of April 14, 2018.This information is current as

TherapyInduced by Single-Chain IL-12 Gene Tumor-Specific CD8 T Cell Responsethe Generation of a Protective

-Inducible Protein-10 Is Essential forγIFN-

N. LodeHomann, Gerhard Gaedicke, Ralph A. Reisfeld and Holger Ursula Pertl, Andrew D. Luster, Nissi M. Varki, Dirk

http://www.jimmunol.org/content/166/11/6944doi: 10.4049/jimmunol.166.11.6944

2001; 166:6944-6951; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2001 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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IFN-g-Inducible Protein-10 Is Essential for the Generation of aProtective Tumor-Specific CD8 T Cell Response Induced bySingle-Chain IL-12 Gene Therapy1

Ursula Pertl,* Andrew D. Luster, † Nissi M. Varki, ‡ Dirk Homann,* Gerhard Gaedicke,§

Ralph A. Reisfeld,* and Holger N. Lode2§

The successful induction of T cell-mediated protective immunity against poorly immunogenic malignancies remains a majorchallenge for cancer immunotherapy. Here, we demonstrate that the induction of tumor-protective immunity by IL-12 in a murineneuroblastoma model depends entirely on the CXC chemokine IFN-g-inducible protein 10 (IP-10). This was established by in vivodepletion 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 ofscIL-12-mediated antiangiogenesis and T cell chemotaxis in mice receiving s.c. injections of scIL-12-producing NXS2 cells. Thesefindings were extended by data demonstrating that IP-10 is directly involved in the generation of a tumor-protective CD81 Tcell-mediated immune response during the early immunization phase. Four lines of evidence support this contention: First, A/Jmice 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, CD81 T cell-mediated MHC class I Ag-restricted tumor cell lysiswas inhibited in such mice. Third, intracellular IFN- g expressed by proliferating CD81 T cells was substantially inhibited inIP-10-depleted, scIL-12 NXS2-vaccinated mice. Fourth, systemic tumor protective immunity was completely abrogated in micedepleted of IP-10 in the early immunization phase, but not if IP-10 was depleted only in the effector phase. These findings suggestthat IP-10 plays a crucial role during the early immunization phase in the induction of immunity against neuroblastoma by scIL-12gene therapy. The Journal of Immunology,2001, 166: 6944–6951.

W e recently demonstrated that effective tumor-protec-tive immunity can be achieved in a poorly immuno-genic, syngeneic tumor model of murine neuroblas-

toma after the transduction of a fusion gene encoding a linearizedsingle chain (sc)3 IL-12 (scIL-12) into NXS2 neuroblastoma cells.In fact, s.c. vaccination with live, scIL-12-producing NXS2 neu-roblastoma cells not only resulted in tumor rejection at the primaryvaccination site but also completely protected such mice fromchallenges with lethal doses of wild-type tumor cells as indicatedby the absence of disseminated metastases (1).

IL-12 is known to have multifunctional activities (2), includingimmunomodulation (1, 3–7) and antiangiogenesis (8–10). Themode of action responsible for the antitumor effects induced bylocal scIL-12 includes inhibition of primary tumor growth (11) as

well as inhibition of metastasis. IL-12 and its downstream medi-ator, IFN-g can elicit diverse immunomodulatory effects becauseof acquired and innate immune mechanisms. IL-12-mediated in-nate immune responses include stimulation of NK cell cytotoxicityand subsequent activation of neutrophils and macrophages. This inturn leads to the production of superoxides and nitric oxide, whichare involved in mechanisms that were shown to control tumorgrowth in animal models (12–15). However, the eradication ofdisseminated metastasis requires adaptive antitumor immune re-sponses, which are initiated by IL-12 via induction of a CD81 Tcell-mediated immune response. This is achieved by augmentingcytotoxicity of primed CD81 T cells or Th1 differentiation of na-ive CD41 T cells that can subsequently provide help for tumor-specific priming of CTLs. A third mechanism of action of IL-12 isprovided by the inhibition of tumor-induced neovascularization,i.e., antiangiogenesis. Experiments in vitro and in vivo have doc-umented that the antiangiogenic effects of IL-12 are indirect andrequire the participation of IFN-g which, in turn, stimulates secre-tion of IFN-g-inducible protein 10 (IP-10). This chain of eventsidentified IP-10 as a key player in IL-12-mediated antiangiogen-esis (8, 10, 16).

IP-10 is a CXC chemokine that has been shown to induce che-motaxis of activated T cells and to inhibit angiogenesis (17–19). Itis produced by activated monocytes, fibroblasts, endothelial cells,epithelial cells, and keratinocytes. IP-10 binds to a seven-trans-membrane G protein-coupled receptor, CXCR3, expressed on ac-tivated T cells, leading to chemotaxis (20). IP-10 has two potentialantitumor effects: antiangiogenesis and immunomodulation, simi-lar to what has been described for IL-12. These observations stim-ulated a number of investigators to determine the role of IP-10 inIL-12-mediated antitumor effects focusing primarily on the growth

*Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037;†Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Al-lergy and Immunology,Massachusetts General Hospital, Harvard Medical School,Charlestown, MA 02129;‡University of California, Cancer Center 0961, La Jolla, CA92093; and§Charite Children’s Hospital, Berlin, Germany

Received for publication December 28, 2000. Accepted for publication March22, 2001.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by a grant from the Culpepper Medical Foundation (toA.D.L.), National Institutes of Health, National Cancer Institute Grants CA83140 (toR.A.R.) and CA69212 (to A.D.L.), and Deutsche Forschungsgemeinschaft, Emmy-Noether Lo Grant 635/2 (to H.N.L.).2 Address correspondence and reprint requests to Dr. Holger N. Lode, Charite Chil-dren’s Hospital, Augustenburgerplatz 1, 13353 Berlin, Germany. E-mail address:holger.lode@charite.de3 Abbreviations used in this paper: sc, single chain; IP-10, IFN-g-inducible protein10; m, mouse.

