Oliver W. Press
From the Departments of Biological Structure and Medicine of the
University of Washington; the Fred Hutchinson Cancer Research Center;
and Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle,
WA.
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ABSTRACT |
CD20 is a nonglycosylated 33 to 37 kD phosphoprotein involved in
B-cell signaling that subserves important functions in the regulation
of B-cell proliferation and differentiation. In addition, this B-cell
surface antigen has been shown recently to be an effective target for
immunotherapy of B-cell malignancies using chimeric (mouse/human) or
radiolabeled murine monoclonal anti-CD20 antibodies. In this report we
show that extensive crosslinking of CD20 with murine anti-CD20
monoclonal antibodies (MoAbs) in the presence of either goat anti-mouse
IgG or Fc receptor (FcR)-expressing cells directly inhibits B-cell
proliferation, induces nuclear DNA fragmentation, and leads to cell
death by apoptosis. The apoptotic effects of these MoAbs can be
inhibited by chelation of extracellular or intracellular
Ca2+ by EGTA or Bapta AM, indicating that
anti-CD20-mediated apoptosis may be related to changes in
Ca2+ concentration. These findings suggest that ligation
of CD20 in vivo by anti-CD20 antibodies in the presence of
FcR-expressing cells may initiate signal transduction events that
induce elevation of [Ca2+]i and lead to
apoptosis of malignant B cells, thereby contributing to the impressive
tumor regressions observed in mouse models and clinical trials using
anti-CD20 MoAbs.
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INTRODUCTION |
CD20 IS A nonglycosylated 33 to 37 kD
phosphoprotein expressed on greater than 95% of normal and neoplastic
B cells. It is expressed on the cell surface from the pre-B stage of
development until terminal differentiation to plasma cells occurs and
has been used as one of the most reliable markers of the B-cell
lineage.1 Monocytes, resting and activated T cells, null
cells, and nonlymphoid cells are uniformly CD20-negative.2
The predicted amino acid sequence of CD20 suggests a structure
containing four transmembrane-spanning regions with both amino and
carboxyl termini located on the cytoplasmic side of the plasma
membrane.3-5 Current studies suggest that CD20 is a B-cell
surface protein with the capacity to serve as a calcium channel,
initiate intracellular signals, and modulate cell growth and
differentiation.6 Ligation of CD20 with anti-CD20 monoclonal antibodies (MoAbs) has been shown to activate tyrosine and
serine/threonine protein kinases, which are noncovalently associated
with CD20, thereby inducing tyrosine phosphorylation of phospholipase
C-gamma (PLC ). In addition, hypercross-linking CD20 with anti-CD20
MoAbs plus a secondary goat anti-mouse (GAM) antibody has been shown to
mobilize calcium from intracellular stores.7,8 Anti-CD20
MoAbs have been shown to exert variable effects on the proliferation of
B cells after binding to the CD20 antigen. The 1F5 anti-CD20 MoAb
stimulates B-cell cycle transition from G0 to G1,9 whereas
another anti-CD20 MoAb (B1) inhibits B-cell progression from the G1
phase of the cell cycle into the S/G2+M stages following mitogen
stimulation. MoAb binding also inhibits B-cell differentiation and
Epstein-Barr virus (EBV)- or pokeweed mitogen (PWM)-induced Ig
secretion.10,11 These studies have shown the importance of
CD20 in important regulatory signals for B cells, although the precise
role of CD20 remains unclear.
The ubiquitous expression of CD20 at high surface densities on
malignant human B cells, and its lack of internalization after MoAb
binding,12 has suggested its utility as a tumor target for
immunotherapy of B-cell lymphomas.13,14 Studies have
documented the efficacy of this approach with objective tumor responses
in 25% to 50% of patients treated with unmodified anti-CD20
MoAbs.13-15 Even more impressive results have been recorded
using radioiodinated I-131-anti-CD20 MoAbs, which have induced
objective responses in 75% to 95% of patients treated with relapsed
B-cell lymphomas.16-20 Although the antitumor effects of
unmodified anti-CD20 MoAbs were initially attributed solely to
complement-mediated cytolysis and antibody-mediated cellular
cytotoxicity (ADCC),21-23 the magnitude of the tumoricidal
effects observed have exceeded those expected, particularly in settings
where unmodified murine MoAbs (which generally fix human complement
poorly and mediate ADCC poorly) have been used. Furthermore, objective
regressions of lymphomas have been observed in 25% to 40% of patients
administered murine anti-CD20 MoAbs trace-labeled with imaging doses of
Iodine-131 believed inadequate to mediate radiation-induced tumor
regressions18,19 (and O.W. Press and M. Corcoran,
unpublished observations, July 1993).
Accumulating evidence from in vitro studies,6,9 animal
tumor models,14,24 and early clinical
trials21,23 suggests that a substantial portion of the
tumoricidal effect of anti-CD20 MoAbs may be mediated by mechanisms
independent of complement, ADCC, or radioactive emissions. Indeed,
ligation of CD20 by MoAbs has been shown to disrupt normal signal
transduction in B lymphoma cells, which have subsequently been shown to
regress, presumably because of induction of cell cycle arrest
and/or apoptosis initiated by cross-linking of CD20 by the
MoAbs.7,25-28
Apoptosis is usually defined as physiological or programmed cell death
and is characterized by specific morphological features, including
cellular and nuclear pyknosis, cytoplasmic blebbing, and margination of
condensed chromatin at the periphery of the nuclear envelope. In
addition, internucleosomal DNA fragmentation typically occurs,
resulting in the production of characteristic "DNA ladders" when
nuclear extracts are subjected to agarose gel electrophoresis.29,30 Cross-linking of many B-cell surface antigens, including surface IgM (sIgM), CD19, CD22, and major histocompatibility complex (MHC) class II have been reported to initiate cell cycle arrest and/or apoptosis in mouse and human B cells. Soluble anti-sIgM antibodies induce apoptosis in Ramos cells,31 and anti-MHC class II MoAbs induce apoptotic death of murine splenic B cells.32 Cross-linking of CD19 and CD22 with their respective MoAbs can also induce apoptosis in B cells, but
only if the cross-linking is amplified by secondary GAM
antibodies.33 Because CD20 is a component of a B lymphocyte
signal transduction complex involved in the regulation of B lymphocytes
following activation similar to the one perturbed by anti-sIgM
antibodies, we decided to test the ability of anti-CD20 MoAbs to induce
apoptotic cell death in malignant human B cells.
