Download Neuroblastoma Neuro-2a Cells Fas/APO

Induction of Antitumor Immunity with
Fas/APO-1 Ligand (CD95L)-Transfected
Neuroblastoma Neuro-2a Cells
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of June 14, 2017.
Motomu Shimizu, Adriano Fontana, Yasutaka Takeda,
Hideki Yagita, Takayuki Yoshimoto and Akio Matsuzawa
J Immunol 1999; 162:7350-7357; ;
http://www.jimmunol.org/content/162/12/7350
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The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 1999 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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References
Induction of Antitumor Immunity with Fas/APO-1 Ligand
(CD95L)-Transfected Neuroblastoma Neuro-2a Cells1
Motomu Shimizu,2* Adriano Fontana,† Yasutaka Takeda,‡ Hideki Yagita,§
Takayuki Yoshimoto,¶ and Akio Matsuzawa|
T
he Fas/Apo-1 (CD95) receptor/ligand system induces apoptosis and plays an important role in the immune system
(1–5). Fas is related to members of the TNF/nerve growth
factor receptor family (6), and Fas ligand (FasL)3 is a member of
the TNF/nerve growth factor family (3). Fas and FasL are broadly
distributed on lymphoid and nonlymphoid cells (3, 6). FasL is also
expressed at the sites of immune privilege such as testis (7), eye (8,
9), and placenta (10). Immunologically privileged tissues constitutively express FasL, and infiltrating Fas1 T cells and Fas1 granulocytes rapidly undergo FasL-induced apoptosis. Thus, the tissue
is protected from immune-mediated damage. Similarly, allograft
rejection of pancreatic islets is prevented with engineered myoblasts expressing FasL (11). Overexpression of FasL in mouse
ankle joints ameliorated collagen-induced arthritis, providing evidence for an antiinflammatory role of FasL in vivo (12). However,
using the same strategy to abrogate the immune response to islets
cells, allograft rejection was not always prevented by overexpres-
*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; ‡Department of Surgery, Institute of
Medical Science, University of Tokyo, Tokyo, Japan; ¶Department of Allergology,
Institute of Medical Science, University of Tokyo, Tokyo, Japan; and \Laboratory
Animal Research Center, Institute of Medical Science, University of Tokyo, Tokyo,
Japan
Received for publication October 14, 1998. Accepted for publication March 26, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
sion of FasL in the transplantation (13–15). A number of tumors
including nonlymphoid tumors have been observed to express constitutively functional FasL (16 –29). These tumor cells are capable
of killing Fas1 Jurkat lymphocytes in vitro. Thereby, FasL positive tumors may impair the tumor immune surveillance. In contrast, tumors expressing FasL can induce granulocyte-mediated tumor rejection (30, 31). Therefore, these reports are inconsistent
with regard to the role of FasL in the regulation of the immune
response and cause much controversy.
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 Fas1 lymphoma cells (Yac-1) implanted i.p. into mice
(33). Culture supernatant of neuroblastoma Neuro-2a cells transfected with murine FasL cDNA (Neuro-2a1FasL) contained FasL
and transduced a potent apoptotic signal to Fas1Yac-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, Neuro2a1FasL, induced potent antitumor immunity associated with interference of tumor growth.
1
This work was supported in part by a Grant-in-Aid from the Ministry of Education,
Science, Sports, and Culture of Japan (072724219).
Materials and Methods
2
Mice
3
Abbreviations used in this paper: FasL, Fas/Apo-1 ligand (CD95L); FSC, forward
scatter; mFasL, FasL in membrane form; MMC, mitomycin C; Neuro-2a1FasL, neomycin resistance and FasL cDNA-transfected Neuro-2a tumor; Neuro-2a1Neo, neomycin resistance cDNA-transfected Neuro-2a tumor; SSC, side scatter.
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).