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00

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of primary tumors in naive mice and the inhibition of neovascu-larization or chemotaxis (3, 18, 19, 21).

Here, we demonstrate for the first time that IP-10 is a key playerin mediating systemic tumor-protective immunity induced byscIL-12 gene therapy by directly affecting Th1-type CD81 T cellsand by playing a crucial role in the early immunization phase ofvaccination with scIL-12.

Materials and MethodsConstruction and characterization of sc mouse (m) IL-12

The construction of scIL-12 was done as described previously (1). Briefly,the cDNAs encoding the p35 and p40 chains of mIL-12 were generatedfrom Con A-stimulated mouse splenocytes by RT-PCR. The cDNA for thescIL-12 fusion protein was constructed by linkage of p35 and p40 subunitswith a synthetic linker. To assure secretion in eukaryotic cells, this con-struct was joined at its 59SmaI site with the 39BalI site of a p24 leadersequence, thereby replacing the amino-terminal arginines of p35 with twoglycines downstream from the cleavage site. After introduction into thepBK-CMV vector (Stratagene, La Jolla, CA) using theNotI andXhoI re-striction sites, NXS2 cells were transfected and stable clones selected in thepresence of 500mg/ml G418 (Sigma, St. Louis, MO).

The mIL-12 protein content produced by NXS2 cells was measured byan ELISA for mIL-12-p70 (Genzyme, Cambridge, MA). Two NXS2 clonessecreting 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 spe-cific IL-12 activity of the scIL-12 construct was one-sixth that of themIL-12 standard (Hoffmann-La Roche, Nutley, NJ), as determined by itsability to induce mIFN-g after incubation with splenocytes, as describedpreviously (1). One NXS2 clone transduced with the empty vector wasused for control experiments. All clones revealed the same proliferationcharacteristics in vitro as NXS2 wild-type cells.

Cell lines and neuroblastoma model

Syngeneic female A/J, C.B-17scid/scidand C.B-17scid/beigemice wereobtained at 6–12 wk of age from The Jackson Laboratory (Bar Harbor,ME). They were housed in the pathogen-free mouse colony at our institu-tion, and all animal experiments were performed according to the NationalInstitutes of Health guide for “The Care and Use of Laboratory Animals.”The murine NXS2 wild-type, NXS2 pBK-CMV, and NXS2 scIL-12 neu-roblastoma cell lines were cultured and used for vaccination experiments,as described previously (1). Briefly, primary tumors were induced by s.c.injection of 2 3 106 NXS2 pBK-CMV cells and their growth comparedwith that of NXS2 scIL-12 cells producing high and low amounts of scIL-12. Tumor growth was determined by microcaliper measurements, andtumor volume was calculated according to the formula1⁄2 (length) 3(width)2. Liver metastases were induced by lateral tail vein injection of 53104 NXS2 wild-type cells, and mice were sacrificed for evaluation after28 days.

Determination of tumor-induced angiogenesis in Matrigel

Matrigel (Becton Dickinson Labware, Bedford, MA), a product derivedfrom the Engelbreth-Holm Swarm tumor (22) consisting of laminin, col-lagen IV, heparin sulfate, proteoglycan, and nidogen/entactin, liquefies at4°C and rapidly polymerizes at 37°C following s.c. injection. Thus, Ma-trigel plugs were induced by s.c. injection of a mixture of liquefied Ma-trigel (0.4 ml) and 13 105 NXS2 pBK-CMV cells or NXS2 scIL-12 cellsin 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 onday 6, after which Matrigel plugs were harvested and weighed. Tumor-induced angiogenesis was quantified by determination of the hemoglobincontent in Matrigel plugs by the method described by Drabkin and Austin(23). Briefly, Matrigel plugs were minced and liquefied in 0.3 ml of PBSat 4°C overnight. Subsequently, hemoglobin content was determined byincubation of 0.1 ml of Matrigel with 1 ml of Drabkin reagent for 15 minat room temperature. Insoluble tissue was removed by centrifugation(14,0003 g for 5 min) and the concentration of hemoglobin calculatedaccording to a calibration curve obtained from serial dilutions of a hemo-globin standard (Sigma). Hemoglobin (Hb) concentrations and weights ofMatrigel plugs were used to calculate mg Hb/g Matrigel.

In vivo depletion of mIP-10 and mIFN-g

Two hamster anti-mouse IP-10 mAbs (1F11 and 1B9) have been generatedby standard techniques. The specificity of these mAbs was tested in directELISA and by immunoblot with other available mouse chemokines, in-

cluding monokine induced by IFN-g (Mig), KC, stromal cell-derived fac-tor-1, eotaxin, macrophage-inflammatory protein (MIP)-1a, MIP-1b, andmonocyte chemoattractant protein-5. Both mAbs were highly specific formIP-10 and did not cross-react with other murine chemokines tested. Thehybridomas have been adapted to serum-free medium and grown in Cell-Max cartridges (Cellco, Kensington, MD), and the mAbs were purified byprotein G affinity chromatography. Purified 1F11 and 1B9 IgG were bothcapable of neutralizing mIP-10-induced chemotaxis and calcium flux re-sponses of mCXCR3 transfected 300–10 cells (24). For in vivo depletionof IP-10 in Matrigel experiments, 250mg of the monoclonal anti-IP-10 Abs1F11 or 1B9 were added to the Matrigel plugs, followed by i.p. injectionof another 250mg of these Abs on days 0, 1, 2, and 4. In addition to themAbs, polyclonal rabbit anti-mouse IP-10 serum, kindly provided by J. M.Farber (National Institutes of Health, Bethesda, MD) was used in Matrigelexperiments. For this purpose, 0.1 ml of anti-IP-10 antiserum was added toliquefied Matrigel followed by i.p. injection of 0.3 ml of antiserum on days0, 1, 2, and 4, similar to 1F11 and 1B9 mAbs.