Our results indicate that anti-CD20 MoAbs can directly inhibit the
proliferation of malignant B cells independent of complement-mediated lysis or ADCC and that apoptosis can be induced in both B-cell lines
and normal tonsil B cells if the CD20 cross-linking is amplified by a
GAM antibody or by incubation with FcR-bearing accessory cells. The
apoptotic effects of anti-CD20 MoAbs are markedly attenuated by
chelation of Ca2+ with EGTA or Bapta AM, an intracellular
calcium chelator. These results indicate that anti-CD20-mediated
apoptosis is related to changes in intracellular Ca2+
concentration and that in vivo ligation of malignant B cells by
anti-CD20 MoAbs followed by FcR-mediated cross-linking by macrophages or other accessory cells may be responsible for a substantial proportion of the cytotoxic effects of anti-CD20 MoAbs observed in
clinical trials.
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MATERIALS AND METHODS |
Cells.
The CD20-expressing human Burkitt's lymphoma cell lines, Ramos, Daudi,
and Raji, were obtained from the American Type Culture Collection
(ATCC; Bethesda, MD) and maintained in log-phase growth in RPMI-1640
supplemented with 12% FBS, 2 mmol/L glutamine, 1 mmol/L sodium
pyruvate, 100 U/mL penicillin, and 100 µg/mL streptomycin. Normal
tonsil B cells were isolated from human tonsils and cultured as
previously described.34 The mouse fibroblast
Ltk cell line transfected with human FcRII receptor
(CDw32) was obtained from ATCC with the kind permission of Dr Jacques
Banchereau35 and maintained in complete RPMI-1640 medium
supplemented with 1 × HAT
(Hypoxanthine + aminopterin + thymidine) medium.
Antibodies and reagents.
The anti-CD20 MoAb 1F5 (IgG2a) and the control anti-CD3 MoAb 64.1 (IgG2a) were produced and purified as previously
described.9,21,36 The anti-CD20 MoAb B1(IgG2a) was
purchased from Coulter Corporation (Miami, FL). F(ab )2
fragments of goat anti-human cell surface IgM antibody (anti-sIgM) and
F(ab)2 fragments of goat anti-mouse IgG
F(ab)2 antibodies were purchased from Jackson
ImmunoResearch Laboratories, Inc (West Grove, PA). F(ab)2
fragments of a GAM antibody were obtained from Pierce Chemical Co
(Rockford, IL). Herbimycin A and genistein were obtained from
Calbiochem (San Diego, CA). Bapta AM was obtained from Molecular Probes
(Eugene, OR).
In vitro cell proliferation assay.
The effects of anti-CD20 MoAbs on malignant B-cell growth in vitro were
determined by assessing [3H]-thymidine incorporation in
Ramos, Daudi, and Raji cells with and without preincubation with
anti-CD20 MoAbs 1F5 or B1.24 Briefly, 5 × 103
to 5 × 104 cells were resuspended in 100 µL culture
medium and plated in 96-well flat-bottom microtiter plates. After
incubating cells at 37°C for 24 hours with anti-CD20 MoAbs, 0.5 µCi
of [3H]-thymidine/well was added, and cells were cultured
for an additional 18 hours. Cells were then harvested onto glass fiber
filters with an automated harvesting system from Skatron Inc (Sterling,
VA), and [3H]-thymidine uptake was assayed with a 4000 series liquid scintillation counter (Downers Grove, IL). In some
experiments, cells coated with anti-CD20 MoAbs were further
cross-linked using an F(ab)2 GAM reagent. In these
experiments, 5 × 104 cells/100 µL were incubated with
10 µg/mL anti-CD20 MoAbs at 4°C for 30 minutes, washed, incubated
with 50 µg/mL GAM for 24 hours at 37°C, pulsed with 0.5 µCi
[3H]-thymidine/well for 18 hours, harvested, and counted.
Flow cytometric analysis of apoptosis and cell cycle arrest using
propidium iodide staining.
Flow cytometric analysis of cellular DNA was performed following
propidium iodide staining according to the method of Fried et
al.37 Briefly, 106 Ramos cells were incubated
with anti-CD20 MoAbs or control antibodies in the presence or the
absence of GAM, washed in phosphate-buffered saline (PBS), and then
gently resuspended in 0.5 mL of a hypotonic fluorochrome solution (50 µg/mL propidium iodide in 0.1% sodium citrate plus 0.1% Triton
X-100; Sigma Chemical Co, St Louis, MO). Samples were stored in the
dark at 4°C until flow cytometric analysis of individual nuclei using
a FACScan flow cytometer (FACScan; Becton Dickinson, San Jose, CA)
could be performed. The percentage of cells that were apoptotic was
measured as described by Nicoletti et al.38 Briefly,
cellular debris was excluded from analysis by raising the forward
scatter threshold, and the DNA content of intact nuclei was recorded on
a logarithmic scale. Apoptotic cell nuclei containing hypodiploid DNA
were enumerated as a percentage of the total population. After
measurement of apoptotic cells, the distribution of cells in each phase
of the cell cycle was determined using the same samples by the
cytometric method of Fried et al.37,39
In some experiments, Ramos cells were first incubated overnight at
37°C with herbimycin A (0.125 to 0.5 µg/mL) or genistein (2.5 to 10 µg/mL) before washing and incubating with anti-CD20 plus GAM. In
other experiments, the calcium chelators EGTA (3 mmol/L) and Bapta AM
(5 µmol/L) were added to cell cultures 30 minutes before anti-CD20
MoAbs to determine the effects of perturbations in intracellular
Ca2+ concentrations on anti-CD20-induced apoptosis.
Light scatter analysis of apoptotic cells by flow cytometry.
