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-Inducible Protein-10 Is Essential for the Generation of a Protective Tumor-Specific CD8 T Cell Response Induced by Single-Chain IL-12 Gene Therapy1
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Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037;
Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy and Immunology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129;
University of California, Cancer Center 0961, La Jolla, CA 92093; and
Charité Children’s Hospital, Berlin, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible protein 10
(IP-10). This was established by in vivo depletion of IP-10 with mAbs
in mice vaccinated against NXS2 neuroblastoma by gene therapy with a
linearized, single-chain (sc) version of the heterodimeric cytokine
IL-12 (scIL-12). The efficacy of IP-10 depletion was indicated by the
effective abrogation of scIL-12-mediated antiangiogenesis and T cell
chemotaxis in mice receiving s.c. injections of scIL-12-producing NXS2
cells. These findings were extended by data demonstrating that IP-10 is
directly involved in the generation of a tumor-protective
CD8+ T cell-mediated immune response during the early
immunization phase. Four lines of evidence support this contention:
First, A/J mice vaccinated with NXS2 scIL-12 and depleted of IP-10 by
two different anti-IP-10 mAbs revealed an abrogation of
systemic-protective immunity against disseminated metastases. Second,
CD8+ T cell-mediated MHC class I Ag-restricted tumor cell
lysis was inhibited in such mice. Third, intracellular IFN-
expressed by proliferating CD8+ T cells was substantially
inhibited in IP-10-depleted, scIL-12 NXS2-vaccinated mice. Fourth,
systemic tumor protective immunity was completely abrogated in mice
depleted of IP-10 in the early immunization phase, but not if IP-10 was
depleted only in the effector phase. These findings suggest that IP-10
plays a crucial role during the early immunization phase in the
induction of immunity against neuroblastoma by scIL-12 gene
therapy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-12 is known to have multifunctional activities (2),
including immunomodulation (1, 3, 4, 5, 6, 7) and antiangiogenesis
(8, 9, 10). The mode of action responsible for the antitumor
effects induced by local scIL-12 includes inhibition of primary tumor
growth (11) as well as inhibition of metastasis. IL-12 and
its downstream mediator, IFN- can elicit diverse immunomodulatory
effects because of acquired and innate immune mechanisms.
IL-12-mediated innate immune responses include stimulation of NK cell
cytotoxicity and subsequent activation of neutrophils and macrophages.
This in turn leads to the production of superoxides and nitric oxide,
which are involved in mechanisms that were shown to control tumor
growth in animal models (12, 13, 14, 15). However, the eradication
of disseminated metastasis requires adaptive antitumor immune
responses, which are initiated by IL-12 via induction of a
CD8+ T cell-mediated immune response. This is
achieved by augmenting cytotoxicity of primed
CD8+ T cells or Th1 differentiation of naive
CD4+ T cells that can subsequently provide help
for tumor-specific priming of CTLs. A third mechanism of action of
IL-12 is provided by the inhibition of tumor-induced
neovascularization, i.e., antiangiogenesis. Experiments in vitro and in
vivo have documented that the antiangiogenic effects of IL-12 are
indirect and require the participation of IFN- which, in turn,
stimulates secretion of IFN--inducible protein 10 (IP-10). This
chain of events identified IP-10 as a key player in IL-12-mediated
antiangiogenesis (8, 10, 16).
IP-10 is a CXC chemokine that has been shown to induce chemotaxis of activated T cells and to inhibit angiogenesis (17, 18, 19). It is produced by activated monocytes, fibroblasts, endothelial cells, epithelial cells, and keratinocytes. IP-10 binds to a seven-transmembrane G protein-coupled receptor, CXCR3, expressed on activated T cells, leading to chemotaxis (20). IP-10 has two potential antitumor effects: antiangiogenesis and immunomodulation, similar to what has been described for IL-12. These observations stimulated a number of investigators to determine the role of IP-10 in IL-12-mediated antitumor effects focusing primarily on the growth of primary tumors in naive mice and the inhibition of neovascularization or chemotaxis (3, 18, 19, 21).