In vaccination experiments, 250mg of anti-IP-10 mAb was added to thetumor inoculum and injected on days 3, 8, and 17 i.p. For depletion in theimmunization phase, 250mg of anti-IP-10 mAb were injected i.p. on day0 followed by 100mg on days 2 and 4. Depletion of IP-10 in the earlyeffector phase was accomplished by 250mg of anti-IP-10 injected i.p. onday 7 followed by 100mg of mAb i.p. on days 10 and 13 and in the lateeffector phase by 250mg of anti-IP-10 i.p. on day 10 followed by 100mgof mAb on days 13 and 16. Rabbit anti-mouse IP-10 serum was not usedin this set of experiments because of lethal serum reactions in all animalsobserved after the third injection.

In experiments with hamster mAbs and rabbit antiserum, equal amountsof normal hamster serum (Harlan Bioproducts, Indianapolis, IN) or normalrabbit serum (Dako, Carpinteria, CA) were applied as negative controls,respectively. In vivo depletion of mIFN-g was accomplished by addition of0.5 mg of rat anti-mouse IFN-g mAb to the tumor inoculum and additionali.p. injections of 1 mg each on days 3, 7, and 18. Rat anti-mouse IFN-gmAb was obtained from ascites generated with the R4-6A2 hybridoma cellline, kindly provided by S. Webb (The Scripps Research Institute, LaJolla, CA).

Histology and immunohistochemistry

Tumor-induced angiogenesis was determined by histological analysis ofneovascularization and tumor cell morphology in Matrigel experiments. Tothis end, Matrigel plugs containing NXS2 pBK-CMV cells or NXS2 scIL-12-producing cells were harvested in the presence or absence of IP-10depletion on day 6, fixed in 10% buffered formalin, sectioned, and stainedwith hematoxylin and eosin. The immunophenotype of NXS2 scIL-12-induced leukocyte infiltrations was determined 6 days after s.c. inoculationof 2 3 106 NXS2 scIL-12 in the presence or absence of IP-10 depletion andcompared with NXS2 pBK-CMV controls. Frozen sections of tumor tissuewere fixed in cold acetone for 10 min followed by removal of endogenousperoxidase with 0.03% H2O2 (30 min, room temperature). Nonspecificbinding was blocked with 10% species-specific serum in 1% BSA/PBS.Biotinylated anti-mouse CD45 mAb (BD PharMingen, La Jolla, CA), bi-otinylated CD8, CD4, Mac1, and NK cell-specific rabbit anti-asialo GM1

antiserum (Wako, Richmond, VA) were diluted to 10mg/ml in 10% goatserum (1% BSA/PBS, pH 7.4) and overlaid onto serial sections. Slideswere incubated in a humid chamber for 30 min followed by the applicationof biotinylated goat anti-rabbit Ab onto slides that were preincubated withanti-asialo GM1 antiserum for 10 min. Streptavidin-labeled HRP was addedand the slides incubated for 10 min, followed by substrate developmentwith the AEC (amino ethyl carbazole) kit (Vector Laboratories, Burlin-game, CA) and a hematoxylin nuclear counterstain. All incubations werefollowed 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 per-formed, as described previously (25). The amplification of a 302-bp frag-ment of mIP-10 was performed in a 25-ml PCR mixture consisting of 20mM Tris-HCl (pH 8.4), 50 mM KCl, 0.2 mM deoxynucleotide triphos-phate, 2 mM MgCl2, 2.5 UTaqPolymerase (Life Technologies, Rockville,MD), and 0.5mM sense and antisense oligonucleotide primers. PCRs wereconducted in the Mastercycler gradient (Eppendorf, Hamburg, Germany)for 30 cycles (96°C, 15 s; 62°C, 30 s; 72°C, 90 s). The primer sequenceswere as follows: mIP-10 sense: ACC ATG AAC CCA AGT GCT GC;mIP-10 antisense: AGT TAA GGA GCC CTT TTA GAC C. PCR productswere separated on 1% agarose gels by electrophoresis. The identity of302-bp fragments was verified by DNA sequencing. The integrity of thecDNA used for PCR amplification was determined by amplification of a299-bp fragment of mouse GAPDH as described previously (25).

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Determination of secreted IL-12

Blood plasma from naive mice or mice inoculated with 23 106 IL-12-producing NXS2 cells or NXS2 pBK-CMV cells was obtained by retro-orbital puncture with heparinized capillaries (Fischer Scientific, Pittsburgh,PA), followed by centrifugation (2000 rpm for 5 min). IL-12 p70 wasmeasured 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 ofRBC with ACK lysis buffer (Life Technologies) and cultured for 5 days inDMEM medium containing, 10% FCS, 1% glutamine, 1% penicillin/strep-tomycin in the presence of 0.04% T-stim (Collaborative Biomedical Prod-ucts, Bedford, MA), 100 IU human IL-2/ml and irradiated NXS2 tumorcells at a tumor cell:effector cell ratio of 1:100. After 5 days, effector cellswere used for the cytotoxicity assay or for subsequent separation into sub-populations. Pure (.95%) CD41 and CD81 T cells were prepared byMACS (Miltenyi Biotec, Auburn, CA). Briefly, mouse splenocytes wereincubated with either anti-mCD8 or anti-mCD4 Ab microbeads (MiltenyiBiotec). MACS was performed according to manufacturer’s guidelines.For the subsequent51Cr release assay, NXS2 target cells were incubated inthe presence of 0.5 mCi of Na2