Apoptotic cells were detected by flow cytometry using the distinct
forward and 90° light scatter characteristics of apoptotic and viable
cells according to Knox et al.40 By flow cytometry, apoptotic cells cause lower forward light scatter (caused by cell shrinkage) and higher side scatter (caused by increased granularity of
the cell, presumably as a result of chromatin condensation and
fragmentation) than their viable counterparts. Thus, apoptotic and
viable cell populations can be clearly identified and large numbers of
cells reproducibly and rapidly counted. The results were expressed as
"percentage apoptosis" by dividing the number of apoptotic cells
enumerated by the total number of cells counted in each culture.
DNA analysis by agarose gel electrophoresis.
Fragmented DNA was isolated and analyzed from Ramos cells after
incubation with and without antibodies as described by Gottschalk et
al.41 Briefly, 2 to 5 × 106 Ramos cells were
incubated with or without 10 µg/mL 1F5 or B1 in the presence or
absence of 50 µg/mL GAM for 24 hours, then lysed in 0.5 mL of lysis
buffer (0.6% sodium dodecyl sulfate (SDS) + 10 mmol/L EDTA, pH
7.0). NaCl was added to a concentration of 1 mol/L and mixed by
inversion. The mixture was left at 4°C for  12 hours and then
centrifuged at 12,000 g for 15 minutes at 4°C. Fifty
micrograms per milliliter RNAase A (Sigma) was added to the supernatant
and incubated at 37°C for 30 minutes. The supernatant was then
extracted with a 1:1 mixture of phenol and chloroform, precipitated
with 75% ethanol, and resuspended in TE buffer (Tris buffer + EDTA).
The sample was then subjected to electrophoresis on a 2% agarose gel
containing 0.5 µg/mL ethidium bromide.
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RESULTS |
Effects of anti-CD20 MoAbs on B-cell proliferation.
The effects of anti-CD20 MoAbs on B-cell proliferation were studied by
incubating malignant human B-cell lines with 3H-thymidine
in the presence or absence of the anti-CD20 MoAb 1F5. The proliferation
of Ramos cells was progressively inhibited by increasing concentrations
of 1F5, with maximal inhibition at concentrations 1 µg/mL (Fig
1A). To determine whether this inhibition was a result of the fixation
of complement derived from fetal bovine serum (FBS) in the culture
medium, we repeated this experiment using FBS that had been
heat-inactivated at 56°C for 30 minutes (Fig
1B). These experiments suggested that both
complement-mediated and complement-independent mechanisms contributed
to the observed inhibition of B-cell proliferation. In the absence of
detectable complement activity, Ramos cell proliferation was inhibited
by up to 53% at high 1F5 concentrations (P < .001; Fig
1B). All subsequent experiments were performed using heat-inactivated
FBS to further characterize the complement-independent mechanisms of
B-cell killing induced by anti-CD20 MoAbs. Interestingly, we found that
the antiproliferative effect of 1F5 was related to cell density and to
the degree of saturation of this antibody on the target cell surface.
When the cell density was higher than 2 × 105 cells/mL
and 1F5 was used at subsaturating concentrations, the inhibitory effect
of 1F5 decreased to as low as 4 ± 3.5% (P > .05). However, the inhibitory effect of anti-CD20 MoAbs could be elicited even under these suboptimal conditions if 1F5 was cross-linked on the
cell surface using a GAM antibody (81 ± 2.2% inhibition, P < .001, data not shown). These results indicate that
under optimal conditions, anti-CD20 MoAbs alone are sufficient to
inhibit Ramos cell growth by ligation of the CD20 antigen, but under
suboptimal conditions augmentation of this effect by hypercross-linking
CD20 with GAM is required to inhibit cell growth. Another anti-CD20 MoAb, B1, had similar inhibitory effects on Ramos cells, and both MoAbs
also inhibited the proliferation of other B lymphoma cell lines (Raji
and Daudi, data not shown) in a similar fashion.
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| Fig 1.
Inhibitory effect of anti-CD20 MoAb 1F5 on Ramos cells
measured by tritiated thymidine incorporation. 104 cells in
100 µL per well were incubated continuously with 0.0001, 0.001, 0.01, 0.1, 1, 10, 100 µg/mL 1F5 in medium containing FBS (A) or medium
containing inactivated FBS (B) at 37°C for 24 hours, followed by
addition of 0.5 µCi [3H]-thymidine per well and
incubating for another 18 hours. Cells incubated in medium without 1F5
were used as control. Data are representative of three concordant
experiments. Similar results were obtained with the B1 anti-CD20
antibody (not shown).
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Induction of apoptosis in Ramos cells by binding of anti-CD20 MoAbs.
To test whether anti-CD20 MoAbs are capable of inducing B-cell death by
apoptosis after inhibiting cell growth, three corroborative assays were
used; propidium iodide (PI) staining of cell DNA after incubation of
cells with anti-CD20 or control MoAbs, DNA fragmentation as assessed by
agarose gel electrophoresis, and flow cytometric light scatter
analysis. In all these assays, incubation of Ramos B cells with
F(ab)2 fragments of an anti-sIgM antibody was used as a
positive control because multiple studies have documented the reliable
induction of Ramos cell apoptosis with this reagent.31,33 In the PI staining assay, the percentage of apoptotic nuclei detected by PI staining and flow cytometric analysis after 24 hours in culture
increased from 2.7 ± 0.3% in the negative control (MoAb 64.1) to
30.6 ± 0.2% (P < .001) in the presence of anti-sIgM
antibodies (Fig 2A). Soluble anti-CD20
antibodies (1F5 and B1), did not induce significant apoptosis by
themselves under these conditions. However, each of these MoAbs clearly
and reproducibly induced cell death when cross-linked by a GAM
antibody; apoptosis occurred in 22.2 ± 1.9% of cells by 1F5 + GAM
and in 27.7 ± 3.5% of cells with B1 + GAM (compared to
5.25 ± 1.3% apoptosis in control cultures incubated with MoAb
64.1 + GAM, P < .001; Fig 2A). A kinetic analysis of the
induction of apoptosis by CD20 cross-linking indicated that DNA
fragmentation could be detected as early as 1 hour after incubation
with anti-CD20 MoAbs + GAM, with near maximal levels of apoptosis
achieved after 12 to 24 hours of incubation (Fig 2B).
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| Fig 2.
