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Immunology |
(IFN-) in Tumor Immunity
-deficient Mice1
Department of Oncology, Biomedical Research Center, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871 [C.N., Y.U., M.I., N.Y., T.M., P.G., M.T., S.O., H.F., T.H.], and Department of Pathology, Sumitomo Hospital, Osaka 563-0005 [T.T.], Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-deficient (IFN--/-) mice induce potent in vitro immune responses such as anti-allo mixed lymphocyte reaction and CTL responses, whereas they often fail to exhibit in vivo immunity. Here, we investigated whether there exists a defect in tumor rejection responses and if so, which process of responses is impaired. IFN--/- and wild-type (WT) BALB/c mice were immunized with attenuated syngeneic CSA1M tumor cells. The capacity of T cells to mediate tumor protection was examined in Winn assays to assess the growth of tumor cells admixed with tumor-sensitized T cells. Splenic T cells from both groups of mice exhibited comparable levels of tumor-neutralizing activity. When portions of immunized mice were directly challenged with viable tumor cells, tumor rejection was induced only in WT mice. CD4+ and CD8+ T-cell infiltration were observed at the site of tumor challenge in WT mice, whereas such a T-cell infiltration did not occur in IFN--/- mice. Similarly, splenic T cells from interleukin 12-treated CSA1M-bearing IFN--/- and WT mice neutralized tumor cells at comparable efficacies in Winn assays. However, the migration of these T cells to tumor masses and the resultant interleukin 12-induced tumor regression took place in WT mice, but neither intratumoral T-cell infiltration nor tumor regression occurred in IFN--/- mice. These results indicate a critical requirement for IFN- in the process of inducing T-cell migration to tumor sites rather than of generating antitumor protective T cells. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, a cytokine secreted by activated T cells and natural killer cells, has multiple immunoregulatory effects on various cell types (1
, 2)
, including the capacity to stimulate the activation of CTLs (3
, 4)
, natural killer cells (5)
, and macrophages (6, 7, 8)
and to induce/enhance the expression of class II MHC antigens (9)
. Through these effects, it has been well appreciated that this cytokine has an important role in the manifestation of cell-mediated inflammatory responses.
IFN- can profoundly affect immune responses in vitro and in vivo. To determine how essential this cytokine is for the normal development and function of the immune system, IFN--/-3
(10)
and IFN-R-/- mice (11)
were prepared, and various in vitro and in vivo immune responses in these mice were examined. Surprisingly, previous studies (10
, 11)
showed that T-cell responses are not impaired in IFN--/- (10)
or IFN-R-/- mice (11)
. Rather, T cells from IFN--/- mice exhibited considerably enhanced anti-alloantigen responses in vitro. Consistent with this, rejection responses of class I and class II MHC-disparate allografts were not affected in IFN--/- (12)
or IFN-R-/- (13)
mice. However, these mutant mice exhibited a profound defect in natural resistance against intracellular pathogens (10
, 11)
that requires the participation of macrophages. Even in allograft responses, the rejection of grafts disparate only in class II MHC was greatly impaired in IFN--deficient (14
, 15)
or IFN--neutralized (16)
mice. Thus, except for the rejection of class I-disparate allografts involving the participation of potent CD8+ CTL, mice deficient in IFN- function have a profound defect in the in vivo immunity including rejection responses and protection against infectious diseases.
Tumor rejection is a sequence of inflammatory responses in which T cells function as a conductor as well as a member of effectors. Taken into consideration that as powerful CD8+ CTLs as those induced in anti-allo class I MHC responses would not be generated in tumor immune responses, IFN- might be important for inducing tumor rejection. However, the importance for IFN- in tumor immunity has been investigated only in a few studies (17)
. Particularly, it is poorly understood to what extent IFN- is essential and at what process of antitumor responses this cytokine is required. The present study addressed these issues and revealed the following. T cells with the capacity to reject tumor cells in vivo are generated in lymphoid organs such as spleens from tumor-immunized or tumor-bearing IFN--/- mice. These T cells can induce comparable degrees of tumor-neutralizing activity to those of WT T cells when artificially admixed with tumor cells in Winn assays. However, they have a fundamental defect in the migration to tumor-challenged sites in tumor-immunized mice or to tumor masses in tumor-bearing mice. Because of this defect, they are unable to function for tumor cell/mass eradication. The results indicate a thus far undescribed aspect of the requirement for IFN- in tumor immunity.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mice.
