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Immunology |
Departments of Surgery [F. T., W. H., M. T. L., H. T.] and Molecular Genetics and Biochemistry [F. T., W. H., P. D. R., M. T. L., H. T.], University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, and Division of Host Defenses, Institution for Advanced Medical Sciences, Hyogo College of Medicine, Nishinomiya, Hyogo 663-8501, Japan [H. O.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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inducing T-cell growth factors, IL-2, and IL-12 in this culture system
and compared them with that mediated by IL-18. Neither IL-2 nor IL-12
induced tumor-specific cytolytic T cells to the same degree as that
mediated by IL-18. Efficacy of this system in induction of
tumor-specific CTLs was also observed in the system using MC38
adenocarcinoma cells. These results are consistent with the notion that
IL-18 induces tumor-specific immunity through enhancing NK activity,
which in turn mediates tumor cell death and activates and primes DCs. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-inducing factor," was cloned from
mice with fulminant hepatitis induced by the administration of
Propionibacterium acnes and lipopolysaccharide
(1)
. IL-18 stimulates NK cells, T cells, B cells, and
cells of the monocyte lineage to express IFN- at high levels
(1
, 2)
. Furthermore, IL-18 plays an important role in
T-cell proliferation (1)
, CTL activation (3)
,
and enhancement of NK cell activity primarily through Fas-FasL
mechanism (2
, 4, 5, 6)
. Systemic administration of rIL-18 is
associated with significant in vivo antitumor effects that
seem to be primarily mediated by enhanced NK activity (7
, 8) . We also have reported that established tumors could be
successfully treated by i.t. injection of recombinant adenoviral
vectors expressing biologically active mIL-18 (Ad.PTH.IL-18; Ref.
9
). Although the mechanism of the antitumor effect has not
been fully explained, we have shown that this antitumor effect is
completely abrogated by depletion of asialo GM-1-positive cells, most
likely NK cells (9)
. Recent studies, including one using IL-18-deficient mice, support the notion that IL-18 plays an important role in the development of cellular immunity (Th1 response) following antigen presentation (10 , 11) . We have reported that mice treated with i.t. injection of Ad.PTH.IL-18 rejected a subsequent rechallenge with tumor cell, suggesting induction of effective tumor immunity with this treatment (9) . In the present study, we analyzed the role of NK cells stimulated with IL-18 in CTL induction against tumor cells in an in vitro system consisting of live tumor cells, NK cells, DCs, and T cells with or without rmIL-18. This in vitro analysis system suggests that NK cells stimulated by IL-18 play a significant role in inducing tumor-specific immunity through activation and antigen-loading DCs, which are the primary antigen-presenting cells eliciting an effective adoptive immune response (12 , 13) .
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tumor Cell Lines and Animals.
MCA205, a methylcholanthrene-induced murine fibrosarcoma cell line, and
MC38, a murine colon adenocarcinoma, were generous gifts from Dr.
S. A. Rosenberg (National Cancer Institute, Bethesda, MD). The D122
highly metastatic variant of 3LL tumor cells was kindly provided by L.
Eisenbach (Weizmann Institute of Science, Rehovot, Israel). YAC-1 was a
generous gift of W. Chambers (University of Pittsburgh, Pittsburgh,
PA). These cell lines were maintained in RPMI 1640 supplemented with
10% heat-inactivated fetal bovine serum, 2 mM glutamine,
100 mg/ml streptimycin, 100 IU/ml penicillin, and 5 x 10-5 M 2-ME (all from Life Technologies, Inc.,
Grand Island, NY), referred to henceforth as CM. Primary cultures of
syngeneic fibroblasts were obtained from the lungs of C57BL/6 mice
(14)
. Wild-type C57BL/6 mice were purchased from Taconic
Farms (Germantown, NY). The scid/scid C57BL/6 mice were
purchased from The Jackson Laboratory (Bar Harbor, MA). These animals
were maintained in the animal facility at the Center for Biotechnology
and Bioengineering, University of Pittsburgh, and were used for
experiments when they were 7–12 weeks of age. All of the animals were
ear-tagged, randomized before experiments, and treated and examined in
a blinded fashion.
Cell Preparation.
