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Immunology |
Divisions of Surgical Oncology [K. J. A., A. R., L. H. B., C. M. V., F. C. E., V. B. D., S. M., J. S. E.], Hematology/Oncology [A. R., J. A. G.], Pathology [S. D. N., P. S.], and Experimental Radiation Oncology [W. H. M.], University of California Los Angeles, Los Angeles, California 90095, and the Division of Molecular Immunology, Center for Biomedical Science, School of Medicine, Chiba University, Chiba 260, Japan [T. N., M. T.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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14 T-cell receptor and CD1 knockout mice, also failed
to abrogate the response to AdVmIL-12. Histological and
immunohistochemical studies of AdVmIL-12-treated tumors showed
extensive inhibition of neovascularization and a marked decrease in
factor VIII-stained endothelial cells. Our studies indicate that
the antitumor response of AdVmIL-12 is independent of direct cytotoxic
cellular immunity (specifically, the function of NK T
cells) and suggest that the initial mechanisms of
AdVmIL-12-mediated tumor regression involve inhibition of angiogenesis. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, which has been identified as an important downstream mediator
of the IL-12 antitumor response (5, 6, 7, 8, 9, 10, 11, 12, 13)
. Although the
immune mechanisms stimulated by IL-12 have been studied extensively,
there is considerable controversy regarding the specific mechanisms by
which IL-12 inhibits tumor growth. Investigations into the precise
molecular and cellular events that mediate tumor regression after IL-12
treatment have been fueled by the demonstration of higher in
vivo efficacy compared with other antitumor cytokines
(13, 14, 15)
.
Studies examining the antitumor mechanisms of IL-12 have
implicated various cellular mediators including CD8 T cells, NK cells,
and NK T cells (5
, 13
, 16
, 17)
. NK T cells are a
recently described T-cell subset that recognizes a limited array of
peptide and nonpeptide antigens presented by the nonpolymorphic
MHC-like molecule CD1 (18
, 19)
. NK T cells express a
limited repertoire of TCR genes [mostly
V14 in mice and V24
in humans (19
, 20)
]. NK T cells readily respond to
cytokine stimulation (IL-12) and to ligand-mediated activation
[-galactosylceramide (21
, 22)
].
-Galactosylceramide, a glycolipid with the unique ability to
specifically activate V14 NK T cells, has
been shown to stimulate NK T cell-mediated antitumor responses
that are dependent on CD40-CD40 ligand and B7-1/CD28 interactions and
on perforin-mediated cytotoxicity (22, 23, 24)
. In one tumor
model, there was an absolute requirement of NK T cells for
IL-12-mediated antitumor responses, and perforin-dependent cytotoxicity
was suggested to mediate the response (25)
. In
another study, NK T cells were shown to be required for both
IL-12-mediated IFN- production and tumor-associated cytotoxicity
(26)
.
Cytokine gene therapy is becoming a promising weapon in the armamentarium against cancer (27) . Adenoviral-based cytokine gene therapy has many advantages over other forms of cytokine delivery (28) . Adenoviral vectors allow local, high-efficiency, but transient transgene expression, generating high-level but self-limited cytokine production in treated tumors. Adenoviral vectors are capable of transducing nondividing cells, increasing the number of transduction targets in a heterogeneously growing population of tumor cells.
We tested AdVmIL-12 in a murine HCC model. Our goals were to test the efficacy of AdVmIL-12 against hepatocellular tumors and to elucidate the mechanisms mediating this response. We were specifically interested in determining the requirement for NK T cells and in identifying possible non-immune mechanisms that contribute to the response. Our studies demonstrate that cytotoxic immune effector mechanisms are not required for AdVmIL-12-mediated antitumor responses in murine HCC models and suggest that AdVmIL-12 mediates tumor regression by inhibition of angiogenesis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice and Cell Lines.
The 6–9-week-old C57BL/6, CB17 SCID, CB17 SCID/beige, and
C129/RAG-2-immunodeficient mice were bred and maintained in a specific
pathogen-free colony at the Experimental Radiation Oncology Animal
Facility at UCLA. NK cell-defective C57BL/6/beige,
C57BL/6/perforin-knockout, and C57BL/6/CD28-knockout mice were obtained
from The Jackson Laboratory (Bar Harbor, ME).