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:
[email protected]
Copyright © 1999 by The American Association of Immunologists
0022-1767/99/$02.00
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Fas/Apo-1 (CD95)-Fas ligand (FasL) system has been implicated in the suppression and stimulation of immune responses. We
examined the induction of antitumor immunity with neuroblastoma Neuro-2a cells transfected with FasL cDNA (Neuro2a1FasL). Neuro-2a1FasL cells expressed FasL on the cell surface and secreted soluble FasL. Histologic and flow cytometric
analyses revealed that Neuro-2a1FasL cells caused neutrophils to infiltrate into the injected site, resulting in strong inflammation.
Neutrophil infiltration was inhibited by treatment with anti-FasL mAb and did not occur in Fas-deficient lpr mice. Normal
syngeneic mice rejected Neuro-2a1FasL cells after the inflammation and acquired tumor-specific protective immunity. CD81 T
cells were responsible for the antitumor immunity. Neuro-2a1FasL cells formed tumors after far longer latency compared with
mock-transfected Neuro-2a1Neo cells in nude mice, and immune competent mice rejected Neuro-2a cells but not sarcoma S713a
cells when they were injected with Neuro-2a1FasL cells in a mixture. These results suggest that neutrophils attracted through the
Fas-FasL system may impair tumor cells by inflammation at the initial step, followed by development of CD81 T cell-dependent
tumor-specific antitumor immunity, leading to complete eradication of tumor cells. Importantly, the treatment with Neuro2a1FasL cells exhibited therapeutic efficacy against growing tumors. The Journal of Immunology, 1999, 162: 7350 –7357.
The Journal of Immunology
7351
Tumors
FasL cDNA was obtained from C57BL mice. Neuro-2a (35), mock (neomycin-resistant cDNA)-transfected Neuro-2a (Neuro-2a1Neo), and
Neuro-2a1FasL cells (33) were maintained in DMEM (Iwaki, Tokyo) containing 10% heat-inactivated FBS, 4.5 g/L glucose, 100 U/ml penicillin,
and 100 mg/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 (Neo1Fas1) and N1d (Neo1) cells were produced from murine hepatoma MH134 cells (Fas2) (34). These three tumor cell lines were passaged by culture in RPMI 1640 medium containing 10% FBS, 2-ME (5 3
1025 M), and kanamycin. S713a sarcoma cells were maintained in RPMI
1640 medium containing 5% FBS, penicillin, and streptomycin.
Cytotoxicity test
In vivo antitumor activity
Neuro-2a cells were incubated with mitomycin C (MMC) (60 mg/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 Neuro2a1FasL, MMC-treated Neuro-2a1FasL, or MMC-treated Neuro-2a (106)
cells on day 214 and challenged i.d. with Neuro-2a or S713a cells (5 3
105) on day 0. Neuro-2a1FasL 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, Neuro2a1FasL (5 3 105) were mixed with Neuro-2a (5 3 105) or S713a (5 3
105) tumor cells and injected i.d. into A/J mice. In a therapeutic experiment, A/J mice injected i.d. with 5 3 105 Neuro-2a or S713a cells on day
0 were treated by injecting viable Neuro-2a1FasL 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 3 b)½ ]. Mice were observed
for tumor appearance and growth for .60 days after inoculation of tumor
cells.
1
1
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-2a1FasL cells on day 214, i.p. administrated with 0.5 mg of purified anti-CD4 mAb on days 23 and 21 to deplete CD41 T cells or 0.2
ml of ascitic anti-CD8 mAb on days 23 and 0 to deplete CD81 T cells, and
i.d. inoculated with 5 3 105 Neuro-2a cells on day 0. CD41 T cells were
reduced from 18% to 0.4% and CD81 T cells from 10.5% to 0.1% in the
spleen of these mice.
