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Department of Cancer Therapeutics, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan;
Clinical Immunology, Department of Internal Medicine, University Hospital, Zürich, Switzerland;
Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan;
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Department of Surgery, Institute of Medical Science, University of Tokyo, Tokyo, Japan;
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Department of Allergology, Institute of Medical Science, University of Tokyo, Tokyo, Japan; and
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Laboratory Animal Research Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neuroblastoma is the most common childhood malignant solid tumor arising from the sympathetic nervous system and characterized by a diversity of clinical behavior, ranging from spontaneous remission to rapid tumor progression (32). Thus, murine neuroblastoma Neuro-2a was selected as a model of human neuroblastoma and studied for the development of effective immunotherapy.
We have studied the cytotoxic potential of soluble recombinant FasL in Fas+ lymphoma cells (Yac-1) implanted i.p. into mice (33). Culture supernatant of neuroblastoma Neuro-2a cells transfected with murine FasL cDNA (Neuro-2a+FasL) contained FasL and transduced a potent apoptotic signal to Fas+Yac-1 cells. The soluble FasL was a full-length molecule (40 kDa) and not a proteolytically cleaved form. However, the effect of FasL on Yac-1 cells was assessed within 1 day after FasL injection. In the current study, we demonstrate that tumor cells expressing FasL, Neuro-2a+FasL, induced potent antitumor immunity associated with interference of tumor growth.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A/J (H-2a, syngeneic to Neuro-2a tumor cells), C3H/HeJ (C3H) (H-2k), C3H/HeJ-lpr/lpr (C3H-lpr) and BALB/c nu/nu (H-2d) mice were purchased from SLC (Hamamatsu, Japan). Double mutant mice, C3H-gld/gld lpr/lpr (C3H-gld/lpr), were constructed from C3H-gld and C3H-lpr and maintained in Laboratory Animal Research Center, Institute of Medical Science, University of Tokyo (34).
Tumors
FasL cDNA was obtained from C57BL mice. Neuro-2a (35), mock (neomycin-resistant cDNA)-transfected Neuro-2a (Neuro-2a+Neo), and Neuro-2a+FasL cells (33) were maintained in DMEM (Iwaki, Tokyo) containing 10% heat-inactivated FBS, 4.5 g/L glucose, 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Rockville, MD) at 37°C under 5% CO2. A Neuro-2a tumor mass was aseptically removed 3 wk after i.d. injection into A/J mice and gently dissociated in PBS with a cell strainer (Falcon, Becton Dickinson, Mountain View, CA). After washing with PBS, tumor cells were cultured and used for the in vivo implantation. F6b (Neo+Fas+) and N1d (Neo+) cells were produced from murine hepatoma MH134 cells (Fas-) (34). These three tumor cell lines were passaged by culture in RPMI 1640 medium containing 10% FBS, 2-ME (5 x 10-5 M), and kanamycin. S713a sarcoma cells were maintained in RPMI 1640 medium containing 5% FBS, penicillin, and streptomycin.
Cytotoxicity test
Cell-free culture supernatants were collected after about 6 x 106 Neuro-2a+FasL and Neuro-2a+Neo cells were incubated in 20 ml of DMEM containing 1% FBS for 2–3 days. F6b or N1d cells suspended in 100 µl of culture medium were incubated with each of the supernatants for 18–20 h in a 96-well microtiter plate. Anti-FasL mAb (clone K10, mouse IgG2b) (36) was harvested as ascites from SCID mice and purified with ammonium sulfate precipitation. For anti-FasL mAb treatment, the supernatant was preincubated with anti-FasL mAb in the well for 30 min at room temperature followed by the addition of tumor cells. After 1-day culture, the cells were incubated with 20 µl of MTT (5 mg/ml) for 2–3 h, and then formazan was produced, which was solubilized with 50 µl of the lysing buffer composed of 50% N,N-dimethylforamide and 20% SDS (34). The OD of each well was measured at 570 nm with an automatic microplate reader. Cell viability was calculated as follows: cell viability = [(OD of supernatant + tumor cells)/(OD of tumor cells)] x 100 (%).