C57BL/6/V14 NK T cell-deficient mice were
the generous gift of Dr. Masaru Taniguchi (Chiba University, Chiba,
Japan). C57BL/6/CD1-deficient mice were the generous gift of Dr. Luc
Van Kaer (Vanderbilt University, Nashville, TN). All mice obtained from
outside sources were bred and housed in a quarantine area in the
specific pathogen-free colony at UCLA. All animal studies were
conducted in accordance with the UCLA Animal Care Policy as prescribed
by the Chancellor’s Animal Research Committee. BWIC3 and its
derivative cell line Hepa 1-6 are well-characterized murine HCC lines,
and B16 is a well-characterized murine melanoma cell line (31
, 32)
. All cell lines were obtained from the American Type Culture
Collection (Manassas, VA). Hepa 1-6 was maintained in vitro
in RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD) with 10% FCS
(Gemini Products, Calabasas, CA) and 1% (v/v) penicillin,
streptomycin, and fungizone (Gemini Products; complete media). BWIC3
and B16 were maintained in vitro in DMEM (Life Technologies,
Inc.) complete media. Human embryonal kidney 293 cells provided by Dr.
Kohnosuke Mitani (UCLA, Los Angeles, CA) were passaged similarly
in DMEM media. Tumors for in vivo studies were maintained
through serial passage of single cell suspensions in the subcutis of
mice as described previously (33)
. Tumors used to generate
single cell suspensions were resected from their s.c. position in
euthanized mice, mechanically dispersed, and enzymatically digested for
2 h with DNase I (0.1 mg/ml; Sigma) and collagenase D (1 mg/ml;
Boehringer Mannheim, Indianapolis, IN) in 40 ml of AIM-V media (Life
Technologies, Inc.). The single cell suspensions were then washed three
times with PBS and either injected into mice to maintain in
vivo tumor or used for in vitro studies.
Treatment and Monitoring of Established Tumors.
All tumors were established in the dorsal flanks of mice by s.c.
injection of serially passaged cells (4–5 x 106 cells/injection) suspended in PBS. After 1–2
weeks of in vivo growth, tumor-bearing mice were randomized
into groups with size-matched tumors (average mean tumor volume,
40–200 mm3
) and treated intratumorally
with either AdVmIL-12 or PBS as a control. As a positive control,
tumor-bearing wild-type mice were treated in parallel with either
AdVmIL-12 or PBS. Tumor growth was assessed by calipers, taking the
mean of two perpendicular diameters at the time of intratumoral
treatment and every 3–4 days thereafter. Tumor volume was estimated by
the formula 4/3 
r3
.
In Vivo Depletion of CD4 and CD8 T-Cell Subsets.
In vivo monoclonal antibody ablation of CD8 (clone 2.43;
ATCC TIB 210) or CD4 (clone GK1.5; ATCC TIB 207) T-cell subsets was
performed by i.p. injection on days 5, 3, and 1 before tumor
inoculation and every 7 days thereafter (0.5 mg
antibody/mouse/injection). Antibody suspensions were purified from
hybridoma supernatants by passage through protein G columns according
to the manufacturer’s instructions (Pierce, Rockford, IL). Eluted
immunoglobulins were dialyzed against PBS and stored at 4°C in 1
mg/ml suspensions. CD4 and CD8 T-cell depletion was confirmed by flow
cytometric analysis of splenocytes from depleted mice on the day of
tumor challenge.
Splenocyte Harvest.
Spleens were harvested from euthanized mice, mechanically dispersed,
and treated with ACK buffer (0.15 M
NH4Cl, 10 mM
KHCO3, and 0.05 mM NaEDTA) to deplete
RBCs. The RBC-depleted splenocyte populations were then resuspended and
washed three times in PBS. Washed cells were then used for additional
studies.
Flow Cytometric Analysis.