BALB/c mice were purchased from Shizuoka Laboratory Animal Center (Hamamatsu, Japan). IFN--/- BALB/c mice (BALB/c-Ifngtm1Ts; Ref. 10
) were obtained from The Jackson Laboratory (Bar Harbor, ME). These mice were bred in our laboratory and used at 6–9 weeks of age.
Reagents.
Murine rIL-12 was provided by 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, CSA1M tumor cells were treated in vitro with 100 µg/ml MMC for 60 min. IFN--/- or WT BALB/c mice were inoculated i.p. with 106 (unless otherwise indicated) MMC-treated tumor cells three times at 1-week intervals. To prepare tumor-bearing mice, mice were inoculated s.c. with viable CSA1M tumor cells (1 x 106/mouse) and used at 2–3-week, tumor-bearing stages.
Preparation of Samples to Assess IL-2 Concentrations: Whole Spleen Cell Culture System.
Unfractionated spleen cells, obtained from a pool of three to four tumor-immunized or tumor-bearing mice, were cultured without addition of exogenous tumor antigens in 24-well culture plates (Corning 25820; Corning Glass Works, Corning, NY) at a concentration of 5 x 106 cells/well in a volume of 1 ml of RPMI 1640 supplemented with 10% FCS as described previously (19
, 20)
. After incubation at 37°C in a humidified incubator (5% CO2) for 24 h, culture SN was harvested by centrifugation and stored at -20°C until use.
Measurement of IL-2 Concentration.
IL-2 concentration was measured by ELISA. Mouse rIL-2 was kindly provided by Shionogi Research Laboratories (Osaka, Japan). The mouse IL-2 ELISA system was prepared using two types of antimouse IL-2 mAbs (JES6-1A12 and biotinylated JES6-5H4; purchased from PharMingen, San Diego, CA).
Preparation of a T-Cell-enriched Population.
Spleen cells were depleted of B cells by immunomagnetic negative selection, as described (21)
. Briefly, spleen cells were incubated with magnetic particles bound to goat antimouse immunoglobulin (Advanced Magnetics, Cambridge, MA). Surface immunoglobulin-negative cells were obtained by removing cell-bound magnetic particles with a rare earth magnet (Advanced Magnetics) and used as a T-cell-enriched population (>90% purity).
Tumor-Neutralization Test (Winn Assay).
A splenic T cell-enriched population from normal or tumor-sensitized mice was admixed with viable tumor cells and the mixture was inoculated s.c. into IFN--/- BALB/c recipient mice. Tumor growth was measured and expressed as the mean ± SE of five mice/group.
IL-12 Treatment.
rIL-12 (0.5 µg/time) was administered i.p. to tumor-bearing mice or mice receiving tumor immunization three or five times every other day.
A Lymphoid Cell Migration Assay.
The assay system was essentially the same as described previously (22)
. Staining of 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 250 µl of diluent were allowed to react with 5 x 10-6 M PKH-26 dissolved in 1 ml of diluent for 5 min at 37°C. Labeling was stopped by adding 2 ml of FCS, and cells were washed five times with RPMI 1640 containing 10% FCS. Mice with similar tumor sizes (
7 mm in diameter) were used as recipients for this assay. PKH-26-labeled spleen cells (3 x 107 cells in 250 µl of RPMI 1640) were injected i.v. into recipient (IL-12-untreated, homologous tumor-bearing) mice. Twenty four h 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/section.
Histological Examination.
Tumor masses were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with H&E for histological examination.
Staining Procedure of Immunohistochemical Examination.