In vivo antitumor activity
Neuro-2a cells were incubated with mitomycin C (MMC) (60
µg/ml) at 37°C for 40 min to deteriorate in vivo transplantability.
In an immunoprophylactic experiment, A/J mice were injected i.d. with
viable Neuro-2a+FasL, MMC-treated Neuro-2a+FasL, or MMC-treated
Neuro-2a (106) cells on day -14 and challenged i.d. with
Neuro-2a or S713a cells (5 x 105) on day 0.
Neuro-2a+FasL and Neuro-2a cells were injected at different sites;
either rostral and caudal sites about 2 cm distant from each other in
the right flank or right and left flank, respectively. For
investigation of the influence of FasL-expressing cells on tumor
formation, Neuro-2a+FasL (5 x 105) were mixed with
Neuro-2a (5 x 105) or S713a (5 x
105) tumor cells and injected i.d. into A/J mice. In a
therapeutic experiment, A/J mice injected i.d. with 5 x
105 Neuro-2a or S713a cells on day 0 were treated by
injecting viable Neuro-2a+FasL or MMC-treated Neuro-2a
(106) cells on days 1 and 3. Antitumor activity was
assessed by both tumor size and tumor incidence (tumor-bearing
mice/total mice). The tumor size was expressed as the square root of
tumor area defined as the product of two perpendicular diameters
[(a x b)
]. Mice were observed for tumor
appearance and growth for >60 days after inoculation of tumor cells.
In vivo inactivation of CD4+ and CD8+ T cells with anti-CD4 and anti-CD8 mAbs
Anti-CD4 (GK1.5) and anti-CD8 (2.43) mAbs were harvested as ascites from nude mice. Anti-CD4 mAb was used after purification with ammonium sulfate precipitation. Mice were immunized by i.d. injection of 106 Neuro-2a+FasL cells on day -14, i.p. administrated with 0.5 mg of purified anti-CD4 mAb on days -3 and -1 to deplete CD4+ T cells or 0.2 ml of ascitic anti-CD8 mAb on days -3 and 0 to deplete CD8+ T cells, and i.d. inoculated with 5 x 105 Neuro-2a cells on day 0. CD4+ T cells were reduced from 18% to 0.4% and CD8+ T cells from 10.5% to 0.1% in the spleen of these mice.
Flow cytometry of cells infiltrated with Neuro-2a+FasL
Mice were i.d. injected with 106 Neuro-2a+FasL
cells, a small but palpable tissue mass which formed at the injected
site was excised 24 h later, and single-cell suspensions were
prepared by teasing tissue masses with a cell strainer (Falcon) in PBS
containing 0.6 mM EDTA. After washing with PBS, cells were incubated
with anti-CD16/CD32 (Fc
RIII/II) (PharMingen, San Diego, CA) to
block FcR-mediated binding of Ab and then stained with
FITC-anti-Gr-1 (Ly-6G) (rat IgG2b) (Caltag, South San Francisco,
CA) (37), FITC-anti-Mac-1 (CD11b) (rat IgG2b) (Caltag) (38),
FITC-anti-CD8 (Becton Dickinson), and/or biotin-anti-Fas Jo2
(hamster IgG) mAbs (PharMingen) and PE-strepavidin (Life Technologies),
PE-anti-CD4 mAb (Becton Dickinson), and analyzed with a FACSCalibur
(Becton Dickinson). Biotin-hamster IgG and FITC-rat IgG2b (Caltag) were
used as isotype control.
