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-Inducible Protein-10 and Another Encoding IL-12, Results in Marked Antitumoral Synergy1
Departments of
*
Medicine and
Genetics, Facultad de Medicina, Universidad de Navarra, Pamplona, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible protein-10 (IP-10)
chemokine (AdCMVIP-10). Injection of AdCMVIP-10 into s.c. tumor nodules
derived from the CT26 murine colorectal adenocarcinoma cell line
displayed some antitumor activity but it was not curative in most
cases. Previous studies have shown that injection of similar s.c. CT26
tumor nodules with adenovirus-encoding IL-12 (AdCMVIL-12) induces tumor
regression in nearly 70% of cases in association with generation of
antitumor CTL activity. AdCMVIP-10 synergizes with the antitumor effect
of suboptimal doses of AdCMVIL-12, reaching 100% of tumor eradication
not only against injected, but also against distant noninjected tumor
nodules. Colocalization of both adenoviruses at the same tumor nodule
was required for the local and distant therapeutic effects.
Importantly, intratumoral gene transfer with IL-12 and IP-10 generated
a powerful tumor-specific CTL response in a synergistic fashion, while
both CD4 and CD8 T cells appeared in the infiltrate of regressing
tumors. Moreover, the antitumor activity of IP-10 plus IL-12 combined
gene therapy was greatly diminished by simultaneous in vivo depletion
of CD4+ and CD8+ T cells but was largely
unaffected by single depletion of each T cell subset. An important role
for NK cells was also suggested by asialo GM1 depletion experiments.
From a clinical point of view, the effects of IP-10 permit one to lower
the required gene transfer level of IL-12, thus preventing
dose-dependent IL-12-mediated toxicity while improving the therapeutic
efficacy of the elicited antitumor response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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inducible protein-10
(IP-10)5 (1, 2), also called Crg-2 (3, 4) in mice, is a
chemokine that belongs to the CXC family known to stimulate the CXCR3
chemokine receptor (5, 6, 7). The pattern of CXCR3 expression
explains that IP-10 attracts, at least in vitro, only activated but not
resting T lymphocytes and NK cells (5, 8, 9, 10). Stably
transfected tumor cell lines expressing IP-10 were rejected through an
immune system-mediated mechanism (11). However, it has
been recently shown that IP-10 also displays antitumoral properties
related to its ability to impair tumoral angiogenesis
(12, 13, 14). Such an effect seems not to be mediated by CXCR3
(15) and has been found to be important for the
antitumoral effects of IL-12 in some models (16).
Therefore, IP-10 has been involved on the interface of the immune (T
and NK recruitment to the malignancies) and nonimmune antitumor
mechanisms (angiostatic effect), making the IP-10 gene a good
therapeutic candidate to be delivered into malignant cells.
Other chemokine genes such as the one encoding lymphotactin
(17) and macrophage inflammatory protein-1
(18) have been transfected into tumor cells showing that
although they attracted T lymphocytes to the malignant tissue, they
failed to induce rejection (17, 18). However, combination
of those chemokines with other cytokines or costimulatory molecules
that ultimately result in lymphocyte activation such as IL-2, B7-1
(CD80), and IL-12 resulted in a marked antitumor effect (17, 19, 20).
IL-12 is a cytokine naturally produced by macrophages (21)
and dendritic cells (22) and plays a key role in the
induction of cellular immune responses (23). IL-12 has
been found to mediate potent antitumor effects that are the result of a
pleyade of actions involving not only the induction of CTL,
Th1-mediated immune responses, and NK activation (21), but
also impairment of tumor vascularization (24).
Unfortunately, the first attempts to test IL-12 in the clinic underwent
failure due to unacceptable dose-related toxicities leading to some
fatalities (25, 26). Thus, viral transfer of IL-12 genes
into tumors holds promise to be an efficacious alternative as proved in
several animal models (27, 28, 29, 30). It is noteworthy that
IL-12 triggers important secretion levels of IFN- from T and NK
cells, which in turn lead to induction of IP-10 in many cell types
(21). Because IL-12 toxicity is reported to be
largely related to IFN- hyperproduction, it is reasonable to assume
that enrichment of the malignant environment directly with IP-10 could
enhance the therapeutic effects of low doses of IL-12, thus permitting
antitumor activity, while avoiding toxicity due to excessive IFN-
production.
