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(Journal of Leukocyte Biology. 2002;72:864-873.)
© 2002 by Society for Leukocyte Biology

A role for endogenous IL-12 in tumor immunity: IL-12 is required for the acquisition of tumor-migratory capacity by T cells and the development of T cell-accepting capacity in tumor masses

Yasuhiro Uekusa*, Ping Gao*, Nobuya Yamaguchi*, Michio Tomura*, Takao Mukai*, Chigusa Nakajima*, Masayuki Iwasaki*, Noritame Takeuchi*, Takahiro Tsujimura{dagger}, Mitsuhiro Nakazawa{ddagger}, Hiromi Fujiwara* and Toshiyuki Hamaoka*

* Department of Oncology, Osaka University Graduate School of Medicine, and
{ddagger} Second Department of Oral and Maxillo-Facial Surgery, Osaka University Faculty of Dentistry, Osaka University, Suita, Japan; and
{dagger} Department of Pathology, Sumitomo Hospital, Osaka, Japan

Correspondence: Dr. Hiromi Fujiwara, Department of Oncology (C6), Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail: hf{at}ongene.med.osaka-u.ac.jp


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ABSTRACT
 
Interleukin (IL)-12 plays a central role in the initiation and regulation of T cell-mediated immune responses. The present study investigated how IL-12, endogenously produced during tumor vaccination, functions for anti-tumor immune responses. Mice were given anti-IL-12 monoclonal antibody during immunization with attenuated syngeneic tumor cells. Splenic T cells from anti-IL-12-treated immunized mice exhibited comparable levels of tumor-neutralizing activity with those from tumor-immunized mice without anti-IL-12 treatment. When these two groups of mice were directly challenged with viable tumor cells, tumor rejection was induced only in anti-IL-12-untreated mice. T cell infiltration was observed at the site of tumor challenge in these mice, whereas such a T cell infiltration did not occur in anti-IL-12-treated mice. The tumor-migratory capacity was directly assessed by transferring spleen cells from tumor-immunized mice into syngeneic, tumor-bearing recipient mice and by quantitating donor cells migrating into recipients’ tumor masses. T cells from anti-IL-12-treated tumor-immunized mice were found to exhibit a markedly reduced tumor-migratory capacity when compared with that of anti-IL-12-untreated mice. Moreover, the migration of T cells from anti-IL-12-untreated mice to tumor masses prepared in anti-IL-12-treated mice was severely reduced. These results indicate that endogenously produced IL-12 has dual roles in anti-tumor-immune resistance: One is to confer T cells with a tumor-migratory capacity, and the other is to allow tumor masses to develop the capacity to accept tumor-migrating T cells.

Key Words: T cell migration • interleukin (IL)-12 • tumor immunity


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INTRODUCTION
 
Interleukin (IL)-12 has pleiotropic effects on T cells as well as natural killer (NK) cells [1 2 3 4 ]. IL-12 is produced by antigen-presenting cells (APC) during their interaction with antigen-specific T cells [5 , 6 ], and this interaction allows T cells to acquire IL-12 responsiveness including IL-12 receptor (IL-12R) expression [7 8 9 10 ]. Through acting on T cells that have expressed IL-12R, this cytokine functions for development of T helper cell type 1 (Th1) cells and production of interferon-{gamma} (IFN-{gamma}) by Th1 cells [11 12 13 14 ].

After IL-12 was demonstrated to possess the capacity to promote a Th1-mediated inflammatory response, this cytokine has also been reported to exhibit potent anti-tumor efficacy in a number of mouse tumor models [15 16 17 18 ]. In some but not all tumor models, T cells from tumor-bearing mice were shown to express IL-12R [19 ], and administration of recombinant (r)IL-12 into these tumor-bearing mice resulted in regression of growing tumors in a T cell-dependent manner [17 , 18 ]. Although it is obvious that IL-12 has an anti-tumor effect, such an effect has been recognized for exogenous rIL-12 used as an immunomodulatory agent. Regarding the production of endogenous IL-12, only a few papers [20 , 21 ] showed intratumoral expression. However, it remains unclear whether IL-12 is endogenously produced in tumor-bearing hosts as a result of the host’s anti-tumor-immune response and whether endogenous IL-12 contributes to the development of T cell-mediated tumor protection.

