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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 12 (December 15), 1998:
pp. 4750-4757
Immunotherapy of B-Cell Lymphoma With CD3x19 Bispecific Antibodies:
Costimulation via CD28 Prevents "Veto" Apoptosis of
Antibody-Targeted Cytotoxic T Cells
By
Peter T. Daniel,
Arne Kroidl,
Joachim Kopp,
Isrid Sturm,
Gerhard Moldenhauer,
Bernd Dörken, and
Antonio Pezzutto
From the Max Delbrück Center for Molecular Medicine,
Berlin-Buch, Germany; Department of Hematology, Oncology, and Tumor
Immunology, Charité-Campus Berlin-Buch, Humboldt University,
Berlin-Buch, Germany; Tumorimmunology Program, German Cancer Research
Center, Heidelberg, Germany.
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ABSTRACT |
Bispecific antibodies (CD3x19) against the CD3 -chain of the
T-cell-receptor/CD3 complex and the CD19 antigen on B cells can target
polyclonal, nontumor-specific T cells to B lymphoma cells. This induces
T-cell activation, and generation of cytotoxic T cells (CTLs). These
polyclonal CTLs, targeted by the CD3x19 bispecific antibodies, can lyse
CD19+ B-lymphoma cells. In a xenotransplant model in
severe combined immunodeficiency deficient (SCID) mice, we
and others observed that CD28 triggering is required for efficient
elimination of B-lymphoma cells and cure from the tumor in addition to
CD3x19 administration. We also showed that the activation and targeting of CTLs to the target cell by signal one alone, ie, the CD3x19 mab,
induces T-cell death by apoptosis. In blocking experiments we showed
that this "veto" apoptosis is mediated by the CD95/Fas ligand.
Addition of anti-CD28 (signal 2) renders the T cells resistant for veto
apoptosis both in vitro and in vivo. We therefore conclude that the
role of costimulation in immunotherapy with bispecific antibodies or
other T-cell-based immune strategies is not only to facilitate T-cell
activation but also to prevent T-cell deletion by apoptosis.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
BISPECIFIC ANTIBODIES have been generated
to circumvent the problem of antigen recognition in T-cell-based
immunotherapy models. Such antibodies consisting of an
anti-CD3 -part (OKT3) and a tumor-cell recognizing moeity can build
a bridge between a T cell and the tumor cell to induce T-cell
activation by CD3 cross-linking. Previous reports have shown that
bispecific antibodies against the CD3 -chain and the pan-B-cell
antigen CD19 (CD3x19) efficiently induce cytotoxic T cells in a
syngeneic situation that then can kill malignant B
cells.1-3 In vivo studies using a CD3x30 bispecific
antibody in a severe combined immunodeficiency disorder (SCID)-mouse
model for CD30+ Hodgkin's lymphoma showed that efficient
elimination of established tumors was achieved only when a second
bispecific antibody against CD30 and CD28 (CD28x30) was administered
during T-cell targeting.4
It is generally accepted that T-cell activation requires two distinct
signals. The first signal depends on the ligation of the T-cell
receptor (TCR)/CD3 complex and the CD4 or CD8 coreceptors.5 The second signal can be provided by cell surface molecules that mediate essential costimulatory signals, thereby complementing the
TCR/CD3-mediated events.6,7 CD28 is such a potent
costimulatory molecule, and ligation of CD28 with agonistic antibodies
or its natural ligands (B7.1 (CD80) and B7.2 (CD86)) synergizes with TCR-mediated signaling to initiate and maintain T-cell
responses.7,8 Recently, ligation of CD28 by agonistic
antibodies has been shown to prevent activation-induced cell death
(AICD) of naive T cells during primary stimulation.9 This
was related to the upregulation of bcl-xL, a potent
apoptosis-preventing member of the bcl-2 gene family.
We previously described that cytotoxic T cells undergo AICD on
target-cell recognition.10 Thus, the cells hit and kill but then die by AICD. This may have importance in limiting potentially dangerous immune responses, eg, in the control of autoimmunity. We
coined the term "veto" apoptosis to describe this phenomenon in
analogy to the classical veto T-cell suppression.11 Veto apoptosis, like other forms of T-cell AICD,12,13 is
mediated by the Fas ligand.10 Costimulation of the T cells
by B7.1 during target-cell recognition could overcome the veto
apoptosis.10 We therefore asked the question whether
targeting a T cell to a B-lymphoma cell by bispecific antibodies as
well induces veto apoptosis and if cytotoxic T-cell (CTL) apoptosis can
be prevented by costimulation via agonistic antibodies against the CD28
antigen. This is of particular importance in view of the ongoing or
planned clinical trials based on T-cell targeting, because such an
event would preferentially lead to T-cell deletion in contrast to
tumor-cell elimination.