The following reagents were purchased to perform immunohistochemical examination: biotinylated antimouse CD4 and anti-CD8 mAbs (PharMingen); biotinylated rat IgG (Jackson Immuno Research, West Grove, PA); and Histofine SA-PO kit and Histofine DAB kit (Nichirei Co. Ltd., Tokyo, Japan). Samples were fixed in 2% paraformaldehyde for 6–12 h at 4°C and then washed sequentially with PBS containing 10, 15, and 20% sucrose for 6 h each at 4°C. The samples were embedded in OCT compound 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 antibody was added. After preincubation with 4% BSA solution, the tissues were overlaid with various biotinylated antibodies 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 washing an additional three times, the labeling was visualized with 0.03% 3,3'-diaminobenzidine tetrahydrochloride solution containing 0.1% hydrogen peroxide for 5 to 6 minutes.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-/- Mice.-/- mice exhibit enhanced proliferative responses after stimulation with mitogen or allogeneic cells (MLR). We confirmed that the MLR by IFN--/- T cells is higher than that by WT T cells. (Fig. 1A)
. Fig. 1B
also shows that enhanced MLR of IFN--/- T cells is accompanied by their higher capacity to produce IL-2.
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-/- T cells, we investigated whether T cells from IFN--/- mice also exhibit higher antitumor in vitro T-cell responses than WT T cells. Our previous study provided an experimental system to assess the in vitro reactivity of tumor-sensitized T cells (19)
. The study showed that spleen cells from tumor-bearing mice or mice immunized with attenuated tumor cells contain both tumor-sensitized T cells and APCs presenting tumor antigens and that culturing these spleen cells results in IL-2 production through T cell-APC interactions without addition of exogenous tumor antigens (19)
. Using this whole spleen cell culture system, we compared the capacity to induce IL-2 production between IFN--/- and WT T cells. BALB/c WT and IFN--/- mice were inoculated s.c. with 106 viable CSA1M tumor cells or immunized i.p. with 106 MMC-treated syngeneic CSA1M tumor cells three times at 1-week intervals. Two to three weeks after tumor cell inoculation or 1 week after the third immunization, spleens were harvested, and spleen cells were cultured for 24 h. Fig. 2
shows that IL-2 production induced by spleen cells from either CSA1M tumor-bearing or CSA1M tumor-immunized IFN--/- mice is comparable with (Fig. 2A)
or higher than (Fig. 2B)
that induced by those from WT mice. Taken together, the results show that T cells in IFN--/- mice have the capacity to mount a higher in vitro response than WT T cells.
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-/- and WT Mice.-/- mice with tumor cells generates T cells capable of promoting in vivo tumor protection, we assessed the capacity of T cells from CSA1M-immunized IFN--/- mice to neutralize viable CSA1M tumor cells in the in vivo Winn assay. Splenic T cells from WT and IFN--/- mice immunized to CSA1M were admixed with 106 viable CSA1M tumor cells, and the mixture was inoculated s.c. into BALB/c IFN--/- recipient mice. As shown in Fig. 3A
, spleen cells from immunized IFN--/- mice produced comparable levels of tumor neutralization to those observed for WT spleen cells. 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 E:T ratios. Instead of T-cell enrichment, T-cell elimination from donor cell inoculum abrogated tumor-neutralizing activity by spleen cells (data not shown). Thus, antitumor T cells that function for tumor rejection can be normally generated in IFN--/- mice.
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-/- Mice to Reject Directly Challenged Tumor Cells.-/- and WT mice immunized to CSA1M by inoculating attenuated CSA1M cells via the i.p. route can reject tumor cells directly challenged at the s.c. site. WT and IFN--/- mice were immunized with 104–106 CSA1M tumor cells three times and 1 week later, challenged with 106 viable CSA1M 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 Fig. 4
, all WT mice immunized with 105 or 106 tumor cells rejected challenged tumor cells. In contrast, IFN--/- mice immunized with the same numbers of tumor cells failed to reject tumor cells, although tumor growth rate was slightly lower in a 106-immunization group than in a non-immunized group. Thus, although WT and IFN--/- mice can similarly generate T cells capable of eradicating 106 admixed tumor cells, IFN--/- mice fail to reject tumor cells directly challenged.