Bone marrow-derived DCs cultured with GM-CSF and IL-4 for 6 days and
hepatic MNCs and were prepared as described previously, respectively
(14
, 15)
.
Flow Cytometry.
For phenotypic analysis, DCs were stained with PE- or FITC-conjugated
mAbs against murine cell surface molecules (CD11c, CD80, CD86, Gr-1,
H-2Kb, I-Ab, and appropriate isotype controls; all were from
PharMingen, San Diego, CA). Lymphoid cells harvested from lymph nodes
were stained with CD4 and CD8. They were examined with the FACScan
(Becton Dickinson, Sunnyvale, CA).
Cytokine Release or CTLs Assay in Vitro.
To examine the role of NK cells in CTL induction in vitro,
splenocytes from C57BL/6 SCID mice (NK-rich cells) were dissociated in
CM to yield a single-cell suspension. The percentage of NK cells was
determined using mAb (41–50%). After erythrocyte lysis, cells of
4 x 105/ml were plated into
24-well plates. Inguinal lymph nodes, abdominal wall lymph nodes, and
axial lymph nodes of naive C57BL/6 mice were collected and suspended in
CM. These cells of 2 x 106/well
were added to NK cells. The percentage of T cells was determined using
antimouse CD8 and antimouse CD4 mAbs (31–40% and 37–51%,
respectively). Day 6 DCs and tumor cells were resuspended and plated
2 x 105/well and 2 x 104/well, respectively. On day 4, the
number of tumor cells reached 1 x 106. rhIL-2 was added to the culture medium at a
very low final concentration of 25 IU/ml. The rmIL18 was added at
graded doses of 0–250 ng/ml in a final volume of 2.4 ml/well. In some
experiments, NK cells were cultured with rIL-18 (250 ng/ml) and rIL-2
(25 IU/ml) for 4 days to analyze the cytolytic activity against MCA205
cells. In some experiments, MCA205 cells were treated with UV at 1.52
mW/cm2 for 30 min and added at 2 x 106/ml (0.5 ml) immediately after
treatment. Forty-eight h after UV treatment, 73% of the MCA205 cells
were confirmed to be apoptotic by Annexin V expression. In some
experiments, either NK cells or DCs were plated in a well using a
0.4-µm pore size transwell (Corning Coster, Cambridge, MA) to prevent
direct cell-to-cell contact. Forty h after the culture, 0.4 ml of
supernatant was collected for ELISA analysis for IFN- (PharMingen).
The lower detection limit of these assays was 15 pg/ml. For cytotoxic
assay, cells were cultured for 4 days and collected and tested for
cytotoxic activity. Viable lymphoid cells were counted using trypan
blue-exclusion method and used as effector cells for the standard 4-h
51Cr release assay against target cells. In
brief, 106 cells of each target were labeled with
100 µCi of
Na251CrO4
for 1 h, rinsed twice, and plated with target cells at an
appropriate E:T ratio in 96-well round-bottomed plates. The supernatant
(100 µl) was collected after a 4-h incubation, and the radioactivity
was counted with in gamma counter. The percent-specific lysis was
calculated using the following formula: % specific lysis = 100 x (experimental release - spontaneous release)/(maximal release - spontaneous
release). In some experiments, 51Cr release assay
was performed with 50 µl of blocking antibodies against
H-2Kb (HB41/B896) and H-2Kd
(HB159) to examine class I restriction in cell killing [both HB41/B896
and HB159 were kindly provided by E. Gorelik (University of Pittsburgh
Cancer Institute, Pittsburgh, PA)].
Cell Sorting.
After preincubation with antimouse CD32/16 (2.4G2) mAb, liver MNCs were
stained with FITC-conjugated antimouse CD3
(145–2C11) and
phycoerythrin-conjugated antimouse NK1.1 (PK136). All mAbs were
obtained from PharMingen. For sorting, a FACS Vantage (Becton Dickinson
Co., San Jose, CA) was used. In experiments to evaluate CTL induction,
sorted NK cells or NKT cells in this way were used in place of
splenocytes harvested from SCID mice as described above.
Statistical Evaluation.