51CrO4 (Amersham, Cleveland, OH) for 2 hat 37°C and washed three times and seeded onto a flat-bottom 96-well plateat a density of 5000 cells/well. Effector cells were added at various E:Tratios in a final volume of 200ml/well and incubated for 8 h. MHC classI-Ag restriction was determined by addition of anti-H2Kk (clone 36-7-5)and anti-H2Dd (clone 34-2-12) Abs (25mg/ml; BD PharMingen). Total51Cr release was induced with 10ml of SDS (10%). Supernatants werecollected from each well for determination of51Cr release. The percentageof target cell lysis was calculated as follows: experimental release (cpm)2spontaneous release (cpm)/total release (cpm)2 spontaneous release(cpm) 3 100 5 percent cytotoxicity. The results were expressed as meanvalue6 SD of at least three experiments.

Flow cytometry

Staining of cell surface Ag and intracellular Ags was performed as de-scribed previously (26). Cell cultures from spleens of control mice, immu-nized mice, and IP-10-depleted mice were labeled with CFSE (MolecularProbes, Eugene, OR) and for tumor-specific stimulation incubated for 5days with irradiated NXS2 tumor cells at a tumor/effector cell ratio of1:100 in DMEM medium supplemented with 10% FCS, 2 mM glutamine,100 IU/ml penicillin, 100mg/ml streptomycin, and 0.04% T-stim (Collab-orative Biomedical Products). For staining of intracellular cytokines, cellswere stimulated for the final 5 h with 5 ng/ml PMA (Sigma) and 500 ng/mlionomycin (Sigma). All stimulated cultures contained 1mg/ml brefeldin A(Sigma) to block protein transport into post-Golgi compartments and allowcytokines to accumulate within cells. Surface staining with PE-labeled anti-CD8 mAb was followed by intracellular staining with APC-labeled anti-IFN-g mAb (BD PharMingen). Cells were analyzed on a FACScan flowcytometer with CellQuest software (Becton Dickinson, MountainView, CA).

Statistics

The statistical significance of differential findings between experimentalgroups of animals was determined by the two-tailed Student’st test. Thenonparametric Wilcoxon test was used to determine the statistical signif-icance of hepatic metastases. Findings were regarded as significant if two-tailed p values were#0.05.

ResultsEffect of scIL-12 production on local antitumor response

The involvement of distinct immune effector cells in the antitumorresponse induced by local scIL-12 production was analyzed in im-munocompetent A/J mice. These data were compared with scid/scid mice and those obtained in scid/beige mice to assess the roleof T cells and NK cells, respectively, in this tumor model. NXS2cells producing IL-12 were rejected in A/J mice, irrespective of thecytokine’s secretion rate (Fig. 1A). This was in contrast to scid/scidand scid/beige mice that only revealed a reduction in continuouss.c. tumor growth but not a complete tumor rejection (Fig. 1B).These findings indicate the involvement of the T cell compartmentin the rejection of NXS2 scIL-12 cells, an observation reportedpreviously (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 micelacking both T and NK cells are at least twice those observed inscid/scid mice lacking mature T cells (Fig. 1B). Differences in s.c.tumor growth attributable to variable cell proliferation rates ofeach cell clone investigated could be excluded by determination of[3H]thymidine incorporation over time, which revealed no differ-ence between each NXS2 cell clone (data not shown). Interest-ingly, a reduction in tumor growth dependent on the scIL-12 se-cretion rate of NXS2 scIL-12 cells also was observed in scid/beigemice (Fig. 1B), defective in both T and NK cell compartments,accompanied by impaired chemotaxis and motility of macro-phages. These findings strongly suggest the involvement of anti-angiogenesis, an additional effector function to immunomodula-tion induced by IL-12.

scIL-12-induced inhibition of angiogenesis in neuroblastoma ismediated by IP-10

Consistent with the scIL-12-induced reduction of tumor growth inimmunodeficient scid/beige mice, a dramatic, local antiangiogeniceffect was revealed by Matrigel plug assays. To this end, the he-moglobin content in Matrigel plugs was determined after s.c. in-oculation of a mixture of Matrigel with NXS2 cells carrying theempty vector (NXS2 pBK-CMV) and compared with Matrigelcontaining scIL-12-producing NXS2 cells. Importantly, scIL-12inhibited NXS2 neuroblastoma-induced neovascularization, as in-dicated by a 1.5–2.0 log decrease in hemoglobin content of NXS2scIL-12 vs NXS2 pBK-CMV empty vector controls (Fig. 2A). Thisfinding was further supported by histological analyses of Matrigelplugs, which in the case of NXS2 scIL-12 cells revealed an almost

FIGURE 1. Effect of local scIL-12 production on s.c. tumor growth inimmunocompetent and immunodeficient mice. Immunocompetent A/Jmice (n5 4; A) and T cell-deficientscid/scidmice (n5 4; B, open sym-bols) or T cell and NK cell-deficientscid/beigemice (n 5 4; B, filledsymbols) were injected s.c. with 23 106 NXS2 cells producing either high(‚, Œ), or low amounts (ƒ,�) of scIL-12 and compared with NXS2 pBK-CMV empty vector controls (E,F). Tumor growth was evaluated by mi-crocaliper measurements performed two to three times per week. In ex-periments with A/J mice (A), the difference between the experimentalgroups and the control group is statistically significant (day 5;p # 0.0001).In experiments with scid/beige mice and scid/scid mice (B), the differencebetween all experimental and control groups is statistically significant(scid/beige: day 8,p # 0.05; day 21,p # 0.001;scid/scid: day 12,p #

0.05; day 20,p # 0.001).