Flow cytometry of cells infiltrated with Neuro-2a1FasL
Mice were i.d. injected with 106 Neuro-2a1FasL 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 (FcgRIII/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),
FIGURE 1. Characterization of Neuro-2a1FasL cells. A, Supernatants
from Neuro-2a1FasL and Neuro-2a1Neo cells were incubated with F6b
and N1d cells. Supernatant from Neuro-2a1FasL cells against F6b cells
(F), supernatant from Neuro-2a1Neo cells against F6b cells (E), supernatant from Neuro-2a1FasL cells against N1d cells (), and supernatant
from Neuro-2a1Neo cells against N1d cells (‚). B, Supernatant from
Neuro-2a1FasL 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 (p1, p , 0.001; p2, p , 0.0001; p3,
p , 0.00001; and p4, p , 0.000001 by Students’ t test vs tumor control),
respectively.
FITC-anti-CD8 (Becton Dickinson), and/or biotin-anti-Fas Jo2 (hamster
IgG) mAbs (PharMingen) and PE-strepavidin (Life Technologies), PE-antiCD4 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-2a1FasL, Neuro-2a1Neo, or Neuro-2a cells at the age of 6 – 8 wk.
Some A/J mice were injected i.p. with 125 mg of anti-FasL (K10) 8 h and
30 min before the transplantation of Neuro-2a1FasL 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
mm, and stained with hematoxylin and eosin for microscopy.
Results
Characterization of FasL expressing in Neuro-2a1FasL cells
Culture supernatants from Neuro-2a1FasL and Neuro-2a1Neo
cells were incubated with the hepatoma cells Fas1 F6b and Fas2
N1d cells to examine the induction of apoptosis by FasL (Fig. 1A).
Supernatant from Neuro-2a1FasL but not Neuro-2a1Neo cells
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Cell-free culture supernatants were collected after about 6 3 106 Neuro2a1FasL and Neuro-2a1Neo cells were incubated in 20 ml of DMEM
containing 1% FBS for 2–3 days. F6b or N1d cells suspended in 100 ml 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 ml of MTT (5 mg/ml) for 2–3 h, and then
formazan was produced, which was solubilized with 50 ml 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 5 [(OD of
supernatant 1 tumor cells)/(OD of tumor cells)] 3 100 (%).
7352
ANTITUMOR IMMUNITY INDUCED BY FAS LIGAND
FIGURE 3. Rejection of Neuro-2a1FasL cells but not Neuro-2a1Neo
and parent Neuro-2a cells. A/J mice were i.d. injected with Neuro2a1FasL (1 3 106) (n 5 8), Neuro-2a1Neo (5 3 105) (n 5 8), or
Neuro-2a (5 3 105) cells (n 5 8). p, p , 0.01 by Mann-Whitney test vs
Neuro-2a control.
Histology
FIGURE 2. Expression of FasL in Neuro-2a1FasL, Neuro-2a1Neo,
and Neuro-2a cells. Cells were stained with anti-FasL mAb and then FITCanti-mouse IgG (shaded area) or FITC-anti-mouse IgG alone and autofluorescence (zzz). The FACS histogram of Neuro-2a1FasL cells stained with
isotype control using mouse IgG2b was the same as that of FITC control.
induced the cytotoxicity against F6b cells. The cytotoxic activity
of supernatant from Neuro-2a1FasL 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-2a1FasL
cells were stained with anti-FasL K10 but not with control mouse
IgG2b (Fig. 2). These results evidenced that Neuro-2a1FasL cells
secreted soluble FasL into culture medium and expressed FasL on
the surface. Fas and FasL were not detected in Neuro-2a cells.
Neuro-2a1FasL cells did not express Fas and were resistant to
FasL-mediated killing (data not shown).