To study the effects of transducing tumor cells in vivo with the IP-10 gene, we have generated a recombinant defective adenovirus encoding for the chemokine (AdCMVIP-10). Intratumoral injection of high doses of AdCMVIP-10 had only mild antitumor activity. Nonetheless, such treatment synergized with adoptive T cell therapy mediated by short-term cultured antitumor T lymphocytes and importantly with suboptimal levels of IL-12 gene transfer within the same tumor nodule. Gene transfer by coinjection into tumors of IP-10 and IL-12 recombinant adenoviruses resulted in a potent enhancement of tumor-specific CTL activity, which could account, at least in part, for the improved therapeutic effects.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Five- to 8 wk-old female BALB/c mice were purchased from Harlan (Barcelona, Spain). Six-week female BALB/cnu/nu mice were obtained from Harlan (Barcelona, Spain) and housed in pathogen-free conditions.
Cells and Abs
The 293 cell line (adenoviral E1 transformed human embryonic kidney cells) and the HepG2 cell line (human hepatoblastoma) were obtained through American Type Culture Collection (Manassas, VA). The CRE8 selective cell line was kindly provided by Dr. S. Hardy (University of California, San Francisco, CA). It has a ß-actin-based expression cassette driving a Cre recombinase gene with an N-terminal nuclear localization signal stably integrated into 293 cells (31). The BALB/c (H-2d) mouse-derived CT26 tumor cell line is an undifferentiated murine colorectal adenocarcinoma (32) that was established from an N-nitroso-N-methylurethan-induced transplantable tumor, obtained from Dr. K. Brand (Max-Planck-Institut fur Biochemie, München, Germany). P815 and YAC-1 cells were obtained through American Type Culture Collection. The CRE8, 293, and HepG2 cell lines were cultured in DMEM supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 100 U/ml streptomycin, and 100 µg/ml penicillin (complete medium). CT26 cells were cultured in RPMI 1640 medium identically supplemented. All cell culture reagents were obtained from Life Technologies (Basel, Switzerland). Hybridomas GK1-5 and H35.17.2 (American Type Culture Collection) were used to obtain anti-CD4 and anti-CD8 Abs from ascitic fluid that was obtained from pristane-primed nude mice injected i.p. with 106 hybridoma cells. Asialo GM1 antiserum was obtained from Wako (Osaka, Japan). Anti IP-10 mAb and anti IP-10 polyclonal anti-serum were purchased from R&D Systems (Minneapolis, MN) and PeproTech (London, U.K.). FITC- and PE-tagged anti-CD4, anti-CD8, anti-CD3, and anti-Pan-NK (DX5) mAbs were obtained from PharMingen (San Diego, CA).
Construction of recombinant adenoviral vectors
Recombinant adenovirus carrying murine IP-10 under the control
of CMV promoter was constructed using Cre-lox recombination
system (31). Splenocytes were obtained from an 8-wk female
BALB/c mouse, cultured with complete medium in
10-cm2 culture dish (Techno Plastic Products,
Trasadingen. Switzerland), and stimulated for 3 h with LPS (20
ng/ml) (Sigma, Madrid, Spain). Subsequently, total cellular RNA was
isolated with Ultraspec (Biotecx Laboratories, Houston, TX), and
amplified by RT-PCR using specific primers for murine IP-10 (mIP-10)
(33). The 311-bp fragments corresponding to mIP-10 cDNA
were cloned into pGEM-T vector system (Promega, Madison, WI) and
sequenced (3, 4). The fragment containing the mIP-10 cDNA
was SphI/SalI excised from pGEM-T/mIP-10, filled
in by Klenow, and blunt-end ligated into BglII-digested
alkaline phosphatase-treated pAdlox. CRE8 cells were
CaPO4 cotransfected with
SfiI-predigested pAdlox/mIP-10 and 
5 DNA
(31). After 10 days, lysate from the cotransfected cells
was used to infect CRE8 cells to eliminate the 5 virus contamination
(two similar passages were performed to assure purity). Lysate from the
last passage was used to infect 293 cells to amplify AdCMVIP-10.
Packaged viral DNA was prepared as described by Hardy et al.
(31) and, when digested with BsaBI, confirmed
the expected pattern upon 1% agarose electrophoresis analysis (all
samples were 5 DNA-free).
Recombinant adenovirus carrying IL-12 (AdCMVIL-12) has been previously described (27). Briefly, an expression cassette of IL-12 under the control of CMV promoter was constructed encompassing IL-12 p35 cDNA, an internal ribosomal entry site, IL-12 p40 cDNA, and a polyadenylation signal. Recombinant adenovirus encoding the IL-12 cassette of expression (AdCMVIL-12) was generated by cotransfection of 293 cells according to standard procedures (34). Adenovirus carrying LacZ reporter gene under the control of CMV promoter (AdCMVLacZ) was produced similarly (35). All recombinant adenoviruses were isolated from a single plaque, expanded in 293 cells, and purified by double cesium chloride gradient ultracentrifugation (35). Purified virus was extensively dialyzed against 10 mM Tris/1 mM MgCl2 and stored in aliquots at 80°C, and it was carefully titred by plaque assay.