Statistical analysis was performed using a repeated measure ANOVA
method when comparing the in vivo tumor growth and cytotoxic
activity in an individual group. The unpaired two-tailed Student’s
t test was used to compare cytokine expression. Differences
were considered significant when the P was <0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression, and Induction
of a Potent and Specific CTL Response in Vitro.
, the cytolytic activity against MCA205 cells were induced 24 h
after culture in the presence of rIL-18 and gradually reduced
afterward. On day 4, the cytolytic activity of the effector cells
cultured with IL-18 was the same as those without IL-18. These results
indicate that the NK cytolytic activity against MCA205 cells was
rapidly enhanced following stimulation with IL-18.
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3
, tumor cell death was enhanced by the addition of rmIL-18 in a
dose-dependent manner only when both NK cells and IL-18 were included.
Thus, the immediate cytotoxic effect seems to be mediated by NK cells
stimulated with rmIL-18, consistent with the results shown in Fig. 1
,
and was not significantly affected by the presence of T cells or DCs
(Figs. 2
3)
.
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in the
culture was enhanced by rmIL-18 in a dose-dependent manner
(4380 ± 68 pg/ml, 3880 ± 115 pg/ml,
2972 ± 312 pg/ml, and 1097 ± 287 pg/ml
in the presence of rmIL-18 at 250 ng/ml, 50 ng/ml, 10 ng/ml and 0,
respectively). Although the potent CTL activity against MCA205 was
induced in this culture system by rmIL-18 in a dose-dependent fashion,
no cytolytic activity was observed against NK-sensitive YAC-1 cells
(Fig. 4A)
. These findings suggest that IL-18 enhances NK cytolytic
activity, promotes tumor cell destruction, and stimulates IFN-
production in the culture system used.
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. These results suggest that neither IL-2 nor IL-12
induces tumor-specific cytolytic T cells as efficiently as IL-18 does
in this coculture system under these conditions.
Specific Immunity Is Induced Only When All Components (NK Cells,
DCs, T Cells, and IL-18) Are Present without a Restriction of Direct
Contact among Other Cell Components in this Coculture System.
To examine the role of each individual cell type and cytokine in this
culture system to generate potent CTLs, we examined the activity of
CTLs induced in the culture system lacking an individual component.
Significant CTL activity was induced (P < 0.01 for all others) against MCA205 cells only when all factors were
included in the system (Fig. 5A)
. The cytolytic activity was not generated against YAC-1
cells (data not shown). Furthermore, the cytolytic activity was
abrogated when either NK cells or DCs were separated using the
transwell system, which allowed only the exchange of soluble factors
(Fig. 5B)
. These data indicate that the MCA205-specific CTLs
were induced only when all factors (NK cells, DCs, T cells, and IL-18)
were present in the absence of barrier and in direct contact with each
other.
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. Cytotoxicity of the effector cells was significantly
enhanced only against MCA205 cells but not against any other cell lines
(P < 0.01 for all). When the effector cells
were incubated with anti-MHC class I antibody, the cytolytic activity
generated was significantly inhibited (Fig. 6B)
. These
results strongly suggest that cytolytic activity of the effector cells
is MCA205 specific and the recognition of the target cells by the
effector cells is restricted MHC class I manner in this system.
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. This suggests that NK cells, but not NKT cells, play the central and
important role for induction of CTLs in this coculture system.
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, the cytolytic activity of CTLs obtained
in this coculture was observed against MC38 cells, but not NK-sensitive
YAC-1 cells. This cytolytic activity was enhanced by rmIL-18 in a
dose-dependent manner. Furthermore, IL-12 or IL-2 failed to generate
CTLs in this coculture at the level achieved with IL-18. These results
support that this approach can be effective for inducing potent CTLs
against weakly immunogenic tumors. Only modest cytolytic activity was
induced for the B16 melanoma (data not shown).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have previously reported that i.t. injection of adenovirus
vector-expressing IL-18 induces specific immunity but depletion of
asialo GM1-positive cells, primarily NK cells, which
substantially abrogate the IL-18-mediated antitumor effects.
