Alterations to the tumor microenvironment following localized irradiation may influence the effectiveness of subsequent immunotherapy. The objective of this study was to determine how IFN-γ influences the inflammatory response within this dynamic environment following radiotherapy. B16/OVA melanoma cells were implanted into C57BL/6 (wild-type (WT)) and IFN-γ-deficient (IFN-γ−/−) mice. Seven days after implantation, mice received 15 Gy of localized tumor irradiation and were assessed 7 days later. Irradiation up-regulated the expression of VCAM-1 on the vasculature of tumors grown in WT but not in IFN-γ−/− mice. Levels of the IFN-γ-inducible chemokines MIG and IFN-γ-inducible protein 10 were decreased in irradiated tumors from IFN-γ−/− mice compared with WT. In addition to inducing molecular cues necessary for T cell infiltration, surface MHC class I expression is also up-regulated in response to IFN-γ produced after irradiation. The role of IFN-γ signaling in tumor cells on class I expression was tested using B16/OVA cells engineered to overexpress a dominant negative mutant IFN-γ receptor (B16/OVA/DNM). Following implantation and treatment, expression of surface class I on tumor cells in vivo was increased in B16/OVA, but not in B16/OVA/DNM tumors, suggesting IFN-γ acts directly on tumor cells to induce class I up-regulation. These increases in MHC class I expression correlated with greater levels of activated STAT1. Thus, IFN-γ is instrumental in creating a tumor microenvironment conducive for T cell infiltration and tumor cell target recognition.

It is well accepted that infiltration of effector T cells into tumors is required for successful antitumor immune responses (1, 2). However, limitations in trafficking and retention of specific T cells within the tumor are important factors behind the inability of immunotherapies to control tumor growth. Having infiltrated the tumor, the effector functions of CTLs may be additionally hampered by the presence of immunosuppressive mediators within this microenvironment (3). For these reasons, immunotherapy used alone, even in relatively immunogenic tumors such as melanomas, has been effective in only a small percentage of patients (4). Thus, there is considerable interest in combining immunotherapy with other treatment modalities to improve treatment outcomes (5). Radiation is an important treatment for the local control of cancer based on its ability to directly kill tumor cells. However, there is increasing evidence that localized irradiation of the tumor may also modify the tumor microenvironment and generate inflammatory cytokines (6), which can increase the robustness of the immune response. Before rational clinical protocols combining these therapies can be devised, it is essential to better understand how such cytokines, generated in response to ionizing radiation, can affect the generation and function of immune and malignant cells present within the tumor microenvironment.

IFN-γ is one such proinflammatory cytokine that plays an important role in immune responses to tumors (7). Tumor-specific CTL effector functions are dependent on IFN-γ signaling (8, 9). IFN-γ has pleiotropic effects in the tumor microenvironment, including the inhibition of cell proliferation and angiogenesis (10). Some of these antitumor effects of IFN-γ are through direct signaling within the tumor cells by inducing caspase activation, surface MHC class I expression, and the up-regulation of IFN-γ-inducible genes, including ones for the antiangiogenic chemokines MIG and IFN-γ-inducible protein 10 (IP-10)3 (11, 12). Because of these broad effects, determining how tumor cells respond to IFN-γ is critical for effective antitumor responses (13). Radiotherapy has been demonstrated to cause inflammation, a potentially beneficial state in which IFN-γ is undoubtedly involved. However, there is limited information on the exact role of this cytokine in tumors treated by local irradiation. By determining what immune parameters critical to antitumor responses are controlled by IFN-γ, treatments can be tailored to effectively use immunotherapies in combination with radiation therapy.

We have evaluated the role of IFN-γ in the tumor microenvironment using two complementary strategies in which the cytokine is incapable of exerting its effects on the tumor cells and the tumor vasculature. In the first system, radiation-induced alterations to the microenvironment were assessed in IFN-γ-deficient (IFN-γ−/−) mice. The absence of the cytokine allowed us to accurately determine the importance of IFN-γ following irradiation by assessing which aspects of the immune response are diminished in these deficient mice compared with wild-type (WT) mice and therefore dependent on IFN-γ. Whereas the complete absence of IFN-γ in the tumor is valuable for assessing the effects of the cytokine on all of the cell types, we were also interested in determining the importance of the tumor cell’s ability to respond to IFN-γ directly. To create a system in which the tumor cell generated no response to the cytokine, we used a tumor cell line transfected with a dominant negative IFN-γ receptor mutant (B16/OVA/DNM) (14). The use of this cell line allowed us to precisely assess whether the ability of the tumor to respond to IFN-γ is crucial for immune responses following localized irradiation. Through the use of these two approaches, we have determined how IFN-γ conditions the tumor microenvironment for effective immune responses. We report that IFN-γ plays a role in T cell trafficking to the tumor microenvironment through the up-regulated expression of the adhesion molecule VCAM-1 on tumor vasculature and chemoattractants MIG and IP-10. The ability of the tumor cell to respond to IFN-γ was found to be important for induction of surface MHC class I expression. Taken together, IFN-γ appears to condition the tumor microenvironment by several mechanisms which facilitate enhanced CTL trafficking and recognition of tumor cells in the context of radiation.