Loss of tumorigenic potential of Neuro-2a cells by FasL
transfection
To investigate the functional consequences of FasL expressed by
Neuro-2a cells, syngeneic A/J mice were injected with 106 Neuro2a1FasL cells (Fig. 3). All of the 19 injected mice rejected Neuro2a1FasL tumor cells after formation of a small but clearly palpable tissue mass on days 1– 4 (Table I). When 106 Neuro-2a1FasL
cells were injected into A/J (n 5 19) and C3H (n 5 10) mice, a
clearly swelled mass as huge as 6 3 6 to 8 3 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 3 1 mm) in A/J mice (n 5 7) and occurred in neither C3H-lpr
(, 1 3 1 mm) (n 5 3) nor C3H-gld/lpr (, 1 3 1 mm) (n 5 6)
mice, which carry a functional defect in Fas alone and both FasL
and Fas (34), respectively, indicating that the Fas-FasL system is
As mentioned above, a palpable swelling developed immediately
at the site where Neuro-2a1FasL 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-2a1FasL cells and underwent
swelling (Fig. 4A). Some neutrophils appeared to be in contact
with tumor cells. On day 5 of Neuro-2a1FasL 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-2a1Neo (Fig. 4B) or Neuro-2a cells (data not shown).
More importantly, Neuro-2a1FasL cells induced neither swelling
nor leukocyte infiltration in C3H-lpr (data not shown) and C3Hgld/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).
Flow cytometry of cells infiltrating around Neuro-2a1FasL cells
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-2a1FasL cells and analyzed by flow cytometry. The
isolated cells were smaller than Neuro-2a1FasL 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) 5 22, side scatter (SSC) 5
21) and a low peak of lymphoid-like cells (FSC 5 73, SSC 5 41).
The histogram of Neuro-2a1FasL cells showed a major peak of
tumor cells (FSC 5 105, SSC 5 85), a minor peak of cell debris
(FSC 5 19, SSC 5 20), and a bottom (FSC 5 60, SSC 5 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 antiGr-1 (Ly-6G) mAb specific for granulocytes (37) and anti-Mac-1
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directly involved in this event. In contrast, tumor growth occurred
without such instant mass formation accompanied in all the mice
injected with 5 3 105 Neuro-2a1Neo or parent Neuro-2a cells
(Fig. 3 and Table I).
The Journal of Immunology
7353
Table I. Induction and tumor specificity of protective immunitya
Immunization
Neuro-2a1FasL (right flank)
Neuro-2a1FasL (right flank)
MMC-Neuro-2a1FasL
MMC-Neuro-2a
PBS
PBS
Neuro-2a1FasL
PBS
Challenge
Neuro-2a (right flank)
Neuro-2a (left flank)
Neuro-2a
Neuro-2a
Neuro-2a1Neo
Neuro-2a
S713a
S713a
Tumor Incidence (%)
2/19 (11)*
1/7 (14)‡
5/7 (71)
5/5 (100)
5/5 (100)
13/13 (100)
8/8 (100)
8/8 (100)
Tumor Size (mm)
17.9 (n 5 2), 0 (n 5 17)†
17.3 (n 5 1), 0 (n 5 6)§
15.4 6 3.7 (n 5 5),0 (n 5 2)¶
17.5 6 3.6
17.0 6 1.4
20.2 6 2.2
18.2 6 2.9
19.1 6 2.8
a
A/J mice were injected with Neuro-2a1FasL (106), MMC-Neuro-2a1FasL (106), MMC-Neuro-2a cells (106), or PBS on day 214 and challenged with Neuro-2a (5 3 105),
Neuro-2a1Neo (5 3 105), or S713a cells (5 3 105) on day 0. Tumor incidence and tumor size were determined on day 21.
*
p , 0.0001 by Fischer’s exact probability test vs PBS1Neuro-2a control.
†
p , 0.0001 by Mann-Whitney test vs PBS 1 Neuro-2a control.
‡
p , 0.0004 by Fischer’s exact probability test vs PBS1Neuro-2a control.
§
p , 0.001 by Mann-Whitney test vs PBS1Neuro-2a control.
¶
p , 0.001 by Mann-Whitney test vs PBS1Neuro-2a control.
