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Introduction |
Interleukin (IL)-12 is a heterodimeric cytokine produced
by APCs, phagocytes, and granulocytes (1). Despite
some in vitro direct effects of IL-12 on T and NK cells,
where it acts as a growth factor, an enhancer of cytotoxicity, and activator of other cytokines, IL-12 activity in vivo
is generally mediated by IFN-
(1). IL-12 has been extensively tested for treatment of experimental tumors, and
with few exceptions was found to cure or improve the survival of mice bearing a variety of tumors (2). The antitumor activity of IL-12 is largely mediated by the IFN-
released at the tumor site through stimulation of macrophages, along with release of other cytokines such as TNF-
,
upregulation of MHC expression on tumor cells, induction
of IFN-inducible protein 10 (IP-10)1 by both tumor cells
(4) and infiltrating T cells, and inhibition of angiogenesis
(5, 6). Exogenous, systemic injection of IFN-
cannot substitute for IL-12 in mediating responses of such magnitude
(7), because in addition to a difference between the two cytokines in half-life, the IFN-
receptor is ubiquitously expressed, whereas IL-12 receptors are expressed only on NK
cells and activated lymphocytes (8). Accordingly, IL-12 exacerbates some autoimmune diseases characterized by local
accumulation and activation of T cells (9).
rIL-12 induces elevated expression of IFN-
in the absence of T cells but is ineffective in curing tumors injected
into nude mice (7). Thus, IL-12-stimulated T cell functions or factors other than IFN-
are needed for antitumor
activity. Nonobese diabetic mice with a disrupted IFN-
gene still develop diabetes, although the onset is delayed
(10), and IL-12 appears to exert an effect that favors disease
progression.
To investigate the antitumor activity of IL-12 in the absence of host-produced IFN-
, IFN-
knockout (GKO)
mice were injected with C26 colon adenocarcinoma cells
transduced or not with IL-12 genes. This tumor is virtually resistant to systemically given rIL-12 (11), but is rejected upon
IL-12 gene transduction (12). Our results point to the critical
role of CD4 T cells and GM-CSF for the IL-12-mediated tumor rejection in the absence of host-produced IFN-
.
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Materials and Methods |
Tumors and Mice.
Colon adenocarcinoma cell line C26 was
derived from BALB/c mice treated with N-nitroso-N-methylurethane (13). C26 cells were transduced with a retroviral vector
bearing IL-12 genes as previously described (12) to obtain C26/
IL-12 cells. IL-12 concentration was determined by two-site
sandwich ELISA using mAb 9A5 against p70 and the peroxidase-conjugated mAb 5C3-POD against p40 (provided by Dr. Luciano Adorini, Roche Milano Ricerche, Milano, Italy). Tumor
cells were cultured in DMEM (GIBCO, Paisley, UK) supplemented with 5% FCS (GIBCO).
BALB/cnAnCr mice (Charles River, Calco, Italy) were maintained at the Istituto Nazionale Tumori under standard conditions according to Institutional guidelines. GKO mice (14) on a
BALB/c background (BALB/c-Ifg<tm1 129>) were purchased
from The Jackson Laboratory (Bar Harbor, ME). Homozygous
mice were identified by PCR of tail-derived DNA using primers
flanking the neo gene (direct: 5'-CAAGTGGCATAGATGTGGAAG-3' and reverse: 5'-GGCAATACTCATGAATGCATCC-3'); the wild-type gene gave an amplified fragment of 340 bp, whereas the disrupted gene gave a 2,400-bp fragment. Homozygous mice were bred and maintained in isolators at the
Charles River Animal Facility.
Tumorigenic activity of control and transduced C26 cells was
assayed in mice injected subcutaneously in the left flank with 5 × 104 or 5 × 105 cells in 0.2 ml. Some mice were injected intraperitoneally over 2 d with 1 µg of rIL-12 (provided by Dr. Maurice
Gately, Hoffmann-La Roche, Nutley, NJ) diluted in HBSS containing 100 µg/ml of mouse serum albumin. Control animals received HBSS only. Some mice were injected intraperitoneally
weekly with 0.2 ml of HBSS containing 300 µg of anti-CD4
(GK1.5 hybridoma, L3T4) or anti-CD8 (53.6.72 hybridoma,
Lyt2) mAbs obtained from American Type Culture Collection (ATCC, Rockville, MD). To neutralize the effect of host-produced GM-CSF, some mice were injected intraperitoneally,
twice per wk, with 0.2 ml of HBSS containing 400 µg of a mixture of two rat anti-mouse GM-CSF mAbs (clones 22E9 and
31G6) obtained from ATCC with permission from Dr. J.A.