C57BL/6J (WT) and B6.129S7-Ifngtm1Ts (IFN-γ−/−) mice were purchased from The Jackson Laboratory. Guidelines for the humane treatment of animals were followed, as approved by the University Committee on Animal Resources. The B16-F0 cell line, a C57BL/6 spontaneously arising melanoma, was obtained from the American Type Culture Collection (CRL 6322). B16-F0 cells transfected to express chicken OVA (B16/OVA) have been previously described (15). B16/OVA were additionally transfected with the pcDNA.mugR overexpression plasmid (gift from Dr. Y. Paterson, University of Pennsylvania, Philadelphia, PA), which encodes a dominant negative mutant IFN-γR under control of the CMV promoter and contains a gene for zeocin resistance (B16/OVA/DNM) (16). Stable B16/OVA/DNM transfectants were generated and cells that expressed the highest levels of IFN-γR as assessed by flow cytometry were selected and further subcloned. B16/OVA/DNM clones were tested in vitro to confirm lack of responsiveness to recombinant mouse IFN-γ, as measured by failure to up-regulate MHC H-2Kb expression.

Bone marrow was harvested from femurs of WT and IFN-γ−/− mice and washed twice in a balanced salt solution. Three × 106 bone marrow cells in HBSS were injected i.v. into lethally irradiated (10 Gy) IFN-γ−/− and WT recipient mice on the same day. After reconstitution, WT→IFN-γ−/− and IFN-γ−/−→WT chimeric mice were allowed to rest for a minimum of 8 wk before experimental use.

One × 105 viable tumor cells were injected i.m. into thighs of all mice. Localized irradiation of tumor-bearing legs was performed as described previously (17). Briefly, each mouse was restrained and received 15 Gy of localized irradiation on day 7 of tumor growth. Mean thigh diameter was determined as previously described (9). Mice were sacrificed for analysis 14 days after tumor initiation.

Tumor vasculature analysis was performed as described previously (17). In brief, small pieces of tumor were excised and incubated in staining buffer. Samples were blocked with Fc block (BD Pharmingen) at 4°C for 10 min. Primary Abs FITC anti-VCAM-1 and PE anti-CD31 (clone 429 and clone MEC13.3; BD Pharmingen) were added directly to the tubes at predetermined concentrations and incubated for an additional 2 h. Samples were washed, mounted on slides, viewed with a fluorescence microscope, and images were acquired using a monochrome charge-coupled device digital camera. Monochrome images of corresponding fields of view were pseudo-colored and overlaid using Image Pro Plus software version 5.0 (Media Cybernetics). The percent VCAM-1+ vessel area was determined on the Ab-stained whole mount preparations. Images were analyzed using Image Pro Plus by segmenting the CD31+ and VCAM-1+ images using a predefined intensity range. From the binary images, the percent vessel areas were computed for multiple regions within each tumor.

Tumors were dissociated using collagenase as previously described (18). These dissociated single-cell suspensions were initially blocked using Fc block and stained with PerCP anti-CD45 (clone 30-F11; BD Pharmingen) to identify host immune cell populations. Phenotypic description of tumor-infiltrating lymphocytes (TILs) was determined on CD45+ populations using the following primary Abs: allophycocyanin anti-CD4 and anti-CD8 (clone GK1.5 and clone 53-6.7; eBioscience). MHC class I expression on tumor cells was determined by gating on CD45 and forward scatter high cells and staining with FITC anti-H-2Kb (clone AF6-88.5; BD Pharmingen). IFN-γ intracellular cytokine staining in the IFN-γ−/− chimeric mice was performed by dissociating the tumor, as stated above, and incubating the cells overnight in medium with 5% FCS and 5 μg/ml each of the peptides pOVA I, pOVAII, and TRP-2. The following day, GolgiPlug (BD Pharmingen) was added for 6 h and the cells were harvested with trypsin, fixed, and permeabilized using 4% paraformaldehyde and stained using PerCP anti-CD45 and PE anti-IFN-γ (clone XMG1.2; BD Pharmingen). Tumor, stromal, and immune cells were distinguished by forward scatter and CD45 expression. Samples were analyzed using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences).