Western blot analysis
HepG2 cells cultured to 75% confluence in 10-cm diameter dishes were infected with AdCMVIP-10 (multiplicity of infection (MOI) = 65), transfected with Padlox/IP-10 (Fugene, Roche, Barcelona, Spain) or left untransfected and maintained in serum-free RPMI 1640 for 36 h. Supernatants were collected and concentrated using Centricom YM-3 (Amicon; Milipore, Madrid, Spain). Recombinant murine IP-10 (R&D Systems) was used as positive control. Proteins were resolved by 15% SDS-PAGE using Tris/Tricine buffer (36) and transfered to Hybond-P membranes (Amersham-Pharmacia Biotech, Madrid, Spain) that were blocked in 5% nonfat milk in PBS 0.1% Tween 20 (PBS-T) overnight at 4°C. After washing four times in PBS-T, the membranes were incubated for 1 h at room temperature in diluted (1/1000) anti-IP-10 goat polyclonal IgG (Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were washed five times in PBS-T and incubated with diluted (1/5000) HRP-conjugated donkey anti-goat IgG (Santa Cruz Biotechnology) for 1 h at room temperature. Blots were developed by enhanced chemiluminescence detection reagents (ECL Plus; Amersham-Pharmacia Biotech).
Chemotaxis assay
Chemotactic activity was measured by migration assays across polycarbonate membranes (37). T cells from BALB/c spleens were enriched by plastic adherence and passage through nylon wool columns. T cells were activated in RPMI 1640, 10% FCS, IL-2 (20 IU/ml) (PeproTech) and Con A (2 µg/ml) (Sigma) for 2 days and then harvested. HepG2 cells at 80% of confluence in a 10-cm diameter dish were infected at a MOI of 65 with AdCMVLacZ, AdCMVmIP-10, or noninfected in RPMI 1640, 0.5% FCS. After 48 h, supernatants from AdCMVLacZ-infected, AdCMVmIP-10-infected, and noninfected cells were collected. Recombinant mIP-10 10 ng/ml protein, rIP-10 plus mAb anti-IP-10 (2 µg/ml), and uninfected cells were added to the lower chamber, and T cells (100 µl at 5 x 106/ml) were resuspended in RPMI 1640 plus 0.5% FCS added to the upper chamber. Migration assays were performed across polycarbonate membranes, 6.5 mm diameter, 10 µm thickness, 5 µm diameter pore size transwell cell culture chambers (Costar, Cambridge, MA). Migration was allowed at 37°C in 5% CO2 atmosphere for 2 h. Filters were then fixed in 1% (v/v) glutaraldehyde in PBS for 1 h and stained in 0.5% (w/v) toluidine blue for 2–4 h. Cell migration was quantified by direct count of cells adhered to the bottom side of the polycabonate filters; 10 microscopic fields per point were counted. Four replicated wells were used for each condition.
In vivo gene therapy of established tumors
Tumors were established by s.c. or intrahepatic implantation of CT26 cells. A total of 5 x 105 cells were injected at the right hind flank of BALB/c syngenic mice. Ten days later, when tumors reached 5–7 mm (in diameter), different recombinant adenoviruses (AdCMVIP-10, AdCMVIL-12, and AdCMVLacZ) at indicated doses were injected intratumorally in a volume of 50 µl diluted in saline buffer (PBS). Tumor growth was monitored twice a week by measuring two perpendicular tumor diameters using a precision caliper. Animals showing severe distress or with tumors that exceeded 1.5 cm in two perpendicular diameters or 2 cm in one diameter were sacrificed for ethical reasons according to institutional guidelines. To induce bilateral tumors, 5 x 105 CT26 cells were injected s.c. into BALB/c mice at both right and left hind flanks.
T cell culture for adoptive therapy
Mice bearing bilateral 5–8 mm (diameter) s.c. CT26 tumors were treated with intratumor injections of 108 pfu of AdCMVIL-12. Draining lymph nodes were removed aseptically 5 days later, and single-cell suspensions were obtained by pressing them mechanically through mesh screens. Lymph node cells were cultured in 24-well plates (Greiner Labortechnik, Frickenhausen, Germany) for 7 days at 5 x 106 cells/well with 2 x 105 CT26 tumor cells/well pretreated for 1 h at 37°C with 150 µg/ml of mytomycin-C (Sigma) (reagent was extensively washed). Cells were cultured for 7 days in complete RPMI 1640 supplemented on day 5 with mIL-2 (8–10 IU/ml) (PeproTech).