Abrams (DNAX Research Institute, Palo Alto, CA).
Morphological Analysis and Immunocytochemistry.
Tumor fragments, tumor draining lymph nodes, and spleens were embedded
in OCT compound (Miles Laboratories, Inc., Elkhart, IN), snap-frozen in liquid nitrogen, and stored at
80°C. Immunochemical analysis using the peroxidase-antiperoxidase (PAP) method was performed as previously described (15). In brief, 5-µm cryostat sections were fixed in acetone and immunostained with rat anti- mouse mAb against CD45 (M1/9.3.4.HL2 hybridoma, T200),
CD8 (53.6.72 hybridoma, Lyt2), CD4 (GK1.5 hybridoma,
L3T4), and Mac-3 (M37/84,6,34 hybridoma), all from ATCC;
GR-1 (RB6-8C5 hybridoma), CD31/PECAM-1 (Mec 13.3 hybridoma), CD51/
v integrin (clone H9.2B8), and CD61/
3 integrin (clone 2C9.G2), all from PharMingen (San Diego, CA); and CD34 (clone 14.7 MEC), from Hycult Biotechnology, B.U.,
Uden, The Netherlands). Sections were preincubated with rabbit
serum and sequentially incubated with optimal dilutions of primary antibodies, rabbit anti-rat IgG (Zymed Laboratories, Inc.,
San Francisco, CA) and rat PAP (Abbot Laboratories, North Chicago, IL). For immunostaining of NK cells, a rabbit anti-asialo
GM1 serum (Wako, Osaka, Japan) was used in combination with
a goat anti-rabbit and goat PAP (Sigma Chemical Co., St. Louis,
MO). Each incubation step lasted 30 min and was followed by a
10-min wash in TBS. Sections were then incubated with 0.03%
H2O2 and 0.06% 3,3'-diaminobenzidine (BDH Chemicals, Poole, England) for 2-5 min, washed in tap water, and counterstained with hematoxylin. The number of immunostained cells
was determined by light microscopy at magnification ×400 in 5 fields on a 1-mm2 grid and is given as cells/mm2 (mean ± SD).
Reverse Transcriptase PCR.
Total cellular RNA was extracted
by cesium chloride gradient, and first-strand cDNA was synthesized
from 1 µg of RNA using Moloney murine leukemia virus reverse
transcriptase (GIBCO BRL, Gaithersburg, MD) for 2 h at 42°C. A
fraction of the cDNA was amplified by PCR with Taq DNA polymerase (Promega Corporation, Madison, WI) using primers specific
for
-actin (Clontech Labs., Inc., Palo Alto, CA), monokine induced
by
-IFN (MIG); (direct, 5'-TCCGCTGTTCTTTTCCTTTTGG-3'; reverse, 5'-TTGAACGACGACGACTTTGGGG-3'), IP-10
(direct, 5'-GCGTTAACCTCCCCATCAGCACCATGAAC-3';
reverse, 5'-CCGCTCGAGGTGGCTTCTCTCCAGTTAAGGA-3'), and IFN-
(direct, 5'-CCGAATTCTGAGACAATGAACGCTACAC-3'; reverse, 5'-GCTCGAGAATCAGCAGCGACTCCTTTTCCG-3'). PCR was carried out in a 50-µl vol (1 µM
primers, 1.25 U Taq DNA polymerase, 1 mM MgCl2) for 25, 30, and 35 cycles (1 min denaturation at 94°C, 1.5 min annealing at
60°C, 2 min extension at 72°C) using a thermal cycler (Perkin-Elmer, Norwalk, CT). One-third of the PCR volume was then analyzed on a 1% agarose gel.
In Situ Hybridization.
The presence of cytokine mRNA was
investigated by in situ hybridization using cDNA probes as previously described (15). MIG (361 bp), IP-10 (344 bp), and GM-CSF (368 bp) probes were prepared by PCR amplification of
murine blast cDNA, using specific primers for MIG and IP-10
(see above) or primers for GM-CSF (Clontech Labs., Inc.). The
products obtained were run on a 1% agarose gel and purified using the Qiaex II gel extraction kit (Qiagen, Hilden, Germany).
Cryostat sections were harvested on RNA-grade slides, air-dried, and fixed in 4% buffered paraformaldehyde for 10 min, and then were dehydrated in ethanol, sequentially rehydrated in PBS/ 50 mM MgCl2, washed in 200 mM Tris-HCl-glycine, acetylated
in 2× SSC, 0.1 M triethanolamine, and 0.5% acetic anhydride
(pH 8.0), washed in 2× SSC, and finally dehydrated in ethanol.