Splenocyte populations and tumor single cell suspensions were obtained
as described above. The following preconjugated monoclonal antibody
antibodies were used: (a) anti-CD4-FITC (Caltag, Burlingame,
CA); (b) anti-CD8-PE (PharMingen, San Diego, CA);
(c) anti-NK1.1-PE (PharMingen); and (d)
anti-CD3-FITC (PharMingen). For NK T cell identification, cells
were double stained with anti-NK1.1 and anti-CD3. After harvest,
splenocytes and tumor samples were resuspended and washed twice in cold
PBS with 2% FCS. Antibody staining of splenocytes and tumor samples
(5 x 105 cells/sample) was
performed in 5–10 µl of antibody solution for 1 h. At the end
of the incubation period, the stained populations were washed three
times in cold PBS with 2% FCS and then resuspended in 1%
paraformaldehyde fixative. Flow cytometric analysis was performed on a
FACScan machine (Becton Dickson, San Jose, CA).
Histological Analysis.
For histological studies, AdVmIL-12-treated and PBS-treated tumors were
harvested, fixed in formalin, embedded in paraffin, sectioned, stained
with H&E, and examined under a light microscope. For
immunohistochemical staining, formalin-fixed paraffin-embedded blocks
were sectioned at 2 µm and placed on positively charged slides.
Slides were baked at 60°C for 30 min, deparaffinized in xylenes, and
rehydrated to tap water through graded ethanols. Slides were then
treated with 3% hydrogen peroxide in methanol for 10 min to quench
endogenous peroxidase activity. The sections were then subjected to
heat-induced antigen retrieval in 0.01 M citrate buffer (pH
6.0) for 25 min in a steamer (Black and Decker) and allowed to cool for
15 min before transferring to 0.01 M PBS. Immunostaining
was performed on a DAKO Autostainer (DAKO Corp., Carpinteria,
CA) using the Envision+ Rabbit Peroxidase Detection System (DAKO
Corp.). The heat-treated slides were incubated with anti-factor VIII
(rabbit anti-von Willebrand factor; DAKO Corp.) primary antibody
(diluted 1:200) for 45 min. After primary antibody treatment, the
slides were rinsed in PBS and incubated with Envision+ anti-rabbit
peroxidase for 30 min. The antibody was then visualized with
diaminobenzidine as the chromogen and counterstained with Harris
hematoxylin.
Quantification of Tumor Vascularity.
Eleven days after treatment, AdVmIL-12-treated and PBS-treated tumors
were harvested, fixed in formalin, and stained with H&E. Slides were
then examined under a light microscope, and the number of blood vessels
per x200 field was counted. Blood vessel counts/field were then
averaged.
Statistical Analysis.
Student’s t test was performed to interpret the
significance between the final tumor volumes of animals treated with
AdVmIL-12 and those treated with PBS. Two-sided Ps reflect
individual comparisons. Tumor growth and vascularity data represent the
mean ± SE.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in which a single intratumoral injection of 1 x 109 pfu of AdVmIL-12 into BWIC3 tumors (average
tumor volume, 200 mm3
) induced a rapid antitumor
response. Increasing the viral dose resulted in more profound antitumor
responses, but doses above 6 x 109 pfu (as a single injection) were uniformly
associated with systemic toxicity and death. These general observations
were drawn from a total of 24 studies using 178 mice. Intratumoral
AdVmIL-12 mediated partial regression in 90% of mice and complete
regressions in 10% of mice. Antitumor responses to intratumoral
AdVmIL-12 treatment were similar in BWIC3 and Hepa 1-6 tumors. All mice
that achieved complete tumor regressions were protected from a
rechallenge with the same tumor at 8 weeks, suggesting the generation
of tumor-specific immunity.
|
. Analysis of the total number of cells in the tumor sample revealed a
dramatic increase in the number of CD4, CD8, NK, and NK T cells
and a decrease in the number of tumor cells in the AdVmIL-12-treated
tumors as compared with the control.