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-/- Mice.
and 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. CSA1M-immunized WT and IFN--/- mice were challenged s.c. with CSA1M cells. The skin including the tumor challenge site was removed 2 days after the tumor challenge and subjected to histological and immunohistochemical examination. As shown in Fig. 5
, the tumor-challenged site in WT mice exhibited strong mononuclear cell infiltration. This cellular infiltration was found to involve the accumulation of CD4+ and CD8+ T cells (Fig. 6)
. In contrast, the skin site of IFN--/- mice exhibits apparently decreased levels of mononuclear cell infiltration compared with that of WT mice (Fig. 5)
. The reduction of cellular infiltration was associated with the detection of only a few CD4+ and CD8+ cells (Fig. 6)
. Thus, IFN--/- 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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-/- Mice.-/- mice along with that in WT mice (Fig. 7)
. In Fig. 7A
, CSA1M-bearing IFN--/- and WT mice that bore different sizes of CSA1M tumors 2 weeks after tumor implantation were treated with IL-12, and in Fig. 7B
, IFN--/- and WT mice bearing comparable sizes of CSA1M tumors different days after tumor implantation received IL-12 treatment. The results of Fig. 7
confirm that CSA1M tumor regression is induced in WT mice after IL-12 administration and demonstrate that the same does not occur in IFN--/- mice. We have shown that tumor regression after IL-12 treatment is associated with a massive T cell (CD4+ and CD8+) infiltration to CSA1M tumor masses (22
, 23
, 26)
. This is again shown in Fig. 8, A
(left panel) and B
. In contrast, cellular infiltration was weak in IFN--/- tumor masses and mostly observed between skin and a tumor mass with unaffected parenchymal structure (Fig. 8A
, right panel).
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-/- Mice.-/- mice to induce IL-12-mediated tumor regression, we first examined whether tumor-sensitized T cells can be generated in tumor-bearing IFN--/- mice. Splenic T cells from CSA1M-bearing IFN--/- and WT mice that have not received IL-12 treatment were subjected to Winn assays (Fig. 3B)
. The results show that both groups of cells neutralize admixed CSA1M tumor cells at the E:T ratio of 10:1 and that there is no substantial difference in the magnitude in the tumor-neutralizing activity. We also examined the tumor-neutralizing capacity of T cells from tumor-bearing IFN--/- and WT mice that have received IL-12 treatment. Both groups of cells exhibited stronger capacities than those from IL-12-untreated mice and neutralized tumor cells, even at the E:T ratio of 1:1 (data not shown). Thus, CSA1M-bearing IFN--/- mice can generate T cells capable of exhibiting comparable magnitudes of tumor-neutralizing activity to those generated in WT mice.
CSA1M-bearing and CSA1M-immunized IFN--/- Mice Have a Defect in Promoting the Recruitment of T Cells to Tumor Masses/Sites.
The reduction of T-cell accumulation at the site of tumor challenge in tumor-immunized IFN--/- mice and in tumor mass from IL-12-treated IFN--/- mice suggests the inability of IFN--/- T cells to migrate to tumor sites/masses. This was directly investigated using a lymphoid cell (T-cell) migration assay that was developed in our laboratory and described previously (22)
. Our previous study (22
, 26
, 27)
showed that spleen cells from CSA1M-bearing mice receiving IL-12 treatment exhibited an enhanced migration into CSA1M tumor masses of CSA1M-bearing IL-12-untreated recipient mice and that almost all migrating cells among a spleen cell inoculum were T cells (22)
. Donor spleen cells were prepared from normal or tumor-bearing IFN--/- or WT mice with or without IL-12 treatment and labeled with a fluorescent dye. These donor cells were transferred into CSA1M-bearing, IL-12-untreated WT mice. Twenty-four h later, the numbers of fluorescein-labeled donor cells were evaluated on the cryostat sections of tumor masses from recipient mice (Fig. 9A)
. The results confirm IL-12-induced enhancement of the migratory capacity of T cells from CSA1M-bearing donor WT mice. In contrast, cells from similarly IL-12-treated CSA1M-bearing IFN--/- mice exhibited a strikingly reduced capacity for the migration to tumor masses of recipient mice.