Histology
A/J, C3H, C3H-lpr, and C3H-gld/lpr mice were i.d. injected with 106 Neuro-2a+FasL, Neuro-2a+Neo, or Neuro-2a cells at the age of 6–8 wk. Some A/J mice were injected i.p. with 125 µg of anti-FasL (K10) 8 h and 30 min before the transplantation of Neuro-2a+FasL cells. The skin at the injected site was examined for the presence or absence of swelling and then cut out 24 h and 3 and 5 days later. The skin samples were fixed in 10% formalin in PBS, processed routinely, embedded in paraffin, sectioned at 3 µm, and stained with hematoxylin and eosin for microscopy.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Culture supernatants from Neuro-2a+FasL and Neuro-2a+Neo cells
were incubated with the hepatoma cells Fas+ F6b and
Fas- N1d cells to examine the induction of apoptosis by
FasL (Fig. 1
A). Supernatant
from Neuro-2a+FasL but not Neuro-2a+Neo cells induced the cytotoxicity
against F6b cells. The cytotoxic activity of supernatant from
Neuro-2a+FasL cells against F6b cells was abrogated by treatment with
anti-FasL mAb but not with control mouse IgG2b (Fig. 1B), indicating that the effector molecule in the
supernatant was FasL. In the flow cytometry, Neuro-2a+FasL cells were
stained with anti-FasL K10 but not with control mouse IgG2b (Fig. 2). These results evidenced that
Neuro-2a+FasL cells secreted soluble FasL into culture medium and
expressed FasL on the surface. Fas and FasL were not detected in
Neuro-2a cells. Neuro-2a+FasL cells did not express Fas and were
resistant to FasL-mediated killing (data not shown).
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To investigate the functional consequences of FasL expressed by
Neuro-2a cells, syngeneic A/J mice were injected with 106
Neuro-2a+FasL cells (Fig. 3). All of the
19 injected mice rejected Neuro-2a+FasL tumor cells after formation of
a small but clearly palpable tissue mass on days 1–4 (Table I). When 106 Neuro-2a+FasL
cells were injected into A/J (n = 19) and C3H
(n = 10) mice, a clearly swelled mass as huge as 6
x 6 to 8 x 8 mm in size appeared at the injected site 24 h
later. This mass formation was inhibited by pretreatment with injection
of anti-FasL mAb K10 (< 1 x 1 mm) in A/J mice
(n = 7) and occurred in neither C3H-lpr (<
1 x 1 mm) (n = 3) nor
C3H-gld/lpr (< 1 x 1 mm)
(n = 6) mice, which carry a functional defect in Fas
alone and both FasL and Fas (34), respectively, indicating that the
Fas-FasL system is directly involved in this event. In contrast, tumor
growth occurred without such instant mass formation accompanied in all
the mice injected with 5 x 105 Neuro-2a+Neo or parent
Neuro-2a cells (Fig. 3 and Table I).
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As mentioned above, a palpable swelling developed immediately at
the site where Neuro-2a+FasL cells were injected. Thus, 106
tumor cells were injected and the injected sites were examined
histologically. At 24 h later, abundant leukocytes, mainly
neutrophils, infiltrated into and around tumor cells in A/J and C3H
mice that were injected with Neuro-2a+FasL cells and underwent swelling
(Fig. 4A). Some neutrophils
appeared to be in contact with tumor cells. On day 5 of Neuro-2a+FasL
cell injection, dead tumor cells and their debris were surrounded by
granulomatous tissues in which many neutrophils were scattered (data
not shown). In contrast, such swelling did not occur and only a few
neutrophils and monocytes were found around tumor cells in A/J mice
injected with Neuro-2a+Neo (Fig. 4B) or Neuro-2a cells (data
not shown). More importantly, Neuro-2a+FasL cells induced neither
swelling nor leukocyte infiltration in C3H-lpr (data not
shown) and C3H-gld/lpr (Fig. 4C) mice.
Thus, Fas played a pivotal role in induction of an inflammatory
response by FasL-transfected tumor cells. Similar massive accumulation
of inflammatory cells consisting of neutrophils has been reported
for other FasL-transfected tumor cells (30, 31).
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To further characterize the cells responsible for the swelling,
free cells were isolated from a tissue mass formed 24 h after
injection of 106 Neuro-2a+FasL cells and analyzed by flow
cytometry. The isolated cells were smaller than Neuro-2a+FasL cells in
size (Fig. 5). The forward and side
scatter histogram of the infiltrated cells consisted of a high peak of
the smallest cells such as erythrocytes and cell debris (forward
scatter (FSC) = 22, side scatter (SSC) = 21) and a low peak of
lymphoid-like cells (FSC = 73, SSC = 41). The histogram of
Neuro-2a+FasL cells showed a major peak of tumor cells (FSC = 105,
SSC = 85), a minor peak of cell debris (FSC = 19, SSC =
20), and a bottom (FSC = 60, SSC = 25). Thus, flow cytometry
was performed by gating the low peak to exclude the smallest cells and
tumor cells. As shown in Fig. 6,
A and B, almost all of these cells (>90%) were
stained with anti-Gr-1 (Ly-6G) mAb specific for granulocytes (37)
and anti-Mac-1 (CD11b) mAb specific for granulocytes, macrophages,
and NK cells (38), indicating that the infiltrating cells were
granulocytes consistent with the histological findings (Fig. 4
).