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complete absence of erythrocytes and the presence of necrotic tu-mor cell islets (Fig. 2C, open arrowhead). This finding is in con-trast to NXS2 pBK-CMV controls that revealed both: viable tumorcell islets (Fig. 2B, arrows) and erythrocytes (Fig. 2B, asterisk).

Two lines of evidence support the involvement of IP-10 in scIL-12-mediated local suppression of neuroblastoma tumor growth.First, analysis of local IP-10 gene expression in animals that weredepleted of scIL-12 indicated increased IP-10 mRNA detectableonly in tumor tissue of mice that received scIL-12-producingNXS2 cells. This in contrast to empty vector controls, mice de-pleted of IL-12 or naive A/J mice (Fig. 3). These findings clearlyindicate scIL-12-dependent production of IP-10 mRNA in the pri-mary tumor’s microenvironment. Second, the antiangiogenic effectof scIL-12-producing NXS2 cells was demonstrated by a decreasein hemoglobin in Matrigel plugs. This antiangiogenic effect wasreversed only in mice receiving both local and systemic adminis-tration of anti-IP-10 Ab, as indicated by both increased hemoglo-bin (Fig. 2A) and by histology demonstrating the reappearance oferythrocytes and the presence of viable tumor islets (Fig. 2D, ar-row and asterisk). These findings were obtained with three differ-ent IP-10 neutralizing reagents, including a polyclonal anti-IP-10antiserum and two anti-IP-10 mAbs, 1F11 and 1B9, that recognizedifferent mIP-10 epitopes. Importantly, these findings demonstratethe efficacy of IP-10 depletion in our experimental model.

Effect of IP-10 depletion on the scIL-12-induced local cellularantitumor response

Based on the antiangiogenic effect induced by local scIL-12 pro-duction and its abrogation by in vivo depletion of IP-10, s.c. tumorgrowth of scIL-12-producing NXS2 cells was analyzed by histo-logical 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 CD41 and CD81

T cells (Fig. 4) but showed no effect on infiltrating Mac11 cells

FIGURE 2. Effect of IP-10 depletion on antiangiogenesis mediated byNXS2 scIL-12 cells. A/J mice were inoculated with Matrigel containingeither NXS2 scIL-12-producing cells or NXS2 empty vector controls.

FIGURE 3. Gene expression of IP-10 at the tumor site in the presenceor absence of scIL-12. IP-10 expression as detected by RT-PCR in scIL-12-producing tumor tissue was determined in the presence or absence ofanti-IL-12 mAb and compared with NXS2 pBK-CMV empty vector con-trols (n 5 3). GAPDH was amplified to demonstrate integrity of cDNAused for the experiments.

IP-10 was depleted in three separate experiments with either monoclonalanti-IP-10 Abs 1F11 (purple bars), or 1B9 (green bars) or polyclonalanti-IP-10 serum (blue bars) and compared with serum controls (NHS) asdescribed inMaterials and Methods(A). The difference between the he-moglobin content of Matrigel plugs of non-IP-10-depleted mice, NXS2pBK-CMV empty vector controls, and IP-10-depleted mice was statisti-cally significant (p# 0.05). For histological analysis, Matrigel plugs wereinjected with scIL-12-producing NXS2 tumor cells in the absence (C) orpresence (D) of anti-IP-10 antiserum and compared with NXS2 pBK-CMVempty vector controls (B). On day 6, Matrigel plugs were removed, fixedin 10% buffered formalin, and stained with hematoxylin and eosin (B–D).Representative areas were photographed at3400 magnification. Arrowsand arrowheads, Viable and necrotic tumor cell islets, respectively.p, Pres-ence of erythrocytes.

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(Fig. 4) and granulocytes (data not shown). Local secretion ofscIL-12 by NXS2 cells in the absence of IP-10 depletion induceda strong inflammatory response concomitant with the absence ofviable tumor cells in the tumor microenvironment (Fig. 4, hema-toxylin and eosin). This was in contrast to controls injected withNXS2 cells carrying only the empty vector.

Effect of IP-10 depletion on systemic protective immunityinduced by the scIL-12 cell vaccine

The role of IP-10 was assessed in the induction of a systemictumor-protective immunity induced by the scIL-12 NXS2 cell vac-cine. For this purpose, A/J mice were vaccinated by s.c. injectionof scIL-12-producing NXS2 cells, challenged 7 days thereafter byi.v. injection of wild-type NXS2 tumor cells, and analyzed fordisseminated liver metastases, in the presence or absence of IP-10.The depletion of IP-10 by two different anti-IP-10 mAbs for theduration of the entire experiment completely abrogated the sys-temic tumor-protective immunity induced by vaccination withscIL-12-producing NXS2 cells and was the same as observed incontrol mice depleted of IFN-g (Fig. 5). This finding suggests thatlocal IP-10 mediates the induction of a systemic tumor-protectiveimmunity.