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MATERIALS AND METHODS |
Cell culture and preparation of T cells for in vitro and in vivo
assays.
All cells were maintained in 1640 RPMI (Seromed-Biochrom, Hamburg,
FRG), 10% heat-inactivated fetal calf serum (FCS; GIBCO-BRL, Karlsruhe, FRG), 2 mmol/L L-Glutamine (GIBCO-BRL, Karlsruhe, FRG), and
penicilline-streptomycin (Seromed-Biochrom, Hamburg, FRG). T cells were
prepared from peripheral blood by means of Ficoll density-gradient
centrifugation. B cells were removed by depletion with anti-CD19
magnetic activated cell sorting (MACS) magnetic beads
(Miltenyi Biotec, Bergisch Gladbach, FRG). Adherent cells were removed
by plastic adherence for 2 hours at 37°C. The purity of the T cells
was controlled by staining with PE-labelled anti-CD3 (Becton Dickinson,
Heidelberg, FRG) and analyzed on a FACSort (Becton Dickinson). The
purity of the T cells was over 94%. T cells were activated in 75 cm2 culture flasks (Nunc, Copenhagen, Denmark) by
immobilized OKT3 (flasks coated overnight at 10 µg/mL antibody in
phosphate-buffered saline [PBS]) in the presence of the agonistic
anti-CD28 mab 15E8 (1µg/mL). 30 U IL-2 (Chiron, Ratingen, FRG) were
added after 24 hours to maintain and expand the T cells for a further 5 days. The cytotoxicity of T cells was then determined in a standard 4-hour 51Cr release assay against Raji or Nalm-6 targets.
Bispecific antibodies.
CD3x19 Quadroma cells were generated and cultured as
described14 in a hollow-fiber bioreactor (Tecnomara,
Fernwald, FRG). The derived antibody mixture was first purified by
affinity-chromatography on a Protein-A Sepharose CL-4B column
(Pharmacia, Uppsala, Sweden) to remove immunoglobulin G1 (IgG1)
parental antibodies. Subsequently, the eluent was subjected to HPLC
purification on a Bakerbond ABx column (JT Baker Inc, Philippsburg, NJ)
that allowed a separation of the bispecific antibody fraction by means
of a morpholinoethane sulfonic acid/sodium acetate gradient. Purity of
the eluted material was assessed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing
conditions as described.14
Immunofluorescence.
B7.1 or B7.2-expression on the B-cell lines Raji and Nalm-6 was
determined by direct staining of 106 cells with
phycoerythrin-labeled anti-CD80 or anti-CD86 mab (Becton-Dickinson). Surface fluorescence was determined using a FACSort (Becton-Dickinson). Dead cells were excluded by propidium iodide (PI) staining.
Measurement of apoptosis: Modified cell-cycle analysis.
Activated T cells (106) were exposed to irradiated target B
cells at a 10:1 ratio. After 24 hours the malignant B cells were removed by depletion with anti-CD19 immunomagnetic beads (Dynal, Chantilly, VA). There was a slight reduction of T-cell numbers in the
range of 5% to 10% after the immunomagnetic depletion, but no
significant difference was observed in T-cell recovery as measured by
absolute numbers of CD2+ cells in the cultures induced with
or without the CD3x19 bs ab in the presence or absence of B-lymphoma
cells. The purity of CD2+ cells was above 97% after MACS
separation. These recovered T cells were pelleted, washed with ice-cold
PBS in U-form 96-well plates, and gently resuspended in 300 µL
hypotonic fluorochrome solution (propidium iodide 50 µg/mL, 0.1 mol/L
sodium citrate plus 0.1% Triton X100) as previously
described.15 After overnight incubation at 4°C in the
dark, the propidium-iodide content of the individual nuclei was
measured on a FACSort (Becton-Dickinson). Cell debris was excluded by
raising the forward-scatter threshold adequately. Apoptotic nuclei
displayed a decreased DNA content below the G1 peak, paralleled by an
increase of the side scatter. Anti-CD95 (anti-APO-1 IgG3) or FII23c
F(ab)2 fragments were produced as previously described.16
Anti-B7.1 antibody BB-1 and control-immunoglobulinM(IgM) were purchased
from Pharmingen (San Diego, CA). CD3x19 bs abs were prepared as
described.1
Measurement of apoptosis: Detection of DNA single-strand breaks by
in situ nick translation (ISNT).