(A) Apoptotic effect of anti-CD20 MoAbs on Ramos cells as
shown by propidium iodide staining. 106 cells/mL were
incubated with 10 µg/mL of the B1 or 1F5 anti-CD20 antibodies or with
the control 64.1 anti-CD3 antibody for 24 hours in the presence or the
absence of GAM cross-linker (50 µg/mL). Cell nuclei were stained with
propidium iodide and analyzed by flow cytometry. Hypodiploid DNA peaks
corresponding to apoptotic nuclei were quantified. Data are
representative of five concordant experiments. (B) Kinetics of
apoptosis induced by CD20 cross-linking. 106 cells/mL were
incubated with or without 10 µg/mL anti-CD20 (B1) MoAb + 50 µg/mL
GAM for 0 to 48 hours. Cell nuclei were stained with propidium iodide
and analyzed by flow cytometry as described above. Anti-sIgM was used
as a positive control. Data are representative of three similar
experiments.
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Two different assays were used to confirm this observation. Classic
nucleosomal DNA ladder patterns were observed in DNA samples from cells
treated by anti-CD20 MoAb (B1) after cross-linking with GAM, as shown
by agarose gel electrophoresis in Fig 3(lane 5). Another anti-CD20 MoAb, 1F5, exhibited similar apoptotic
effects (data not shown). Cells treated by anti-CD20 MoAbs alone did
not consistently display DNA ladder formation (Fig 3, lane 4). Similar results were observed in flow cytometric light scatter analyses in five
consecutive experiments, one of which is shown in Fig 4.
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| Fig 3.
DNA degradation in Ramos cells caused by anti-CD20 MoAb
(B1) plus GAM shown by agarose gel electrophoresis. Ramos cells
(2 × 106) were treated with 10 µg/mL control anti-CD3
MoAb (64.1) or anti-CD20 MoAb (B1) in the presence or the absence of
GAM cross-linker (50 µg/mL) at 37°C for 24 hours. Fragmented DNA
was isolated and analyzed on a 2% agarose gel. Lane 1, molecular
weight markers; lane 2, 64.1; lane 3, 64.1 + GAM; lane 4, B1; lane 5, B1 + GAM; lane 6, anti-sIgM.
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| Fig 4.
Apoptosis of Ramos cells induced by anti-CD20 MoAbs as
shown by flow cytometry with light scatter analysis. 106
cells/mL were incubated with 10 µg/mL antibodies for 24 hours in the
presence or the absence of GAM cross-linker at 50 µg/mL. Pelleted
cells were resuspended in fixative (5% formaldehyde, 1% FBS in pH 7.2 PBS), and analyzed by flow cytometry. Apoptotic cells were identified
and quantified by flow cytometry according to their smaller size and
higher density. The data are representative of five experiments.
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We next tested whether normal human B lymphocytes were also susceptible
to apoptosis after ligation of CD20 by MoAbs. Tonsil B cells were
cultured for 2 days with anti-CD20 MoAbs or a control anti-CD3 MoAb
64.1 in the presence or absence of GAM, followed by detection of
apoptotic cells by PI staining. High rates of spontaneous apoptosis
were observed in tonsil B cells in control cultures incubated alone or
with control MoAb 64.1 (31 ± 0.14%). Incubation with anti-CD20
MoAbs alone slightly reduced the level of spontaneous apoptosis
(28.9 ± 0.042%, P < .05), as previously described for
normal human B cells.42 However, hypercrosslinking of CD20
on tonsil B cells with anti-CD20 + GAM significantly increased the
percentage of tonsil B cells undergoing apoptosis (47.25 ± 0.77%, compared with 35.25 ± 0.92 in control cultures incubated with 64.1 + GAM, P < .001), similar to the effects of
hypercrosslinking CD20 on the B lymphoma cell lines.
Because Fc receptor (FcR) engagement is known to inhibit
anti-Ig-induced B-cell responses,43 we investigated
whether the cytotoxic effect of intact anti-CD20 MoAbs could be caused
by the interaction of the Fc region of the MoAbs with FcRs on Ramos cells. To test this hypothesis we compared the effects of intact anti-CD20 MoAbs with those induced by their F(ab)2
fragments. The results indicated that the inhibitory effect of
F(ab)2 fragments of the B1 anti-CD20 MoAb
(34.00 ± 11.25% of counts per minute [cpm] obtained in control
cultures using MoAb 64.1) were comparable with those of the intact B1
MoAb (44.78 ± 10.39% of control cpm, P > .05).
Furthermore, hypercross-linking CD20 with anti-CD20 MoAbs plus GAM
[goat anti-mouse Ig F(ab)2 for anti-CD20
F(ab)2] augmented the apoptotic effects of both anti-CD20
F(ab)2 and intact anti-CD20 MoAb similarly (data not
shown), indicating that neither binding of the Fc portion of anti-CD20
MoAbs to FcR nor FcR cross-linking was essential for the observed
effects.
Induction of cell cycle arrest.
To determine whether cross-linking CD20 induces cell cycle arrest as
well as apoptosis, we analyzed the distribution of Ramos cells in
various phases of the cell cycle by a flow cytometric method using the
same experimental samples used to measure apoptosis. The results of
four separate experiments are shown in Table
1. As can be seen, in all experiments small
increases in the percentage of cells in the G1 phase of the cell cycle
were observed after incubation with anti-CD20 antibodies, suggesting
that cell cycle arrest was induced in a small fraction of the cells by
CD20 ligation. The magnitude of this effect could be amplified by
crosslinking with anti-CD20 MoAbs + GAM, increasing the percentage of
cells in G1 phase from 30% to 43% and correspondingly decreasing the percentage of cells in S + G2/M phases from 70% to 57% (Table 1).
FcR-expressing cells amplify apoptosis induced by anti-CD20 MoAbs.