Considering these findings, we hypothesized that tumor-specific
immunity could be efficiently promoted by first inducing tumor cell
destruction by NK cells stimulated with IL-18. To examine the role of
IL-18 in inducing specific immunity, we used an in vitro CCS
containing viable tumor cells, NK cells, DCs, and T cells as well as
cytokines, including IL-18, IL-2, or IL-12. The results of this CCS
suggest that IL-18 enhances NK activity, inducing death of viable tumor
cells that in turn seem to be acquired and processed by DCs to promote
CTL generation from näive T cells. As shown in Figs. 1
2
3
, tumor
cells were destroyed by NK cells activated with IL-18, and
tumor-specific CTLs were induced as an apparent consequence. This
NK-mediated destruction of tumor cells was enhanced by the presence of
IL-18 in a dose-dependent manner (Fig. 2
, A–E). As a
result, the specific cytolytic activity of CTLs against MCA205
fibrosarcoma cells was augmented by the presence of IL-18 in a
dose-dependent manner (Fig. 4A)
. As shown in Fig. 1
, NK
cells after coculture of 96 h exhibited marginal cytolytic
activity against tumor cells involved in the coculture. Thus, the
effector cells that showed cytolytic effect against tumor cells after
coculture were considered to be CTL effectors. Similar findings were
also observed when MC38 adenocarcinoma cells were used in the system
(Fig. 8A)
. These findings, indeed, suggest that tumor cell
destruction by NK cells is an important initiating event for inducing
tumor-specific CTLs.
To investigate the role of the individual components involved in this
system for generating MCA205-specific CTLs, we examined a CCS lacking
individual components. As shown in Fig. 3
, A–E, MCA205
cells were completely killed only when both rmIL-18 and NK cells were
present during culture. Presence of DCs in culture did not alter the
extent of MCA205 death and relevant lymphocyte proliferation. However,
the most potent and specific cytolytic activity was observed only when
the culture contained all components, including IL-18, NK cells, DCs,
and T cells. When any of the factors were missing in the culture, the
cytolytic activity was significantly reduced (P < 0.01 for all; Fig. 5A
). To analyze the role of the
direct contact of NK cells with tumor cells, we performed the CCS using
transwells, separating NK cells and their targets. As shown in Fig. 5B
, the cytolytic activity was greatly reduced when direct
contact was inhibited between NK cells and other cells components.
These results would indicate that the presence of NK cells play an
important role in inducing specific immunity against tumor cells.
To determine the role of NK and NKT cells in CCS for efficient and
rapid CTL induction, we sorted NK cells and NKT cells from liver MNCs.
As shown in Fig. 7
, potent cytolytic activity of the effector cells was
induced when NK cells were included in the CCS, but not when NKT cells
were used. Some reports have shown the results consistent with our
data. Local production of IFN- by NK cells, but not NKT cells, was
shown to be important for generating xenospecific CTLs
(23)
. Leite-de-Moraes et al.
(24)
also demonstrated that NKT cells could kill the Fas+
cells following stimulation with IL-18 and IL-12, but cannot do with
IL-18 alone. Thus, it seems that NK cells, but not NKT cells, play an
important role in supplying antigen to DCs by mediating tumor cell
death.
Furthermore, we examined the efficiency of other established and
IFN- inducing T-cell growth factors, IL-2 and IL-12, and compared
them with IL-18 in the induction of cytolytic T cells against MCA205
fibrosarcoma cells and MC38 adenocarcinoma cells, respectively. The
expression of IFN- including either IL-12 or IL-18 in the coculture
is 4869 pg/ml and 3315 pg/ml (when cocultured with MCA205) and 4844
pg/ml and 3751 pg/ml (when cocultured with MC38), respectively. These
data showed that the difference of the effect of IL-12 and IL-18 for
induction of IFN- was considered to be marginal. These results
suggest that neither IL-2 nor IL-12 induces tumor-specific cytolytic T
cells as IL-18 does in this CCS (Figs. 4B
and 8B)
. Effective and rapid generation of such potent cytolytic
activity against tumor cells, and potentially other targets, could be
quite useful for development of adoptive immunotherapies as well as for
generation of T cells important for antigen identification.