Detection of chemokines in tumors was performed by Western blot analysis. The entire tumor was excised and placed directly into a Dounce homogenizer containing lysis buffer (10 mM Tris (pH 8.0,) 0.5% Nonidet P-40, 250 mM NaCl, 10 mM sodium orthovanadate, 100 μM PMSF, 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 μg/ml aprotinin). Insoluble material was removed by centrifugation at 12,000 × g for 20 min. Total protein content was determined using a BCA Protein Assay kit (Pierce). Equivalent amounts of protein from each tumor sample were incubated with excess heparin-conjugated agarose beads (Sigma-Aldrich) to enrich for chemokines (19). Bound proteins were eluted from beads by boiling in Laemmeli’s sample buffer under reducing conditions. Proteins were separated on 15% SDS-PAGE gels and transferred to nitrocellulose membranes. Blots were probed with biotinylated polyclonal Abs to IP-10, MIG, and MIP-1α (R&D Systems). To determine the level of STAT1 phosphorylation in nonimmune cell populations, dissociated tumor suspensions were incubated with magnetic beads conjugated with an anti-CD45 Ab. Whole cell extracts were generated from the CD45+ depleted cell suspension after multiple rounds of incubation in lysis buffer and centrifugation. Equivalent amounts of protein from each tumor sample were separated on 8% SDS-PAGE gels and transferred to nitrocellulose membranes. Blots were probed with polyclonal Abs against pSTAT1 (Y701), total STAT1 (Santa Cruz Biotechnology), and β-actin.

Anti-IFN-γ Western blots were performed on whole tumor homogenate with no prior enrichment. To confirm IFN-γ levels in the serum, mice were bled via the tail vein 9 days postimplantation and serum was extracted. In both cases, equivalent total protein was loaded into each well, separated on 15% SDS-PAGE gels, and transferred to nitrocellulose membranes. Blots were then probed with biotinylated anti-IFN-γ (clone R4-6A2). Secondary Abs conjugated to HRP were added to blots and developed using ECL chemiluminescent substrate (Pharmacia).

TILs were purified from dissociated tumor suspensions using magnetic beads conjugated to anti-Thy-1 (clone T24/40.7) and used as effector cells after an overnight incubation in a standard 6-h chromium release assay. Target cells included B16/OVA cells that had been cultured in vitro in the presence or absence of recombinant mouse IFN-γ at 20 ng/ml for 96 h to maximally induce surface expression of MHC class I. In addition to isolation of TILs, the remaining tumor and stromal cells from the dissociated suspension were cultured overnight. Tumor cells were separated from the other adherent host cells by a gentle trypsinization. Enriched tumor cells were used as targets in a cytotoxicity assay with TILs to determine the lytic susceptibility of the cultured in vivo tumor cells.

IFN-γ production by IFN-γ−/− chimeric mouse splenocytes was evaluated using IFN-γ-specific ELISPOT assays. Multiscreen Immobilon-P plates (Millipore) were coated overnight at 4°C with 4 μg/ml rat anti-mouse IFN-γ (clone AN18.17.24). A total of 5 × 105 spleen cells was added to the top wells and diluted 2-fold down the column. Con A (5 μg/ml) (Sigma-Aldrich) was added to each well. After an 18-h culture, the plates were washed and IFN-γ was detected using biotinylated R4-6A2 Ab and spots were developed using an alkaline phosphatase substrate kit III (Vector Laboratories). Spots were enumerated using an ImmunoSpot plate reader (Cellular Technology).

One day before tumor implantation, mice were injected i.p. with either 0.1 mg of rat IgG (Jackson ImmunoResearch Laboratories) or rat anti-mouse IFN-γ (clone AN18) in 0.2 ml of HBSS. This treatment was repeated three times per week until the end of the experiment. Tumors were implanted and irradiated 7 days later as described above.

A nonparametric rank sum test was used for one-way ANOVA, followed by multiple comparison procedures (Kruskal-Wallis test or Tukey test). Statistical significance was assessed at p < 0.05 for all comparisons.