For adoptive transfer of CD4+ cells, splenocytes from mice who had rejected CT26 s.c. tumor nodules by treatment with AdCMVIP-10 plus AdCMVIL-12 15 days earlier were incubated with anti-CD4-coupled magnetic beads (MiniMACS, Miltenyi Biotech, Bergisch Gladbach, Germany), positively selected by magneting sorting according to manufacturer instructions and injected i.v. to BALB/c nu/nu mice.
In vivo treatment of CT26 tumors by recombinant adenovirus and adoptive transfer of lymphocytes
BALB/c mice, in groups of five to six, received 5 x 105 CT26 cells in 25 µl of PBS injected surgically in the mid-lobe of the liver under general anesthesia. Ten days later, tumor diameters were assessed by surgical examination and injected with 5 x 108 pfu of recombinant adenoviruses or an equal volume (50 µl) of PBS. For cellular adoptive therapy, mice were injected i.v. with 5 x 106 cells from short-term antitumor T lymphocyte cultures 72 h after adenovirus administration. These mice received three i.p. injections of 2 x 104 IU of human rIL-2 (Chiron, Medfield, MA) in PBS on alternate days. Tumor size (mean diameter) was assessed by laparotomy using a precision caliper on day 19 after tumor challenge. Statistic significance of the differences among groups was evaluated by nonorthogonal contrasts.
Peptides
The H-2Ld-restricted peptides AH1 (SPSYVYHQF) (38) and P815AB (LPYLGWLVF) (39) were synthesized by F-moc chemistry as described (40), and purity was confirmed by HPLC.
51Cr release assay
Cytotoxicity was analyzed in conventional 5-h 51Cr release assays as described (41). Briefly, 51Cr-loaded CT26, P815, and YAC-1 cells were incubated with effector cells at different E:T ratios in triplicate wells, and 51Cr release (cpm) into the supernatants was measured in a gamma-counter to calculate percentage specific release as described (41). In some experiments, P815 cells were incubated during the assay with various concentrations of synthetic AH1 or P815AB peptides.
Depletion of lymphocytes and tumor growth
Tumor-bearing mice, four to five in each group, were depleted of CD4+ or CD8+ cells by i.p. injection of 100 µl of anti-CD4 or anti-CD8 ascitic fluid eight times, on days -3, -2, -1, 0, 5, 10, 14, and 21. Animals received treatment by intratumor injection of AdCMVIP-10 (5 x 108 pfu) and AdCMVIL-12 (7, 5 x 107 pfu) on day 0. Tumor growth was assessed twice a week. Experiments were repeated twice.
For NK depletion, anti-asialo GM1 (Wako) was given i.p. (100 µl/dose) when indicated. Gadolinium chloride (Sigma) was given i.v. (20 µg/dose). Depletions were monitored by FACS analysis of PBMC stained with fluorocrome-tagged anti-CD3, anti-CD4, anti-CD8, or anti-Pan-NK (DX5) (PharMingen) (41).
Histology and immunohistochemistry
For hemotoxylin-eosin staining, 4% formaldehyde-fixed tumor nodules were paraffin embedded, and sections of 4–6 µm thickness were stained according to standard procedures. For immunohistochemical staining, tumor tissues were embedded in Tissue-Tek OCT compound (Sakura, Zoeterwoude, The Netherlands), snap frozen in liquid nitrogen, and stored at -80°C. Tissues were sectioned on a cryostat at 4–6 µm, warmed at room temperature, air dried, and fixed in prechilled acetone for 10 min. After rinsing in PBS, endogenous peroxidase activity was neutralized using Dako peroxidase blocking reagent (Dako, Carpinteria, CA). Subsequently, sections were incubated with rat mAbs against mouse CD8 or CD4 (PharMingen) for 1 h at room temperature. Anti-rat IgG peroxidase conjugate (Sigma) was used as secondary Ab according to manufacturer’s instructions. The chromogenic substrate diaminobenzidine (Dako) followed by Mayer’s hematoxylin counterstaining was used to visualize positive reactions. Images are representative of multiple microscopic fields observed in at least three tumors equally treated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IP-10 cDNA was amplified by RT-PCR from total purified BALB/c splenocytes stimulated with LPS (20 ng/ml) for 3 h. A 311-bp product was directly cloned into pGEM-T vector system and sequenced to rule out PCR errors and to confirm the identity of IP-10 cDNA.
To construct the recombinant adenovirus, a strategy based on
Cre-lox-directed recombination was used (31).