To quantify apoptotic cells we used the method as previously described
for detection of DNA single-strand breaks in apoptotic cells.17 Briefly, the cells were fixed with 1%
formaldehyde followed by 70% ethanol. The fixed cells were washed with
nick buffer (50 mmol/L Tris HCl pH 7.8, 5 mmol/L MgCl2, 0.1 mol/L  -mercaptoethanol, 10 µg/mL bovine serum albumin [BSA]) and
incubated with a mixture of 1.3 µL dATP, dCTP, dGTP each 0.2 mmol/L
(Perkin-Elmer-Cetus, Überlingen, FRG), 1.6 µL nick buffer,
0.3 µL 1mmol/L biotinylated-16-dUTP (Boehringer Mannheim, Mannheim,
FRG) and 1 unit E.coli DNA polymerase I (AGS, Heidelberg, FRG) for 90 minutes at 15°C to complete DNA single-strand breaks. The
biotin-labeled cells were stained with avidin-fluorescein
isothiocyanate (FITC) (cell sorter grade; Camon, Wiesbaden, FRG). The
DNA was stained with 1 µg/mL propidium iodide before analysis on a
FACScan (Becton-Dickinson). Percentage of DNA fragmentation corresponds
with percent green fluorescence in the propidium iodide
(PI)+, eg, nuclei-containing population.
Animal experiments.
Animals (C.B-17 scid/scid mice [SCID]) from our own breeding
colony) were kept and treated in accordance with local animal protection laws. The mice were kept in isolators under gnotobiotic conditions. Food and water were autoclaved. The mice were not subjected
to antibiotic drug treatment. No mouse pathogens were detected. For
serology, sterile sentinel mice were added to the colony. These mice
were serologicaly negative for Sendai, PVM, MVM, Reo3, MHV (Corona),
Theiler's GD VII, Polyoma, K-, and m.-Adeno virus. Leakiness was
determined by measuring mouse IgM and mouse IgG and mice expressing
serum titers above 50 µg/ml IgM or IgG were excluded from the study.
Tumor cells were injected subcutaneously in the inguinal region (Raji,
107 per animal) or intraperitoneally (Nalm-6,
107 per animal). Human T cells were prepared and
preactivated in vitro by immobilized OKT3, soluble anti-CD28, and
subsequent culture in low-dose IL-2 as described above. 107
activated T cells were injected together with the Raji cells subcutaneously or, with a delay of 1 day, intraperitoneally in the
Nalm-6 experiments. Antibodies were injected intraperitoneally together
with the T cells at the following doses: CD3x19 bs ab at 200 µg/animal, monospecific anti-CD3 (OKT3) plus anti-CD19 (HD37) at 100 µg/animal, anti-CD28 (15E8) at 50 µg/animal. Serum half-life of the
antibodies was determined as described18,19 and was 7.4 days for the CD3x19 and 8.1 days for the anti-CD28 mab. Peritoneal
washout was performed by rigorously injecting 10 mL ice-cold PBS into
the peritoneal cavity of sacrificed mice. Contaminating murine cells
were removed via MACS with anti-H2D d mab and
strepatavidin-magnetic beads (Miltenyi); Nalm-6 cells were depleted
simultaneously as described above.
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RESULTS |
CD28 costimulation is required for tumor-cell elimination by bispecific
antibodies in vivo.
To determine the in vivo antitumor activity of the CD3x19 bispecific
antibodies, we established xenotransplant models of the Raji Burkitt
lymphoma and Nalm-6 pre-B-ALL in SCID mice. Raji cells, when injected
subcutaneously led to locally growing tumors that, at late stages, also
showed lymphoid dissemination to the inguinal, axillar, thoracic, and
abdominal lymph nodes as described.20 Nalm-6 cells were
injected intraperitoneally and rapidly disseminated to liver, bone
marrow and peripheral blood. The mice rapidly developed hind limb
paralysis due to meningeal infiltration and had to be sacrificed. Human
T cells were preactivated in vitro by immobilized OKT3, soluble
anti-CD28 and low-dose IL-2. These T cells were applied together with
the tumor cells s.c. (Raji) or injected intraperitoneally with a delay
of 1 day (Nalm-6). Control mice received (a) in vitro activated T cells
together with the tumor cells without antibodies or (b) T cells plus
the monospecific anti-CD3 and anti-CD19 antibodies
(Fig 1). These mice showed no increased
survival as compared with mice injected with tumor cells alone.