Slides were then prehybridized for 10 min at 70°C with 2× SSC,
50% formamide, and 500 µg/ml salmon sperm DNA, and hybridized overnight at 42°C with 32P-labeled-specific cDNA
probes (0.5 × 106 cpm/section), 2× SSC, 500 µg/ml yeast
RNA, 5× Denhardt's solution, 10 mM dithiothreitol, and 10%
dextran sulfate. Unbound and nonspecifically bound probes were
removed by sequential washes in 2× SSC, 50% formamide, and
1× SSC, 50% formamide at 45°C, and in 0.1× SSC at room
temperature. Slides were then dehydrated in ethanol, dipped in
autoradiographic emulsion NTB-2 (Eastman Kodak Co., Rochester, NY), and exposed for 24-72 h at 4°C in a light-tight box,
then developed in D19 (Eastman Kodak Co.), fixed in Rapid
Fixer (Eastman Kodak Co.), and counterstained or not with hematoxylin. RNA specific binding was controlled by previous digestion with 100 µg/ml ribonuclease A and 10 U/ml ribonuclease T (Sigma Chemical Co., Poole, England). PstI-digested
pUC9 plasmid fragments were used as negative controls. Cytosmears of LPS + IFN-
-stimulated or PHA + ionomycin-stimulated splenocytes were used as positive controls in each experiment.
Cytokine Production.
In vitro cytokine production by total or
purified T cells isolated from lymph nodes draining the site of tumor injection was induced by culturing lymphocytes (2 × 105/
well) in 96-well flat-bottomed plates precoated or not with 10 µg/ml of anti-CD3 mAb (145-2C11 hybridoma) at 37°C for 18 h. Supernatants were recovered and assayed for IFN-
, GM-CSF,
TNF-
, IL-4, and IL-10 production by specific ELISA (all from
PharMingen). Cytokine levels were calculated using standard
curves constructed using recombinant murine cytokines.
CD8 and CD4 purified lymphocytes were obtained by magnetic cell sorting. In brief, lymphocytes derived from draining
lymph nodes were labeled with rat anti-mouse CD4 (L3T4)- or
CD8 (Ly-2)-conjugated paramagnetic microbeads (MiniMacs;
Miltenyi Biotec., Bergisch Gladbach, Germany) and separated by
a magnetic field using a positive selection column.
Additional experiments were performed with the same splenocytes used for NK cell assay (see below). In those cases, splenocytes from mice treated with rIL-12 or untreated were cultured
in vitro for an additional 18 h in the presence or absence of rIL-12
(10 ng/ml) before CD3 stimulation.
NK Cell Assay.
To measure NK cell activity in response to
IL-12, BALB/c and GKO mice were injected intraperitoneally
with rIL-12 (1 µg/mouse) or HBSS for 2 d, and at day 3 fresh
splenocytes were tested for cytotoxic activity against 51Cr-labeled
YAC cells in the presence of 500 U/ml of human rIL-2 (Chiron-Italia, Milan, Italy).
Mixed Lymphocyte Tumor Culture and Cell-mediated Cytotoxicity
Assays.
Mixed lymphocyte tumor culture was performed in
RPMI 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10% FCS (Hyclone, Logan, UT). Responder lymph
node or spleen cells were stimulated with
-irradiated (15,000 rad) C26 cells. Responders and stimulators were suspended to 2.5 × 105 and 2.5 × 104 cells/ml, respectively, and mixed in a total volume of 2 ml in 24-well plates (Costar, Cambridge, MA). Cultures
were incubated in a humidified atmosphere of 5% CO2 in air. In
cell-mediated cytotoxicity (CMC) assays, C26 cells were the specific target, YAC-1 cells were used as controls for NK cell-mediated lysis, and F1 spontaneously transformed fibroblasts (BALB/c)
were the negative controls for C26 tumor-specific lysis. In some
experiments, additional targets were syngeneic Con A-induced
blast cells pulsed or not with the AH1 peptide, the immunodominant epitope of the C26-associated gp70 antigen (16). Tumor
specific lysis was measured as previously described (11).
In Vitro Macrophage NO2
Production.
Thioglycolate-elicited macrophages were washed from the peritoneal cavity and resuspended
in complete medium. Adherent macrophage monolayers were
obtained by plating cells in 24-well plates at 2 × 106 cells/well for
2 h at 37°C in 5% CO2. Nonadherent cells were removed and
freshly prepared complete medium was added with the indicated
experimental reagents (1 µg/ml LPS, 200 U/ml IFN-
). Nitrite
concentration in the medium was measured by a microplate assay
method (17). In brief, 100-µl aliquots were removed from conditioned medium and incubated with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamine dihydrochloride, 2.5% H3PO4) at room temperature for 10 min. Absorbance at 550 nm was determined in a microplate reader. NO2
concentration was determined using sodium nitrite as a standard.