The present study investigated the potential role of endogenous IL-12 in the development of anti-tumor T cell responses. The results show that neutralization of endogenously produced IL-12 by pretreatment with anti-IL-12 monoclonal antibodies (mAb) during tumor immunization did not affect the generation of T cells with the capacity to inhibit tumor growth as assessed in tumor-neutralization assays (Winn assays). However, tumor-sensitized T cells that developed in IL-12-neutralized hosts failed to migrate from lymphoid organs to tumor sites. Consequently, IL-12-neutralized mice immunized to tumor via an intraperitoneal (i.p.) route exhibited a markedly reduced capacity to reject tumor cells subcutaneously (s.c.) challenged. It was also found that the migration of T cells generated in anti-IL-12-untreated mice to tumor masses prepared in anti-IL-12-treated mice was markedly inhibited. Thus, the results indicate that endogenously produced IL-12 plays critical roles in the acquisition of a tumor-migratory capacity by T cells and the development of a T cell-accepting capacity in tumor masses.


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MATERIALS AND METHODS
 
Tumor cell lines
Two tumor cell lines were used: CSA1M fibrosarcoma [22 ] in BALB/c origin and OV-HM ovarian carcinoma [23 ] in (C57BL/6xC3H/He)F1 (B6C3F1) origin. These tumor cell lines were maintained in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) at 37°C in a humidified atmosphere with 5% CO2.

Mice
Male BALB/c and female B6C3F1 mice were obtained from Shizuoka Experimental Animal Center (Hamamatsu, Japan) and were used at 6–9 weeks of age.

Reagents
Anti-IL-12 mAb (C17.8) [24 ] was prepared from ascitic fluid of hybridoma cells. The purification was performed by precipitation with ammonium sulfate followed by YFLC Gel filtration (Yamazen Corporation, Osaka, Japan). Control rat immunoglobulin G (IgG) was obtained from BioMeda (Foster City, CA). Murine rlL-12 was provided from Genetics Institute Inc. (Cambridge, MA). A fluorescent dye, PKH-26-GL (abbreviated as PKH-26), was purchased from Sigma Chemical Co. (St. Louis, MO).

Preparation of tumor-immunized or tumor-bearing mice
To prepare tumor-immunized mice, tumor cells were treated in vitro with 100 µg/ml mitomycin-C (MMC) for 60 min. Mice were inoculated i.p. with 105 MMC-treated tumor cells three times at 4- or 5-day intervals. To prepare tumor-bearing mice, mice were inoculated s.c. with viable tumor cells (1x106/mouse) and used at 2- to 4-week tumor-bearing stages.

Preparation of T cell-enriched, Thy1+ cell-depleted and NK1.1+ cell-depleted splenocyte populations
Spleen cells were depleted of B cells by immunomagnetic-negative selection, as described [25 ]. Briefly, spleen cells were incubated with magnetic particles bound to goat anti-mouse Ig (Advanced Magnetics, Cambridge, MA). Surface Ig-negative cells were obtained by removing cell-bound magnetic particles with a rare earth magnet (Advanced Magnetics) and were used as a T cell-enriched population (>90% purity). For the preparation of Thy1+ cell- and NK1.1+ cell-depleted populations, spleen cells were incubated with superparamagnetic microbeads conjugated to anti-Thy1 or anti-DX5 mAb (Miltenyi Biotec, Sunnyvale, CA). Labeled cells were separated by magnetic cell sorting using the MiniMACS (Miltenyi Biotec). The nonmagnetic cells were collected after passing through a MiniMACS column. This procedure repeated twice, and the nonmagnetic cells that passed through the second MiniMACS column were used as a Thy1+ (T cell)- or NK1.1+ cell-depleted population.

Tumor-neutralization test (Winn assay)
A splenic T cell-enriched or T cell- or NK1.1+ cell-depleted population from normal or tumor-sensitized mice was admixed with viable tumor cells, and the mixture was inoculated s.c. into normal recipient mice. Tumor growth was measured and expressed as the mean ± SE of five mice/group.

Anti-IL-12 mAb treatment
Anti-IL-12 mAb (1 mg/mouse/time) was injected 1 day before the first and second immunization with tumor cells (twice at a 4- or 5-day interval). Control mice were given the same amount of rat Ig.

rIL-12 treatment
rIL-12 (0.5 µg/time) was administered i.p. to tumor-bearing mice three times every other day.