Eradication of tumorigenic Neuro-2a cells by inflammatory cells
induced with Neuro-2a1FasL cells
The histological and flow cytometric results revealed that Neuro2a1FasL cells can attract neutrophils around them. To determine
whether or not these cells can affect tumor formation, a mixture of
5 3 105 Neuro-2a1FasL and 5 3 105 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 3 105 Neuro-2a1FasL
and 5 3 105 S713a (Fas-negative) cells were i.d. inoculated into
mice (Fig. 8). The growth of S713a cells was not suppressed by
Neuro-2a1FasL cells. These results indicated that the Neuro2a1FasL 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.
FIGURE 4. Histology of the sites where
106 tumor cells were injected 24 h previously.
Large cells on the top are the injected tumor
cells. A, Abundant leukocytes, mostly neutrophils, infiltrate between and around Neuro2a1FasL cells in an A/J mouse. Neutrophils
surround tumor cells and appear to be in contact with tumor cells. Note the larger area infiltrated and far higher density of leukocytes
compared with B and C. The same result was
also observed in C3H mice. B, Small numbers
of neutrophils and monocytes infiltrate around
Neuro-2a1Neo cells in an A/J mouse. C, No
neutrophils are found around Neuro-2a1FasL
cells in a C3H-gld/lpr mouse. Original magnification, 3140.
Incomplete eradication of Neuro-2a1FasL cells in nude mice
To determine whether or not T cells are required for eradication of
Neuro-2a1FasL cells, the tumor cells were implanted into nude
mice. All nude mice suppressed the growth of Neuro-2a1FasL
cells but not of Neuro-2a1Neo cells as shown in Fig. 9. However,
five of six nude mice injected with Neuro-2a1FasL 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 Neuro2a1FasL cells in addition to neutrophils.
Establishment of protective antitumor immunity after eradication
of Neuro-2a1FasL cells
Neuro-2a1FasL cells were eradicated spontaneously and
Neuro-2a cells were also eradicated when injected together with
Neuro-2a1FasL cells. Hereupon, to address whether or not protective immunity was established after rejection of Neuro2a1FasL cells, mice were challenged with Neuro-2a cells 14 days
after injection of Neuro-2a1FasL 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 Neuro2a1Neo cells (data not shown) or PBS alone did not induce antitumor immunity. Moreover, mice were immunized with MMCtreated Neuro-2a1FasL cells as a control (Table I). The antitumor
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(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).
7354
ANTITUMOR IMMUNITY INDUCED BY FAS LIGAND
FIGURE 7. Effect of Neuro-2a1FasL cells on tumor formation by
Neuro-2a cells. Neuro-2a cells (5 3 105) alone and a mixture of Neuro2a1FasL (5 3 105) and Neuro-2a (5 3 105) cells were injected i.d. into A/J
mice. The number of mice is shown. p, p , 0.0001 by Mann-Whitney test
vs Neuro-2a control
immunity induced was significantly less potent in these mice than
in those injected with viable Neuro-2a1FasL cells. MMC-treated
Neuro-2a1FasL cells neither released FasL in vitro nor developed
palpable swelling in vivo. These results indicated that the active
Role of CD41 and CD81 T cells in protective antitumor
immunity
To address whether CD41 or CD81 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 CD42CD82 (data not shown). Anti-CD8 but
not anti-CD4 mAb treatment completely abrogated antitumor immunity, demonstrating that CD81 T cells were required for the
rejection of tumor cells in protective immunity.
Therapeutic effect of Neuro-2a1FasL cells
As Neuro-2a1FasL 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
FIGURE 6. Characterization of cells infiltrated by Neuro-2a1FasL
cells with flow cytometry. The infiltrating cells were prepared from A/J
mice injected with Neuro-2a1FasL cells (106) 24 h before. The cells were
stained with (A) FITC-anti-Gr-1 and PE-Fas, (B) FITC-anti-Mac-1 and
PE-Fas, (C) FITC-rat IgG2b and biotin-hamster IgG 1 PE-avidin (isotype
control), (D) FITC-anti-CD8 and PE-anti-CD4, and (E) autofluorescence.