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-/- or WT mice with or without IL-12 treatment. Their tumor-migratory capacity was assessed by transferring into CSA1M-bearing, IL-12-untreated recipient mice (Fig. 9B)
. Donor cells from tumor-immunized WT mice exhibited the capacity to migrate to tumor masses, which is consistent with the results of Figs. 4
5
6
. Such a migratory capacity was enhanced when donor mice were treated with IL-12 along with tumor immunization. In contrast, cells from tumor-immunized IFN--/- mice exhibited only marginal levels of enhancement in tumor migration compared with those from unimmunized mice. This was also the case when donor IFN--/- mice received the combined treatment of tumor immunization and IL-12 injection. Consistent with the failure to enhance T-cell migration after IL-12 treatment, IFN--/- mice failed to reject directly challenged tumor cells, even after the combined treatment (Fig. 10)
. Taken together, the results indicate that the failure of IFN--/- mice to induce tumor regression after IL-12 treatment and to reject directly challenged tumor cells after tumor immunization with or without IL-12 treatment is ascribed to the inability to promote T-cell trafficking to tumor masses/sites.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-/- and wild-type mice generate comparable magnitudes of tumor-neutralizing T cells. However, tumor-immunized IFN--/- mice fail to reject directly challenged tumor cells. IL-12-mediated regression of growing CSA1M tumors is induced in wild-type but not in CSA1M-bearing IFN--/- mice. Histological examination revealed that T cells do not migrate into the site of tumor cell challenge in tumor-immunized IFN--/- mice or the tumor mass in IL-12-treated IFN--/- mice. Splenic T cells from tumor-immunized or IL-12-treated tumor-bearing WT mice, when i.v. transferred into tumor-bearing recipient mice, migrated to tumor masses in these recipient mice, whereas those from similarly treated IFN--/- mice did not. Thus, the results indicate that IFN- deficiency does not affect the induction of tumor-sensitized T cells but critically influences the acquisition of their trafficking capacity.
IFN- plays a central role in promoting innate and adaptive mechanisms of host defense (28
, 29)
. Evidence that IFN- up-regulates the function of various effector cells in cell-mediated immune responses (3, 4, 5, 6, 7, 8, 9)
has also suggested a critical role of this cytokine in tumor immunity. In fact, this notion was supported recently by the observations that IL-12-induced tumor regression is blocked by neutralizing IFN- produced after IL-12 injections (23
, 30)
. 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
, 31) have suggested that IFN- plays a role in some of the processes such as an intratumoral antitumor effector mechanism. However, it is unclear how IFN- contributes to promoting other processes of tumor immunity, particularly how this cytokine exhibits an effect on the activation of T cells in lymphoid organs and their trafficking to tumor masses.
Regarding the effect of IFN- on T-cell activation, earlier studies showed that IFN- is an important factor in the induction of CTL differentiation (3
, 4)
. This was initially supported by the observation that the development of CTL during MLR is accompanied by the release of IFN- to cultures (32)
. However, two reports described that addition of IFN--neutralizing antibody to the MLR did not reduce CTL generation (33
, 34)
. More critically, IFN- deficiency does not affect T-cell activation but rather enhances anti-allo responses including proliferation and CTL generation (10)
. We have confirmed these (Fig. 1)
and further showed that the induction of antitumor T cells as evaluated by an in vitro culture system (19
, 20)
is not affected by IFN- deficiency (Fig. 2)
. Our present results showed that splenocytes from CSA1M tumor-bearing or tumor-immunized IFN--/- mice induced comparable levels of cytokine (IL-2) production to those of WT splenocytes. More importantly, IFN--/- 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 WT and IFN--/- T cells (Fig. 3)
. These results indicate that IFN- is not required for inducing the sensitization/activation of T cells with the capacity to eradicate tumor cells in vivo and that such T cells can be generated in lymphoid organs of IFN--/- mice similarly in those of WT mice.