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. AdVmIL-12 also retained potent
antitumor activity in NK cell-defective beige mice (Fig. 3C)
, in T-cell- and B-cell-deficient SCID (Fig. 3D)
and RAG-2-deficient (Fig. 3E)
mice,
and in SCID/beige (Fig. 3F)
mice deficient in
functional T, B, and NK cells. Thus, T, B, or NK cells are not required
for AdVmIL-12-mediated antitumor activity.
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14 NK T-cell knockout
mice. The lack of V14 (Fig. 4A)
or CD1-dependent (Fig. 4B)
NK T cells
failed to abrogate the response to intratumoral AdVmIL-12 treatment of
Hepa 1-6 tumors. Therefore, AdVmIL-12-mediated antitumor responses
appear to be independent of the effector functions of NK T cells.
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. Thus, common immune cell costimulatory
and cytotoxic effector mechanisms are not required for the generation
of AdVmIL-12-mediated antitumor responses.
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. H&E-stained AdVmIL-12-treated tumors show decreased blood
vessel formation and extensive cellular necrosis compared with an
abundance of blood vessel infiltration and rich tumor growth in the
PBS-treated tumors (Fig. 6A)
. Immunohistochemical staining
of AdVmIL-12-treated tumors using a monoclonal antibody to factor VIII,
an endothelial cell marker, revealed decreased endothelial cell
infiltration and blood vessel density in AdVmIL-12 treated tumors (Fig. 6B)
. This is in marked contrast with staining of PBS-treated
tumors (Fig. 6B)
, which clearly demonstrates neovascular
endothelial cell staining with anti-factor VIII antibody. The marked
decrease in neovascularization of AdVmIL-12-treated tumors suggests
that antiangiogenic mechanisms initiate the antitumor responses
produced by intratumoral AdVmIL-12.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Various investigators have explored the antitumor effect of direct
intratumoral AdVmIL-12 treatment of established tumors. Transgenic mice
developing spontaneous breast tumors given a single intratumoral
injection of AdVmIL-12 showed tumor regressions in 75% of treated mice
(36)
. Notably, IFN- was produced in significant
quantities within IL-12-treated tumors. Complete responders rejected a
subsequent rechallenge, suggesting the development of immunological
memory. Although it was not directly explored in the current studies,
the mechanism of this immunological memory may be cross-presentation of
tumor-derived antigenic epitopes released by dying tumor cells, leading
to a delayed immunological response. In another study, intratumoral
AdVmIL-12 was shown to significantly increase the survival of mice
bearing intrahepatic MCA-26 tumors in a metastatic colon cancer model
(37)
. A single intratumoral injection of AdVmIL-12 was
able to generate tumor regressions in a dose-dependent manner in mice
bearing murine bladder tumors, with mice receiving 1 x 109 pfu of virus consistently achieving complete
responses (16)
. Complete responders rejected a subsequent
rechallenge. These antitumor responses were shown to be systemic by
experiments in which contralateral uninjected tumors regressed after
intratumoral AdVmIL-12 treatment of similar tumors growing in the
opposite flank. Intratumoral AdVmIL-12 was also shown to generate tumor
regressions in a s.c. colon cancer (CT-26) model with 76% of treated
mice achieving a complete response, and complete responders also
rejected a subsequent rechallenge (38)
. Nasu et
al. (39)
examined the effect of intratumoral
AdVmIL-12 on the growth of orthotopically placed prostate tumors and
showed significant tumor regressions, decreased numbers of
pre-established pulmonary metastases, and prolonged survival in
AdVmIL-12-treated mice.
NK T cells have been proposed as a requisite cell type in
IL-12-mediated tumor regression. Kawamura et al.
(26)
showed that IL-12-stimulated liver and spleen
mononuclear populations from mice with a targeted gene mutation in CD1
were less cytotoxic against YAC-1 and P815 tumor cell lines than
against wild-type mononuclear cells after i.p. injection of
recombinant IL-12. CD1 is a MHC-like molecule required for positive
selection of CD1-dependent NK T cells during immune cell ontogeny
(19)
. This group also showed that serum IFN- levels
were lower in CD1-deficient mice than in wild-type mice after i.p.