Coinjection (intraperitoneally) of CD3x19 bs ab together with the T
cells led only to a slightly prolonged survival of mice transplanted
with the Nalm-6 lymphoma cells. Nevertheless, 4 of 10 of the mice
injected with Raji cells were cured by injection of the bispecific
antibody plus T cells. In contrast to this limited survival improvement
with the bs ab CD3x19 alone, the coinjection of anti-CD28 with the
bispecific CD3x19 antibody protected all mice injected with Raji (Fig
1a) and 9 of 10 mice that received Nalm-6 from tumor growth and
dissemination (Fig 1b). The surviving mice were maintained for a
further 6 months and remained disease free during this time. In the
above experiments, the T cells were activated in vitro because SCID
mice transplanted intraperitoneally or intravenously with resting human
peripheral blood lymphocytes showed no antitumor response on
anti-CD3x19 injection. This may be due to the limited recirculation and
survival of human lymphocytes in SCID mice.17 In addition,
there was no significant difference observed in survival (or kinetics
of tumor outgrowth) in control mice injected with tumor cells,
monospecific antibodies, and anti-CD28 as compared with those who
received no anti-CD28 (data not shown). This rules out a nonspecific,
CD3x19-independent enhancement of alloreactivity by the anti-CD28 mab
as cause for the antitumor response mediated by CD3x19 plus anti-CD28
combination therapy.
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| Fig 1.
Antitumor effect of bispecific CD3x19 in xenotransplanted
SCID mice. (a) 107 Raji Burkitt lymphoma were injected
subcutaneously together with 107 activated T cells. CD3x19
and anti-CD28 (clone 15E8) were injected intraperitoneally at the same
time (b) 107 Nalm-6 cells were injected intraperitoneally.
Activated T cells (107), CD3x19, and anti-CD28 were
injected intraperitoneally 24-hours later.  , tumor cells alone;  ,
tumor cells plus monospecific control antibodies (anti-CD19 [HD37]
plus anti-CD3 [OKT3], 100 µg each as single dose);  , tumor cells
plus CD3x19 (200 µg single dose);  , tumor cells plus CD3x19 (200 µg single dose) plus anti-CD28 (clone 15E8, 50 µg single dose).
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CD3x19 bispecific antibodies induce apoptosis in the
antibody-targeted cytotoxic T cells.
Because we observed in previous experiments that CTLs can undergo
apoptosis on target-cell contact,10 we tested if this was
also the case in T-cell targeting with CD3x19 bs ab. T cells were
activated polyclonally with plastic-immobilized OKT3 mab plus anti-CD28
mab and maintained in IL-2-containing medium for 5 days. This
stimulation induced optimal T-cell proliferation as measured by
3H-thymidine uptake. The addition of anti-CD28 to the
CD3-cross-linking also yields an improved cytotoxicity of the activated
T cells.1 The activated T cells were then exposed to
allogeneic Raji target cells and cytotoxicity against Raji was measured
in a standard 51Cr-release assay. Bispecific CD3x19 ab was
added in solution to the effector/target cell mixture and triggered
specific lysis of the Raji Burkitt lymphoma target cells at
concentrations above 0.05 µg/mL (data not shown). Addition of
anti-CD28 ab to the CD3x19-targeted T-cell/Raji coculture did improve
killing of the Raji target cells. Similar data were obtained with
Nalm-6 as target cell (data not shown). To test if the T cells
themselves underwent activation-triggered apoptosis in this system, we
assessed T-cell apoptosis by measuring nuclear DNA content of the T
cells. A large percentage of the T cells was induced to apoptosis by
the T-cell targeting during coculture with the target cells
(Fig 2). The addition of CD3x19 bispecific
ab to the target cells induced apoptosis in 65% of the T cells when
Nalm-6 was employed as target (Fig 2a) and 30% of the T cells in the
case of Raji target cells (Fig 2b). The addition of anti-CD28 reduced
the amount of T-cell apoptosis in both systems by about 50%.
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| Fig 2.
Effect of anti-CD28 on CTL apoptosis during T-cell
targeting. T cells were generated by activation of peripheral blood T
cells by CD3 cross-linking and culture for 7 days in IL-2-supplemented
medium (30 U/ml). T cells were then cultured for 24 hours at an E/T
ratio of 10:1 in the absence or presence of CD3x19 bs ab (10 µg/mL).