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Results |
Accelerated Tumor Take and Reduced Tumor Regression in
GKO Mice Bearing the IL-12-producing C26 Carcinoma.
We previously reported that C26 cells transduced with
IL-12 genes (C26/IL-12) showed reduced tumor take
(<50%) and delayed onset in BALB/c mice injected at a
dose of 5 × 104 cells (the LD100 for nontransduced C26),
whereas a dose of 5 × 105 cells resulted in initial tumor
take followed by regression in 80-100% of injected mice
(12). In GKO mice injected with 5 × 104 cells, C26/IL-12
tumor onset was accelerated but tumors developed in only
40% of the mice (Fig. 1 A). At a dose of 5 × 105, C26/IL-12
cells formed tumors in all GKO mice with onset similar to
that in BALB/c mice, but only a few GKO mice were able
to reject the initial tumor (Fig. 1 B). An enhanced rate of
C26/IL-12 tumor take was also observed in BALB/c mice
treated with neutralizing mAb to IFN-
(data not shown).
These results indicate that the antitumor activity initiated
by IL-12 released at the tumor site is only partially dependent on host-produced IFN-
, although IFN-
appears to
be important in the early response to the tumor.

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Fig. 1.
Tumorigenicity and
latency of C26/IL-12 cells injected
into BALB/c (open circles) and
GKO mice (closed circles) at doses
of 5 × 104 (A) and 5 × 105 (B).
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Unimpaired Memory and CTL Responses and Increased GM-CSF Production in GKO Mice.
BALB/c mice that did not
develop tumors were susceptible to subsequent challenge
with parental C26 cells, whereas mice that remained tumor-free after rejecting incipient C26/IL-12 tumors were
resistant to challenge (Table 1). These results are consistent with our previous suggestion that debulking of an incipient
tumor allows a prolonged exposure of tumor-associated antigens to host T cells, whereas an immune response rapid
enough to inhibit initial tumor take might avoid T cell activation (18). Indeed, all GKO mice that lacked the early
response but still rejected C26/IL-12 cells were resistant to
C26 challenge (Table 1). Furthermore, splenocytes from
resistant GKO mice were cytotoxic against C26 but not
against F1 transformed fibroblasts (Table 1). We previously showed that the TCRV
repertoire of CTL recognizing
C26 is restricted mainly to V
6 (19); immunohistochemical analysis of the site of tumor challenge revealed positive
staining for both CD8 and V
6 in most infiltrating lymphocytes (Fig. 2). Together, these results suggest that an IL-12-producing tumor can be rejected despite the lack of
IFN-
through a mechanism that clearly involves T cell activation since memory T cells and CTLs were detected both in vivo and in vitro.
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Table 1
Tumorigenicity of C26/IL-12 and Induction of both Memory Response and CMC to Parental C26 Cells in BALB/c and
GKO Mice
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Fig. 2.
Immunostaining of lymphocytes infiltrating the site of C26
challenge. Tumor sections from BALB/c (A) and GKO mice (B and C)
that had already rejected C26/IL-12 cells were immunostained for TCR
V 6 (A and B) and for CD8 T cells (C). The distribution of TCR V 6+
around a neural structure (B) is similar to that of CD8+ lymphocytes (C).
Frozen sections were immunostained using the PAP method and counterstained with hematoxylin. Original magnification: ×300.
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We compared C26 and C26/IL-12 cells for CTL induction in BALB/c and GKO mice injected in the footpad
with irradiated cells followed by restimulation of lymphocytes from the draining popliteal nodes with C26 cells in
vitro. C26/IL-12 primed mice of both strains for CTL induction with similar efficacy, whereas C26 cells induced a
slightly reduced CTL activity in GKO mice (Fig. 3). Analysis of cytokine release by primed T cells using the same
lymphocytes stimulated with mAb to CD3 coated on plastic revealed threefold higher levels of GM-CSF produced
by lymphocytes from GKO mice than by lymphocytes
from BALB/c mice primed with C26/IL-12 (Fig. 4 A).
Analysis of CD4 and CD8 T lymphocytes sorted with
magnetic beads before CD3 stimulation showed that CD8
T cells produced the higher absolute amount of GM-CSF,
whereas the relative contribution of CD4 lymphocytes to
the difference in GM-CSF production between BALB/c
and GKO mice was greater (Fig. 4 B). IL-4 and IL-10 were undetectable, whereas TNF-
levels were unchanged in
both BALB/c and GKO lymphocytes (data not shown).