Importantly, about 20% of the cells were positive for anti-Fas Ab,
albeit the possibility that Fas on the cells might be occupied
with FasL. The cells did not stain with either anti-CD4 or
anti-CD8 mAbs (Fig. 6D).
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The histological and flow cytometric results revealed that
Neuro-2a+FasL cells can attract neutrophils around them. To determine
whether or not these cells can affect tumor formation, a mixture of
5 x 105 Neuro-2a+FasL and 5 x 105
Neuro-2a cells was inoculated into A/J mice (Fig. 7). Tumor formation was completely
suppressed in five (83%) and delayed in one (17%) of six mice. To
test the tumor specificity, a mixture of 5 x 105
Neuro-2a+FasL and 5 x 105 S713a (Fas-negative) cells
were i.d. inoculated into mice (Fig. 8).
The growth of S713a cells was not suppressed by Neuro-2a+FasL cells.
These results indicated that the Neuro-2a+FasL cell-induced
inflammatory cells can reject the parent Neuro-2a tumor but not S713a
sarcoma of different origin. It was thus suggested that FasL-producing
cells might eradicate tumorigenic cells by attracting tumoricidal cells
in a tumor Ag-specific manner.
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To determine whether or not T cells are required for eradication
of Neuro-2a+FasL cells, the tumor cells were implanted into nude mice.
All nude mice suppressed the growth of Neuro-2a+FasL cells but not of
Neuro-2a+Neo cells as shown in Fig. 9.
However, five of six nude mice injected with Neuro-2a+FasL cells
developed tumors after a latent period as long as 3 wk. Thus, it became
clear that T cells are essential for complete rejection of
Neuro-2a+FasL cells in addition to neutrophils.
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Neuro-2a+FasL cells were eradicated spontaneously and Neuro-2a
cells were also eradicated when injected together with Neuro-2a+FasL
cells. Hereupon, to address whether or not protective immunity was
established after rejection of Neuro-2a+FasL cells, mice were
challenged with Neuro-2a cells 14 days after injection of Neuro-2a+FasL
cells (Table I). They manifested a strong immunoprotective effect
(rejection rate: 17 of 19, 89%) for >60 days after inoculation of
Neuro-2a cells. In contrast, immunization with MMC-treated Neuro-2a or
MMC-treated Neuro-2a+Neo cells (data not shown) or PBS alone did not
induce antitumor immunity. Moreover, mice were immunized with
MMC-treated Neuro-2a+FasL cells as a control (Table I). The antitumor
immunity induced was significantly less potent in these mice than in
those injected with viable Neuro-2a+FasL cells. MMC-treated
Neuro-2a+FasL cells neither released FasL in vitro nor developed
palpable swelling in vivo. These results indicated that the active
production of FasL was necessary for the induction of antitumor
immunity. When challenge with Neuro-2a cells was given at the site
either ipsilateral or contralateral to the immunized site, the same
degree of antitumor immunity was observed (Table I), indicating that
Neuro-2a+FasL cells can induce systemic immunity. Importantly, S713a
cells formed tumors at the same growth rate in both immunized and
control mice in support of the induction of tumor-specific antitumor
immunity with Neuro-2a+FasL cells (Table I).
Role of CD4+ and CD8+ T cells in protective antitumor immunity
To address whether CD4+ or CD8+ T cells
were responsible for tumor-specific antitumor immunity, the immunized
mice were injected with anti-CD4 or anti-CD8 mAb before
challenging with Neuro-2a cells (Table II). Flow cytometric analysis indicated
that Neuro-2a cells were CD4-CD8- (data not
shown). Anti-CD8 but not anti-CD4 mAb treatment completely
abrogated antitumor immunity, demonstrating that CD8+ T
cells were required for the rejection of tumor cells in protective
immunity.