IL-12. These data are contrary to our data that show retention of
AdVmIL-12 antitumor activity in the same CD1-deficient mice. Cui
et al. (25)
and Taniguchi et al.
(40)
examined the effect of recombinant IL-12 in mice with
a targeted mutation in the V14 TCR gene that
preferentially lack V14 NK T cells, the
predominant NK T-cell subtype. Their studies showed that the
antitumor effect of i.p. recombinant murine IL-12 against B16 was
abrogated in V14 NK T cell-deficient
mice and was restored in RAG mice genetically engineered to
preferentially develop V14 NK T cells but lack
T, B, and NK cells (40)
. The authors suggested that there
is an absolute requirement for NK T cells to mediate the antitumor
effects of IL-12 because IL-12 efficacy was abrogated in
V14 NK T cell-deficient mice and
completely restored in mice harboring V14
NK T cells as their only lymphoid cell type. This in
vivo data are contrary to our data, although we used the same
V14 NK T cell-deficient mice used in
their study. In vitro studies showed that concanamycin A, a
known inhibitor of perforin-mediated killing, could inhibit
V14 NK T-cell cytotoxicity (41)
.
To test the hypothesis that V14 NK T
cells require perforin to mediate cytotoxic antitumor responses to
IL-12, we used perforin knockout mice and were consistently able to
generate potent antitumor responses to intratumoral AdVmIL-12. The
discrepancy between these two reports and our data may be explained by
a difference in study design and IL-12 delivery. Their studies, which
examined the effect of recombinant IL-12 delivered systemically,
differed dramatically from our studies using AdVmIL-12 delivered
intratumorally with respect to the amount and duration of IL-12
presence in the treated tumors. The high-level of IL-12 produced in
tumors treated intratumorally with AdVmIL-12 may directly initiate
antiangiogenic mechanisms, obviating the need for specific cellular
subsets to generate tumor regressions. The absolute requirement of
NK T cells for IL-12 efficacy is not supported by our data.
NK T cells have the unique ability to respond to nonpeptide
antigens, namely gylcolipids, through their invariant TCR (22
, 42)
. The glycolipid, -galactosylceramide, when presented in
the context of the MHC-like molecule CD1, has been shown to
specifically activate V14 NK T cells
(23
, 43)
. This -galactosylceramide-mediated activation
was shown to be dependent on B7-1/CD28 interactions (23)
.
To test the possibility that V14 NK T
cells are stimulated in vivo by IL-12 and generate antitumor
responses through glycolipid/CD1-dependent mechanisms, we used mice
with a targeted gene mutation in CD28. In our studies, AdVmIL-12
effectively mediated antitumor responses in CD28 knockout mice, further
supporting the NK T cell independence of the antitumor effects of
IL-12.
Antiangiogenesis is an antitumor mechanism that nonspecifically impairs tumor growth by limiting access of vital nutrients and metabolites to rapidly growing tumors, leading to cellular apoptosis and tumor regression (44) . Inhibition of angiogenesis is suggested in established tumors by the identification of poor tumor neovascularization on histological examination and by demonstrating an objective decrease in endothelial cell infiltration into tumors using immunohistochemistry and specific endothelial cell markers (45, 46, 47, 48) . Our studies show that treatment of established hepatomas with AdVmIL-12 results in marked inhibition of neovascularization with a distinct decrease in the number of blood vessels and factor VIII-staining endothelial cells in treated tumors.
The antiangiogenic effects of IL-12 have been well documented.
One of the earliest reports of the antiangiogenic effects of IL-12 was
by Voest et al. (49)
, who showed that
recombinant murine IL-12 significantly inhibited corneal
neovascularization in C57BL/6, SCID, and beige mice. IFN-
antibody treatment abolished this effect, underscoring the importance
of IFN- in IL-12-dependent antiangiogenesis. Notably, as in our
studies, IL-12 was effective in SCID and beige mice, which
argues for a similar antiangiogenic mechanism in our model. Since then,
many reports have more clearly defined the mechanism by which IL-12
inhibits angiogenesis. Sgadari et al. (50)
and
Angiolillo et al. (51)
showed that IL-12
induces the expression of IP-10 from mouse splenocytes, a known
inhibitor of angiogenesis. In this study anti-IFN- antibody
completely abolished the antiangiogenic effect of IL-12, whereas
antibody to IP-10 resulted in significant but partial inhibition of
IL-12-induced antiangiogenesis. These data suggest that IFN- is a
requisite mediator of IL-12-mediated antiangiogenic effects, with IP-10
being an important downstream regulator of IFN--dependent
antiangiogenesis. In another study, Dias et al.