To evaluate the time interval critical for IP-10-mediated induc-tion of systemic protective immunity by scIL-12 gene therapy,three additional IP-10 depletion schedules were tested. Anti-IP-10mAb was administered either in the immunization phase (days0–4) or the early (days 7–28) and late (days 10–28) effectorphases (Table I). In the group of mice that received the anti-IP-10Ab only during the immunization phase (day 0–4), four of fourmice developed metastases after immunization with the cellularscIL-12 vaccine as compared with immunized controls that werecompletely protected from metastases. This finding was identicalwith results obtained with a depletion schedule covering the entireduration of the experiment (Fig. 5), indicating that the presence of

IP-10 during the immunization phase is critical for the induction ofprotective immunity. However, if IP-10 was depleted during theearly effector phase, only two of four mice developed metastases,and if IP-10 was depleted in the late effector phase, only one offour mice developed metastases. These observations suggest thatIP-10 plays a crucial role in the generation of effector T cellsduring the early immunization phase.

The contention that IP-10 mediates the induction of protectiveantitumor immunity by scIL-12-producing NXS2 cells was furthersupported by data from two sets of experiments. First, the ampli-tude of the CTL response was analyzed in IP-10-depleted animals,and compared with undepleted controls. Depletion was accom-plished with 250mg of anti-IP-10 mAb 1B9 added to the tumorinoculum and injected on days 3 and 8 i.p. Effector cells wereisolated from A/J mice vaccinated with scIL-12 NXS2 cells eitherin the presence or absence of IP-10 depletion, 14 days after vac-cination and used against NXS2 target cells in a standard51Crrelease assay at a fixed E:T cell ratio of 100:1. The cytolytic re-sponse in vitro observed in bulk cultures prepared from A/J micevaccinated with scIL-12 NXS2 was almost completely inhibited bythe depletion of IP-10 in vivo (Fig. 6A). This response was furthercharacterized by the isolation of CD81 and CD41 T cell subpopu-lations from these same mice, which revealed primarily an MHCclass I Ag-restricted CD81 T cell response with some cytolyticactivity in the CD41 T cell compartment (Fig. 6B), an observationthat was reported previously (1). However, mice vaccinated withscIL-12 NXS2 and depleted of IP-10 revealed a clear inhibition ofthe MHC class I Ag-restricted CD81 T cell response in contrast tothe cytolytic CD41 T cell compartment, which was not affected byIP-10 depletion (Fig. 6B).

Second, we determined the effect of IP-10 depletion in scIL-12NXS2-vaccinated mice on the number of proliferating CD81 Tcells producing IFN-g. This analysis revealed a 2-fold reduction ofsuch CD81 T cells in mice depleted of IP-10 in contrast to non-depleted controls (Fig. 7), suggesting a direct involvement of IP-10in the generation of a Th1-type T cell-mediated antitumor immuneresponse.

DiscussionThe induction of an effective cellular immune response againstsyngeneic tumors, which includes a local increase in inflammatory

FIGURE 4. Histological and immunohistochemical analyses of scIL-12-induced leukocytic infiltrates in the presence or absence of anti-IP-10mAb. Four days after s.c. inoculation of scIL-12-producing NXS2 cells andsubsequent IP-10-depletion, tumors were removed and subjected to histo-logical analysis. Sections of paraffin embedded or frozen tumor tissue fromeach group were stained with hematoxylin and eosin (H&E) (top row) andanalyzed for T cells with anti-CD4 mAb (second row) and anti-CD8 mAb(third row) or for macrophages with Mac-1 mAb (bottom row). Tumor fociand infiltrates are depicted at3400 magnification. Brown coloring indi-cates positive staining for each marker, respectively.

FIGURE 5. Effect of IP-10 depletion on experimental liver metastasis inscIL-12-vaccinated mice. In three separate experiments, A/J mice wereinoculated s.c. with either scIL-12-producing cells or NXS2 pBK-CMVempty vector controls. Experimental liver metastases were induced on day7 by i.v. injection of 53 104 NXS2 wild-type cells. Two groups of animalsvaccinated with scIL-12-producing NXS2 cell were injected with neutral-izing mAbs (scIL-121 mAb) specific for mIP-10 (1F11:n 5 10,f; 1B9:n 5 4,o) and one group with mAbs against mIFN-g (R4-6A2:n 5 4, M).Systemic, hepatic metastases were assessed by counting the number ofmetastatic foci on the surface of the liver. Bars represent average numbersof metastatic foci per group6 SE Differences in the number of metastaticliver foci of scIL-12 NXS2-vaccinated mice and IP-10- or IFN-g-depletedgroups of mice were statistically significant (p,p , 0.05).

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Th1 cytokines in the tumor microenvironment followed by a sys-temic protective immunity, is a promising approach for cancerimmunotherapy. IL-12 has proven to be an excellent candidate forachieving this goal (4, 5, 27–35). We previously demonstrated thatNXS2 neuroblastoma cells that were genetically engineered to se-crete a linearized version of mIL-12 can induce a T cell-dependentprotective immunity in a poorly immunogenic tumor model of mu-rine neuroblastoma in syngeneic A/J mice (1). The finding of aCD81 T cell-mediated tumor-protective immunity was in contrastto an NK cell-mediated local antitumor effect induced in the sametumor model by IL-2 gene therapy (36) and with a tumor-specificAb-IL-2 fusion protein (25), in both cases concomitant with theabsence of a T cell memory. These findings emphasize the superiorefficacy of IL-12 over IL-2 in priming CD81 T cell responsesagainst poorly immunogenic neuroblastoma tumors. Here, wedemonstrate for the first time that the induction of a CD81 T cell-mediated tumor-protective immunity by scIL-12 gene therapy isentirely dependent on the production of CXC chemokine IP-10.This contention was supported by three lines of evidence. First,abrogation of the T cell-mediated protective immunity induced byscIL-12 gene therapy with two anti-IP-10 mAbs resulted in thereappearance of disseminated liver metastases in immunized ani-mals (Fig. 5). Second, MHC class I Ag-restricted target cell lysisby CD81 T cells was inhibited by depletion of IP-10 (Fig. 6).Third, the number of proliferating CD81 T cells producing IFN-grevealed a 2-fold reduction in mice depleted of IP-10. Further-more, tumor-protective immunity was only entirely abrogated ifIP-10 was depleted in the immunization phase before tumor cellchallenge, but not if IP-10 was depleted in the effector phase.