Control cultures were performed with medium alone (ie, only T cells
plus [a] Nalm-6 or [b] Raji), monospecific control antibodies plus
tumor cells (OKT3 and HD37 at 5 µg/mL), anti-CD28 (15E8, 1 µg/mL),
IgM-control mab (G155-228, 10 µg/ml), or anti-B7.1 (BB-1, 10 µg/ml). After incubation, the remaining B lymphoma cells were removed
from the coculture by immunomagnetic depletion with anti-CD19 and
anti-CD20 as described.10 T-cell apoptosis was measured on
the single cell level as described.15
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Nalm-6 is essentially void of B7.1 (CD80) and B7.2 (CD86) expression
(Fig 3). It was therefore tempting to
speculate that the lower rate of T-cell AICD observed during targeting
to Raji cells as compared with Nalm-6 could be attributed to the
B7-expression of the Raji cells. Differences in CD3 crosslinking were
unlikely to be involved in this effect because both lines express
comparable amounts of CD19 and yield comparable amounts of
51Cr release when tested in CD3x19-mediated T-cell
targeting. Thus, we added a blocking anti-B7.1 (BB1) mab to the
T-cell/target-cell coculture system. The anti-B7.1 mab and an
isotype-matched IgM control mab had no intrinsic effect on T-cell
death. Whereas blocking of B7.1 had no effect on the apoptosis of T
cells targeted to (B7 ) Nalm-6 cells, CD3x19-induced
T-cell AICD in the presence of Raji cells was nevertheless increased
suggesting that endogenous B7.1 can prevent target-induced T-cell AICD
(Fig 2b) . None of the antibodies used had a stimulatory or inhibitory
influence on T-cell apoptosis in the absence of B-lymphoma target cells (data not shown).
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| Fig 3.
B7 expression on Raji and Nalm-6. Raji and Nalm-6 were
stained for B7.1 (CD80) and B7.2 (CD86) expression by the use of
phycoerythrin-labeled antibodies. Surface fluorescence was analyzed on
FACSort cytometer. Dead cells were excluded by staining with propidium
iodide.
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Veto apoptosis is mediated by the Fas ligand.
The CD95/Fas ligand (FasL) has been implicated in T-cell
AICD.12 We previously observed that AICD of CTLs, which we
termed veto apoptosis, is mediated in part by autocrine production of the FasL by the T cells on target-cell contact.13 The
addition of F(ab)2 fragments of anti-CD95 (anti-APO-1 IgG3) ab could
inhibit T-cell AICD by preventing binding of the FasL to the CD95
antigen.12 We therefore investigated if the target-induced
veto apoptosis is, like other forms of AICD, as well mediated by the
FasL. We used Nalm-6 as target cells, exposed them to activated T cells and added control F(ab)2 or anti-CD95 F(ab)2 to the CD3x19-triggered cultures. Although 61% of the T cells were induced to apoptotic death
by the CD3x19 bs ab in the presence of Nalm-6 targets, the addition of
anti-CD95 F(ab)2 fragments decreased T-cell AICD to 32%. No reduction
was observed with the control F(ab)2 (Fig
4).
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| Fig 4.
Fas ligand expression and function. Peripheral T cells
were activated by CD3-crosslinking and were maintained for 7 days in
IL-2-containing medium (30 U/ml). T cells were then cultured for 24 hours at an E/T ratio of 10:1 in the absence or presence of CD3x19 bs
ab (10 µg/mL). Control cultures were performed with medium alone (ie,
only T cells plus Nalm-6), monospecific control antibodies (OKT3 and
HD37 at 5 µg/mL), or control F(ab) fragments (FII23c IgG3, 5 µg/mL19). Anti-CD95 F(ab)2 fragments (anti-APO-1
IgG3, 5 µg/mL) were used to block interaction between CD95/Fas and
Fas ligand. After incubation, the remaining B lymphoma cells were
removed from the coculture by immunomagnetic depletion with anti-CD19
and anti-CD20 as described.10 T-cell apoptosis was measured
on the single-cell level as described.15
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CD28 costimulation prevents activation-induced cell death in vivo.
The previous experiments showed that T-cell activation by CD3x19 bs ab
alone leads to AICD and that CD28-mediated costimulation protects from
T-cell AICD in vitro during target-cell contact. In addition, we knew
that costimulation through CD28 drastically improved the antitumor
effect of the CD3x19 bispecific ab in the SCID xenotransplant models.