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Fig. 3.
CTL activity after in
vivo priming of BALB/c (open
symbols) and GKO mice (closed
symbols) with irradiated C26 (triangle) or C26/IL-12 cells (circle).
Target cells are indicated on top
of the figure.
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Fig. 4.
CD3-stimulated GM-CSF production by BALB/c (white
bars) or GKO (black bars) lymphocytes from popliteal lymph nodes draining the site of C26/IL-12 injection (A). Mean ± SD of three independent
experiments. Release of GM-CSF upon CD3 stimulation by purified
CD4 and CD8 lymphocytes (B).
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NK Cytotoxicity and Macrophage NO2
Production in Response to IL-12 Stimulation in GKO Mice.
To analyze the
nature of the early defective response in GKO mice, NK
activity in response to rIL-12 was tested in mice injected or
not with rIL-12 (1 µg/mouse per day) for 2 d; at day 3, fresh splenocytes were tested for cytotoxic activity against
YAC cells. NK cytotoxicity was indeed reduced, but not
abrogated, in GKO mice injected with rIL-12 (Fig. 5 A) although splenocytes from these mice showed increased
GM-CSF production upon CD3 stimulation (Fig. 5 B).

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Fig. 5.
NK-mediated cytotoxicity of YAC cells (A) and
splenocyte GM-CSF production
(B) after in vivo stimulation with
rIL-12. Splenocytes were freshly
isolated from BALB/c (white
bars) and GKO (black bars) mice
treated twice with 1 µg of rIL-12, and GM-CSF production
was measured by ELISA after
18 h incubation in wells coated
with mAb to CD3.
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To test GKO macrophage function, NO2
production
was measured in thioglycolate-elicited macrophages treated
with LPS in the presence or absence of IFN-
(Table 2).
Although GKO macrophages in the absence of exogenous
IFN-
were less responsive than their BALB/c counterparts to LPS, they promptly recovered the ability to produce NO2
after addition of IFN-
. Consistent with this
result, injection of mice with rIL-12 primed BALB/c (20),
but not GKO macrophages, against the subsequent exposure to LPS (Table 2). Thus the absence of IFN-
impairs
the NO response of macrophages, and IL-12 does not restore this response.
Immunohistology of Leukocytes Infiltrating the C26/IL-12
Tumor.
Reduced NK activity in the presence of an intact
and robust CTL response might explain the accelerated onset of C26/IL-12 tumor formation when 5 × 104 cells are
injected, but not the reduced rejection of the same cells injected at a dose of 5 × 105. To gain some insight into the
tumor-associated events occurring in vivo, the entire site of
tumor injection, the incipient tumors when present, and
the draining lymph nodes were dissected at different time
points and analyzed by immunocytochemistry. Immunostaining of infiltrating leukocytes indicated a profound reduction in lymphocyte number, mainly CD8, and in NK
cells, but an increased number of macrophages and granulocytes in the C26/IL-12 tumors from GKO mice (Table
3). This effect was clearly due to the lack of IFN-
, since
treatment of BALB/c mice with mAb to IFN-
produced
the same effect (Table 3). Thus, in GKO mice, NK cells
and CTLs are induced but apparently cannot infiltrate the
C26/IL-12 tumors.
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Table 3
Immunocytochemical Characterization of Leukocytes Infiltrating C26/IL-12 Colon Carcinoma Injected into BALB/c, GKO, or
Anti-IFN- -treated BALB/c Mice
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Tumor-associated and Systemic Expression of IP-10 and
MIG.
An antiangiogenic activity has been described elsewhere for IL-12 (21), which is mediated through IFN-
and its induced chemokines IP-10 and MIG. PCR analysis
of RNA extracted from C26/IL-12 tumors and draining
nodes for expression of IP-10 and MIG revealed a slight reduction in IP-10 levels and undetectable MIG expression in both tumors and nodes of GKO mice (Fig. 6). In situ
hybridization confirmed these results and revealed induction of IP-10 and MIG expression in BALB/c mice injected with C26/IL-12 cells (Fig. 7). The effect of IL-12
released by C26/IL-12 tumor cells on IP-10 and MIG was
systemic since upregulation of both molecules was detected
in spleen sections from BALB/c but not GKO mice (data not shown).

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Fig. 6.