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As Neuro-2a+FasL cells conferred strong antitumor immunity on
mice, we addressed the possibility of their application to tumor
therapy. Mice were injected with Neuro-2a cells, followed by treatment
with Neuro-2a+FasL or MMC-treated Neuro-2a cells 1 and 3 days later
(Fig. 10). Tumor growth was not
influenced at all by treatment with MMC-treated Neuro-2a cells. Tumor
formation did not occur at all during 3-wk observation in 5 (38%) of
13 mice treated with Neuro-2a+FasL cells, and tumor growth was delayed
in the others compared with control mice. All mice that did not develop
tumors rejected the second challenge with Neuro-2a cells. Thus,
immunologic memory was established in these mice. To test whether or
not the therapeutic effect was tumor specific, mice were injected with
S713a cells, followed by treatment with Neuro-2a+FasL cells. However,
the growth of S713a tumors was not affected at all (Fig. 8). These
results suggested that tumor cells expressing both soluble and
membrane-bound FasL might manifest a suppressive effect on growing
tumors through a tumor-specific antitumor mechanism.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 2, 3, and 10 and Table I).
Neuro-2a+FasL cells were found to induce protective and therapeutic
immunity against parent Neuro-2a cells (Table I and Fig. 10). Thus,
FasL produced locally acted as an immunostimulatory rather than an
immunosuppressive cytokine, and promoted potent antitumor immunity.
FasL produced by melanoma cells has been shown to abrogate the
antitumor immune response (16). The findings presented here show the
opposite, namely FasL-mediated enhancement of tumor immune
surveillance. Of note is that Neuro-2a cells transfected with FasL cDNA
produce FasL in membrane form (mFasL), which is the bioactive form in
contrast to the cleaved soluble peptide. In contrast, Kayagaki et al.
(36) reported polymorphism of murine FasL with different biological
activity. We showed that less potent FasL that was derived from C57BL
mice induced remarkable antitumor immunity (Table I and Fig. 10).
However, immunological privilege was also proved using less potent FasL
(7, 9, 11). Thus, the two opposite phenomena, immunostimulation and
immunological privilege (immunosuppression), may not be explained by
polymorphism in FasL.
Seino et al. (30) and Arai et al. (31) reported that FasL induced
granulocyte-mediated inflammation, leading to tumor rejection. Ligation
of Fas can induce secretion of IL-8 (39), which might contribute to the
ensuing of inflammation. Histological examination and flow cytometric
analysis revealed the prompt infiltration of neutrophils into the i.d.
site where Neuro-2a+FasL cells were injected (Figs. 4 and 6). These
neutrophils partly expressed Fas on the surface. In addition,
infiltration of neutrophils into the tumor site was not observed in
Fas-deficient mice. These results suggested that the host response
resulting in tumor rejection may be initiated by Fas-FasL
system-mediated recruitment of neutrophils to the tumor.
To elucidate the antitumor mechanism of FasL-producing tumor cells,
S713a sarcoma cells mixed with Neuro-2a+FasL cells were inoculated into
mice (Fig. 8). Neuro-2a+FasL cells did not inhibit the growth of S713a
tumors despite the fact that they exhibited strong antitumor activity
against parent Neuro-2a cells. Direct killing of S713a by FasL did not
occur because S713a is a Fas- tumor. These results were
very important for elucidating the role of neutrophils in antitumor
response. Neuro-2a+FasL cells release FasL, which is known to have
chemotactic activity to neutrophils (40). The neutrophils that were
massively accumulated in the tumor site were in close contact with
tumor cells (Fig. 4A). Neutrophils may release various
cytokines including proinflammatory cytokines (IL-1, IL-8, TNF) (41)
and damage tumor cells (42, 43). If the neutrophils exert their
antitumor activity indirectly through the cytokines, by-stander tumor
S713a tumor cells may also undergo damage. However, Neuro-2a+FasL cells
did not affect S713a cells at all (Fig. 8). Thus, the direct contact
between neutrophils and tumor may be necessary for neutrophils to
attack tumor cells. Neutrophils displayed enzyme- and nitric
oxide-mediated lytic activity against tumor cells (43, 44). In
contrast, neutrophils discriminated between G-CSF-producing and
G-CSF-nonproducing tumor cells and repressed only the former cells by
direct contact with them (42). The rejection of adenocarcinoma
engineered to release IL-2 or TNF-
was associated with the massive
presence of neutrophils that were in close contact with tumor cells
(45). These results taken together suggested that antitumor activity of
neutrophils may be mainly dependent on their direct contact with tumor
cells but not on their indirect effects. Neutrophils attracted by FasL
may exert antitumor activity after their adhesion to tumor cells. A
further study is needed to elucidate the FasL-mediated antitumor
mechanism.