(8)
demonstrated, in a murine breast cancer model, that
IL-12 down-regulates vascular endothelial growth factor levels in
treated tumors and that this down-regulation is likely mediated through
IFN- produced by IL-12 stimulation. This study also showed that
IL-12 treatment was associated with a marked decrease in matrix
metalloproteinase 9, an enzyme important in endothelial cell migration
and angiogenesis, and an increase in the expression of tissue inhibitor
of metalloproteinase 1, a known inhibitor of matrix metalloproteinase 9
(52)
. In a recent study by Gee et al.
(12)
, IL-12 was shown to inhibit the growth of K1735
melanoma tumors in C3H/HeN mice in an IFN--dependent manner. Using
in vivo Matrigel assays, IFN- blockade was shown to
abrogate IL-12-induced inhibition of neovascularization in K1735
implants. Interestingly, IL-12-induced IFN- was also shown to
produce direct cellular apoptosis independent of the hypoxia-related
apoptosis associated with antiangiogenesis. These studies suggest that
IFN- produced intratumorally in response to IL-12 treatment is
capable of inhibiting tumor growth through two independent mechanisms:
(a) directly through induction of tumor cell apoptosis; and
(b) through inhibition of angiogenesis producing cellular
hypoxia and hypoxia-related apoptosis.
In conclusion, our studies show that in vivo transduction of growing murine hepatocellular tumors with AdVmIL-12 leads to profound tumor growth regression. This AdVmIL-12-mediated regression could not be abrogated in models designed to inhibit NK T-cell function and other cytotoxic immune mechanisms. AdVmIL-12 treatment was associated with a rapid infiltration of lymphoid cells and clearly demonstrable inhibition of tumor-associated neovascularization. These data clearly demonstrate that NK T cells are not absolutely required for IL-12-mediated antitumor responses and suggest that mechanisms independent of direct cellular cytotoxicity initiate the effects of IL-12 demonstrated in murine HCC models.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Presented in part at the Owen Wagensteen
Surgical Forum of the 84th Annual Clinical Congress of the
American College of Surgeons, October 26, 1998, Orlando, Florida. This
study was supported in part by NIH/National Cancer Institute Grants RO1
CA79976, RO1 CA77623, T32 CA75956, and K12 CA76905; the Monkarsh Fund;
and the Stacey and Evelyn Kesselman Research Fund. 
2 To whom requests for reprints should be
addressed, at Division of Surgical Oncology, Room 54–140 Center for
the Health Sciences, University of California Los Angeles School
of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095-1782. Phone:
(310) 825-2644; Fax: (310) 825-7575; E-mail: jeconomou{at}mednet.ucla.edu
3 The abbreviations used are: IL-12, interleukin
12; HCC, hepatocellular carcinoma; SCID, severe combined
immunodeficient; RAG-2, recombinase activating gene 2; NK, natural
killer; AdVmIL-12, adenoviral vector expressing murine IL-12; TCR,
T-cell receptor; pfu, plaque-forming unit(s); UCLA, University of
California Los Angeles; IP-10, IFN-inducible protein 10.
Received 5/18/00. Accepted 9/21/00.
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REFERENCES |
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Y. Nishioka, H. Wen, K. Mitani, P. D. Robbins, M. T. Lotze, S. Sone, and H. Tahara Differential effects of IL-12 on the generation of alloreactive CTL mediated by murine and human dendritic cells: a critical role for nitric oxide J. Leukoc. Biol., May 1, 2003; 73(5): 621 - 629. [Abstract] [Full Text] [PDF]
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