Thereby, IP-10 cDNA plus the encapsidation adenoviral signal was
introduced into the defective adenoviral genome (5) by
cotransfection into Cre-expressing CRE8 cells, placing the IP-10 cDNA
under the transcriptional control of CMV promoter as shown in Fig. 1
A. Nonrecombinant helper
adenovirus was purged by repeated infection of CRE8 cells, and,
subsequently, recombinant adenovirus (AdCMVIP-10) was purified and
produced by infection of 293 cells, as described in Materials and
Methods.
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B). Slight
immunoblot IP-10 reactivity was also observed in HepG2 cells
transfected with liposome-coated IP-10-encoding plasmid
(PAdlox/IP-10). The culture supernatant of AdCMVIP-10-infected cells contains IP-10-dependent chemotactic activity for activated T cells
To verify that IP-10 expressed by AdCMVIP-10 was functional, HepG2 cells were infected at MOI = 65 and the culture supernatants were harvested 48 h later. Chemotactic activity for Con A T cells blasts was assessed by quantifying T cell migration across 5-µm pore polycarbonate membranes in a transwell cell culture chamber assay to measure the response to chemotactic stimuli placed into the lower chamber.
As shown in Fig. 2, supernatants from
AdCMVIP-10-infected cells attracted Con A blasts well above control
levels. Such an activity was abrogated by addition of a neutralizing
anti-IP-10 mAb and it was not present in the supernatants of
AdCMVLacZ-infected HepG2 cells. Our Con A-stimulated polyclonal T cell
populations encompassed approximately equal numbers of
CD4+ and CD8+ T cells. When
using magnetic bead-purified CD4+ and
CD8+ cells from such Con A blasts, we obtained
data consistent with previous observations (8) that
indicate a much stronger effect on the activated
CD4+ cell subset (data not shown).
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To explore whether AdCMVIP-10 had therapeutic effects against tumors, the CT26 colon adenocarcinoma cell line was s.c. injected into syngenic BALB/c mice. On day 9, nodules ranging from 4 to 8 mm in diameter were injected with 109 pfu of AdCMVIP-10, a control adenovirus (AdCMVLacZ), or saline buffer.
AdCMVIP-10 induced complete regressions of malignant tumors in 2 of 14
animals, whereas all tumors were lethal in the control groups. Fig. 3A shows the individual follow
up of the diameter of the tumor nodules. In some animals treated with
AdCMVIP-10, a tendency to delay tumor growth in comparison to control
animals was noted.
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Intratumoral AdCMVIP-10 fosters the therapeutic efficacy of systemic T cell adoptive therapy
Because IP-10 expression can result in the attraction of activated T lymphocytes into the malignant tissue, we reasoned that AdCMVIP-10 infection of hepatic tumor nodules could potentiate the effect of adoptive transfer of antitumor T lymphocytes.
To study this issue, T cell cultures were obtained from the lymph nodes of mice who had rejected CT26 tumors upon intratumoral injection of an adenovirus-encoding murine IL-12 (AdCMVIL-12) by 7-day coculture with mitomycin-C-treated CT26 cells. Such short-term T cell cultures displayed potent CTL activity against CT26 and contained both activated CD4+ and CD8+ T cells, as we have previously reported (42).
Those T cell cultures were used for i.v. injection to treat tumor
nodules derived from injection of CT26 cells into the mid-lobe of the
liver in such a way that they gave rise to tumor nodules (4–8 mm in
diameter) 8 days later. As shown in Fig. 4, adoptive transfer of 5 x
106 of such T cells did not show any effect
against those CT26 liver tumor nodules. Under similar conditions,
intratumor injection of 109 pfu of AdCMVIP-10
achieved only one tumor rejection of 11 mice treated. In contrast, 5 of
14 mice receiving both intratumor AdCMVIP-10 (109
pfu) on day 8 and antitumor T cells (5 x
106 i.v.) 36 h later showed complete
regression of their tumors when surgically inspected 12 days later. Our
results indicated a moderate synergistic effect of adoptive T cell
therapy and gene transfer of IP-10 into established liver tumor
nodules, which resulted in long-term survival in some cases (Fig. 4B). These data are reminiscent of our previous study with
AdCMVIL-12 plus adoptive T cell therapy (42), although the
synergy with adoptive T cell transfer displayed by IL-12 in the same
system was more intense.
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We have described a potent antitumor therapeutic effect of AdCMVIL-12 against CT26-derived tumors when used at doses of 109 and 108 pfu (42). Under such conditions, AdCMVIL-12 directly injected into the tumor nodules led to eradication in 60–80% of the cases. When dealing with CT26 tumors, IL-12 gene transfer elicited an antitumor immune response that was CD8+ T cell but not CD4+ T cell dependent.