As shown in the above experiments, apoptosis of Ramos cells can only be
reliably initiated by anti-CD20 MoAbs if they are cross-linked with
GAM. To determine whether similar cross-linking might conceivably occur
in vivo under clinical conditions in which anti-CD20 MoAbs are
administered to patients bearing B-cell malignancies, we investigated
whether functional cross-linking of Ramos cells coated with anti-CD20
MoAbs might be achieved by binding of the Fc domain of these antibodies
to FcR of adjacent cells. To test this possibility, we incubated
Ramos cells with anti-CD20 MoAbs in the presence or absence of mouse
fibroblasts transfected with the human Fc receptor
(FcRII/CDw32).35 As indicated in Fig 5, these cells were capable of providing
functional cross-linking and inducing apoptosis of Ramos cells bearing
surface anti-CD20 MoAbs. The percentage of apoptotic nuclei increased
from 6.05 to 7.35 ± 0.81% for two negative controls to 25.30 ± 0.60% (P < .001) and 14.10 ± 0.39%
(P < .001) for anti-CD20 MoAbs B1 and 1F5, respectively.
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| Fig 5.
Apoptosis of Ramos cells incubated with anti-CD20 MoAbs
in the presence of FcR-expressing accessory cells. Each well of a 24-well plate was coated with 5 × 105 FcR-expressing
accessory cells at 37°C overnight. 106 Ramos cells were
added to each coated well with PBS, control anti-CD3 antibody (64.1),
or anti-CD20 MoAbs. After 24 hours incubation at 37°C, cells were
stained with PI and analyzed by flow cytometry. Anti-CD20 MoAbs (B1 and
1F5) plus GAM and anti-sIgM antibody were used as positive control in
the absence of FcR-expressing accessory cells. Data are
representative of four concordant experiments.
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Effect of inhibitors of protein kinases on apoptosis.
Experiments by other investigators7 as well as our own
unpublished results have shown that the CD20 molecule is noncovalently associated with both tyrosine and serine kinases. To test whether these
protein kinases are involved in the cascade of events induced by CD20
cross-linking and leading to apoptosis, we exposed Ramos cells to the
tyrosine kinase inhibitor herbimycin A and the nonspecific protein
kinase inhibitor genistein before CD20 hypercross-linking with
anti-CD20 MoAbs + GAM. Unexpectedly, both inhibitors appeared to
augment the induction of apoptosis by anti-CD20 MoAbs + GAM in a
dose-dependent manner, rather than inhibiting it as anticipated (Fig
6). In three concordant experiments,
herbimycin increased the percentage of fragmented DNA by 57.5 ± 5.8%
compared with cultures incubated with B1 + GAM without herbimycin.
Also, a slight increase in apoptosis of Ramos cells was observed with
herbimycin A and genistein by themselves, as has previously been
reported for Jurkat cells treated with genistein.44
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| Fig 6.
Effect of protein kinase inhibitors on apoptosis induced
by CD20 cross-linking. 106 Ramos cells/mL were incubated
overnight with 0.125 to 0.5 µg/mL herbimycin A or 2.5 to 10 µg/mL
genistein at 37°C. After washing twice with medium, cells were
cultured with or without 10 µg/mL B1 anti-CD20 MoAb + 50 µg/mL
GAM for 24 hours. Cell nuclei were stained with PI and analyzed by flow
cytometry as described above. Cells treated with inhibitors only were
used as controls. All wells contained comparable concentrations of
dimethyl sulfoxide, which was used as a diluent to dissolve herbimycin
A and genistein. The data are representative of three experiments.
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Correlation of anti-CD20-mediated apoptosis with increases in
intracellular Ca2+ concentrations.
Because anti-sIgM-induced apoptosis of lymphoma cells has been
reported to be dependent on increases in
[Ca2+]i and to be inhibited by calcium
chelators,40 we chose to investigate the role of
intracellular calcium fluxes on anti-CD20-mediated apoptosis. Our
unpublished results as well as reports by others7 indicate
that ligation of CD20 with soluble anti-CD20 MoAbs alone is
insufficient to mediate detectable increases in
[Ca2+]i. However, hypercross-linking CD20
with anti-CD20 MoAbs and a GAM antibody7 does induce
reproducible elevations of [Ca2+]i. These
results suggested that anti-CD20-mediated apoptosis, like
anti-sIgM-mediated apoptosis, may be influenced by fluctuations in
[Ca2+]i. To test this hypothesis, we
incubated Ramos cells in the presence and absence of the extracellular
Ca2+ chelator EGTA and the intracellular Ca2+
chelator Bapta AM in the presence of anti-sIgM or anti-CD20 MoAbs + GAM
(Fig 7). Calcium chelation was found to
inhibit apoptosis of Ramos cells induced by either anti-sIgM or
anti-CD20 MoAbs + GAM, though inhibition of apoptosis was not complete
in either case, suggesting that other signaling events may also be
involved.
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| Fig 7.
Ca2+ chelators inhibit apoptosis induced by
hypercross-linking CD20. 106 Ramos cells/mL were incubated
with 3 mmol/L EGTA or 3 mmol/L EGTA plus 5 µmol/L Bapta AM for 30 minutes at 37°C before incubation with 10 µg/mL antibodies for 24 hours in the presence or absence of GAM cross-linker (50 µg/mL). Cell
nuclei were stained with PI and analyzed by flow cytometry. Hypodiploid
DNA peaks corresponding to apoptotic nuclei were quantified, and
results presented as antibody-mediated apoptosis minus spontaneous
apoptosis. Cells that were not pretreated with either EGTA or Bapta
were used as controls. Data are representative of four experiments.