FIGURE 8. Absence of antitumor effect of Neuro-2a1FasL cells
against S713a cells. A mixture of S713a (5 3 105) and Neuro-2a1FasL
(5 3 105) cells were injected i.d. into A/J mice (M) (n 5 8). A/J mice were
injected with S713a cells (5 3 105), followed by treatment with Neuro2a1FasL cells (106) (F) (n 5 8) and PBS (E) (n 5 8) on days 1 and 3.
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FIGURE 5. FSC and SSC histograms of cells infiltrated by Neuro2a1FasL cells and of Neuro-2a1FasL cells. The infiltrating cells were
prepared from A/J mice injected with Neuro-2a1FasL cells (106) before
24 h. Neuro-2a1FasL cells were cultured in vitro and measured with a
FACS. Shown are the contour plot (zzz) of infiltrating cells and Neuro-2a
cells1FasL cells and the gated area (—).
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-2a1FasL 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-2a1FasL cells
(Table I).
The Journal of Immunology
7355
FIGURE 9. Suppression of Neuro-2a1FasL but not Neuro-2a1Neo tumor growth in nude mice. BALB/c nude mice were i.d. inoculated with 106
Neuro-2a1FasL (n 5 6) or 106 Neuro-2a1Neo (n 5 6) on day 0.
Discussion
In this study, we showed that in contrast to FasL negative tumors,
tumor cells that express FasL on cell surface and release FasL were
rejected in a syngeneic system (Figs. 1, 2, 3, and 10 and Table I).
Neuro-2a1FasL 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
Table II. Characterization of antitumor effector T cells induced by
immunization with Neuro-2a1FasL cellsa
Immunization
mAb Treatment
Tumor Incidence
(%)
Tumor Size
(mm)
1
1
1
–
Anti-CD4
Anti-CD8
–
–
0/8 (0)*
8/8 (100)
0/6 (0)†
7/7 (100)
0‡
19.0 6 4.8
0‡
18.2 6 1.8
a
Two weeks after i.d. injection with Neuro-2a1FasL (106) cells, mice were i.d.
challenged with Neuro-2a (5 3 105). Some mice were treated with anti-CD4 and
anti-CD8 mAb to deplete CD41 and CD81 T cells, respectively, before challenge.
Tumor incidence and tumor size were determined at 3 wk after the challenge.
*
p , 0.0004; and †, p , 0.002 by Fischer’s exact probability test vs Neuro-2a
control.
‡
p , 0.0001 by Mann-Whitney test vs Neuro-2a control.
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-2a1FasL 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-2a1FasL cells were
inoculated into mice (Fig. 8). Neuro-2a1FasL 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 Fas2
tumor. These results were very important for elucidating the role of
neutrophils in antitumor response. Neuro-2a1FasL 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-2a1FasL 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
Downloaded from http://www.jimmunol.org/ by guest on June 14, 2017
treatment with Neuro-2a1FasL 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-2a1FasL 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 Neuro2a1FasL 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.
FIGURE 10. Treatment of Neuro-2a tumor-bearing mice with Neuro2a1FasL cells. A/J mice were injected with Neuro-2a cells (5 3 105),
followed by treatment with Neuro-2a1FasL cells (106) (F) (number of
mice, n 5 13), MMC-Neuro-2a cells (106) (M) (n 5 6), and PBS (E) (n 5
8) on days 1 and 3. Tumor incidence is presented at each time point in the
figure. p, p , 0.0001 by Mann-Whitney test including tumor-bearing mice
vs Neuro-2a control.
7356
Acknowledgments
We thank Prof. S. Fujimoto, Kochi Medical School, for his generous supply of S713a sarcoma.
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