In Fig. 5, we show that lower doses of
AdCMVIL-12 (7.5 x 107 pfu) given to s.c.
tumor nodules that were allowed to grow for 9 days (4–8 mm diameter)
only cured 4 of 11 cases with some delay of tumor growth in two
additional mice. In striking contrast, combination of this suboptimal
dose of AdCMVIL-12 (7.5 x 107 pfu) plus
5 x 108 pfu of AdCMVIP-10 consistently
resulted in tumor regression of every treated tumor (10 of 10 cases).
Such an outstanding result did not reflect transgene-unrelated effects
caused by adenovirus combination because similar doses of AdCMVIP-10
and AdCMVLacZ did not show any significant change in tumor progression
beyond a small delay in tumor growth in 3 of 10 cases if compared with
intratumor injection of saline buffer. Moreover, in an additional set
of experiments (Fig. 6) combination of
AdCMVIL-12 (7.5 x 107 pfu or 5 x
107 pfu) with AdCMVLacZ (5 x
108 pfu) resulted in 40% tumor regression, while
the same doses of AdCMVIL-12 combined with AdCMVIP-10 (5 x
108 pfu) caused 100% tumor disappearance. These
results together with those of Fig. 5 indicate that the synergistic
effect of AdCMVIP-10 on the antitumoral activity generated by
AdCMVIL-12 is not transgene independent but mediated by IP-10
expression. In addition, repeated peritumoral injection of an antiserum
anti-IP-10 delayed the rejection of tumors treated by AdCMVIP-10
plus AdCMVIL-12 for about 2 wk (data not shown).
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To ascertain as to whether adenoviral gene transfer of IP-10 and
IL-12 required expression of the therapeutic genes on the same tumor
nodule to display synergistic effects, we conducted experiments in
which two tumor nodules were generated by CT26 s.c. inoculation into
opposite flanks of the same mouse (Table I). Treatment with AdCMVIL-12 at
suboptimal doses (7.5 x 107 pfu) led to
tumor nodule regression of the treated site in only two of six cases,
whereas the untreated site lethally progressed in every case. When
AdCMVIP-10 (5 x 108 pfu) and AdCMVIL-12
(7.5 x 107 pfu) were injected in different
nodules, two of six tumors regressed in the IL-12-transfected side and
one of six regressed in the IP-10-transfected nodules. Interestingly,
coinjection of the same doses of AdCMVIP-10 and AdCMVIL-12 led to tumor
regression in every case (five of five cases) both at injected and at
the untreated tumor location. Thus, synergy was greatly dependent on
colocalization of both transgene products and thereby it could mediate
a potent therapeutic effect against distant untreated tumors nodules.
This is considered important to treat widespread metastatic
disease.
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In the setting of CT26-derived s.c. tumor nodules treated with
5 x 108 pfu of AdCMVIP-10 and 7.5 x
107 pfu of AdCMVIL-12 given in a single bolus, we
next studied the requirement of T cells for the observed therapeutic
effect. Specific depletion of either CD4+ or
CD8+ cells with specific mAb 3 days before
intratumor treatment with the adenovirus combination resulted in a
minor decrease of the antitumor activity (Fig. 7, A and B).
However simultaneous depletion of CD4+ and
CD8+ T cells resulted in lethal tumor progression
in seven of nine mice, despite having received the AdCMVIP-10 plus
AdCMVIL-12 combination (Fig. 7C). In BALB/c nude mice,
intratumor treatment with AdCMVIP-10 plus AdCMVIL-12 also lacked
antitumor activity (Fig. 7, D and E), confirming
the T cell absolute requirement. These results are in contrast with
depletion experiments in mice whose tumors had been treated with
AdCMVIL-12 alone. In this case, only CD8+ T cells
are absolutely required for the antitumor effect, indicating a
selective effect of IP-10 on activated CD4+ T
lymphocytes (27). Nonetheless, the nature of the effector
cells under CD8+ depletion was not clear, and
experiments depleting NK cells with asialo GM1 and macrophages with
gadolinium chloride were conducted (Fig. 8A). We found that depletion
of asialo GM1+ cells allowed the progression of
two of four tumors treated with AdCMVIP-10 plus AdCMVIL-12. In
contrast, gadolinium chloride did not impair the therapeutic effects
(data not shown). Interestingly, under combined depletion of
macrophages, NK, and CD8 cells, a residual antitumor activity was
observed.
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B) show that the adoptive transfer of
CD4+ cells clearly delayed the progression of the
tumors nodules but did not cure them. Taken together, our data suggest
that AdCMVIP-10 plus AdCMVIL-12 therapeutic effects are a result of
combined functions of T cells (CD4+ and
CD8+), NK cells, and nonimmune mechanisms
possibly related to tumor vasculature.