We therefore assessed whether such a protection from AICD also occurs
in vivo. Because the Nalm-6 model completely depends on anti-CD28 for
tumor prevention we selected it for the in vivo apoptosis assays.
Groups of three SCID mice were injected intraperitoneally with Nalm-6
cells alone or in combination with T cells, CD3x19 bs ab, with or
without anti-CD28. After 16-hour incubation in vivo, the T cells were recovered by peritoneal washout and T-cell apoptosis was determined, after removal of the contaminating murine cells and Nalm-6 cells, by
ISNT staining for DNA strand breaks. The average of background apoptosis of the T cells in the absence of Nalm-6 or antibodies was
9%. When the T cells were injected together with Nalm-6 and the
monospecific control antibodies, the apoptosis rate of the T cells was
8%. In the presence of the CD3x19 antibody and Nalm-6, 30.7% of the T
cells showed apoptotic DNA strand breaks that could be decreased to
14.4% (P < .01) in the presence of anti-CD28 mab (Table 1). The half life of the antibodies
was 7.1 days for the CD3x19 bs ab and 8.2 days for 1.5 E8 anti CD28 mab
(data not shown).
Although these data are consistent with the overall hypothesis that
CD28 triggering prevents T-cell apoptosis during T-cell targeting, the
effect also could have been the consequence of the upregulation of
adhesion molecules on T-cell activation, which may impact on the
ability to retrieve these cells. To exclude such an effect we counted
the absolute numbers of cells recovered from the peritoneal cavity and
stained with anti-CD2 mab to identify the human T cells. Absolute
numbers of T cells recovered from the peritoneal cavity 16 hours after
injection are shown in Table 2. The T-cell
recovery ranged from 29% to 38% and there was no significant
difference between animals injected with T cells alone, T cells plus
tumor cells plus monospecific antibodies, or the bs ab in the presence
or absence of CD28.
These data clearly show that the recovery rate of T-cell numbers,
despite low, is not influenced by the activation procedure during the
first 16 hours. Thus, this in vivo experiment excludes an effect of the
activation procedure on the T-cell recovery and therefore strongly
supports, together with the data shown in Table 1, our hypothesis that
induction of T-cell apoptosis as well occurs in vivo after T-cell
targeting by bispecific antibodies.
To exclude that the induction of apoptosis occurs only at high
concentrations of bs ab, we performed titration experiments in which
groups of three SCID mice were injected intraperitoneally with
irradiated Nalm-6 B lymphoma cells and activated T cells (Fig 5). Control animals were injected with
the monospecific control antibodies (anti-CD19 [HD37] and anti-CD3
[OKT3]) that were titrated from 100 µg down to 12.5 µg each. The
other groups were treated with CD3x19 bs ab (200 µg, titrated down to
25 µg), or CD3x19 (200 µg, titrated down to 25 µg) plus anti-CD28
(50 µg/mL). An additional group of animals received anti-CD28 mab
alone, ie, without anti-CDx19 or the CD3 and CD19 monospecific control
mabs. In this group the anti-CD28 mab was titrated from 200 µg per
animal down to 25 µg per animal. After 16 hours, the cells were
washed out of the peritoneal cavity, murine peritoneal cells and B
cells were depleted as described above and cells were stained for DNA strand breaks by means of ISNT. Apoptosis of the CD3x19-targeted T
cells was detected down to a CD3x19 concentration of 25 µg per mouse.
The anti-CD28 mab that was added at a fixed amount of 50 µg per mouse
could suppress the induction of T cell apoptosis at all CD3x19
concentrations. No changes in T-cell background apoptosis were observed
in the group treated with the monospecific CD3 and CD19. CD28 mab alone
did neither enhance nor decrease T-cell apoptosis when applied in the
absence of CD3x19 bs ab.
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| Fig 5.
Titration of CD3x19 bs ab and apoptosis induction in
vivo. In analogy to the experiment shown in Table 1, induction of
T-cell apoptosis was measured on the single-cell level by ISNT after
CD3x19 bs ab targeting in vivo. Groups of three SCID mice were injected
intraperitoneally with irradiated Nalm-6 (107) and
activated T cells (5 × 107 ). Control groups
were mock-injected with PBS (not shown, values were in the range of the
anti-CD3 + anti-CD19 group), anti-CD28 (white circles; mab titrated
from 200 down to 25 µg per mouse), or monospecific control antibodies
(white squares, anti-CD19 [HD37] plus anti-CD3 [OKT3], titrated
from 100 µg down to 12.5 µg each). The other groups were treated
with CD3x19 bs ab (black squares; titrated from 200 µg down to 25 µg), or CD3x19 plus anti-CD28 (black circles; 200 µg CD3x19
titrated down to 25 µg plus anti-CD28 at a fixed amount of 50 µg/animal). After 16 hours, the cells were washed out of the
peritoneal cavity, B cells were depleted as described above, and cells
were stained for DNA strand-breaks using ISNT. Percentages of
strand-break-positive cells as a marker for apoptosis were measured on
the single level using a FACSort flow cytometer.