Here, we demonstrate for the first time that IP-10 is a key player in mediating systemic tumor-protective immunity induced by scIL-12 gene therapy by directly affecting Th1-type CD8+ T cells and by playing a crucial role in the early immunization phase of vaccination with scIL-12.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The construction of scIL-12 was done as described previously (1). Briefly, the cDNAs encoding the p35 and p40 chains of mIL-12 were generated from Con A-stimulated mouse splenocytes by RT-PCR. The cDNA for the scIL-12 fusion protein was constructed by linkage of p35 and p40 subunits with a synthetic linker. To assure secretion in eukaryotic cells, this construct was joined at its 5' SmaI site with the 3' BalI site of a p24 leader sequence, thereby replacing the amino-terminal arginines of p35 with two glycines downstream from the cleavage site. After introduction into the pBK-CMV vector (Stratagene, La Jolla, CA) using the NotI and XhoI restriction sites, NXS2 cells were transfected and stable clones selected in the presence of 500 µg/ml G418 (Sigma, St. Louis, MO).
The mIL-12 protein content produced by NXS2 cells was measured by an
ELISA for mIL-12-p70 (Genzyme, Cambridge, MA). Two NXS2 clones
secreting either low (0.5 ng/106 cells/24 h) or
high levels (0.7 ng/106 cells/24 h) of scIL-12
were chosen for vaccination experiments. The specific IL-12 activity of
the scIL-12 construct was one-sixth that of the mIL-12 standard
(Hoffmann-La Roche, Nutley, NJ), as determined by its ability to induce
mIFN- after incubation with splenocytes, as described previously
(1). One NXS2 clone transduced with the empty vector was
used for control experiments. All clones revealed the same
proliferation characteristics in vitro as NXS2 wild-type cells.
Cell lines and neuroblastoma model
Syngeneic female A/J, C.B-17 scid/scid and C.B-17 scid/beige mice were obtained at 6–12 wk of age from The Jackson Laboratory (Bar Harbor, ME). They were housed in the pathogen-free mouse colony at our institution, and all animal experiments were performed according to the National Institutes of Health guide for "The Care and Use of Laboratory Animals." The murine NXS2 wild-type, NXS2 pBK-CMV, and NXS2 scIL-12 neuroblastoma cell lines were cultured and used for vaccination experiments, as described previously (1). Briefly, primary tumors were induced by s.c. injection of 2 x 106 NXS2 pBK-CMV cells and their growth compared with that of NXS2 scIL-12 cells producing high and low amounts of scIL-12. Tumor growth was determined by microcaliper measurements, and tumor volume was calculated according to the formula 1/2 (length) x (width)2. Liver metastases were induced by lateral tail vein injection of 5 x 104 NXS2 wild-type cells, and mice were sacrificed for evaluation after 28 days.
Determination of tumor-induced angiogenesis in Matrigel
Matrigel (Becton Dickinson Labware, Bedford, MA), a product derived from the Engelbreth-Holm Swarm tumor (22) consisting of laminin, collagen IV, heparin sulfate, proteoglycan, and nidogen/entactin, liquefies at 4°C and rapidly polymerizes at 37°C following s.c. injection. Thus, Matrigel plugs were induced by s.c. injection of a mixture of liquefied Matrigel (0.4 ml) and 1 x 105 NXS2 pBK-CMV cells or NXS2 scIL-12 cells in the presence or absence of 0.1 ml of anti-IP-10 mAbs (1F11, 1B9), anti-IP-10 antiserum, or normal serum as a control. Mice were sacrificed on day 6, after which Matrigel plugs were harvested and weighed. Tumor-induced angiogenesis was quantified by determination of the hemoglobin content in Matrigel plugs by the method described by Drabkin and Austin (23). Briefly, Matrigel plugs were minced and liquefied in 0.3 ml of PBS at 4°C overnight. Subsequently, hemoglobin content was determined by incubation of 0.1 ml of Matrigel with 1 ml of Drabkin reagent for 15 min at room temperature. Insoluble tissue was removed by centrifugation (14,000 x g for 5 min) and the concentration of hemoglobin calculated according to a calibration curve obtained from serial dilutions of a hemoglobin standard (Sigma). Hemoglobin (Hb) concentrations and weights of Matrigel plugs were used to calculate mg Hb/g Matrigel.