MIG and IP-10 expression as detected by reverse
transcriptase PCR in C26/IL-12
tumors (T) and draining lymph
nodes (LN) from BALB/c and
GKO mice. -actin was used to
control cDNA quality, and IFN-
primers were used to confirm the
lack of IFN- expression in
GKO mice.
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Fig. 7.
In situ hybridization of IP-10 (A and B) and MIG (C and D)
probes with C26/IL-12 tumors from GKO (A and C) and BALB/c mice
(B and D). (A) In GKO mice, IP-10 hybridization was weak and mostly
restricted to tumor cells; whereas (B) C26/IL-12 tumors growing in
BALB/c mice showed high level expression of IP-10 in tumor cells, some
reactive cells, endothelium and epithelial cells of the subcutaneous sweat
glands (SG). (D) Several lymphocytes of the inflammatory infiltrate were
positive for MIG expression in BALB/c, whereas (C) MIG expression
was undetectable in GKO. Positive cells were identified by the presence
of a high accumulation of cytoplasmic black granules. Hematoxylin counterstaining. Original magnification: A-C, ×400; D, ×1,000.
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Vascularization of the C26/IL-12 Tumor.
The parental
C26 tumor grew in both BALB/c or GKO mice without
differences; in comparison to them, C26/IL-12 tumors
were characterized by vessels with an enlarged lumen filled
with granulocytes and macrophages in BALB/c mice (Fig.
8), whereas in GKO mice, tumor vessels were more numerous (Table 4), although generally negative for expression of the angiogenesis-associated integrin
v
3 (22), and
were usually thinner in structure (Fig. 8).

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Fig. 8.
CD31 (PECAM-1) immunostaining of blood vessels associated with C26 (A and B) and C26/IL-12 (C-F) tumors in GKO (A-D) and
BALB/c (E and F) mice. Vessels of the C26/IL-12 tumors in BALB/c mice were less numerous and showed an enlarged lumen as compared with those in
GKO mice, which were more similar to those in C26 tumors, although less numerous. Large areas of tumor necrosis are more evident in C26/IL-12 tumors from BALB/c where the host response was stronger. PAP immunostaining, hematoxylin counterstain. Original magnification: A, C, and E, ×400;
B, D, and F, ×1,000.
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Differential Requirement for CD4 T Cells and for GM-CSF
in BALB/c and GKO Mice for C26/IL-12 Tumor Rejection.
Analysis of leukocyte infiltration (Table 3) indicated
that CD4 cells were more numerous than CD8 lymphocytes in tumors from GKO mice, in contrast to tumors
from BALB/c mice in which CD8 cells predominate. Unlike BALB/c mice, in which rejection of C26/IL-12 tumors was CD4 independent, GKO mice required CD4
lymphocytes to reject the C26/IL-12 tumor (Fig. 9).
Moreover, CD4 T cell depletion was associated with disappearance or further reduction of the few CD8 T lymphocytes that infiltrated the C26/IL-12 tumor in GKO mice
(Table 5). Both BALB/c and GKO mice required CD8 T
lymphocytes for tumor rejection (Fig. 9). These results suggest that some factor(s) essential for CD8 T cell recruitment
or survival at the tumor site in the absence of IFN-
is produced by CD4 cells. This factor was identified as GM-CSF,
whose expression was higher in GKO than BALB/c mice
in response to C26/IL-12 cells, a result confirmed by in
situ hybridization of tumor-draining lymph nodes (Fig. 10,
A-D), and was not detectable in tumor sections from
CD4-depleted GKO mice (Fig. 10 G). Moreover, treatment of BALB/c and GKO mice with mAbs against GM-CSF before injection with C26/IL-12 cells abrogated tumor inhibition in GKO but not in BALB/c mice (Fig. 9 C).

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Fig. 9.
Tumorigenicity and
latency of C26/IL-12 tumor cells
injected at a dose of 5 × 104 cells
into BALB/c or GKO mice depleted of CD4 (A) or CD8 (B) T
cells or treated with mAbs
against GM-CSF (C).
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Table 5
Immunocytochemical Characterization of Leukocytes Infiltrating C26/IL-12 Colon Carcinoma Injected into CD4-depleted BALB/c
and GKO MICE
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Fig. 10.
Detection of GM-CSF expression by in situ hybridization. Tumor-draining lymph nodes (A-D) and C26/IL-12 tumors (E-G) were collected from BALB/c (A, B, and E), GKO (C, D, and F), and CD4-depleted GKO mice (G). Tumor-draining lymph nodes from BALB/c mice revealed
GM-CSF expression in macrophages of the subcapsular sinus and in scattered lymphocytes of the T cell-dependent area, whereas the nodes from GKO
mice were characterized by a much higher number of strongly positive lymphocytes in the paracortex. GM-CSF was expressed by scattered cells of the
inflammatory infiltrate of BALB/c tumors (E), but by a large number of cells in tumors from GKO mice (F). Such positive cells disappeared after CD4 T
cell depletion (G). F, primary follicles; PC, paracortical area; S, subcapsular sinus. [32P]dCTP-labeled GM-CSF autoradiography. Hematoxylin counterstain.