In agreement with these experiments, specific immunostaining of frozen
tumor sections treated 11 days earlier by a combination of AdCMVIP-10
plus AdCMVIL-12 showed a marked infiltration of
CD4+ lymphoid cells (Fig. 9A) with foci of
CD8+ infiltrate (Fig. 9B). Such
results are reminiscent of the reported infiltrate induced by
AdCMVIL-12 alone, which consists also of CD4+ and
CD8+ T cells (Ref. 43 and our own
results; data not shown).
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Splenocytes harvested from mice who have received intratumor
treatment with AdCMVIP-10 plus suboptimal AdCMVIL-12 2–3 wk earlier
displayed a potent lytic activity against CT26 after 6 days
restimulation in vitro with mitomycin-C-treated CT26 cells. This
activity was not detected in the spleen of mice who had been
intratumorally treated with the same doses of either AdCMVIP-10 or
AdCMVIL-12 (Fig. 10A). The
CTL activity was directed, at least in part, to the described Moloney
murine leukemia virus env gene-encoded tumor-associated Ag expressed by
CT26 (38), because these lymphocyte cultures lysed P815
cells pulsed with the AH1 antigenic determinant presented by
H-2Ld (Fig. 10B). Specificity of
cytotoxicity was confirmed against nonpulsed P815, P815 pulsed with
P815AB (control peptide), and the NK-sensitive target YAC-1 (Fig. 10B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although our data demonstrate that functional murine IP-10 can be detected in the supernatant of cells infected in vitro with AdCMVIP-10, only a mild effect on established CT26-derived s.c. malignant nodules was noted after the injection of relatively large doses of the adenovirus. Despite the lack of measurable effects on the macroscopic tumor growth, histological examination of treated tumors disclosed areas of necrosis and vascular damage, consistent with the reported data for intratumoral injection of the recombinant protein (12). In our hands, supernatant from cells infected in vitro with AdCMVIP-10 attracted CD4+ T cells with higher intensity than CD8+ T cells, an effect that could be abrogated with anti IP-10 Abs. The preferential attraction of CD4+ cells is in accordance with published data (8).
Intratumoral injection of AdCMVIL-12 (an adenovirus encoding for both
chains of IL-12) at doses equal to those shown in Fig. 3 for AdCMVIP-10
induced tumor regression in 
70% of the cases when treating
comparable tumor nodules associated with a marked increase in antitumor
CTL activity (27). Because such lymphocyte cultures
contained both activated CD4+ and
CD8+ T cells, it was reasoned that IP-10 would
probably help the homing of antitumor lymphocytes into the tumor
tissue. Our data shows that IP-10 gene transfer into large
well-established CT26 carcinomas growing in the liver had a synergistic
effect with the adoptive transfer of short-term anti-tumor T
lymphocyte cultures, an effect that was not observed with the control
adenovirus-encoding ß-galactosidase gene. It is conceivable that
IP-10 expressed by the malignant tissue attracts
CD4+ cells and CTLs that in turn execute the
antitumor effect.
These data are in agreement with previous reports in which intratumor injection of fibroblasts retrovirally modified with lymphotactin lack antitumor effects unless cotransfected with the immunostimulatory factor IL-2 (17). It was concluded that lymphotactin induced a tumor infiltrate but failed to expand and activate an antitumor immune response powerful enough to destroy the malignant inoculum. Similar pieces of information have been raised with the use of an adenovirus encoding for both IL-12 and lymphotactin, which were studied in comparison with adenovirus encoding separately each factor (20). In summary, in those studies lymphotactin increased the effects of IL-12 by attracting a more intense lymphoid infiltrate.
Because AdCMVIP-10 showed some synergy with adoptive transfer of lymphocytes derived from mice whose tumors were treated with AdCMVIL-12, the potential synergy of both adenoviruses when used together was explored. To this end, a suboptimal dose of AdCMVIL-12 was chosen. We show that simultaneous injection of AdCMVIP-10 along with AdCMVIL-12 induced tumor regressions in 100% of the cases using both optimal and suboptimal intralesional AdCMVIL-12 doses. This finding is important for two reasons: 1) in our CT26 model AdCMVIL-12 by itself failed to induce complete regressions in 20–30% of the cases, and 2) IL-12 can induce lethal shock (25), and reducing IL-12 doses, based in its synergistic effect with chemokines, might allow dose reduction with similar or even better effects in the absence of toxicity. Because we have not detected serious toxicity up to 1011 pfu of AdCMVIP-10 given intratumorally (data not shown), we believe that gene transfer of IP-10 will permit the use of safer doses of IL-12 without toxicity related to IP-10.