In vivo depletion of mIP-10 and mIFN-
Two hamster anti-mouse IP-10 mAbs (1F11 and 1B9) have been
generated by standard techniques. The specificity of these mAbs was
tested in direct ELISA and by immunoblot with other available mouse
chemokines, including monokine induced by IFN- (Mig), KC, stromal
cell-derived factor-1, eotaxin, macrophage-inflammatory protein
(MIP)-1
, MIP-1
, and monocyte chemoattractant protein-5.
Both mAbs were highly specific for mIP-10 and did not cross-react with
other murine chemokines tested. The hybridomas have been adapted to
serum-free medium and grown in CellMax cartridges (Cellco, Kensington,
MD), and the mAbs were purified by protein G affinity chromatography.
Purified 1F11 and 1B9 IgG were both capable of neutralizing
mIP-10-induced chemotaxis and calcium flux responses of mCXCR3
transfected 300–10 cells (24). For in vivo depletion of
IP-10 in Matrigel experiments, 250 µg of the monoclonal
anti-IP-10 Abs 1F11 or 1B9 were added to the Matrigel plugs,
followed by i.p. injection of another 250 µg of these Abs on days 0,
1, 2, and 4. In addition to the mAbs, polyclonal rabbit anti-mouse
IP-10 serum, kindly provided by J. M. Farber (National Institutes
of Health, Bethesda, MD) was used in Matrigel experiments. For this
purpose, 0.1 ml of anti-IP-10 antiserum was added to liquefied
Matrigel followed by i.p. injection of 0.3 ml of antiserum on days 0,
1, 2, and 4, similar to 1F11 and 1B9 mAbs.
In vaccination experiments, 250 µg of anti-IP-10 mAb was added to the tumor inoculum and injected on days 3, 8, and 17 i.p. For depletion in the immunization phase, 250 µg of anti-IP-10 mAb were injected i.p. on day 0 followed by 100 µg on days 2 and 4. Depletion of IP-10 in the early effector phase was accomplished by 250 µg of anti-IP-10 injected i.p. on day 7 followed by 100 µg of mAb i.p. on days 10 and 13 and in the late effector phase by 250 µg of anti-IP-10 i.p. on day 10 followed by 100 µg of mAb on days 13 and 16. Rabbit anti-mouse IP-10 serum was not used in this set of experiments because of lethal serum reactions in all animals observed after the third injection.
In experiments with hamster mAbs and rabbit antiserum, equal amounts of
normal hamster serum (Harlan Bioproducts, Indianapolis, IN) or normal
rabbit serum (Dako, Carpinteria, CA) were applied as negative controls,
respectively. In vivo depletion of mIFN- was accomplished by
addition of 0.5 mg of rat anti-mouse IFN- mAb to the tumor
inoculum and additional i.p. injections of 1 mg each on days 3, 7, and
18. Rat anti-mouse IFN- mAb was obtained from ascites generated
with the R4-6A2 hybridoma cell line, kindly provided by S. Webb (The
Scripps Research Institute, La Jolla, CA).
Histology and immunohistochemistry
Tumor-induced angiogenesis was determined by histological analysis of neovascularization and tumor cell morphology in Matrigel experiments. To this end, Matrigel plugs containing NXS2 pBK-CMV cells or NXS2 scIL-12-producing cells were harvested in the presence or absence of IP-10 depletion on day 6, fixed in 10% buffered formalin, sectioned, and stained with hematoxylin and eosin. The immunophenotype of NXS2 scIL-12-induced leukocyte infiltrations was determined 6 days after s.c. inoculation of 2 x 106 NXS2 scIL-12 in the presence or absence of IP-10 depletion and compared with NXS2 pBK-CMV controls. Frozen sections of tumor tissue were fixed in cold acetone for 10 min followed by removal of endogenous peroxidase with 0.03% H2O2 (30 min, room temperature). Nonspecific binding was blocked with 10% species-specific serum in 1% BSA/PBS. Biotinylated anti-mouse CD45 mAb (BD PharMingen, La Jolla, CA), biotinylated CD8, CD4, Mac1, and NK cell-specific rabbit anti-asialo GM1 antiserum (Wako, Richmond, VA) were diluted to 10 µg/ml in 10% goat serum (1% BSA/PBS, pH 7.4) and overlaid onto serial sections. Slides were incubated in a humid chamber for 30 min followed by the application of biotinylated goat anti-rabbit Ab onto slides that were preincubated with anti-asialo GM1 antiserum for 10 min. Streptavidin-labeled HRP was added and the slides incubated for 10 min, followed by substrate development with the AEC (amino ethyl carbazole) kit (Vector Laboratories, Burlingame, CA) and a hematoxylin nuclear counterstain. All incubations were followed by three wash steps with PBS (pH 7.1).