In our previous studies (17), we had performed initial experiments indicating that local tumor radiation could up-regulate VCAM-1 expression on tumor vessels. We have confirmed and extended this observation to include the involvement of the proinflammatory cytokine IFN-γ in this process. To examine how radiation affects VCAM-1 expression in the tumor microenvironment, we removed B16/OVA tumors initiated in C57BL/6 mice (WT) 7 days after 15 Gy of radiation was delivered to the tumor-bearing leg and performed whole mount histology using anti-CD31 (green) and anti-VCAM-1 (red). Tumors that were untreated did not contain vessels expressing VCAM-1 (Fig. 1,A), whereas vessels in irradiated tumors were found to express VCAM-1, even 7 days after treatment (Fig. 1,C). To determine the role of IFN-γ in this up-regulation, IFN-γ-deficient mice (IFN-γ−/−) were given the same inoculum of B16/OVA cells as WT mice and were either left untreated or treated with 15 Gy of radiation in the tumor-bearing leg 1 wk after tumor implantation. Whole mount histology for VCAM-1 was performed on tumors from IFN-γ−/− mice 7 days after treatment. As seen in WT mice, unirradiated tumors grown in IFN-γ−/− mice did not express VCAM-1 on vasculature (Fig. 1,B). In contrast to the irradiated tumors in WT mice, VCAM-1 expression was noticeably absent on vessels in irradiated tumors grown in IFN-γ−/− mice (Fig. 1 D). This marked difference in VCAM-1 expression between WT and IFN-γ−/− suggests an important role for this cytokine in the composition of the tumor microenvironment.

FIGURE 1.

VCAM expression on tumor vasculature is altered in the absence of IFN-γ. Whole mount staining of B16/OVA tumors treated with 0 Gy (A and B) or 15 Gy (C and D) of radiation from WT (A and C) or IFN-γ−/− (B and D) mice. Overlaid images on corresponding fields are pseudo-colored green for CD31 and orange for VCAM-1. Bar represents 50 μm in all images; representative data from three separate experiments.

FIGURE 1.

VCAM expression on tumor vasculature is altered in the absence of IFN-γ. Whole mount staining of B16/OVA tumors treated with 0 Gy (A and B) or 15 Gy (C and D) of radiation from WT (A and C) or IFN-γ−/− (B and D) mice. Overlaid images on corresponding fields are pseudo-colored green for CD31 and orange for VCAM-1. Bar represents 50 μm in all images; representative data from three separate experiments.

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T cells and NK cells are thought to be the significant producers of IFN-γ in the tumor microenvironment, but other cell types, including macrophages and fibroblasts, have been reported to produce this cytokine under certain circumstances (20, 21, 22). Due to the importance of IFN-γ in the induction of adhesion molecules on tumor vasculature, we next addressed whether hemopoietic cell-derived IFN-γ is primarily responsible for the activated phenotype observed on blood vessels. To generate a system in which IFN-γ is not produced by hemopoietic cells, we generated bone marrow chimeric mice in which IFN-γ expression is restricted to radioresistant host cells. Recipient WT mice were given a whole body dose of 10 Gy of irradiation and reconstituted with bone marrow from IFN-γ−/− mice (IFN-γ−/−→WT) and allowed to rest for a minimum of 8 wk. Reciprocal chimeric mice (WT→IFN-γ−/−) were also generated to determine whether hemopoietic cell-derived IFN-γ is sufficient to activate tumor vasculature in irradiated tumors in the absence of IFN-γ production from other host cells. Both sets of chimeric mice were implanted with B16/OVA cells and given 15 Gy of tumor irradiation on the same schedule for treatment and analysis as described earlier. Untreated tumors grown in either IFN-γ−/−→WT or WT→IFN-γ−/− mice did not contain vessels that up-regulated VCAM-1 expression (Fig. 2, A and B), once again revealing the requirement for an inflammatory insult such as localized radiation for activation of tumor vasculature. Irradiated tumors grown in mice whose IFN-γ production was restricted to donor hemopoietic cells (WT→IFN-γ−/−) had vessels that expressed VCAM-1 (Fig. 2,D), suggesting that IFN-γ production by only hemopoietic cells is adequate to activate vessels. However, in the reciprocal chimeras, we observed some vascular expression of VCAM-1 in mice whose donor hemopoietic-derived cells were incapable of producing IFN-γ (IFN-γ−/−→WT; Fig. 2,C). The extent of VCAM-1 expression on the vessels in each of the experimental groups was quantified by determining the percentage of the vessel area that was VCAM-1 positive (Fig. 3) and demonstrated a significant difference between the radiation-treated mice whose hemopoietic cells are incapable of producing IFN-γ compared with those in which only the hemopoietic cells can produce IFN-γ. To determine whether this low level of VCAM expression could be due to the remaining cells capable of making IFN-γ, for example, due to a low level of residual hemopoietic cells in the chimeras, we performed several additional assays. First, we confirmed that B16 cells do not themselves make IFN-γ since none was detectable by ELISA in the supernatant of B16 cells grown in vitro (data not shown). Second, spleen cells from the chimeric animals were stimulated with Con A and assayed for IFN-γ production by ELISPOT assay, which revealed the presence of low numbers of IFN-γ-producing cells. Finally, intracellular staining of CD45+ cells within the tumor also revealed that a small percentage of these cells (1%) were IFN-γ-producing cells. These data suggest that the low level of VCAM-expressing vessels in the IFN-γ−/−→WT mice was due to IFN-γ production by the remaining WT cells that were not completely eliminated in the chimeric mice and highlight the importance of even small amounts of IFN-γ.