Original magnification: A and C, ×250; G, ×400; B, D-F, ×1,000.
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Discussion |
Direct comparative data obtained using the same tumor
transduced with several different cytokine genes, including
IL-12, and using rIL-12 systemically, have shown that this
cytokine is optimal in inducing antitumor activity in preclinical settings (23). The effects of IL-12 have been found
to be IFN-
dependent in several models (1). IFN-
has
been strongly associated with a favorable outcome of therapy given as rIL-12 (24) or as tumor cell vaccines based on
IL-12 transduction (25). The intensity of IL-12-induced
tumor inhibition is more directly associated with IFN-
production than with CTL activity (26). Although IFN-
has been associated with the antitumor activity of cytokines other than IL-12 (15), and even with the cure of metastasis by adoptive transfer of CD8 T cells (27), IFN-
-secreting
tumor cells were not inhibited to the same extent as were
IL-12-secreting tumors. This observation might rest in the
lower efficiency of IFN-
released by engineered tumor
cells as compared with IFN-
physiologically produced as a
secondary cytokine by infiltrating leukocytes and providing
the correct immunological context (28). More likely, IL-12
induces secondary factors (in addition to IFN-
) different
from those induced by IFN-
, depending on the type of leukocytes that infiltrate tumors transduced with either cytokine. In the TSA mammary tumor model, for example,
TSA/IL-12 is rich in polymorphonuclear leukocytes that
are rare or absent in TSA/IFN-
(29). Depletion experiments have also underscored the role of granulocytes in cytokine-activated regression of established tumors (23).
Our study aimed to identify factors that might functionally substitute for IFN-
rather than simply cooperate with
it in mediating IL-12 antitumor effects. The use of GKO
mice ensured the complete absence of IFN-
throughout
the experiments, a condition more difficult to obtain by using neutralizing antibodies. Since the first description of
these mice (14), most of the studies to delineate the immunological defect have focused on the antimicrobial response, a setting in which IL-12 is an early mediator (30). The emerging picture is that GKO mice are susceptible to
acute infection because of a defective early response, but
are capable of a specific immune response and of a late response against chronic infection (30, 31). In these mice, no
upregulation of Th2 cytokine mRNA was detected, but
IL-12 was shown to enhance rather than suppress the Th2
type response (32), despite the recent description of IFN-
-dependent regulation of IL-12R
2 (33). Our analysis of
the impaired immune response of GKO mice to C26 carcinoma cells engineered to produce IL-12 revealed the expected defective early response and unimpaired CTL response as well as several unexpected findings. First, ~50%
of both BALB/c and GKO mice did not develop tumors
after injection of 5 × 104 C26/IL-12 cells, but only GKO
mice mounted a memory response. This result is compatible with the notion of an absent early response that allows
late T cell activation, since the other 50% of GKO mice
had tumors that formed more quickly than did those in
BALB/c mice. CTL activation and infiltration of the challenge site by oligoclonal V
6 lymphocytes in GKO mice
support such a notion, in accord with previous findings on
tumors engineered to produce IL-2 at high (strong early
NK response but no memory) and low (no early NK response but memory) levels (34). Thus, the rejection of incipient tumors is generally associated with immune memory (35), as also seen in BALB/c mice injected with 5 × 105 C26/IL-12 cells (Table 1 and reference 12).
Second, we identified GM-CSF as a possible substitute
for IFN-
in maintaining the immune response still present
in GKO mice. This identification is based on repeated
findings from different experiments in which GM-CSF was
tested by ELISA in lymphocytes from lymph nodes draining the injection sites of either live or irradiated C26/IL-12
cells, as well as from spleens of mice injected with rIL-12.