RT-PCR for the detection of IP-10
Isolation of mRNA from tumor tissues and synthesis of cDNA was performed, as described previously (25). The amplification of a 302-bp fragment of mIP-10 was performed in a 25-µl PCR mixture consisting of 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 0.2 mM deoxynucleotide triphosphate, 2 mM MgCl2, 2.5 U Taq Polymerase (Life Technologies, Rockville, MD), and 0.5 µM sense and antisense oligonucleotide primers. PCRs were conducted in the Mastercycler gradient (Eppendorf, Hamburg, Germany) for 30 cycles (96°C, 15 s; 62°C, 30 s; 72°C, 90 s). The primer sequences were as follows: mIP-10 sense: ACC ATG AAC CCA AGT GCT GC; mIP-10 antisense: AGT TAA GGA GCC CTT TTA GAC C. PCR products were separated on 1% agarose gels by electrophoresis. The identity of 302-bp fragments was verified by DNA sequencing. The integrity of the cDNA used for PCR amplification was determined by amplification of a 299-bp fragment of mouse GAPDH as described previously (25).
Determination of secreted IL-12
Blood plasma from naive mice or mice inoculated with 2 x 106 IL-12-producing NXS2 cells or NXS2 pBK-CMV cells was obtained by retroorbital puncture with heparinized capillaries (Fischer Scientific, Pittsburgh, PA), followed by centrifugation (2000 rpm for 5 min). IL-12 p70 was measured in plasma by using an ELISA kit (Genzyme, Cambridge, MA), according to the manufacturer’s instructions (assay sensitivity, 5 pg/ml).
Cytotoxicity assays
Effector cells were prepared from mouse spleen cells by hypotonic lysis of RBC with ACK lysis buffer (Life Technologies) and cultured for 5 days in DMEM medium containing, 10% FCS, 1% glutamine, 1% penicillin/streptomycin in the presence of 0.04% T-stim (Collaborative Biomedical Products, Bedford, MA), 100 IU human IL-2/ml and irradiated NXS2 tumor cells at a tumor cell:effector cell ratio of 1:100. After 5 days, effector cells were used for the cytotoxicity assay or for subsequent separation into subpopulations. Pure (>95%) CD4+ and CD8+ T cells were prepared by MACS (Miltenyi Biotec, Auburn, CA). Briefly, mouse splenocytes were incubated with either anti-mCD8 or anti-mCD4 Ab microbeads (Miltenyi Biotec). MACS was performed according to manufacturer’s guidelines. For the subsequent 51Cr release assay, NXS2 target cells were incubated in the presence of 0.5 mCi of Na251CrO4 (Amersham, Cleveland, OH) for 2 h at 37°C and washed three times and seeded onto a flat-bottom 96-well plate at a density of 5000 cells/well. Effector cells were added at various E:T ratios in a final volume of 200 µl/well and incubated for 8 h. MHC class I-Ag restriction was determined by addition of anti-H2Kk (clone 36-7-5) and anti-H2Dd (clone 34-2-12) Abs (25 µg/ml; BD PharMingen). Total 51Cr release was induced with 10 µl of SDS (10%). Supernatants were collected from each well for determination of 51Cr release. The percentage of target cell lysis was calculated as follows: experimental release (cpm) - spontaneous release (cpm)/total release (cpm) - spontaneous release (cpm) x 100 = percent cytotoxicity. The results were expressed as mean value ± SD of at least three experiments.
Flow cytometry
Staining of cell surface Ag and intracellular Ags was performed
as described previously (26). Cell cultures from spleens
of control mice, immunized mice, and IP-10-depleted mice were labeled
with CFSE (Molecular Probes, Eugene, OR) and for tumor-specific
stimulation incubated for 5 days with irradiated NXS2 tumor cells at a
tumor/effector cell ratio of 1:100 in DMEM medium supplemented with
10% FCS, 2 mM glutamine, 100 IU/ml penicillin, 100 µg/ml
streptomycin, and 0.04% T-stim (Collaborative Biomedical Products).