FIGURE 2.

IFN-γ production by hemopoietic cells contributes to VCAM-1 expression on tumor vessels. Whole mount staining of B16/OVA tumors grown in IFN-γ−/−→WT (A and C) or WT→IFN-γ−/− (B and D) bone marrow chimeric mice treated with 0 Gy (A and B) or 15 Gy (C and D) of radiation. Overlaid images on corresponding fields are pseudo-colored green for CD31 and orange for VCAM-1. Bar represents 50 μm in all images. In the top right corner of each image is listed the donor into host chimeric model.

FIGURE 2.

IFN-γ production by hemopoietic cells contributes to VCAM-1 expression on tumor vessels. Whole mount staining of B16/OVA tumors grown in IFN-γ−/−→WT (A and C) or WT→IFN-γ−/− (B and D) bone marrow chimeric mice treated with 0 Gy (A and B) or 15 Gy (C and D) of radiation. Overlaid images on corresponding fields are pseudo-colored green for CD31 and orange for VCAM-1. Bar represents 50 μm in all images. In the top right corner of each image is listed the donor into host chimeric model.

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FIGURE 3.

Quantification of VCAM-1 expression on tumor vessels. Images such as those shown in Fig. 2 were analyzed using Image-Pro software to determine the area that was CD31+ (vessel area) and the area that was VCAM-1+. The percentage of CD31+ vessel area that was also VCAM-1+ was calculated. Each point represents separate images, which were obtained from multiple areas in multiple tumors. The horizontal bars represent the medians for each group. Using the Kruskal-Wallis test, the 15-Gy treated groups were statistically significant (p < 0.05).

FIGURE 3.

Quantification of VCAM-1 expression on tumor vessels. Images such as those shown in Fig. 2 were analyzed using Image-Pro software to determine the area that was CD31+ (vessel area) and the area that was VCAM-1+. The percentage of CD31+ vessel area that was also VCAM-1+ was calculated. Each point represents separate images, which were obtained from multiple areas in multiple tumors. The horizontal bars represent the medians for each group. Using the Kruskal-Wallis test, the 15-Gy treated groups were statistically significant (p < 0.05).

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Although T cells are found to infiltrate tumors in IFN-γ−/− mice, there is a general decrease in the overall density of these cells in untreated and irradiated tumors when compared with the infiltration observed in WT mice (data not shown). In addition to the lack of VCAM-1 expression on tumor vessels, diminished levels of T cell chemoattractants could also contribute to the deficiency in overall tumor infiltration. The IFN-γ-inducible chemokines MIG (CXCL9) and IP-10 (CXCL10) are known to be important T cell chemoattractants to sites of inflammation and in protective antitumor responses (19, 23). We sought to determine whether localized irradiation can affect the levels of these chemokines in the tumor and whether the presence of IFN-γ can influence this aspect of antitumor immunity. In addition, because B16/OVA cells produce MIG and IP-10 in vitro in response to rIFN-γ (Fig. 4,B), we generated a clone of B16/OVA that overexpressed a dominant negative mutant IFN-γ receptor chain (B16/OVA/DNM) (14) so as to determine the contribution of tumor cell-derived IFN-γ-induced MIG and IP-10 levels in vivo following localized irradiation. Chemokine levels were detected using Western blots of SDS-PAGE-separated whole tumor lysates that had been incubated with heparin-binding agarose beads to enrich for MIG and IP-10 (24). Equivalent amounts of protein from each tumor sample were used for the purification. B16/OVA tumors grown in WT mice contained MIG and IP-10, whose levels were slightly increased upon tumor irradiation (Fig. 4,A, lanes 1 and 2). As expected, tumors grown in IFN-γ−/− mice expressed low levels of these chemokines even when irradiated (Fig. 4,A, lanes 3 and 4). The low levels can be attributed to the low in vitro basal production of MIG and IP-10 by B16/OVA cells (see Fig. 4,B). B16/OVA/DNM tumors did not contain nearly as high levels of MIG and IP-10 as B16/OVA tumors despite their growth in WT mice that were capable of producing IFN-γ (Fig. 4,A, lanes 5 and 6). As expected, IFN-γ treatment does not affect the expression of these chemokines by the B16/OVA/DNM tumor cells growing in vitro (Fig. 4 B, lanes 3 and 4). These results suggest that the production of MIG and IP-10 by the tumor cells themselves in response to IFN-γ is responsible for the increase following irradiation. Interestingly, irradiation also enhanced the expression of MIP-1α in an IFN-γ-dependent fashion, although this chemokine has not been reported to be inducible by IFN-γ, and may represent an indirect effect of IFN-γ.