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DISCUSSION |
CD20 was the first human B-cell differentiation antigen to be
identified by an MoAb after the identification of surface
immunoglobulin. Accumulating evidence indicates that this molecule is
an initiating component of signal transduction pathways that regulate
B-cell proliferation and differentiation and that ligation of CD20 by MoAbs can inhibit B-cell proliferation and
differentiation.6 Our results confirm and extend these
findings by showing clearly and reproducibly that hypercross-linking of
CD20 with anti-CD20 MoAbs plus a secondary GAM antibody not only
inhibits B lymphoma cell growth, but also induces cell death by
apoptosis. More importantly, our data show that similar
hypercross-linking of CD20 can be achieved by incubation of anti-CD20
MoAb-coated lymphoma cells with FcR-expressing accessory cells and that
under these conditions, apoptosis occurs in the absence of GAM. These
findings suggest that anti-CD20-mediated apoptosis may occur in vivo
in lymphoma patients treated with anti-CD20 antibodies and that this
mechanism may be partially responsible for the clinical remissions
observed in these clinical trials.13,15,16,18 Two
preliminary abstracts published by other groups suggests that our
findings regarding apoptosis are reproducible in other laboratories
using other anti-CD20 antibodies.25,27
The intracellular signaling events that culminate in apoptosis are not
fully understood and appear to vary for different stimuli and for
different cell lines.45 Our experiments and those of others7 have shown that cross-linking of CD20 with
anti-CD20 MoAbs induces phosphorylation of tyrosine and
serine/threonine kinases and their substrates including PLC1, but it
is insufficient to mobilize intracellular calcium stores unless
hypercross-linking of anti-CD20 with GAM antibody is performed. Because
our studies also showed that apoptosis can be induced in Ramos cells
incubated with anti-CD20 MoAbs + GAM but not by soluble anti-CD20 MoAbs alone, we hypothesized that calcium fluxes were involved in the intracellular signaling pathways leading to apoptosis. Our results confirm this supposition and indicate that anti-CD20-mediated apoptosis can be effectively inhibited by the Ca2+
chelators EGTA and Bapta AM, which prevent the increases in
[Ca2+]i normally induced by
hypercross-linking CD20 with anti-CD20 MoAbs + GAM. These observations
are also consistent with recent reports that anti-sIgM-mediated
apoptosis in group I MUTU cells40 and CD3/T-cell receptor
complex-mediated apoptosis in immature thymocytes are mediated by
increases in intracellular calcium concentrations.46,47
There are at least two potential mechanisms for the induction of
changes in cytosolic calcium that may mediate the apoptotic effects
observed using anti-CD20 MoAbs. The first explanation derives from
studies by Tedder et al suggesting that the CD20 molecule, with its
four membrane spanning regions, functions as a plasma membrane calcium
channel when multimeric complexes of CD20 are formed by
cross-linking.6,48 It has been convincingly shown that some
anti-CD20 MoAbs (eg, B1) inhibit B-cell progression from the G1 phase
of the cell cycle into the S/G2+M stages following mitogen
stimulation,10,11 and it has been proposed that this inhibition of cell cycle progression derives from the ability of
anti-CD20 MoAbs to sustain conductive calcium channel activity, because
cell cycle progression is dependent on low levels of cytosolic calcium.
The current studies suggest that sustained calcium channel conductance
induced by CD20 hypercross-linking may also lead to apoptosis, at least
under the conditions used in these experiments.
Increased levels of cytosolic calcium may also be derived from the
release of calcium stores from the endoplasmic reticulum through a
protein tyrosine kinase (PTK) dependent signaling pathway initiated by
anti-CD20 MoAb binding, a phenomenon that is accentuated when CD20 is
hypercross-linked with GAM.7 Previous reports (as well as
our unpublished data) have shown that binding of anti-CD20 MoAbs
increases the phosphorylation of CD20-associated serine and tyrosine
kinases and their substrates, including PLC. Differential effects of
phosphorylation of PLC have been reported, which appear to be
dependent on the extent of CD20 cross-linking.7 When CD20
was bound by anti-CD20 MoAbs alone, phosphorylation of PLC occurred, but increases in [Ca2+]i required
further cross-linking of CD20 with a secondary GAM antibody. These
observations imply that the signals derived from binding of anti-CD20
MoAbs alone may differ either qualitatively or quantitatively from
signals emanating from CD20 hyper-crosslinking with anti-CD20 MoAbs + GAM. This differential signaling may explain apparently contradictory
observations with the 1F5 anti-CD20 MoAb, which stimulates cell cycle
progression of normal, resting B cells9 but suppresses
growth of malignant B-cell lymphomas in vitro and in vivo (J.A.
Ledbetter and C. Siegall, unpublished observations, September
1997) and causes apoptosis when further cross-linked by a
secondary antibody.
We used the protein kinases inhibitors, herbimycin A and genistein, in
preliminary experiments to test whether protein tyrosine kinases are
involved in the signaling pathways leading to apoptosis induced by CD20
hypercross-linking. These inhibitors consistently failed to inhibit the
apoptotic effects of anti-CD20 + GAM, and in fact augmented the
process. PTK inhibitors (Herbimycin A and tyrphostin AG135) have also
been reported by others to have no effect on apoptosis of Burkitt's
lymphoma cell lines induced by other stimuli.40 At least
two explanations can be proposed to explain these findings. First, it
is possible that PTKs are not actively involved in the signaling
pathways involved in CD20-mediated apoptosis, explaining the failure of
kinase inhibitors to abrogate the phenomenon. As mentioned above, it is
possible that the CD20 molecule functions as a calcium channel, thereby
autonomously generating the calcium flux that terminates in apoptosis
after anti-CD20 hypercross-linking. Alternatively, it is conceivable that CD20 hypercross-linking activates several different signaling pathways, some of which may be mediated by protein kinases with proapoptotic downstream effects and some of which generate
antiapoptotic signals. The net effect of CD20 ligation may depend on a
complex summation or interplay of a multitude of positive and negative signals.49 Because the inhibitors genistein and herbimycin
A are nonspecific and inhibit different protein kinases to variable degrees,50 it is plausible that these inhibitors abrogated
the messages transduced by the putative antiapoptotic kinases to a greater degree than those from the postulated proapoptotic kinases. The
net result of such preferential inhibition would be an augmentation of
anti-CD20-mediated apoptosis by the nonspecific kinase inhibitors, as
observed in our experiments (Fig 6). A similar hypothesis has been
proposed by others for anti-sIgM-mediated apoptosis in B cells49 and to describe the effects of protein kinase
inhibitors on positive and negative signaling pathways associated with
other surface molecules (eg, CD19, CD2233). Further
experiments with more selective protein kinase inhibitors or antisense
oligonucleotides will be necessary to clarify the roles of protein
kinases in CD20-mediated apoptosis.