A lymphoid cell migration assay
The assay system was essentially the same as previously described [26 ]. Staining spleen cells with a fluorescent dye (PKH-26) was performed according to the manufacturer’s recommended procedure. Briefly, spleen cells suspended to a concentration of 5 x 107/ml in 1 ml diluent were allowed to react with 5 x 10-6 M PKH-26 dissolved in 1 ml diluent for 5 min at 37°C. Labeling was stopped by adding 2 ml FCS, and cells were washed five times with RPMI 1640 containing 10% FCS. Mice with similar tumor sizes (approximately 7 mm in diameter) were used as recipients for this assay. PKH-26-labeled spleen cells (3x107 cells in 250 µl RPMI-1640 medium) were injected intravenously (i.v.) into recipient (IL-12-untreated, homologous tumor-bearing) mice. Twenty-four hours after injection, tumor masses were removed, and cryostat sections were prepared. The entry of fluorescence-labeled donor cells was quantified under a fluorescence microscopy and expressed as the mean cell number ± SE per section.

Histological examination
Tumor masses were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for histological examination.

Staining procedure of immunohistochemical examination
The following reagents were purchased to perform immunohistochemical examination: biotinylated anti-mouse CD4 and anti-CD8 mAb (PharMingen, San Diego, CA); biotinylated rat IgG (Jackson ImmunoResearch, West Grove, PA); Histofine SA-PO kit and Histofine 3,3'-diaminobenzidine tetrahydrochloride (DAB) kit (Nichirei Co. Ltd., Tokyo, Japan). Samples were fixed in 4% paraformaldehyde for 6–12 h at 4°C and then washed sequentially with phosphate-buffered saline (PBS) containing 10, 15, and 20% sucrose for 6 h each at 4°C. The samples were embedded in Tissue-Tek OCT compound (Sakura Finetechnical Co. Ltd., Tokyo, Japan) and frozen at -80°C. Cryostat sections (5 µm) were cut, air-dried, and then washed three times with PBS. The sections were incubated in PBS containing 10% hydrogen peroxide at room temperature for 30 min for blocking endogenous peroxidase activity before a biotinylated Ab was added. After preincubation with 4% bovine serum albumin solution, the tissues were overlaid with various biotinylated Ab and incubated in a humidified chamber at room temperature for 2 h. After washing three times, the sections were incubated with peroxidase-conjugated streptavidin solution for 30 min. After additional three-time washing, the labeling was visualized with 0.03% DAB solution containing 0.1% hydrogen peroxide for several minutes.

Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was prepared from cytokine-stimulated T cells by the acid guanidium-thiocyanate-phenol-chloroform method. Total RNA (1 µg) was reverse-transcribed into cDNA in a total volume of 20 µl using random primers and SUPERSCRIPTTMII RNase H- RT (Life Technologies, Rockville, MD). PCR amplification was carried out in a total volume of 50 µl PCR Gold Buffer (x1; PE Applied Biosystems, Branchburg, NJ) containing 1.0 µl first-strand cDNA, 1.5 mM MgCl2, 0.25 mM of each dNTP, 2 µM of each primer, and 0.5 U Ampli Taq Gold DNA polymerase (PE Applied Biosystems). The following oligonucleotides were used: IL-12 p35 sense primer 5'-CTCCTAAACCACCTCAGTTTGGCCAGGGTC-3', IL-12 p35 antisense primer 5'-TAGATGCTACCAAGGCACAGGGTCATCATC-3', IL-12 p40 sense primer 5'-CACTCATGGCCATGTGGGAGCTGGAGAAAG-3', IL-12 p40 antisense primer 5'-TCCGGAGTAATTTGGTGCTTCACACTTCAG-3', ß-actin sense primer 5'-AGAAGAGCTATGAGCTGCCTGACG-3', and ß-actin antisense primer 5'-CTTCTGCATCCTGTCAGCAATGCC-3'. Cycle parameters were: annealing, 45 s at 60°C (IL-12 p35 and p40) or 55°C (ß-actin); elongation, 1 min at 72°C; and denaturation, 30 s at 94°C. Resulting PCR products were separated in 2% agarose gel and visualized by SYBR Green staining. Sequences of the IL-12 p35, IL-12 p40, and ß-actin (for standardization) were amplified out of each cDNA batch with 30, 30, and 18 amplification cycles, respectively.