FIGURE 4.

IFN-γ signaling in irradiated tumors influences chemokine expression levels. A, Tumors were removed from mice that were untreated or irradiated. Whole tumor lysates were enriched for chemokines by incubating with heparin-conjugated agarose beads overnight at 4°C. Heparin-binding proteins were removed by boiling in sample buffer under reducing conditions and separated on a 15% SDS-PAGE gel. Blots were probed with mAbs to the listed proteins. Chemokine levels were assessed in at least three tumors from each group; a representative blot from one experiment is shown. B, Lysates from tumor cells grown in vitro and treated or not with IFN-γ were prepared and analyzed as described above.

FIGURE 4.

IFN-γ signaling in irradiated tumors influences chemokine expression levels. A, Tumors were removed from mice that were untreated or irradiated. Whole tumor lysates were enriched for chemokines by incubating with heparin-conjugated agarose beads overnight at 4°C. Heparin-binding proteins were removed by boiling in sample buffer under reducing conditions and separated on a 15% SDS-PAGE gel. Blots were probed with mAbs to the listed proteins. Chemokine levels were assessed in at least three tumors from each group; a representative blot from one experiment is shown. B, Lysates from tumor cells grown in vitro and treated or not with IFN-γ were prepared and analyzed as described above.

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Having determined that the presence of IFN-γ in the tumor microenvironment alters the expression of adhesion molecules on vasculature and chemokines, correlating with an increase in the number of infiltrating T cells (17), we next examined what role this cytokine played in MHC class I expression on tumor cells following localized radiation. In order for CTL to effectively control tumor growth, they require tumor cell expression of MHC class I. Exposure to rIFN-γ and ionizing radiation in vitro is known to increase surface expression of class I on tumor cells (25), thereby potentially increasing their recognition by CTL. We demonstrated that IFN-γ treatment of B16/OVA cells in vitro markedly up-regulated their expression of H-2Kb molecules, but, as expected, had no effect on B16/OVA/DNM (Fig. 5, A–D).

FIGURE 5.

Irradiation-induced up-regulation of H-2Kb is partially mediated by IFN-γ. In vitro grown B16/OVA (A and C) or B16/OVA/DNM (B and D) cells were untreated (A and B) or treated with IFN-γ (C and D) and stained with Abs against H-2Kb (filled gray lines) and isotype-matched Ab (open white lines). E, Bars represent the mean fluorescence intensity (±SE) of three experiments in which single-cell suspensions of B16/OVA and B16/OVA/DNM tumors treated with 0 Gy (▪) or 15 Gy (□) of radiation from WT or IFN-γ−/− mice were stained with Abs to CD45 and H-2Kb. Shown are the mean fluorescence intensities (MFI) of the CD45 (tumor) cells. Statistical analysis was done using the Tukey multiple comparison test.

FIGURE 5.

Irradiation-induced up-regulation of H-2Kb is partially mediated by IFN-γ. In vitro grown B16/OVA (A and C) or B16/OVA/DNM (B and D) cells were untreated (A and B) or treated with IFN-γ (C and D) and stained with Abs against H-2Kb (filled gray lines) and isotype-matched Ab (open white lines). E, Bars represent the mean fluorescence intensity (±SE) of three experiments in which single-cell suspensions of B16/OVA and B16/OVA/DNM tumors treated with 0 Gy (▪) or 15 Gy (□) of radiation from WT or IFN-γ−/− mice were stained with Abs to CD45 and H-2Kb. Shown are the mean fluorescence intensities (MFI) of the CD45 (tumor) cells. Statistical analysis was done using the Tukey multiple comparison test.