For staining of intracellular cytokines, cells were stimulated for the
final 5 h with 5 ng/ml PMA (Sigma) and 500 ng/ml ionomycin
(Sigma). All stimulated cultures contained 1 µg/ml brefeldin A
(Sigma) to block protein transport into post-Golgi compartments and
allow cytokines to accumulate within cells. Surface staining with
PE-labeled anti-CD8 mAb was followed by intracellular staining with
APC-labeled anti-IFN- mAb (BD PharMingen). Cells were analyzed
on a FACScan flow cytometer with CellQuest software (Becton Dickinson,
Mountain View, CA).
Statistics
The statistical significance of differential findings between
experimental groups of animals was determined by the two-tailed
Student’s t test. The nonparametric Wilcoxon test was used
to determine the statistical significance of hepatic metastases.
Findings were regarded as significant if two-tailed p values
were 
0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The involvement of distinct immune effector cells in the antitumor
response induced by local scIL-12 production was analyzed in
immunocompetent A/J mice. These data were compared with scid/scid mice
and those obtained in scid/beige mice to assess the role of T cells and
NK cells, respectively, in this tumor model. NXS2 cells producing IL-12
were rejected in A/J mice, irrespective of the cytokine’s secretion
rate (Fig. 1
A). This was in
contrast to scid/scid and scid/beige mice that only revealed a
reduction in continuous s.c. tumor growth but not a complete tumor
rejection (Fig. 1B). These findings indicate the involvement
of the T cell compartment in the rejection of NXS2 scIL-12 cells, an
observation reported previously (1). Here, we extended
these findings by establishing a role for NK cells in local tumor
growth. Specifically, the dose-dependent growth rates of NXS2 scIL-12
cells in scid/beige mice lacking both T and NK cells are at least twice
those observed in scid/scid mice lacking mature T cells (Fig. 1B). Differences in s.c. tumor growth attributable to
variable cell proliferation rates of each cell clone investigated could
be excluded by determination of [3H]thymidine
incorporation over time, which revealed no difference between each NXS2
cell clone (data not shown). Interestingly, a reduction in tumor growth
dependent on the scIL-12 secretion rate of NXS2 scIL-12 cells also was
observed in scid/beige mice (Fig. 1B), defective in both T
and NK cell compartments, accompanied by impaired chemotaxis and
motility of macrophages. These findings strongly suggest the
involvement of antiangiogenesis, an additional effector function to
immunomodulation induced by IL-12.
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Consistent with the scIL-12-induced reduction of tumor growth in
immunodeficient scid/beige mice, a dramatic, local antiangiogenic
effect was revealed by Matrigel plug assays. To this end, the
hemoglobin content in Matrigel plugs was determined after s.c.
inoculation of a mixture of Matrigel with NXS2 cells carrying the empty
vector (NXS2 pBK-CMV) and compared with Matrigel containing
scIL-12-producing NXS2 cells. Importantly, scIL-12 inhibited NXS2
neuroblastoma-induced neovascularization, as indicated by a 1.5–2.0
log decrease in hemoglobin content of NXS2 scIL-12 vs NXS2 pBK-CMV
empty vector controls (Fig. 2A). This finding was further
supported by histological analyses of Matrigel plugs, which in the case
of NXS2 scIL-12 cells revealed an almost complete absence of
erythrocytes and the presence of necrotic tumor cell islets (Fig. 2C, open arrowhead). This finding is in contrast to NXS2
pBK-CMV controls that revealed both: viable tumor cell islets (Fig. 2B, arrows) and erythrocytes (Fig. 2B,
asterisk).
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). These findings clearly
indicate scIL-12-dependent production of IP-10 mRNA in the primary
tumor’s microenvironment. Second, the antiangiogenic effect
of scIL-12-producing NXS2 cells was demonstrated by a
decrease in hemoglobin in Matrigel plugs. This antiangiogenic effect
was reversed only in mice receiving both local and systemic
administration of anti-IP-10 Ab, as indicated by both increased
hemoglobin (Fig. 2
A) and by histology demonstrating the
reappearance of erythrocytes and the presence of viable tumor islets
(Fig. 2D, arrow and asterisk). These findings were obtained
with three different IP-10 neutralizing reagents, including a
polyclonal anti-IP-10 antiserum and two anti-IP-10 mAbs, 1F11
and 1B9, that recognize different mIP-10 epitopes. Importantly, these
findings demonstrate the efficacy of IP-10 depletion in our
experimental model.