Whereas i.p. immunization of WT and IFN--/- mice with tumor cells resulted in comparable levels of antitumor effector generation in spleens, a fundamental difference was observed in the capacity to reject tumor cells directly challenged at the s.c. site between these two groups of mice. Our results showed that the inability of IFN--/- mice to reject challenged tumor cells was attributable to the failure of effector T cells generated in lymphoid organs to migrate to the challenge site of tumor cells. A similar defect was observed in tumor-bearing IFN--/- mice receiving IL-12 treatment. IFN--/- and WT mice both generated tumor-neutralizing T cells during the tumor-bearing state (Fig. 3B)
. IL-12 treatment up-regulated the antitumor capacity of T cells from both groups of mice so that they can completely neutralize tumor cells at the E:T ratio of 1:1.4
Nevertheless, tumor regression after IL-12 treatment was induced in WT but not in IFN--/- mice. As has been shown (22, 23, 24, 25)
, tumor regression in WT mice was associated with a massive T-cell accumulation in tumor masses. In contrast, only a few infiltrating cells were seen in IFN--/- tumor masses.
Furthermore, the defect in T-cell migration to tumor sites was functionally demonstrated using the in vivo lymphoid cell migration assay (22)
. Our previous studies demonstrated that donor spleen cells from IL-12-untreated, CSA1M-bearing mice failed to migrate to tumor masses of CSA1M-bearing recipient mice, whereas donor cells from IL-12-treated, CSA1M-bearing mice exhibited enhanced migration to recipients’ tumor masses (22
, 26
, 27)
. In this assay, migrating cells out of a spleen cell inoculum was found to be mostly CD4+ or CD8+ T cells (22)
, showing T-cell migration to tumor masses. This study confirmed that donor spleen cells (virtually, T cells) from IL-12-treated CSA1M-bearing WT mice, when transferred i.v. into CSA1M-bearing recipient mice, displayed the migration to recipients’ tumor masses. In contrast, donor cells from IL-12-treated, CSA1M-bearing IFN--/- mice failed to exhibit the migration. Similarly, donor cells from tumor-immunized WT mice exhibited enhanced migration to tumor masses in recipient mice, whereas cells from tumor-immunized IFN--/- mice failed to show the migration.
It should be noted that unlike donor cells from IL-12-untreated tumor-bearing mice, cells from the IL-12-untreated, tumor-immunized WT mice have the capacity to migrate to tumor masses. Considering the up-regulatory effect of IL-12 on T-cell migration in tumor-bearing mice, we also examined whether administration of IL-12 into tumor-immunized mice enhance the capacity of T cells to migrate to tumor sites. Whereas tumor immunization followed by IL-12 injection in WT mice further enhanced the migration compared with tumor immunization alone, T cells from tumor-immunized IFN--/- mice failed to show the migration after IL-12 injection along with tumor immunization (Fig. 9B)
. Consistent with this, the failure of IFN--/- mice to reject directly challenged tumor cells was not corrected, even after IL-12 administration along with tumor immunization (Fig. 10)
.
The migration of leukocytes including T cells into sites of inflammation is a multistep process mediated by a series of cellular and molecular interactions (35
, 36)
. We have demonstrated that T-cell migration to tumor sites depends on the interaction between vascular cell adhesion molecule-1/intercellular adhesion molecule-1 and VLA-4/LFA-1 (22)
. Although the requirement for adhesion molecules in the migration process has been well appreciated (37, 38, 39)
, recent studies have shown that chemokines and their receptors also play a fundamental role in leukocyte migration (40, 41, 42)
. It is increasingly becoming evident that chemokine receptors function to induce affinity modulation of adhesion molecules (35
, 43, 44, 45, 46)
. In this regard, we found recently that there is no substantial difference between IFN--/- and WT T cells in the induction of IL-12 receptor and adhesion molecule such as LFA-1 and VLA-4 after T-cell receptor triggering and IL-12 stimulation.5
Considering the role of chemokine receptors in the functional activation of adhesion molecules, however, further studies will be required to compare the expression of chemokine receptors as well as the adhesive capacity of LFA-1 and VLA-4 between IFN--/- and WT T cells.