DCs play an important role as primary antigen-presenting cells to
initiate and maintain T-cell responses. We and others have shown that
DCs pulsed with tumor-associated antigens (25, 26, 27)
, tumor
lysates (28)
, or RNA (29
, 30)
allow selection
and/or activation of specific CTLs. DCs present exogeneous antigens on
MHC class I molecules to induce priming or tolerance of CD8+ cells
(31)
. As a source of the antigens, DCs phagocyte apoptotic
cells to provide antigenic peptides and necrotic cells to be matured
(32)
. In this study, we have demonstrated that DCs are an
important cell population in generating effective CTLs against tumor in
combination with IL-18 and NK cells in this CCS (Fig. 5)
. As shown in a
previous study (33)
, we have demonstrated here that CTL
induction against tumor cells was suppressed when DCs were absent (Fig. 5A)
or separated (Fig. 5B)
in the coculture well.
At the beginning of the coculture, the day 6 DC showed 85% of MHC
class II, 81% of CD80, 81% of CD86, and 68% of CD11c. Our data have
demonstrated that CD11c, CD80, and CD86 expression of DCs after
coculture with apoptotic cells or without coculture was not altered
within 48 h from the coculture, thereafter increased when
cocultured with apoptotic cells (34)
. Although the
addition of rmIL-18 does not directly affect surface markers of DCs
(data not shown), DC functions are affected by contact with tumor
cells. We have recently identified dynamic changes in chemokine
receptor expression (up-regulation of CCR7) on DCs following contact
with apoptotic tumor (34)
. Also, we demonstrated in this
study that effective CTLs were not generated when DCs were separated in
the coculture using transwell, showing that direct contact with other
elements in the coculture could be important for generating CTLs. Taken
together, IL-18 seems to induce more frequent and effective tumor cell
death through enhancing NK activity, enabling, in turn, DCs to serve as
effective antigen-presenting cells, and then inducing potent and
specific immunity. Thus, the implications are that IL-18/NK/DC plays a
critical inductive and interactive role in promoting tumor-specific
immunity. Furthermore, such T cells might be useful to be used in the
clinic for adoptive immunotherapy and the research for identification
of novel tumor associated antigens.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grant PO1-CA59371 (to
M. T. L., P. D. R., and H. T.), a Career Development Award from
the American Society of Clinical Oncology (to H. T.), and The Uehara
Memorial Foundation (Tokyo, Japan) Postdoctoral Research Fellowship (to
F. T.). 
2 To whom requests for reprints should be
addressed, at Department of Surgery and Bioengineering, The Institute
of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai,
Minato-ku, Tokyo, 108-8639, Japan. Phone: 81-3-5449-5345; Fax:
81-3-5449-5444; E-mail: tahara{at}ims.u-tokyo.ac.jp
3 The abbreviations used are: IL, interleukin; NK,
natural killer; DC, dendritic cell; i.t., intratumoral; CM, complete
medium; MNC, mononuclear cell; CCS, cooperative coculture system;
GM-CSF, granulocyte machrophage-colony-stimulating factor; r,
recombinant; mAb, monoclonal antibody.
Received 1/ 4/00. Accepted 7/ 5/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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production by T cells. Nature (Lond.), 378: 88-91, 1995.[Medline]
interferon induction found in the livers of mice causes endotoxic shock. Infect. Immun., 63: 3966-3972, 1995.[Abstract]
-inducing factor/IL-18 administration mediates IFN-- and IL-12-independent antitumor effects. J. Immunol., 160: 1742-1749, 1998.-inducing factor/interleukin-18 in mice bearing syngeneic Meth A sarcoma malignant ascites. Cancer Immunol. Immunother., 43: 361-367, 1997.[Medline]
inducing factor, interleukin-18 (IL-18). Gene Ther., 6: 808-815, 1999.[Medline]
production and activates IRAK and NF
B. Immunity, 7: 571-581, 1997.[Medline]
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) versus all others lacking an individual element
(**, P < 0.01 for all).
B, effector cells were obtained from coculture
separating either NK cells or DCs using a transwell system as described
in "Materials and Methods." On day 4 of coculture, effector cells
were collected and cytolytic activity was assessed against MCA205 cells
and YAC-1 cells at various E:T ratios. UC, upper chamber
separated cells. Data represent the mean ± SD of
cytotoxicity. There is a statistically significant difference between
the group with no separation and a group with either NK cells or DCs
separated (**, P < 0.01).