Neuro-2a+FasL cells formed tumors after a far longer latent period
compared with Neuro-2a+Neo cells in nude mice (Fig. 9), indicating that
neutrophils cannot eradicate tumor cells completely without T cells. In
support of this, immune competent A/J mice rejected Neuro-2a+FasL cells
inoculated alone (Fig. 3), Neuro-2a cells injected as a mixture with
Neuro-2a+FasL cells (Fig. 7) and even growing Neuro-2a cells after
treatment with Neuro-2a+FasL cells (Fig. 10). As a result, they
acquired tumor-specific immunological memory, because they could not
reject S713a sarcoma cells (Table I). CD8+ T cells were
responsible for the established antitumor immunity (Table II). In
addition, neutrophils were required to induce antitumor effector
CD8+ T cells in mice (45) and rats (46). These results
suggest that neutrophils alone can inhibit growth of Neuro-2a+FasL
cells at least temporarily and that CD8+ T cells are
essential for the complete rejection of Neuro-2a+FasL cells.
Neuro-2a+FasL cells not only express FasL on the cell surface but also
release the bioactive soluble full-length FasL (Figs. 1 and 2) (33). We
have recently reported that i.v. but not i.p. injection of
Neuro-2a+FasL-derived FasL induced apoptosis of liver cells with liver
failure within 2 h of injection (33). However, we confirmed that
i.d. injection of Neuro-2a+FasL (106) cells caused neither
clinically detrimental effects such as loss of body weight nor
impairment of liver function as determined by serum
glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, and
total bilirubin levels 24 h after the injection in wild-type mice
(data not shown). Thus, toxicity of Neuro-2a+FasL cells may require
significant levels of mFasL in the serum, while local expression of
mFasL at the injection site or in different tissues may be relatively
harmless.
The genetic modification of tumor cells for the local expression of
cytokines IL-1, IL-2, IL-4, IL-12, IFN-, and TNF has recently been
studied as a novel approach to the active immunotherapy of cancer (47).
Immunization with the modified tumor cells promoted elimination of
tumors by various effector cells. Neuro-2a+FasL cells induced
CD8+T cell-dependent protective immunity (Table II) and
therapeutic activity in tumor-bearing mice (Fig. 10). Tumor cells
expressing FasL were used as a good vehicle for continuous local
delivery of FasL because Neuro-2a+FasL cells were completely rejected
in mice. This study indicates that gene transfer of FasL to tumors may
generate potent antitumor immunity leading to regression of tumor
cells. This pathway may be amplified by direct action of FasL on tumors
expressing Fas such as gliomas (48).
On the basis of the present and previous results (30, 31), we hypothesize that membrane-bound and soluble FasL may cause neutrophils to infiltrate tumor sites by its chemokine-like activity and eradicate tumor cells by a local inflammatory reaction in a nonspecific manner, which results in the establishment of specific antitumor immunity. The mechanism for this process remains to be elucidated, and this strategy may be applicable to cancer therapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Motomu Shimizu, Department of Cancer Therapeutics, Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo, 113-8613 Japan. E-mail address:
3 Abbreviations used in this paper: FasL, Fas/Apo-1 ligand (CD95L); FSC, forward scatter; mFasL, FasL in membrane form; MMC, mitomycin C; Neuro-2a+FasL, neomycin resistance and FasL cDNA-transfected Neuro-2a tumor; Neuro-2a+Neo, neomycin resistance cDNA-transfected Neuro-2a tumor; SSC, side scatter.