These findings suggest a role for IP-10 in the generation of a tu-mor-specific Th1-type CD81 T cell immune response during theimmunization phase.

Previous observations of an antitumor response against plasmo-cytoma and mammary adenocarcinoma cells that were geneticallyengineered to secrete mIP-10 focused on a local antitumor re-sponse induced by IP-10 (3). This study established that the anti-tumor properties of IP-10 are mediated by thymus-derived cells.However, the role of IP-10 in the induction of protective immunitywas not addressed by these studies. We also demonstrated in ourtumor model the involvement of both major functional propertiesof IP-10, T cell chemotaxis (3, 7) and antiangiogenesis (16, 37), inthe scIL-12-mediated local antitumor effect (Figs. 1–3). Interest-ingly, depletion of IP-10 efficiently inhibited both scIL-12-medi-ated chemotaxis and antiangiogenesis, but did not impede the com-plete rejection of scIL-12-producing primary tumors inimmunocompetent mice (data not shown). This was in contrast todepletion of IFN-g or IL-12, which resulted in continuous s.c.tumor growth by scIL-12-producing neuroblastoma cells (data notshown). A similar finding was reported in a syngeneic model ofrenal cell carcinoma (RENCA) in which the effect of systemicIL-12 injection on partial regression of s.c. primary tumors of

FIGURE 6. Vaccination with scIL-12 NXS2 cells induces an IP-10 me-diated CTL response against NXS2 target cells. Splenocytes (A) and CD81

and CD41 T cells (B) obtained from mice vaccinated with 23 106 NXS2scIL-12-producing cells in the presence and absence of IP-10 depletionwith 1B9 mAb were obtained 14 days after vaccination and compared withsuch cells obtained from empty vector controls. Depletion was accom-plished with 250mg of anti-IP-10 mAb 1B9 added to the tumor inoculumand injected on days 3 and 8 i.p. Cytotoxic activity was determined in an8-h 51Cr release assay against NXS2 target cells at a fixed E:T cell ratio of100:1. MHC class I-Ag restriction was determined by addition of anti-H2Kk and anti-H2Dd mAb (25 mg/ml).

FIGURE 7. Determination of CD81 T cells of the Th1 type in the pres-ence or absence of IP-10 determined by intracellular staining of IFN-g inproliferating CD81 T cells. Splenocytes from mice vaccinated with scIL-12-producing NXS2 cells were analyzed 14 days after immunization. Cellswere labeled with CFSE, cultured for 5 days, and analyzed by FACS asdescribed inMaterials and Methods. Representative histograms show thepresence or absence of IFN-g signals (x-axis) in proliferating CD81 T cells(y-axis) of vaccinated mice with (CandD) and without (AandB) depletionof IP-10. Signals obtained without PHA stimulation are shown as negativecontrols (AandC). Numbers indicate the percentage of proliferating CD81

T cells producing IFN-g. The average percentage6 SE of proliferatingCD81 T cells producing IFN-g (n 5 4) is shown in a bar graph (E). Thedifference in numbers of proliferating CD81 T cells producing IFN-g be-tween the NXS2 scIL-12 immunized mice in the presence or absence ofIP-10 depletion is statistically significant (p,p , 0.0001).

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BALB/c mice remained unaffected by the depletion of IP-10 (7).This observation and our findings support the contention that animmunological and not an antiangiogenic mechanism is involvedin IL-12-mediated local tumor rejection, which is independent ofIP-10. This may involve other IL-12 mediators downstream, suchas Mig, IFN-g, or a direct effect of IL-12, per se. The persistenceof Mac-1-positive cells in the local microenvironment of scIL-12-producing primary neuroblastoma tumors, even in the absence ofIP-10, suggest a role for macrophages in scIL-12-mediated, IP-10-independent rejection of primary tumors, especially because theseeffector cells are also known to be directly activated by IL-12 andIFN-g.

We further extended these findings by establishing a role forIP-10 in the induction of systemic tumor-protective immunity thatdepended on CD81 T cells of the Th1 type. The mechanisms in-volved in IP-10-mediated induction of a vaccination effect byscIL-12 gene therapy are related to the induction and/or priming ofCD81 T cells of the Th1 type by this chemokine. This contentionis supported by two observations. First, scIL-12 NXS2-vaccinatedmice depleted of IP-10 during the immunization phase had a de-crease in the tumor-specific cytolytic response and had fewerIFN-g secreting CD81 T cells after Ag-specific stimulation. Sec-ond, IP-10 depletion only entirely abrogated the protective effectof scIL-12 if the anti-IP-10 mAb was injected within the first 4days after immunization.