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Based on the antiangiogenic effect induced by local scIL-12
production and its abrogation by in vivo depletion of IP-10, s.c. tumor
growth of scIL-12-producing NXS2 cells was analyzed by histological and
immunohistochemical analyses in mice depleted of IP-10. Tumors induced
by scIL-12-producing NXS2 cells in IP-10-depleted mice revealed
decreases in infiltrating CD4+ and
CD8+ T cells (Fig. 4) but showed no effect on infiltrating
Mac1+ cells (Fig. 4) and granulocytes (data not
shown). Local secretion of scIL-12 by NXS2 cells in the absence of
IP-10 depletion induced a strong inflammatory response concomitant with
the absence of viable tumor cells in the tumor microenvironment (Fig. 4, hematoxylin and eosin). This was in contrast to controls injected
with NXS2 cells carrying only the empty vector.
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The role of IP-10 was assessed in the induction of a systemic
tumor-protective immunity induced by the scIL-12 NXS2 cell vaccine. For
this purpose, A/J mice were vaccinated by s.c. injection of
scIL-12-producing NXS2 cells, challenged 7 days thereafter by i.v.
injection of wild-type NXS2 tumor cells, and analyzed for disseminated
liver metastases, in the presence or absence of IP-10. The depletion of
IP-10 by two different anti-IP-10 mAbs for the duration of the
entire experiment completely abrogated the systemic tumor-protective
immunity induced by vaccination with scIL-12-producing NXS2 cells and
was the same as observed in control mice depleted of IFN- (Fig. 5). This finding suggests that local
IP-10 mediates the induction of a systemic tumor-protective
immunity.
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). In the group of mice
that received the anti-IP-10 Ab only during the immunization phase
(day 0–4), four of four mice developed metastases after immunization
with the cellular scIL-12 vaccine as compared with immunized controls
that were completely protected from metastases. This finding was
identical with results obtained with a depletion schedule covering the
entire duration of the experiment (Fig. 5), indicating that the
presence of IP-10 during the immunization phase is critical for the
induction of protective immunity. However, if IP-10 was depleted during
the early effector phase, only two of four mice developed metastases,
and if IP-10 was depleted in the late effector phase, only one of four
mice developed metastases. These observations suggest that IP-10 plays
a crucial role in the generation of effector T cells during the early
immunization phase.
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A). This response was further
characterized by the isolation of CD8+ and
CD4+ T cell subpopulations from these same mice,
which revealed primarily an MHC class I Ag-restricted
CD8+ T cell response with some cytolytic activity
in the CD4+ T cell compartment (Fig. 6B), an observation that was reported previously
(1). However, mice vaccinated with scIL-12 NXS2 and
depleted of IP-10 revealed a clear inhibition of the MHC class I
Ag-restricted CD8+ T cell response in contrast to
the cytolytic CD4+ T cell compartment, which was
not affected by IP-10 depletion (Fig. 6B).
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. This analysis
revealed a 2-fold reduction of such CD8+ T cells
in mice depleted of IP-10 in contrast to nondepleted controls (Fig. 7), suggesting a direct involvement of
IP-10 in the generation of a Th1-type T cell-mediated antitumor immune
response.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Second, MHC class I
Ag-restricted target cell lysis by CD8+ T cells
was inhibited by depletion of IP-10 (Fig. 6). Third, the number of
proliferating CD8+ T cells producing IFN-
revealed a 2-fold reduction in mice depleted of IP-10. Furthermore,
tumor-protective immunity was only entirely abrogated if IP-10 was
depleted in the immunization phase before tumor cell challenge, but not
if IP-10 was depleted in the effector phase. These findings suggest a
role for IP-10 in the generation of a tumor-specific Th1-type
CD8+ T cell immune response during the
immunization phase.