Thus, it is obvious that IFN- deficiency affects a critical step of tumor immunity in which tumor-sensitized T cells migrate from lymphoid organs to tumor sites. Our earlier study (26)
showed that administration of anti-IFN- mAb prevents the induction of vascular cell adhesion molecule-1/intercellular adhesion molecule-1 expression on intratumoral vasculature, preventing the induction of T-cell acceptability of tumor masses. Instead, the present study illustrated that T cells from tumor-immunized or tumor-bearing IFN--/- mice, even after IL-12 treatment, fail to migrate to wild-type tumor masses to which tumor-sensitized wild-type T cells can gain access. These results clearly indicate that IFN- deficiency affects the acquisition of the trafficking capacity in T cells themselves. This also verifies the importance for taking into consideration a process of intratumoral T-cell migration in attempt to enhance tumor immunity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan. 
2 To whom requests for reprints should be addressed, at Department of Oncology, Biomedical Research Center, Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-3982; Fax: 81-6-6879-3989; E-mail: hf{at}ongene.med.osaka-u.ac.jp
3 The abbreviations used are: IFN--/-, IFN--deficient; IFN-R-/-, IFN- receptor-deficient; rIL, recombinant IL; WT, wild type; MMC, mitomycin C; APC, antigen-presenting cell; mAb, monoclonal antibody; MLR, mixed lymphocyte response; LFA, leukocyte function antigen; VLA, very late antigen; SN, supernatant.
4 C. Nakajima, H. Fujiwara, and T. Hamaoka, unpublished data.
5 C. Nakajima, M. Iwasaki, T. Uekusa, H. Fujiwara, and T. Hamaoka. IFN- deficiency does not affect the expression of LFA-1 and VLA-4 after T-cell receptor triggering and/or IL-12 stimulation, submitted for publication.
Received 11/22/00. Accepted 2/13/01.
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 J. A. Hollenbaugh and R. W. Dutton IFN-{gamma} Regulates Donor CD8 T Cell Expansion, Migration, and Leads to Apoptosis of Cells of a Solid Tumor. J. Immunol., September 1, 2006; 177(5): 3004 - 3011. [Abstract] [Full Text] [PDF]
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 T. Ono, M. Harada, A. Yamada, M. Tanaka, Y. Takao, Y. Tanaka, T. Mine, K. Sakamoto, T. Nakashima, and K. Itoh Antitumor Effects of Systemic and Local Immunization with a CTL-Directed Peptide in Combination with a Local Injection of OK-432 Clin. Cancer Res., February 15, 2006; 12(4): 1325 - 1332. [Abstract] [Full Text] [PDF]
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S. Yu, G. C. Sharp, and H. Braley-Mullen Thyrocytes Responding to IFN-{gamma} Are Essential for Development of Lymphocytic Spontaneous Autoimmune Thyroiditis and Inhibition of Thyrocyte Hyperplasia J. Immunol., January 15, 2006; 176(2): 1259 - 1265. [Abstract] [Full Text] [PDF]
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M. Moeller, N. M. Haynes, M. H. Kershaw, J. T. Jackson, M. W. L. Teng, S. E. Street, L. Cerutti, S. M. Jane, J. A. Trapani, M. J. Smyth, et al. Adoptive transfer of gene-engineered CD4+ helper T cells induces potent primary and secondary tumor rejection Blood, November 1, 2005; 106(9): 2995 - 3003. [Abstract] [Full Text] [PDF]
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 Q. Li, A. L. Carr, E. J. Donald, J. J. Skitzki, R. Okuyama, L. M. Stoolman, and A. E. Chang Synergistic Effects of IL-12 and IL-18 in Skewing Tumor-Reactive T-Cell Responses Towards a Type 1 Pattern Cancer Res., February 1, 2005; 65(3): 1063 - 1070. [Abstract] [Full Text] [PDF]