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RESULTS
 
Expression of mRNAs for IL-12 p35 and p40 in lymphoid organs from tumor-immunized mice
Our previous studies demonstrated that spleen cells of tumor-immunized mice contain tumor-sensitized T cells and APC presenting tumor antigens [27 ] and that upon in vitro cultures, T cells produce various T cell cytokines such as IL-2 and IFN-{gamma} [27 , 28 ], and APC produce IL-12 [29 ] through T cell-APC interactions. To determine whether IL-12 expression is induced in vivo in lymphoid organs of mice receiving tumor vaccination, we examined the expression of mRNA for IL-12 p35 and p40. BALB/c and B6C3F1 mice were immunized i.p. with 105 MMC-treated syngeneic tumor cells (CSA1M for BALB/c and OV-HM for B6C3F1) three times at 1-week intervals. One day after the third immunization, spleens were harvested. Total RNA wa0s isolated from spleen cells of normal and tumor-immunized mice and subjected to RT-PCR. As shown in Figure 1A , freshly prepared spleen cells from tumor-immunized but not from normal mice express mRNAs for IL-12 p35 and p40 subunits. Although IL-12 p70 was not detected in plasma from tumor-bearing mice (data not shown), the above results suggest that IL-12 is endogenously induced in lymphoid organs from tumor-immunized mice.



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  		Figure 1. Expression of IL-12 mRNAs by spleen cells from tumor-immunized and tumor-bearing mice. (A) BALB/c and B6C3F1 mice were immunized i.p. with 105 syngeneic CSA1M and OV-HM tumor cells, respectively, three times at 1-week intervals. One day after the third immunization, spleens were harvested. (B) BALB/c and B6C3F1 mice were inoculated s.c. with 106 syngeneic CSA1M and OV-HM tumor cells, respectively. Spleens were harvested at 4 (CSA1M)- or 3 (OV-HM)-week tumor-bearing stages. Total RNA was isolated from freshly prepared spleen cells. Isolated RNA was subjected to RT-PCR for IL-12 p35, IL-12 p40, and ß-actin (control) mRNA transcripts.

Comparison of the capacity to neutralize tumor cells in Winn assays between tumor-immunized T cells from anti-IL-12-treated and untreated mice
To determine whether neutralization of IL-12 endogenously produced during tumor immunization affects the generation of T cells that can function for tumor protection, we assessed the capacity of T cells from anti-IL-12-treated or untreated tumor-immunized mice to neutralize viable tumor cells in the Winn assay. Anti-IL-12-treated or untreated BALB/c and B6C3F1 mice were immunized with syngeneic tumor cells. One week after the third immunization, spleens were harvested, and T cell-enriched fractions were prepared. Splenic T cells from tumor-immunized mice with or without anti-IL-12 treatment were admixed with 106 viable tumor cells, and the mixture was inoculated s.c. into syngeneic recipient mice. As shown in Figure 2 (A for CSA1M and B for OV-HM), T cells from anti-IL-12-treated, tumor-immunized mice produced comparable levels of tumor neutralization with those observed for T cells from anti-IL-12-untreated, tumor-immunized mice. In fact, there was no substantial difference between two groups of T cells in the capacity to inhibit the growth of admixed tumor cells when evaluated at graded effector T cell:tumor cell (E:T) ratios. To show that tumor neutralization is mediated by T cells but not by NK/NKT cells, we prepared T cell (Thy1+ cell)- and NK1.1+ cell-depleted splenocyte populations and used these as responding effector cells instead of T cell-enriched populations. Before tumor neutralization tests (Fig. 3A ), the responding cells were examined for T cell (CD4+/CD8+ cell) or NK1.1+ cell depletion (Fig. 3B) . Although the data for cell depletion were shown for responding cells from normal mice in Figure 3B , similar patterns of results were obtained for cells from mice immunized in the presence or absence of anti-IL-12 treatment (data not shown). As shown in Figure 3A , tumor neutralization was abrogated or not influenced by elimination of T cells or NK1.1+ cells, respectively, from a donor cell inoculum, indicating T cell mediation of tumor neutralization. Thus, anti-tumor T cells that function for tumor rejection can be normally generated in anti-IL-12-treated mice.



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  		Figure 2. Comparable levels of generation of tumor-neutralizing effector T cells in spleens from anti-IL-12-treated or untreated mice following tumor immunization. Mice were injected i.p. with 1 mg/mouse anti-IL-12 mAb followed by the same treatment 4 days later. These anti-IL-12-treated or untreated (control rat Ig-treated) mice were immunized with 105 MMC-treated tumor cells 1 day after the first anti-IL-12 treatment and were boosted twice at 4- to 5-day intervals. Spleens were harvested 1 week after the third immunization. Graded numbers of splenic T cells from mice immunized to CSA1M (A) or OV-HM (B) following anti-IL-12 treatment were admixed with 106 viable tumor cells, and the mixture was inoculated s.c. into syngeneic recipient mice. [E:T indicates effector (splenic T cells):tumor cells.] The growth of admixed tumor cells was expressed as the mean ± SE of five mice/group.