The combination of IL-12 and IP-10 transferred into the same tumor
nodule displayed antitumor effects against a distant s.c. concomitant
tumor. The relevance of these data is seen when we consider the natural
evolution of metastasic colon cancer in which it will be often
impossible to treat all the malignant sites at once. It is interesting
to consider that synergy of IL-12 and IP-10 only took place when both
recombinant adenoviruses were given to the same nodule, but not when
identical doses were injected into distant tumor nodules. This is in
agreement with the so-called "attraction and activation" hypothesis
(45), which predicts the necessity of colocalization of
immunostimulatory and chemoattractant factors as previously seen for
lymphotactin (17, 20) and macrophage inflammatory
protein-1 (18).
We have found an absolute requirement for T lymphocytes in the antitumor effect of IL-12 plus IP-10 local gene transfer. Total loss of the effect is only seen after double depletion of CD4+ and CD8+ cells indicating that both subsets can independently mediate the effects. This is in contrast with our own data obtained in the same CT26 model when using AdCMVIL-12 at optimal doses in which CD8+ T cells were the only lymphocytes required for the effect (27). Our results pinpoint to a role for CD4+ cells consistent with the observed mass infiltrate of CD4+ and CD8+ cells presumably attracted by IP-10. Results obtained after specific depletion with mAbs are not surprising, for CD4+ T cells have been found to display antitumor properties despite the lack of MHC class II expression on malignant cells (46). The role of CD4+ T cells could be to stimulate tumor cell killing by macrophages (46) and stimulation of dendritic cells to prime CTLs (47).
Cytokine secretion by CD4 T cells also can be activating NK cells to carry out their functions. In accordance to our depletion data with anti-asialo GM1, NK cells probably play a pivotal role in tumor rejection but their contribution is not sufficient, because combined therapy fails in nude mice who have normal NK activity but lack conventional T cells. The role for NK cells in the effects of IP-10 plus IL-12 combined therapy is in agreement with published observations from other groups (48, 49). We cannot rule out a contribution by NK-T cells as suggested by others (50) because it is noteworthy that asialo GM1 antiserum also depletes NK-T cells and possibly other T cell populations (51). In our experimental conditions, although macrophages could exert some antitumor activity, extensive gadolinium chloride treatment do not significantly alter the outcome, indicating that there is not important requirement for these cell populations. Partial efficacy of adoptive transfer of immune CD4 cells to nude mice further show a role for Th cells in the therapeutic activity. Despite the adoptive transfer of high numbers of such T cells, the antitumor effect was not complete, a result that can be interpreted in the sense that local conditioning of malignant tissue by the expression of the IL-12 and IP-10 transgenes is also required.
Our results show a dramatic increase in anti CT26 CTL activity upon intratumoral injection of AdCMVIL-12 plus AdCMVIP-10, which is probably related to an ongoing Th response favored by IP-10-mediated recruitment of activated CD4 cells. Nonetheless, other potential effects of IP-10 could favor CTL generation such as the observed necrosis of malignant tissue that could release tumor Ags in a fashion suitable for presentation by professional APCs (52). Increase in CTL activity has also been reported for the combination of IL-12 and lymphotactin by gene transfer (20).
Antitumor effects of IL-12 plus IP-10 most likely reflect the complexity of an interconnected network of immune and nonimmune mechanisms difficult to dissect and possibly redundant in achieving the outcome of tumor rejection. Regardless of the mechanisms involved, we are reporting a powerful tool to treat experimental murine malignancies by simultaneous adenoviral gene transfer of IP-10 and IL-12. Potency and safety profiles of the approach might make it find a place in immunotherapy of human malignancies.
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Acknowledgments |
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Footnotes |
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2 I.N. and G.M. contributed equally to this study.
3 Address correspondence and reprint requests to Drs. Ignacio Melero or Cheng Qian, Department of Medicine, School of Medicine, University of Navarra, C/Irunlarrea 1, Pamplona 31008, Spain. E-mail address:
4 I.M. and J.P. equally share credit for senior authorship.
5 Abbreviations used in this paper: IP-10, IFN--inducible protein-10; AdCMVIP-10, adenovirus encoding IP-10; AdCMVIL-12, adenovirus encoding IL-12; AdCMVLacZ, adenovirus encoding LacZ; mIP-10, murine IP-10; MOI, multiplicity of infection.
Received for publication August 12, 1999. Accepted for publication January 13, 2000.
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)
and a loxP site, all flanked by the adenoviral inverted
terminal repeats (ITRs). Recombinant adenovirus was created by
cotransfection of pAdlox/IP-10 and 