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To determine whether there was a similar effect in vivo, tumors grown in WT and IFN-γ−/− mice were removed and flow cytometry for class I expression levels on tumor cells was performed using anti-H-2Kb. B16/OVA tumors initiated in IFN-γ−/− mice and B16/OVA/DNM tumor cells implanted in WT mice were used to test the hypothesis that IFN-γ would mediate the up-regulation of class I in tumors following radiation treatment in vivo. Untreated tumors expressed similar levels of H-2Kb under all conditions, even when grown in IFN-γ−/− mice (Fig. 5,E, filled bars). Following irradiation of B16/OVA tumors grown in WT mice, a marked increase in H-2Kb was observed (Fig. 5,E). Up-regulation of MHC class I is likely a result of IFN-γ signaling upon the tumor cells since it was not evidenced to a similar degree in irradiated tumors in IFN-γ−/− mice or B16/OVA/DNM tumors (Fig. 5 E). The presence of IFN-γ signaling in the tumor microenvironment is therefore important in generating a tumor cell phenotype that can be recognized by CTL.

Since the levels of H-2Kb on tumor cells in vivo were altered based on whether they were exposed to radiation and could respond to IFN-γ, we next assessed whether these levels would affect the ability of the cells to serve as targets for CTL in an in vitro chromium release assay (Fig. 6). The source of CTL in this assay was TILs isolated from dissociated tumor suspensions using magnetic beads conjugated to an anti-Thy-1 mAb (26). After the isolation of the TILs, the tumor and remaining host cells were also cultured. Only the adherent cell population, containing primarily tumor cells, was labeled with 51Cr for use as targets and then subsequently cultured with the TILs isolated from the corresponding tumor in the cytotoxicity assay. In addition to the ex vivo tumor cells as targets, TILs were also incubated with in vitro B16/OVA cells that were exposed to rIFN-γ to maximally induce H-2Kb levels and served as the positive control in determining the lytic activity of TILs. These experiments were repeated three times and the results from each are plotted on a single graph demonstrating the reproducibility of these data. Low class I-expressing in vitro B16/OVA targets were not efficiently lysed by TILs isolated from any of the tumors, revealing the importance of class I on tumor cells in eliciting the lytic activity of CTL. TILs isolated from irradiated tumors (Fig. 6, B, D, and F) had slightly greater lytic activity against class Ihigh in vitro targets compared with TILs isolated from untreated tumors (Fig. 6, A, C, and E). IFN-γ−/− TILs were able to lyse targets (Fig. 6, C and D), albeit at a lower level compared with TILs from WT mice, suggesting that lack of this cytokine did not completely abrogate CTL function. Ex vivo tumor cells expressed lower levels of class I compared with the in vitro cells exposed to rIFN-γ and thus were less susceptible to lysis by the TILs isolated from those tumors. The increased level of class I on irradiated B16/OVA tumors in WT mice (Fig. 5,E) resulted in a greater lytic susceptibility of those cells to TILs (Fig. 6,B) compared with unirradiated ex vivo tumor cells grown in WT mice (Fig. 6,A). Ex vivo tumor cell targets from untreated and irradiated IFN-γ−/− mice (Fig. 6, C and D) displayed lower lytic susceptibility compared with targets from WT mice (Fig. 6, A and B), demonstrating that the presence of IFN-γ in the tumor microenvironment affects the ability of CTL to lyse those tumor cells, which correlates with their levels of class I expression. Responsiveness of the tumor cells to IFN-γ is important in regulating their recognition and lysis by CTL, as evidenced by the similarity in lysis between untreated and irradiated ex vivo B16/OVA/DNM tumor cells isolated from WT mice (Fig. 6, E and F).

FIGURE 6.

Lytic activity of TILs from nonirradiated and irradiated tumors. TILs were isolated from B16/OVA (A–D) and B16/OVA/DNM (E and F) tumors treated with 0 Gy (A, C, and E) or 15 Gy (B, D, and F) of radiation from WT (A, B, E, and F) or IFN-γ−/− (C and D) mice. Pooled TILs were incubated in a 6-h 51Cr release assay with the following targets: ♦, B16/OVA + rIFN-γ (class Ihigh); ▴, B16/OVA (class Ilow); •, the ex vivo tumor cells from which the TILs were isolated. Each line represents the results from an individual experiment.

FIGURE 6.

Lytic activity of TILs from nonirradiated and irradiated tumors. TILs were isolated from B16/OVA (A–D) and B16/OVA/DNM (E and F) tumors treated with 0 Gy (A, C, and E) or 15 Gy (B, D, and F) of radiation from WT (A, B, E, and F) or IFN-γ−/− (C and D) mice. Pooled TILs were incubated in a 6-h 51Cr release assay with the following targets: ♦, B16/OVA + rIFN-γ (class Ihigh); ▴, B16/OVA (class Ilow); •, the ex vivo tumor cells from which the TILs were isolated. Each line represents the results from an individual experiment.