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  		Figure 3. Tumor neutralization is mediated by T cells but not by NK1.1+ cells. Thy1+ cell (T cell)- and NK1.1+ cell-depleted splenic populations were prepared from normal spleen cells or cells of B6C3F1 mice sensitized to OV-HM with or without anti-IL-12 treatment as described in Materials and Methods. The cells were admixed with 106 OV-HM tumor cells for tumor neutralization test (A). Portions of the cells were confirmed for the depletion of Thy1+ cells or NK1.1+ cells by flow cytometry analysis with FACSCalibur after staining doubly with allophycocyanin-conjugated anti-B220 and a mixture of FITC-conjugated anti-CD4 plus anti-CD8 mAb (B, left for Thy1+ cell depletion) or with PE-conjugated anti-NK1.1 and FITC-conjugated anti-CD3 mAb (B, right for NK1.1+ cell depletion). The left panels show the staining of the whole (nongated) spleen cells. The right panels represent the staining after gating on cells other than B cells that were also stained with anti-B220. The numbers on each figure are the percentages of the respective mAb-stained cells.

Failure of anti-IL-12-treated, tumor-immunized mice to reject directly challenged tumor cells
We examined whether anti-IL-12-treated and untreated mice immunized to CSA1M or OV-HM by inoculating the respective attenuated tumor cells via the i.p. route can reject tumor cells challenged directly at the s.c. site. Anti-IL-12-treated and untreated mice were immunized with 105 CSA1M or OV-HM tumor cells three times and 1 week later, were challenged with 106 viable tumor cells. One million was the minimum tumor cell number that can form a tumor mass in normal mice at a 100% incidence. As shown in Figure 4 , all mice in an anti-IL-12-untreated group rejected challenged tumor cells. In contrast, anti-IL-12-treated mice failed to reject tumor cells. Thus, although tumor-immunized mice in anti-IL-12-treated and untreated groups can similarly generate T cells capable of eradicating 106 admixed tumor cells, anti-IL-12-treated mice fail to reject tumor cells directly challenged. It should also be noted that tumor growth rate in unimmunized groups in both tumor models is considerably higher in an anti-IL-12-treated than in an untreated group. This is compatible with the notion that endogenous IL-12 may function to inhibit tumor growth.



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  		Figure 4. Failure of anti-IL-12-treated mice to reject directly challenged tumor cells following tumor immunization. Anti-IL-12 treatment and tumor immunization were done in the same protocol as that in Figure 2 . Anti-IL-12-treated and untreated mice were challenged s.c. with 106 viable tumor cells 1 week after the third immunization. Tumor growth was expressed as the mean ± SE of five mice/group.

The reduced levels of T cell accumulation at the site of tumor challenge in anti-IL-12-treated mice
The results of Figures 2 3 4 indicate that T cells with the capacity to eradicate tumor cells are generated in lymphoid organs such as spleens by tumor immunization, but they fail to function for the rejection of s.c.-challenged tumor cells. Considering a possibility of the failure of tumor-sensitized T cells to migrate to tumor sites, microscopic examination of tumor-challenged skin sites was performed. Tumor-immunized mice were challenged s.c. with tumor cells. The skin, including the tumor challenge site, was removed 2 days after the tumor challenge and was subjected to histological and immunohistochemical examination. As shown in Figure 5 , the OV-HM tumor-challenged site in anti-IL-12-untreated mice exhibited strong mononuclear cell infiltration. This cellular infiltration was found to involve the accumulation of CD4+ and CD8+ T cells (Figure 6 ), but the accumulation of NK1.1+ cells was hardly detected (data not shown). In contrast, the skin site of anti-IL-12-treated mice exhibits apparently decreased levels of mononuclear cell infiltration compared with that of anti-IL-12-untreated mice (Fig. 5) . The reduction of cellular infiltration was associated with the detection of only a few CD4+ and CD8+ cells (Fig. 6) . These different T cell-infiltrating patterns in IL-12-treated and untreated mice in the OV-HM model were also observed in the CSA1M model (data not shown). Thus, anti-IL-12-treated mice exhibit apparently reduced levels of T cell accumulation at the site of tumor challenge despite the generation of tumor-sensitized T cells by immunization.