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DISCUSSION |
T-cell activation is initiated by signals transmitted through the
T-cell receptor/signal transduction complex (signal 1). Other
costimulatory receptor-ligand interactions between T-cells and
antigen-presenting cells (APC) are needed, however, for complete activation (signal 2). Signaling by APC that provide the antigen signal
in combination with costimulation, leads to activation, proliferation,
and differentiation. Signaling through the T-cell receptor alone can
lead to anergy5 or AICD.10 Previous in vivo
studies showed that the efficacy of T cells against tumor cells in
immunotherapy approaches can be improved by delivering costimulatory
signals. Costimulation by CD28-mediated signals affects a wide variety
of T-cell activation parameters (reviewed in7,8) and can
protect T cells from AICD.9,10 In bispecific antibody-mediated T-cell targeting, immunodeficient mice were cured
from established human tumors when the mice were treated with both the
CD3xCD30 bs ab and received a costimulatory signal via a second
CD28xCD30 bs ab.21 New strategies for costimulation of T
cells to generate anticancer immunity focus on the generation of B7
constructs that may be delivered to the tumor-cell targets, eg, via
(retro-) viral vectors,10 or by injection of chimeric fusion proteins in which the B7.1 or B7.2 is linked by genetical engineering to molecules such as single-chain antibodies22
which then mediate targeting of the costimulatory construct. In this line, the costimulation via agonistic CD28 antibodies was shown to
overcome anergy induced by signal 1 alone and to allow the activation
of primed or preactivated T cells but not necessarily of the naive T
cells in a syngeneic murine model.23 Such a requirement for
costimulation was shown as well in other studies, in which costimulation via B7/CD28 facilitated the generation of CTLs during CD3-redirected cytotoxicity assays in vitro not only from the CD45RO+ memory population but also from small resting
(CD45RO-) T cells.24
These observations prompted us to investigate the question of whether T
cells targeted to tumor cells by bispecific antibodies, ie, the signal
1 alone, undergo apoptosis on target-cell contact and cross-linking of
the CD3 complex.
We found that a substantial proportion of the activated and
antibody-targeted CTLs dies by apoptosis on target-cell contact. Blocking experiments showed that this is mediated by production of the
FasL on targeting of the T cells to the target cells by the CD3x19 bs
ab. In previous reports, induction of the CD95/Fas ligand (FasL) has
been shown to mediate activation-induced death of T
cells.12 CD95/Fas is a surface receptor that is induced in
lymphoid cells by activation.17,25 In recent studies,
expression of the FasL has been reported at immunologically privileged
sites and on tumor cell lines.26-29 We therefore
investigated whether the target-cell and bispecific antibody-induced
T-cell AICD in our systems could be attributed to the production of
FasL. We previously showed, however, that the expression of FasL on a
tumor cell line is not required to trigger CTLs to die by veto
apoptosis. The T cells themselves produced the FasL on target-cell
contact and this is sufficient to mediate T-cell death.10
We also observed that normal, nonmalignant human B cells and a panel of
B-cell lines do not express the FasL, no matter if resting or
activated.13 Thus, we provided evidence that the FasL is
derived from the T cells and not from the B lymphoma cells. Given the
potential importance of this novel regulatory concept, we would suggest
using the term veto apoptosis for T-cell AICD induced on target-cell
contact.10 The veto apoptosis can be mediated by expression
of the FasL or other molecules on the target cell26-30 or,
as we describe here, by autocrine production of the FasL in the absence
of the costimulatory signal 2. In analogy to the well-established veto
phenomenon,11,31,32 the T-cell activation by signal 1 alone
would lead to activation and induce sensitivity for AICD on target-cell
contact. Apart from the case of T-cell targeting by bispecific
antibodies, such an apoptosis-inducing signal can theoretically be
delivered by each (MHC+) cell of the body that is capable
of antigen presentation.11,31,32 Veto apoptosis appears to
be mediated by additional mechanisms other than CD95/Fas because
blocking with anti-CD95 F(ab)2 fragments did not completely prevent
apoptosis in our systems. Possible additional mechanisms may involve
TNF-receptors or other death receptors such as DR3, or
DR4.33
Our data show that such a mechanism acts also in vivo because CD3x19 bs
ab induced T-cell AICD in xenotransplanted SCID mice. In parallel to
our in vitro data, the administration of anti-CD28 decreased T-cell
AICD in vivo. Nevertheless, it was a possibility that such an induction
of apoptosis on CD3x19-mediated T-cell targeting occurs in vivo only at
high bs ab concentrations. We therefore performed a titration
experiment in which we titrated the bs ab amount from 200 down to 25 µg CD3x19 per mouse. The rate of T-cell apoptosis induced by CD3x19
in the presence of Nalm-6 target cells is declining below 50 µg
CD3x19 bs ab per mouse, but is still detectable at 25 µg bs ab. The
additional application of anti-CD28 mab decreases the rate of T-cell
apoptosis, in analogy to the in vitro experiments, by approximately
50% whereas the injection of anti-CD28 alone at different doses did
not influence background T-cell apoptosis.