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J. Skitzki, R. A. Craig, R. Okuyama, R. N. Knibbs, K. McDonagh, A. E. Chang, and L. M. Stoolman Donor Cell Cycling, Trafficking, and Accumulation during Adoptive Immunotherapy for Murine Lung Metastases Cancer Res., March 15, 2004; 64(6): 2183 - 2191. [Abstract] [Full Text] [PDF]
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K. Chamoto, T. Tsuji, H. Funamoto, A. Kosaka, J. Matsuzaki, T. Sato, H. Abe, K. Fujio, K. Yamamoto, T. Kitamura, et al. Potentiation of Tumor Eradication by Adoptive Immunotherapy with T-cell Receptor Gene-Transduced T-Helper Type 1 Cells Cancer Res., January 1, 2004; 64(1): 386 - 390. [Abstract] [Full Text] [PDF]
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N. M. Haynes, J. A. Trapani, M. W. L. Teng, J. T. Jackson, L. Cerruti, S. M. Jane, M. H. Kershaw, M. J. Smyth, and P. K. Darcy Rejection of Syngeneic Colon Carcinoma by CTLs Expressing Single-Chain Antibody Receptors Codelivering CD28 Costimulation J. Immunol., November 15, 2002; 169(10): 5780 - 5786. [Abstract] [Full Text] [PDF]
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N. M. Haynes, J. A. Trapani, M. W. L. Teng, J. T. Jackson, L. Cerruti, S. M. Jane, M. H. Kershaw, M. J. Smyth, and P. K. Darcy Single-chain antigen recognition receptors that costimulate potent rejection of established experimental tumors Blood, October 16, 2002; 100(9): 3155 - 3163. [Abstract] [Full Text] [PDF]
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J. G. Segal, N. C. Lee, Y. L. Tsung, J. A. Norton, and K. Tsung The Role of IFN-{gamma} in Rejection of Established Tumors by IL-12 : Source of Production and Target Cancer Res., August 15, 2002; 62(16): 4696 - 4703. [Abstract] [Full Text] [PDF]
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R. E. Miller, J. Jones, T. Le, J. Whitmore, N. Boiani, B. Gliniak, and D. H. Lynch 4-1BB-Specific Monoclonal Antibody Promotes the Generation of Tumor-Specific Immune Responses by Direct Activation of CD8 T Cells in a CD40-Dependent Manner J. Immunol., August 15, 2002; 169(4): 1792 - 1800. [Abstract] [Full Text] [PDF]
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R. A. Wilcox, D. B. Flies, H. Wang, K. Tamada, A. J. Johnson, L. R. Pease, M. Rodriguez, Y. Guo, and L. Chen Impaired Infiltration of Tumor-specific Cytolytic T Cells in the Absence of Interferon-{gamma} despite Their Normal Maturation in Lymphoid Organs during CD137 Monoclonal Antibody Therapy Cancer Res., August 1, 2002; 62(15): 4413 - 4418. [Abstract] [Full Text] [PDF]
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 S. E.A. Street, J. A. Trapani, D. MacGregor, and M. J. Smyth Suppression of Lymphoma and Epithelial Malignancies Effected by Interferon {gamma} J. Exp. Med., July 1, 2002; 196(1): 129 - 134. [Abstract] [Full Text] [PDF]
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Y. Uekusa, W.-G. Yu, T. Mukai, P. Gao, N. Yamaguchi, M. Murai, K. Matsushima, S. Obika, T. Imanishi, Y. Higashibata, et al. A Pivotal Role for CC Chemokine Receptor 5 in T-Cell Migration to Tumor Sites Induced by Interleukin 12 Treatment in Tumor-bearing Mice Cancer Res., July 1, 2002; 62(13): 3751 - 3758. [Abstract] [Full Text] [PDF]
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T. Hayashi and D. L. Faustman Development of Spontaneous Uterine Tumors in Low Molecular Mass Polypeptide-2 Knockout Mice Cancer Res., January 1, 2002; 62(1): 24 - 27. [Abstract] [Full Text] [PDF]
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