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  		Figure 5. Decreased levels of cellular infiltration at tumor-challenged sites in anti-IL-12-treated mice. Anti-IL-12-treated and untreated mice were immunized with tumor cells three times and challenged with viable tumor cells. Two days later, the skin, including the tumor-challenged site, was removed and subjected to H&E staining. Original magnification, x400.



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  		Figure 6. Decreased levels of CD4+ and CD8+ T cell accumulation at OV-HM-challenged sites in anti-IL-12-treated mice. Portions of animals prepared in the experiments of Figure 5 were subjected to immunohistochemical examination. Cryostat sections were prepared from the skin, including the OV-HM tumor-challenged site, and were stained for CD4 and CD8. The results are representative of three different sections in each group. Original magnification, x400.

T cells from anti-IL-12-treated, tumor-immunized mice have a defect in the recruitment to tumor masses/sites
The reduction of T cell accumulation at the site of tumor challenge in anti-IL-12-treated, tumor-immunized mice suggests the inability of T cells to migrate to tumor sites. This was directly examined using a lymphoid cell (T cell) migration assay that was developed in our laboratory and previously described [26 ]. Donor spleen cells were prepared from tumor-immunized mice with or without anti-IL-12 treatment. Their tumor-migratory capacity was assessed by transferring into syngeneic tumor-bearing recipient mice and counting migrating cells on cryostat sections of tumor masses in recipient mice (Fig. 7 ). In this assay, most of migrating cells were found to be T cells in a spleen cell inoculum [26 , 30 ]. Donor cells from anti-IL-12-untreated, tumor-immunized mice exhibited the capacity to migrate to tumor masses. In contrast, cells from anti-IL-12-treated tumor-immunized mice exhibited only marginal levels of enhanced migratory capacity compared with those from unimmunized control mice. Taken together, the results indicate that the failure of anti-IL-12-treated mice to reject directly challenged tumor cells after tumor immunization is ascribed to the inability to promote T cell trafficking to tumor masses/sites.



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  		Figure 7. Failure of T cells from anti-IL-12-treated tumor-immunized mice to migrate to tumor masses. Spleens were obtained from unimmunized normal (N) or tumor-immunized (Imm) mice with or without anti-IL-12 treatment. Spleen cells were labeled with PKH-26. Spleen cells (3x107/mouse) were transferred i.v. into syngeneic tumor-bearing mice. Twenty-four hours later, tumor masses were removed, and cryostat sections were prepared. The number of fluorescent dye-positive cells was evaluated under a fluorescence microscopy and expressed as the mean ± SE of three sections per tumor mass. The results are representative of three similar experiments.

Tumor masses prepared in anti-IL-12-treated mice have a defect in the development of the capacity to accept tumor-migrating T cells
Our previous study [31 ] showed that T cell migration to tumor masses depends not only on whether T cells acquire the tumor-migratory capacity but also on whether tumor masses have the capacity to accept T cells. Regarding the latter capacity, our results [31 ] demonstrated a critical role for peritumoral stroma and vasculature in the acceptance of tumor-infiltrating T cells. As in tumor-immunized mice (Fig. 1A) , Figure 1B illustrates that IL-12 is also expressed in CSA1M- and OV-HM-tumor-bearing mice. We determined whether neutralization of IL-12 generated in tumor-bearing mice affects the development of the T cell-accepting capacity in tumor masses. CSA1M and OV-HM tumor masses were prepared in anti-IL-12-treated or untreated mice, and tumor masses were subjected to histological examination. Figure 8 shows that CSA1M and OV-HM tumors developed peritumoral stroma between s.c. tissue and tumor parenchyma when generated in anti-IL-12-untreated, normal mice (upper panels), which is consistent with our previous report [32 ]. In contrast to these tumors, CSA1M and OV-HM tumors growing in anti-IL-12-treated mice failed to induce peritumoral stroma (lower panels).



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  		Figure 8. Decreased development of peritumoral stroma in tumor masses from anti-IL-12-treated recipient mice. Mice were inoculated s.c. with CSA1M or OV-HM tumor cells, and tumor masses were removed 3 weeks (CSA1M) or 2 weeks (OV-HM) after tumor cell implantation. Original magnification, x400.