Such a protection from T-cell AICD through costimulation is, however,
short-lived.13 Protection from AICD lasts at least 3-days
after signal 1 (CD3/TCR crosslinking) and signal 2 (B7.1 recognition),
whereas B7.1-mediated costimulation only marginally protected from AICD
when the antigenic signal was delivered 7-days after the primary
activation and costimulation.10
We believe that our observation is of potential importance for many
immune-based therapeutic strategies. T-cell activation by antigen alone
could preferentially lead to activation and priming for subsequent
T-cell deletion (or anergy induction). Hence, the efficiency of
costimulatory signals in experimental tumor therapy might be explained
not only by a more efficient T-cell activation but also by the
prevention of in vivo T-cell AICD. Interestingly, the endogenous
expression of costimulatory B7 by Raji Burkitt lymphoma cells may be
responsible for the higher efficiency of the CD3x19 bs ab to prevent
tumor growth in the SCID mouse Raji xenotransplant model as compared
with Nalm-6 cells. A blocking experiment showed that the endogenous B7
expression of Raji contributes to the lower T-cell AICD encountered
during T-cell AICD in CD3x19 targeting to Raji. This is supported by
the less pronounced T-cell apoptosis when T cells were targeted to Raji
cells instead of Nalm-6 cells. Our experiments on retrovirally
modified, B7.1-expressing MCF-7 breast cancer cells showed that B7.1
expression on the target cell protected alloreactive CTLs from veto
apoptosis as compared with mock-transfected MCF-7.10
Nevertheless, a significant number of T cells underwent apoptotic cell
death on coculture with the Raji cells in the presence of CD3x19 bs ab.
This might indicate that the B7 expressed by these malignant cells is
not fully functional.
Altogether, our data strongly suggest that costimulation is essential
for T-cell- based immunotherapy such as in the case of T-cell
targeting by bispecific antibodies. Our results help to explain the
requirement of CD28 costimulation for bispecific antibody in tumor
therapy in animal models (present study and4) and lack of
efficiency in CD3x19 alone in clinical studies.34 The
costimulation appears to be required not only for T-cell activation but
also to prevent deletion of the activated T cells by rendering them
apoptosis-resistant. This extends the concept of immune therapy to the
problem of T-cell depletion by the therapeutic stimulus itself and
should motivate to further investigate not only how to activate T cells
but also how to save them from deletion by AICD.
| |
ACKNOWLEDGEMENT |
We thank R.A.W. van Lier, CLB, Amsterdam, The Netherlands and G. Moldenhauer, German Cancer Research Center, Heidelberg, FRG for the
most generous gift of purified 15E8 anti-CD28 antibody and P.H. Krammer
for kindly providing the anti-APO-1 IgG3 antibody. We are grateful to
C.H. Köhne, University of Rostock, FRG, for helpful discussions.
This work was supported by the European Union in the Biomed2 and the
TMR (training and mobility of researchers) program.
| |
FOOTNOTES |
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address correspondence to Peter Daniel, MD, Department of Hematology,
Oncology, and Tumor Immunology, Robert Rössle Klinik,
Charité-Campus Berlin-Buch, Humboldt-University, Lindenberger Weg
80, 13125 Berlin-Buch, Germany; e-mail: pdaniel{at}mdc-berlin.de.
| |
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Abstract
Introduction