Received for publication October 14, 1998. Accepted for publication March 26, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ligation of Fas antigen. J. Immunol. 155:4147.[Abstract]
, IFN-, and TNF-. Lab. Invest. 74:146.[Medline]
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 J Doering, B Begue, M J Lentze, F Rieux-Laucat, O Goulet, J Schmitz, N Cerf-Bensussan, and F M Ruemmele Induction of T lymphocyte apoptosis by sulphasalazine in patients with Crohn's disease Gut, November 1, 2004; 53(11): 1632 - 1638. [Abstract] [Full Text] [PDF]
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R. R. Saff, E. S. Spanjaard, A. M. Hohlbaum, and A. Marshak-Rothstein Activation-Induced Cell Death Limits Effector Function of CD4 Tumor-Specific T Cells J. Immunol., June 1, 2004; 172(11): 6598 - 6606. [Abstract] [Full Text] [PDF]
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D. Samel, D. Muller, J. Gerspach, C. Assohou-Luty, G. Sass, G. Tiegs, K. Pfizenmaier, and H. Wajant Generation of a FasL-based Proapoptotic Fusion Protein Devoid of Systemic Toxicity due to Cell-surface Antigen-restricted Activation J. Biol. Chem., August 22, 2003; 278(34): 32077 - 32082. [Abstract] [Full Text] [PDF]
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S. Buonocore, F. Paulart, A. Le Moine, M. Braun, I. Salmon, S. Van Meirvenne, K. Thielemans, M. Goldman, and V. Flamand Dendritic cells overexpressing CD95 (Fas) ligand elicit vigorous allospecific T-cell responses in vivo Blood, February 15, 2003; 101(4): 1469 - 1476. [Abstract] [Full Text] [PDF]
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P. G. Knox, A. E. Milner, N. K. Green, A. G. Eliopoulos, and L. S. Young Inhibition of Metalloproteinase Cleavage Enhances the Cytotoxicity of Fas Ligand J. Immunol., January 15, 2003; 170(2): 677 - 685. [Abstract] [Full Text] [PDF]
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 F. H. Igney and P. H. Krammer Immune escape of tumors: apoptosis resistance and tumor counterattack J. Leukoc. Biol., June 1, 2002; 71(6): 907 - 920. [Abstract] [Full Text] [PDF]
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A. de Thonel, A. Bettaieb, C. Jean, G. Laurent, and A. Quillet-Mary Role of protein kinase C zeta isoform in Fas resistance of immature myeloid KG1a leukemic cells Blood, December 15, 2001; 98(13): 3770 - 3777. [Abstract] [Full Text] [PDF]
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A. M. Hohlbaum, M. S. Gregory, S.-T. Ju, and A. Marshak-Rothstein Fas Ligand Engagement of Resident Peritoneal Macrophages In Vivo Induces Apoptosis and the Production of Neutrophil Chemotactic Factors J. Immunol., December 1, 2001; 167(11): 6217 - 6224. [Abstract] [Full Text] [PDF]
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 A. M. Hohlbaum, S. Moe, and A. Marshak-Rothstein Opposing Effects of Transmembrane and Soluble FAS Ligand Expression on Inflammation and Tumor Cell Survival J. Exp. Med., April 3, 2000; 191(7): 1209 - 1220. [Abstract] [Full Text] [PDF]
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),
supernatant from Neuro-2a+FasL cells against N1d cells (
), and
supernatant from Neuro-2a+Neo cells against N1d cells (
).
B, Supernatant from Neuro-2a+FasL cells was pretreated
with anti-FasL mAb and control IgG2b, followed by the addition of
F6b cells. The original concentrations of mAb and control IgG2b were 20
mg/ml. The results of one of more than three similar experiments are
presented. Bar and asterisk indicate the SD of the mean and statistical
significance (*1, p < 0.001; *2,
p < 0.0001; *3, p <
0.00001; and *4, p < 0.000001 by Students’
t test vs tumor control), respectively.
) (n = 8). A/J mice were injected with S713a
cells (5 x 105), followed by treatment with
Neuro-2a+FasL cells (106) (•) (n = 8)
and PBS (