Previous observations of an antitumor response against plasmocytoma and
mammary adenocarcinoma cells that were genetically engineered to
secrete mIP-10 focused on a local antitumor response induced by IP-10
(3). This study established that the antitumor properties
of IP-10 are mediated by thymus-derived cells. However, the role of
IP-10 in the induction of protective immunity was not addressed by
these studies. We also demonstrated in our tumor model the involvement
of both major functional properties of IP-10, T cell chemotaxis
(3, 7) and antiangiogenesis (16, 37), in the
scIL-12-mediated local antitumor effect (
Figs. 1–3). Interestingly,
depletion of IP-10 efficiently inhibited both scIL-12-mediated
chemotaxis and antiangiogenesis, but did not impede the complete
rejection of scIL-12-producing primary tumors in immunocompetent mice
(data not shown). This was in contrast to depletion of IFN- or
IL-12, which resulted in continuous s.c. tumor growth by
scIL-12-producing neuroblastoma cells (data not shown). A similar
finding was reported in a syngeneic model of renal cell carcinoma
(RENCA) in which the effect of systemic IL-12 injection on partial
regression of s.c. primary tumors of BALB/c mice remained unaffected by
the depletion of IP-10 (7). This observation and our
findings support the contention that an immunological and not an
antiangiogenic mechanism is involved in IL-12-mediated local tumor
rejection, which is independent of IP-10. This may involve other IL-12
mediators downstream, such as Mig, IFN-, or a direct effect of
IL-12, per se. The persistence of Mac-1-positive cells in the local
microenvironment of scIL-12-producing primary neuroblastoma tumors,
even in the absence of IP-10, suggest a role for macrophages in
scIL-12-mediated, IP-10-independent rejection of primary tumors,
especially because these effector cells are also known to be directly
activated by IL-12 and IFN-.
We further extended these findings by establishing a role for IP-10 in
the induction of systemic tumor-protective immunity that depended on
CD8+ T cells of the Th1 type. The mechanisms
involved in IP-10-mediated induction of a vaccination effect by scIL-12
gene therapy are related to the induction and/or priming of
CD8+ T cells of the Th1 type by this chemokine.
This contention is supported by two observations. First, scIL-12
NXS2-vaccinated mice depleted of IP-10 during the immunization phase
had a decrease in the tumor-specific cytolytic response and had fewer
IFN- secreting CD8+ T cells after Ag-specific
stimulation. Second, IP-10 depletion only entirely abrogated the
protective effect of scIL-12 if the anti-IP-10 mAb was injected
within the first 4 days after immunization.
The detailed function of IP-10 in CD8+ T
cell-mediated immunity after scIL-12 gene therapy in this neuroblastoma
model remains obscure. Thus, direct activation of effector T cells by
IP-10 was analyzed in a culture of splenocytes from mice immunized with
scIL-12-producing NXS2 cells in the presence or absence of increasing
amounts of anti-IP-10 Abs ex vivo. There was no difference in the
number of proliferating
IFN-+/CD8+ T cells in
the presence or absence of IP-10 (data not shown), even at 100 µg/ml
anti-IP-10 Ab. This observation indicates that IP-10 secreted in
such cultures is not activating CD8+ T cells
directly, suggesting indirect mechanisms of IP-10-mediated induction of
CD8+ T cell immunity. In fact, such indirect
mechanisms may involve APCs (38, 39, 40, 41). Immature APCs were
demonstrated to up-regulate chemokine receptors during differentiation
into Langerhans cells (38), and IP-10 Ag fusion proteins
were shown to target Ag to such APCs, resulting in a dramatically
increased Ag-specific immune response (39). It further was
reported that a distinct subset of blood APCs that have picked up Ag in
the periphery, identified as plasmacytoid monocytes, express the IP-10
receptor CXCR3 and home to lymph nodes guided by IP-10 released from
high endothelial venules (40). There, these cells mature
to plasmacytoid dendritic cells characterized by a potent Th1
polarization to subsequently generate an Ag-specific immune response
(41). This, in combination with an IP-10-mediated increase
in T cells provides a most effective milieu for the induction of a
CD8+ T cell response.
Furthermore, IP-10 was reported necessary for effector T cell
trafficking and function and host survival in Toxoplasma
gondii infection (24). Neutralization of IP-10
inhibited the influx of CD4+ and
CD8+ T cells into spleens and livers of mice
infected by T. gondii, resulting in a simultaneous 3-log
increase in the parasite burden and a significant decrease in survival.