Regardless of the mechanism involved, the elevated intracellular
calcium levels induced by CD20 hypercross-linking appear to inhibit
cell cycle progression and trigger apoptosis. Soluble anti-CD20 MoAbs
appear to inhibit proliferation without induction of apoptosis, because
the minuscule cytosolic calcium fluxes induced by soluble anti-CD20
MoAbs alone are insufficient to trigger the latter
phenomenon.7 In contrast, extensive cross-linking of CD20
with anti-CD20 MoAbs plus GAM (or FcR-bearing accessory cells)
appears to surpass the cytosolic calcium threshold required for
induction of apoptosis in a fashion analogous to that shown for
anti-sIgM-mediated apoptosis.40
Several groups have investigated the differential effects of simple
ligation of cell surface molecules by MoAbs (and other ligands) and
contrasted these effects with those observed after extensive
cross-linking of the same surface molecules.33,51-54 Studies with surface Ig have suggested that soluble
antibodies promote B-cell tolerance through clonal anergy,
whereas membrane bound antigens, which confer extensive cross-linking
of ligand-receptor pairs, readily induce B-cell death by
apoptosis.51 Fas-mediated induction of apoptosis is also
dependent on efficient cross-linking of the Fas molecule on the cell
surface.52 Dhein et al reported that F(ab)2
fragments of anti-Fas MoAbs and some isotypes of anti-Fas MoAbs (eg,
IgG2b) were unable to induce apoptosis of target cells, but that the
apoptotic capabilities of these inactive or less active antibody
preparations could be fully reconstituted by hypercross-linking with
protein A, anti-mouse Ig, or anti-mouse Ig
F(ab)2.52 Complementary findings from another
group53 indicate that (1) two different polyclonal
anti-sIgM antibodies administered to severe combined immunodeficiency
(SCID) mice with BCL1 tumors induce dormancy more effectively than two
MoAbs directed against different sIgM epitopes, (2) a mixture of three
anti-sIgM MoAbs mimic the effect of polyclonal antibodies and (3)
hypercross-linking of sIgM with a single MoAb followed by a secondary
anti-Ig antibody increases apoptosis markedly. By analogy with these
studies of sIgM cross-linking, we hypothesize that extensive
cross-linking of CD20 with anti-CD20 MoAbs plus GAM (or
FcR-expressing accessory cells) generates stronger and/or
more sustained signals than simple ligation of CD20, and that the more
extensive cross-linking of CD20 results in increased cytosolic calcium
levels and rapid induction of irreversible cell death by apoptosis.
| |
FOOTNOTES |
Submitted May 7, 1997;
accepted October 15, 1997.
Supported by Grant No. R01 CA55596 from the National Institutes of
Health, Bethesda, MD.
Address reprint requests to Daming Shan, MD, Department of
Biological Structure, University of Washington, PO Box 357420, Seattle,
WA 98195.
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 gratefully acknowledge the secretarial assistance of Patricia Kury
Adam.
| |
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Bcl-2 Antisense (G3139, Genasense) Enhances the in Vitro and in Vivo Response of Epstein-Barr Virus-associated Lymphoproliferative Disease to Rituximab
Clin. Cancer Res.,
May 1, 2003;
9(5):
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[Abstract]
[Full Text]
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S. P. Bohen, O. G. Troyanskaya, O. Alter, R. Warnke, D. Botstein, P. O. Brown, and R. Levy
Variation in gene expression patterns in follicular lymphoma and the response to rituximab
PNAS,
February 18, 2003;
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[Abstract]
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F. Ravandi, M. Talpaz, and Z. Estrov
Modulation of Cellular Signaling Pathways: Prospects for Targeted Therapy in Hematological Malignancies
Clin. Cancer Res.,
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[Abstract]
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J. J. C. Sheu, T. Cheng, H. Y. Chen, C. Lim, and T.-W. Chang
Comparative Effects of Human Ig{alpha} and Ig{beta} in Inducing Autoreactive Antibodies Against B Cells in Mice
J. Immunol.,
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[Abstract]
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O. Manches, G. Lui, L. Chaperot, R. Gressin, J.-P. Molens, M.-C. Jacob, J.-J. Sotto, D. Leroux, J.-C. Bensa, and J. Plumas
In vitro mechanisms of action of rituximab on primary non-Hodgkin lymphomas
Blood,
February 1, 2003;
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[Abstract]
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M. S. Cragg, S. M. Morgan, H. T. C. Chan, B. P. Morgan, A. V. Filatov, P. W. M. Johnson, R. R. French, and M. J. Glennie
Complement-mediated lysis by anti-CD20 mAb correlates with segregation into lipid rafts
Blood,
February 1, 2003;
101(3):
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[Abstract]
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A. D. Kennedy, M. D. Solga, T. A. Schuman, A. W. Chi, M. A. Lindorfer, W. M. Sutherland, P. L. Foley, and R. P. Taylor
An anti-C3b(i) mAb enhances complement activation, C3b(i) deposition, and killing of CD20+ cells by rituximab
Blood,
February 1, 2003;
101(3):
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[Abstract]
[Full Text]
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D. Decaudin, G. Des Guetz, C. Mathiot, J. Dumont, P. Hubert, A. Vincent-Salomon, and P. Pouillart
Absolute lymphocyte count as a predictive factor for response to monoclonal anti-CD20 antibody therapy
Ann. Onc.,
January 1, 2003;
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K Arzoo, S Sadeghi, and H A Liebman
Treatment of refractory antibody mediated autoimmune disorders with an anti-CD20 monoclonal antibody (rituximab)
Ann Rheum Dis,
October 1, 2002;
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[Abstract]
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D. W. Seldin
Techniques for Using Bexxar for the Treatment of Non-Hodgkin's Lymphoma*
J. Nucl. Med. Technol.,
September 1, 2002;
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A. L. Rose, B. E. Smith, and D. G. Maloney
Glucocorticoids and rituximab in vitro: synergistic direct antiproliferative and apoptotic effects
Blood,
August 13, 2002;
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[Abstract]
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T. Igarashi, Y. Kobayashi, M. Ogura, T. Kinoshita, T. Ohtsu, Y. Sasaki, Y. Morishima, T. Murate, M. Kasai, N. Uike, et al.
Factors affecting toxicity, response and progression-free survival in relapsed patients with indolent B-cell lymphoma and mantle cell lymphoma treated with rituximab: a Japanese phase II study
Ann. Onc.,
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M. J. Polyak and J. P. Deans
Alanine-170 and proline-172 are critical determinants for extracellular CD20 epitopes; heterogeneity in the fine specificity of CD20 monoclonal antibodies is defined by additional requirements imposed by both amino acid sequence and quaternary structure
Blood,
May 1, 2002;
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3256 - 3262.