In addition, in situ hybridization showed that GM-CSF was highly expressed in C26/IL-12 tumor sections and
draining nodes from GKO but not BALB/c mice. A functional role for GM-CSF was suggested by experiments in
CD4-depleted GKO mice, where the complete abrogation
of any residual response to C26/IL-12 cells was associated
with a decrease in GM-CSF expression. Experiments using
mAbs to neutralize GM-CSF confirmed this role. Elevated production of GM-CSF by lymphocytes from GKO mice
was not restricted to C26/IL-12 response since a similar result was obtained by injecting the TSA/IL-12 mammary
carcinoma (data not shown). Production of GM-CSF by
tumor-infiltrating lymphocytes has been described as a predictor of tumor response in melanoma patients (36), and we have found that GM-CSF is associated with the therapeutic outcome of mice with C26 lung metastasis after
treatment with a C26/IL-12 cell vaccine (our unpublished
results). In CD4-depleted GKO mice, the decline in GM-CSF levels was accompanied by a reduction in the number
of infiltrating granulocytes. These cells can mediate tumor
cell killing through direct or bystander effects (37) and can
participate in the cross-talk with CD8 T cells, which is instrumental in the rejection of established C26 colon carcinomas transduced to express G-CSF (15) and in the IL-12-
mediated rejection of TSA mammary carcinoma (23). Such
cross-talk was sustained by CD8 cell-produced IFN-
(15),
which is known to maintain granulocyte survival (38), a
function that may well be supported by GM-CSF. The
IFN-
-independent late response to Leishmania donovani
that has been attributed to TNF-
(39) may also require
GM-CSF. Indeed, Taylor and Murray (39) noted that although TNF-
was probably the primary effector component, IL-12-induced GM-CSF in GKO mice likely acts as
a compensatory factor for granuloma assembly in the absence of endogenous IFN-
.
Third, we found that CD4 T cells, which were not necessary for IL-12-mediated tumor rejection in BALB/c
mice or were even suppressive (11, 23), became essential
for the IFN-
-independent response to C26/IL-12 cells.
Although CD8 T lymphocytes produced the higher
amount of GM-CSF, CD4 T cells accounted for the larger difference in GM-CSF production between BALB/c and
GKO lymphocytes. In addition, the CD4/CD8 ratio in
C26/IL-12 tumors in GKO mice was reversed as compared
with that in BALB/c tumors, with CD4 T cells as the predominant lymphocyte population. Depletion of CD4 T
cells in GKO mice abolished the presence of CD8 T cells either directly or indirectly through the break in granulocyte-CD8 cross-talk (34, 40), possibly via the decrease in
GM-CSF and consequent reduction of infiltrating granulocytes.
Finally, our studies revealed a correlation between expression of IFN-
-inducible IP-10 and MIG and tumor
vascularization, although this correlation does not entirely
explain the IL-12-mediated antitumor effect still present in
GKO mice. Although IL-12-mediated antitumor activity
has also been observed in SCID and nu/nu mice, suggesting the activation of some nonimmunological events, nu/nu mice show a weaker response than that seen in euthymic
mice, despite their 10-fold higher levels of serum IFN-
(7). Such a setting should favor induction of IP-10, which
reportedly elicits a potent thymus-dependent antitumor response in vivo (41) and is responsible for T cell recruitment
when expressed at the tumor site (4). Moreover, IP-10 has
been identified as a final inhibitor of neoangiogenesis induced by IL-12 (42). In GKO mice, C26/IL-12 tumors
were characterized by a partial reduction in number of
blood vessels as compared with tumors growing in BALB/c
mice. This may be explained in part by the reduced expression of IP-10 and MIG in GKO mice, but other factors are
undoubtedly required (perhaps GM-CSF), since CD4 T cell depletion in GKO but not in BALB/c mice reestablished vascularization conditions similar to those of IL-12-
nonproducing C26 cells (Table 4). Tumor vessel status
probably depends on a balance of factors that promote and
inhibit neovascularization, as well as on granulocytes, which in the context of a local inflammatory response induce vessel wall injury and compromise the function of the
vasculature and of the underlying tissues (43).
Together, our results point to GM-CSF as a cytokine
that sustains an immune response to IL-12-producing tumors in the absence of IFN-
. Moreover, the results suggest
the existence of an alternative pattern of immune response
that uses different regulatory leukocytes and chemokines to
provide a similar outcome, i.e., tumor rejection.
Address correspondence to Mario P. Colombo, Experimental Oncology D, Istituto Nazionale Tumori, Via
Venezian 1, 20133 Milano, Italy. Phone: 39-2-2390-252; Fax: 39-2-2362-692; E-mail: mcolombo{at}istitutotumori.mi.it
We thank Dr. Giorgio Trinchieri for critically reviewing the manuscript. The technical expertise of Mr.
Ivano Arioli is gratefully acknowledged. We also thank Mr. Mario Azzini for preparation of figures.
This work was supported by special programs on gene therapy from the Associazione Italiana per la Ricerca
sul Cancro and the Italian Ministry of Health (ISS), and by Consiglio Nationale delle Ricerche.
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