The detailed function of IP-10 in CD81 T cell-mediated immu-nity after scIL-12 gene therapy in this neuroblastoma model re-mains obscure. Thus, direct activation of effector T cells by IP-10was analyzed in a culture of splenocytes from mice immunizedwith scIL-12-producing NXS2 cells in the presence or absence ofincreasing amounts of anti-IP-10 Abs ex vivo. There was no dif-ference in the number of proliferating IFN-g1/CD81 T cells in thepresence or absence of IP-10 (data not shown), even at 100mg/mlanti-IP-10 Ab. This observation indicates that IP-10 secreted insuch cultures is not activating CD81 T cells directly, suggestingindirect mechanisms of IP-10-mediated induction of CD81 T cellimmunity. In fact, such indirect mechanisms may involve APCs(38–41). Immature APCs were demonstrated to up-regulate che-mokine receptors during differentiation into Langerhans cells (38),and IP-10 Ag fusion proteins were shown to target Ag to suchAPCs, resulting in a dramatically increased Ag-specific immuneresponse (39). It further was reported that a distinct subset of bloodAPCs that have picked up Ag in the periphery, identified as plas-macytoid monocytes, express the IP-10 receptor CXCR3 and home

to lymph nodes guided by IP-10 released from high endothelialvenules (40). There, these cells mature to plasmacytoid dendriticcells characterized by a potent Th1 polarization to subsequentlygenerate an Ag-specific immune response (41). This, in combina-tion with an IP-10-mediated increase in T cells provides a mosteffective milieu for the induction of a CD81 T cell response.

Furthermore, IP-10 was reported necessary for effector T celltrafficking and function and host survival inToxoplasma gondiiinfection (24). Neutralization of IP-10 inhibited the influx ofCD41 and CD81 T cells into spleens and livers of mice infectedby T. gondii,resulting in a simultaneous 3-log increase in the par-asite burden and a significant decrease in survival. These findingsdemonstrate a critical role for IP-10 in effector T cell trafficking inthis infectious disease model. In contrast to this model with highIP-10 levels in parasite infected organs, tumors and metastasesinduced by NXS2 wild-type cells do not produce IP-10 (Fig. 3 anddata not shown). Therefore, it is unlikely that inhibition of IP-10-mediated effector T cell trafficking to neuroblastoma metastasesprovides a mechanism for the effects observed in this tumor model.However, in contrast to metastases, we clearly demonstrated T celltrafficking to the local vaccination site, whenever NXS2 neuro-blastoma cells were producing scIL-12 (Fig. 4.). In this case, IP-10was highly expressed (Fig. 3), and administration of anti-IP-10Abs clearly decreased the number of T cells at the vaccination site(Fig. 4).

These findings indicate that IP-10 may be involved in the gen-eration of early activated T cells by either recruiting immatureAPC or early activated T cells into the tumor. APCs that pick upAg in the tumor migrate back to the lymph node where they gen-erate activated T cells, which respond to a gradient of IP-10 em-anating from the tumor, leading to T cell infiltration of the tumorand amplification of the response. Thus, inhibition of these pro-cesses by anti-IP-10 Abs would abrogate the generation of tumor-specific CD81 effector T cells. This contention also is consistentwith the observation that IP-10 depletion is only completely ef-fective when applied during the early immunization phase withloss of activity over time (Fig. 5, Table I).

In summary, we have demonstrated that the induction of sys-temic tumor-protective immunity by scIL-12 gene therapy is de-pendent on the CXC chemokine IP-10 during early immunization.Inhibition of IP-10 during the immunization phase resulted in com-plete abrogation of the vaccination effect as indicated by a failureof IP-10-depleted mice to control a subsequent tumor challenge.

Table I. Effect of IP-10 depletion schedules on scIL-12 gene therapy-induced antineuroblastoma immuneresponsesa

DepletionbDay 0–4

Immunization PhaseDay 7–28

Early Effector PhaseDay 10–28

Late Effector Phase

Controlc 4/4d 4/4 4/4scIL-12c 0/4 0/4 0/4scIL-12 1 anti-IP-10c,e 4/4* 2/4** 1/4**

a The effect of three separate depletion schedules was investigated in A/J mice immunized with scIL-12-producing NXS2neuroblastoma cells.

b Three different depletion scheduled were used: 1) during the immunization phase: 250mg of 1B91 anti-IP-10 mAb were injectedi.p. on day 0 and 100mg on days 2 and 4; 2) during the early effector phase, 250mg were injected on day 7 followed by tumor cellchallenge on the same day and then 100mg every third day until day 28; 3) during the late effector phase Ab treatment was startedon day 10 with 250mg and continued every third day with 100mg until day 28.

c Mice (n 5 4) were immunized by s.c. injection with 53 106 NXS2 neuroblastoma cells genetically engineered to secretescIL-12 and 23 106 NXS2 cells carrying the empty vector. Seven days after inoculation, mice received a lethal i.v. challenge with5 3 104 NXS2 wild-type cells. 28 days after vaccination, mice were sacrificed and the presence or absence of macroscopicneuroblastoma metastases on the surface of the liver was determined.

d Number of mice with macroscopic liver metastases from a total of four mice.e The difference between mice injected with scIL-12-producing NXS2 cells depleted of IP-10 and mice only injected with

scIL-12-producing NXS2 cells was statistically significant on days 0–4 (p, p , 0.05), but not on days 7–28 or 10–28 (pp,p . 0.1).

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These findings were supported by a decrease in CD81 T cell ac-tivation in IP-10-depleted mice as defined by inhibition of MHCclass I Ag-restricted tumor target cell lysis and a decrease in thenumber of Ag-specific CD81 T cells producing IFN-g. Taken to-gether, these data describe a novel role for IP-10 in the generationof protective antitumor immunity induced by scIL-12 genetherapy.

AcknowledgmentsWe thank Dr. J. M. Farber for the rabbit anti-mouse IP-10 antiserum andDr. S. Webb for the R4-6A2 hybridoma cell line. We would like to extendour appreciation to Lynne Kottel for the preparation of this manuscript.This is The Scripps Research Institute’s manuscript number 12656-IMM.

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