We examined whether T cells are capable of migrating to tumor masses that are generated in anti-IL-12-treated mice and consequently lack peritumoral stroma. Our previous studies [26 , 30 , 31 ] showed that splenic T cells from CSA1M- or OV-HM-bearing mice receiving IL-12 treatment exhibited a strong capacity to migrate into CSA1M or OV-HM tumor masses. The migratory capacity of these splenic T cells was stronger than that of T cells from tumor-immunized mice as used in Figure 7 . Therefore, splenic T cells from tumor-bearing mice that had received IL-12 treatment in vivo were used as donor cells and transferred into syngeneic tumor-bearing recipient mice that had been untreated or treated with anti-IL-12. As shown in Figure 9 , donor cells from IL-12-treated tumor-bearing mice exhibited high levels of migration into tumor masses in anti-IL-12-untreated recipient mice. In contrast, when portions of the same donor cells were transferred into anti-IL-12-treated recipient mice, their migration was almost completely (CSA1M) or considerably (OV-HM) inhibited. Together with the observation of Figure 7 , these results indicate that neutralization of IL-12 inhibits tumor masses from generating the capacity to accept tumor-migrating T cells as characterized by the development of peritumoral stroma.



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  		Figure 9. Spleen cells from IL-12-treated tumor-bearing mice fail to migrate into tumor masses generated in anti-IL-12-treated mice. IL-12 treatment of donor mice was performed by injecting rIL-12 (0.5 µg/mouse) to 3 weeks CSA1M-bearing or 2 weeks OV-HM-bearing mice three times every other day. Spleen cells from normal mice, tumor-bearing mice, or IL-12-treated tumor-bearing mice were stained with a fluorescent dye. These stained cells (3x107) were used as donor cells and i.v. transferred into syngeneic tumor-bearing recipient mice that had been treated with anti-IL-12 mAb or control rat IgG. Twenty-four hours after the donor cell transfer, cryostat sections of tumors in recipient mice were prepared. The results are representative of two similar experiments.


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DISCUSSION
 
This study showed that anti-IL-12-treated and untreated mice generate comparable magnitudes of tumor-neutralizing T cells following in vivo immunization with tumor cells. However, anti-IL-12-treated, tumor-immunized mice fail to reject directly challenged tumor cells. Histological examination revealed that T cells do not migrate into the site of tumor cell challenge in these anti-IL-12-treated mice. Splenic T cells from tumor-immunized mice, when i.v.-transferred into tumor-bearing recipient mice, migrated to tumor masses in recipient mice, whereas donor T cells prepared from anti-IL-12-treated, tumor-immunized mice did not. Moreover, when donor T cells from IL-12-exposed T cells from tumor-sensitized (tumor-bearing) mice were transferred into anti-IL-12-treated, tumor-bearing recipient mice, the migration to tumor masses in these recipient mice was strikingly decreased compared with that into tumor masses of anti-IL-12-untreated, tumor-bearing recipient mice. These results indicate that neutralization of endogeneously produced IL-12 strikingly affects the acquisition of the tumor-migratory capacity by T cells as well as the development of the T cell-accepting capacity in tumor masses.

IL-12 plays a central role in promoting innate and adaptive mechanisms of host defense through up-regulating the function of various effector cells in cell-mediated immune responses [1 , 2 ]. Such IL-12 effects have also suggested a critical role of this cytokine in tumor immunity. In fact, this notion was recently supported by the observations that administration of rIL-12 to tumor-bearing mice induces tumor regression in a number of tumor models [15 16 17 18 ]. Tumor rejection involves a number of processes: sensitization/activation of effector T cells with tumor antigens in lymphoid organs; migration of T cells together with other effector cells to tumor masses; and tumor cell attack by these tumor-infiltrating effectors. Previous studies [26 , 30 , 31 ] have suggested that IL-12 administered to tumor-bearing hosts functions for some of the above processes, particularly playing an important role in the trafficking of T cells to tumor masses. However, it remains to be solved whether IL-12 is endogenously produced during anti-tumor immune responses and if so, how such endogenous IL-12 works for tumor protection.

Regarding the production of IL-12 in tumor-bearing mice, our previous studies showed that spleen cells from tumor-bearing mice contain tumor-primed T cells and APC binding tumor antigens [27 , 28 ] and that culturing these two populations results in IL-12 production via the CD40-CD40 ligand interaction [29 ]. Our present results demonstrated that IL-12 mRNA expression occurs in spleens from mice receiving tumor vaccination. Taken together, it appears that APC produce IL-12 in lymphoid organs from tumor-bearing as well as tumor-immunized mice by interacting with tumor-sensitized T cells.