[Abstract]
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I. M. Pedersen, A. M. Buhl, P. Klausen, C. H. Geisler, and J. Jurlander
The chimeric anti-CD20 antibody rituximab induces apoptosis in B-cell chronic lymphocytic leukemia cells through a p38 mitogen activated protein-kinase-dependent mechanism
Blood,
February 15, 2002;
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[Abstract]
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G. Cartron, L. Dacheux, G. Salles, P. Solal-Celigny, P. Bardos, P. Colombat, and H. Watier
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Blood,
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[Abstract]
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J. C. Byrd, S. Kitada, I. W. Flinn, J. L. Aron, M. Pearson, D. Lucas, and J. C. Reed
The mechanism of tumor cell clearance by rituximab in vivo in patients with B-cell chronic lymphocytic leukemia: evidence of caspase activation and apoptosis induction
Blood,
February 1, 2002;
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[Abstract]
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J. Golay, M. Lazzari, V. Facchinetti, S. Bernasconi, G. Borleri, T. Barbui, A. Rambaldi, and M. Introna
CD20 levels determine the in vitro susceptibility to rituximab and complement of B-cell chronic lymphocytic leukemia: further regulation by CD55 and CD59
Blood,
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[Abstract]
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B. Bellosillo, N. Villamor, A. Lopez-Guillermo, S. Marce, J. Esteve, E. Campo, D. Colomer, and E. Montserrat
Complement-mediated cell death induced by rituximab in B-cell lymphoproliferative disorders is mediated in vitro by a caspase-independent mechanism involving the generation of reactive oxygen species
Blood,
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[Abstract]
[Full Text]
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D. Shan, A. K. Gopal, and O. W. Press
Synergistic Effects of the Fenretinide (4-HPR) and Anti-CD20 Monoclonal Antibodies on Apoptosis Induction of Malignant Human B Cells
Clin. Cancer Res.,
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S. Alas and B. Bonavida
Rituximab Inactivates Signal Transducer and Activation of Transcription 3 (STAT3) Activity in B-Non-Hodgkin's Lymphoma through Inhibition of the Interleukin 10 Autocrine/Paracrine Loop and Results in Down-Regulation of Bcl-2 and Sensitization to Cytotoxic Drugs
Cancer Res.,
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G. Jung, L. Grosse-Hovest, P. H. Krammer, and H.-G. Rammensee
Target Cell-restricted Triggering of the CD95 (APO-1/Fas) Death Receptor with Bispecific Antibody Fragments
Cancer Res.,
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[Abstract]
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S. Alas, C. Emmanouilides, and B. Bonavida
Inhibition of Interleukin 10 by Rituximab Results in Down-Regulation of Bcl-2 and Sensitization of B-cell Non-Hodgkin's Lymphoma to Apoptosis
Clin. Cancer Res.,
March 1, 2001;
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[Abstract]
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M.-A. Ghetie, H. Bright, and E. S. Vitetta
Homodimers but not monomers of Rituxan (chimeric anti-CD20) induce apoptosis in human B-lymphoma cells and synergize with a chemotherapeutic agent and an immunotoxin
Blood,
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[Abstract]
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K. S. Kan, V. A. Anderson, W. S. Leong, A. M. Smith, A. T. Worth, and G. T. Stevenson
Thioether-Bonded Constructs of Fab'{{gamma}} and Fc{{gamma}} Modules Utilizing Differential Reduction of Interchain Disulfide Bonds
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B. Jahrsdörfer, G. Hartmann, E. Racila, W. Jackson, L. Mühlenhoff, G. Meinhardt, S. Endres, B. K. Link, A. M. Krieg, and G. J. Weiner
CpG DNA increases primary malignant B cell expression of costimulatory molecules and target antigens
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O. W. Press, J. P. Leonard, B. Coiffier, R. Levy, and J. Timmerman
Immunotherapy of Non-Hodgkin's Lymphomas
Hematology,
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P. Colombat, G. Salles, N. Brousse, P. Eftekhari, P. Soubeyran, V. Delwail, E. Deconinck, C. Haioun, C. Foussard, C. Sebban, et al.
Rituximab (anti-CD20 monoclonal antibody) as single first-line therapy for patients with follicular lymphoma with a low tumor burden: clinical and molecular evaluation
Blood,
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S. Mathas, A. Rickers, K. Bommert, B. Dörken, and M. Y. Mapara
Anti-CD20- and B-cell Receptor-mediated Apoptosis: Evidence for Shared Intracellular Signaling Pathways
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Triggering FC{alpha}-Receptor I (CD89) Recruits Neutrophils as Effector Cells for CD20-Directed Antibody Therapy
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J. Honeychurch, A. L. Tutt, T. Valerius, I. A. F. M. Heijnen, J. G. J. Van de Winkel, and M. J. Glennie
Therapeutic efficacy of Fcgamma RI/CD64-directed bispecific antibodies in B-cell lymphoma
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M. S. Kaminski, J. Estes, K. R. Zasadny, I. R. Francis, C. W. Ross, M. Tuck, D. Regan, S. Fisher, J. Gutierrez, S. Kroll, et al.
Radioimmunotherapy with iodine 131I tositumomab for relapsed or refractory B-cell non-Hodgkin lymphoma: updated results and long-term follow-up of the University of Michigan experience
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J. Golay, L. Zaffaroni, T. Vaccari, M. Lazzari, G.-M. Borleri, S. Bernasconi, F. Tedesco, A. Rambaldi, and M. Introna
Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis
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E. E. Idusogie, L. G. Presta, H. Gazzano-Santoro, K. Totpal, P. Y. Wong, M. Ultsch, Y. G. Meng, and M. G. Mulkerrin
Mapping of the C1q Binding Site on Rituxan, a Chimeric Antibody with a Human IgG1 Fc
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L. Heinzerling, R. Dummer, W. Kempf, M. H. Schmid, and G. Burg
Intralesional Therapy With Anti-CD20 Monoclonal Antibody Rituximab in Primary Cutaneous B-Cell Lymphoma
Arch Dermatol,
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Abstract
Introduction
Abstract