These findings demonstrate a critical role for IP-10 in effector T cell
trafficking in this infectious disease model. In contrast to this model
with high IP-10 levels in parasite infected organs, tumors and
metastases induced by NXS2 wild-type cells do not produce IP-10 (Fig. 3
and data not shown). Therefore, it is unlikely that inhibition of
IP-10-mediated effector T cell trafficking to neuroblastoma metastases
provides a mechanism for the effects observed in this tumor model.
However, in contrast to metastases, we clearly demonstrated T cell
trafficking to the local vaccination site, whenever NXS2 neuroblastoma
cells were producing scIL-12 (Fig. 4.). In this case, IP-10 was highly
expressed (Fig. 3), and administration of anti-IP-10 Abs clearly
decreased the number of T cells at the vaccination site (Fig. 4).
These findings indicate that IP-10 may be involved in the generation of
early activated T cells by either recruiting immature APC or early
activated T cells into the tumor. APCs that pick up Ag in the tumor
migrate back to the lymph node where they generate activated T cells,
which respond to a gradient of IP-10 emanating from the tumor, leading
to T cell infiltration of the tumor and amplification of the response.
Thus, inhibition of these processes by anti-IP-10 Abs would
abrogate the generation of tumor-specific CD8+
effector T cells. This contention also is consistent with the
observation that IP-10 depletion is only completely effective when
applied during the early immunization phase with loss of activity over
time (Fig. 5, Table I).
In summary, we have demonstrated that the induction of systemic
tumor-protective immunity by scIL-12 gene therapy is dependent on the
CXC chemokine IP-10 during early immunization. Inhibition of IP-10
during the immunization phase resulted in complete abrogation of the
vaccination effect as indicated by a failure of IP-10-depleted mice to
control a subsequent tumor challenge. These findings were supported by
a decrease in CD8+ T cell activation in
IP-10-depleted mice as defined by inhibition of MHC class I
Ag-restricted tumor target cell lysis and a decrease in the number of
Ag-specific CD8+ T cells producing IFN-. Taken
together, these data describe a novel role for IP-10 in the generation
of protective antitumor immunity induced by scIL-12 gene therapy.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Holger N. Lode, Charité Children’s Hospital, Augustenburgerplatz 1, 13353 Berlin, Germany. E-mail address: holger.lode{at}charite.de
3 Abbreviations used in this paper: sc, single chain; IP-10, IFN--inducible protein 10; m, mouse.
Received for publication December 28, 2000. Accepted for publication March 22, 2001.
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References |
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production. J. Immunol. 153:1697.[Abstract]
produced by tumor-infiltrating cells and its secondary induction of anti-tumor pathways. J. Leukocyte Biol. 62:450.[Abstract]
affect tumorigenicity and response to IL-12 therapy and antiangiogenesis. Immunity 9:25.[Medline]
v antagonist and an antibody-cytokine fusion protein eradicates spontaneous tumor metastases. Proc. Natl. Acad. Sci. USA 96:1591.-inducible protein 10 (IP-10), a member of the C-X-C chemokine family, is an inhibitor of angiogenesis. Biochem. Biophys. Res. Comm. 210:51.[Medline]
-inducible protein-10 (IP-10) chemokine by human neutrophils. Eur. J. Immunol. 27:111.[Medline]
-independent rejection of interleukin 12-transduced carcinoma cells requires CD4+ T cells and granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 188:133. in mediating the antitumor efficacy of interleukin-12. J. Immunother. Emphasis Tumor Immunol. 17:71.[Medline]
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, 
), or low amounts (
,
) of scIL-12 and compared with NXS2
pBK-CMV empty vector controls (
,•). Tumor growth was evaluated by
microcaliper measurements performed two to three times per week. In
experiments with A/J mice (A), the difference between
the experimental groups and the control group is statistically
significant (day 5; p 
; 1B9:
n = 4, 
) and one group with mAbs against
mIFN-
). Systemic, hepatic
metastases were assessed by counting the number of metastatic foci on
the surface of the liver. Bars represent average numbers of metastatic
foci per group ± SE Differences in the number of metastatic liver
foci of scIL-12 NXS2-vaccinated mice and IP-10- or IFN-