The present study demonstrated that endogenously produced IL-12 plays two aspects of roles in tumor immunity by acting on T cells and by functioning for tumor masses. Following tumor immunization, anti-IL-12-treated mice could generate in vivo tumor-neutralizing T cells in spleens, and there was no substantial difference in the magnitude of the tumor-neutralizing capacity between T cells from anti-IL-12-treated and untreated mice. These results indicate that IL-12 is not necessarily required for inducing the sensitization/activation of T cells with the capacity to eradicate tumor cells in vivo. Despite comparable levels of anti-tumor effector generation in spleens, however, a fundamental difference was observed in the capacity to reject tumor cells directly challenged at the s.c. site between IL-12-neutralized and unneutralized groups of mice. The inability of anti-IL-12-treated mice to reject challenged tumor cells was associated with the failure of effector T cells generated in lymphoid organs to migrate to the challenge site of tumor cells. The defect in T cell migration to tumor sites was functionally demonstrated using the in vivo lymphoid cell migration assay [26 ]: Donor cells from anti-IL-12-untreated tumor-immunized mice migrated to tumor masses in recipient mice, whereas cells from anti-IL-12-treated tumor-immunized mice failed to show the migration. Thus, IL-12 endogenously produced during anti-tumor immune responses confers T cells with the capacity to migrate to tumor sites.

The migration of leukocytes including T cells into sites of inflammation is a multistep process mediated by a series of cellular and molecular interactions [33 , 34 ]. We have demonstrated that T cell migration to tumor sites depends on the interaction between vascular cell adhesion molecule 1 (VCAM-1)/intercellular adhesion molecule-1 (ICAM-1) and very late antigen-4/lymphocyte function-associated antigen-1 [26 ]. Although the requirement for adhesion molecules in the migration process has been well appreciated [35 36 37 ], recent studies have shown that chemokines and their receptors also play a fundamental role in leukocyte migration [38 39 40 41 ] by inducing the functional activation of adhesion molecules [33 , 41 42 43 44 ]. It is also evident that among chemokine receptors, CCR5 and CXCR3 are expressed on a Th1 type of T cells infiltrating sites of inflammation [45 , 46 ]. In this regard, we recently found that IL-12 is the essential cytokine that is capable of inducing CCR5 expression on T cell receptor-triggered T cells [47 , 48 ]. These observations are compatible with the notion that neutralization of endogenous IL-12 during tumor immunization prevents CCR5 induction on tumor-sensitized T cells, thereby generating anti-tumor T cells without the capacity to migrate to tumor sites.

Further, we investigated the effect of IL-12 neutralization on the development of tumor masses from the morphological and functional aspects. Without IL-12 neutralization, CSA1M and OV-HM tumors developed peritumoral stroma between s.c. tissue and tumor parenchyma, as previously reported [31 , 32 ]. Our previous study also showed that there is a fundamental difference in the nature of vasculature between peritumoral and parenchymal tumor-associated stroma [31 ]: Vasculature at the former expressed ICAM-1/VCAM-1, whereas that at the latter did not. The development of peritumoral stroma is quite important for the induction of intratumoral T cell migration, as T cells make an entry to tumor masses exclusively at the peritumoral area [31 ], and T cell migration is almost completely inhibited by administration of anti-ICAM-1/anti-VCAM-1 mAb [26 ]. This study showed that neutralization of endogenously produced IL-12 inhibits the development of peritumoral stroma and that T cells fail to migrate to tumor masses lacking peritumoral stroma in anti-IL-12-treated mice. Although it is totally unknown how peritumoral stroma develops, our observations suggest that endogenously produced IL-12 is closely related to the development of this particular organization. This may be compatible with observations that the development of peritumoral stroma requires components involved in inflammatory responses [32 ].

Thus, it is obvious that endogenously produced IL-12 functions for the host’s anti-tumor immune responses via two different mechanisms, one of which is to confer T cells with the tumor-migratory capacity and the other, to function for the development of the T cell-accepting capacity of tumor masses. IFN-{gamma} is also required for mediating these mechanisms [30 , 32 ]. As IFN-{gamma} is produced by T cells following IL-12 stimulation, the primary requirement would be IL-12 production. Considering that IL-12 also has the bioactivity independent of IFN-{gamma} actions, it is possible that a part of the above mechanisms is accomplished by IL-12 per se. Elucidation of the overall IL-12-mediated mechanisms could contribute to a better understanding of our attempt to enhance the efficacy of tumor vaccination.


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ACKNOWLEDGEMENTS
 
This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan. The authors are grateful to Mrs. Mami Yasuda for her secretarial assistance.

Received November 27, 2001; revised July 31, 2002; accepted August 1, 2002.


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