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To determine the effect of IFN-γ on tumor growth, groups of mice were either depleted of IFN-γ by regular injections with the anti-IFN-γ Ab, (clone AN18) or injected with rat IgG as a control. These mice were then challenged with 105 B16/OVA, the tumor-bearing leg was irradiated 7 days later with 15 Gy, and tumor growth was monitored. As can be seen in Fig. 7, the tumors in the IFN-γ-depleted mice grew with significantly faster kinetics than the control mice. In addition, serum from randomly selected control and treated mice was analyzed for the presence of IFN-γ by Western blot, which verified that the IFN-γ levels were markedly reduced in the treated animals (data not shown).

FIGURE 7.

Depletion of IFN-γ slows postirradiation tumor growth in vivo. Groups of five mice each were given rat IgG as a control (solid lines) or anti-IFN-γ (dotted lines) and tumors were implanted in the flanks of mice as described in Materials and Methods, and the leg diameters were measured on a regular basis. Mice were then treated with a single dose of 15 Gy on day 7 after tumor implantation. Statistical analysis comparing tumor growth on days 8–22 was done using the Mann-Whitney U test (p < 0.002).

FIGURE 7.

Depletion of IFN-γ slows postirradiation tumor growth in vivo. Groups of five mice each were given rat IgG as a control (solid lines) or anti-IFN-γ (dotted lines) and tumors were implanted in the flanks of mice as described in Materials and Methods, and the leg diameters were measured on a regular basis. Mice were then treated with a single dose of 15 Gy on day 7 after tumor implantation. Statistical analysis comparing tumor growth on days 8–22 was done using the Mann-Whitney U test (p < 0.002).

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We next investigated the signaling mechanism that might account for the difference in MHC class I expression and the subsequent lytic susceptibility of the tumor cells in vivo. STAT1 is a key transcription factor in IFN-γ signaling processes and is known to regulate expression of class I and caspases in responsive cells (27, 28). Because STAT1 controls the processes within cells that render them susceptible to CTL recognition and lysis, we asked whether differences in active STAT1 could account for the variation in class I expression and lytic susceptibility in the tumor cells following localized radiation. Western blots for phosphorylated STAT1 (pSTAT1) were performed on lysates obtained by dissociating the tumor into a single-cell suspension and removing the immune cells using magnetic beads conjugated to an anti-CD45 mAb. Whole cell lysates were prepared from the remaining tumor and stromal cells and then separated on an SDS-PAGE gel (Fig. 8). Additionally, lysates of the entire tumor were also generated to assess levels of IFN-γ within the tumor microenvironment, as it is known to induce STAT1 activation (29). Irradiated B16/OVA tumors in WT mice had greater levels of pSTAT1 (Fig. 8, lane 2) compared with untreated tumors (lane 1), which may account for the disparity in class I expression and lytic susceptibility of these tumors. Despite the absence of IFN-γ, untreated and irradiated B16/OVA tumors were found to have some low level of pSTAT1 when grown in IFN-γ−/− mice (Fig. 8, lanes 3 and 4). An increase in other cytokines within irradiated tumors grown in IFN-γ−/− mice could explain the slight increase in pSTAT1 compared with untreated tumors grown in the same mice. B16/OVA/DNM tumor cells do not activate STAT1 in response to rIFN-γ in vitro (data not shown), but are nonetheless found to have pSTAT1 in vivo whether untreated or irradiated (Fig. 8, lanes 5 and 6). The effects of IFN-γ on stromal cells may contribute to the levels of pSTAT1 in these tumors. Tumors that could not generate an IFN-γ signal, either due to the absence of the cytokine or a defective receptor, had a lower level of pSTAT1 compared with tumors that were capable of responding to IFN-γ in the microenvironment.

FIGURE 8.

Deficient IFN-γ signaling limits STAT1 phosphorylation within irradiated tumors. Western blot analysis of pSTAT1 and total STAT1 in B16/OVA (lanes 1–4) and B16/OVA/DNM tumors (lanes 5 and 6) grown in WT (lanes 1, 2, 5, and 6) or IFN-γ−/− (lanes 3 and 4) mice that received 0 Gy (lanes 1, 3, and 5) or 15 Gy (lanes 2, 4, and 6) of radiation. Whole cell lysates of tumor after removal of the CD45+ population were run on an 8% SDS-PAGE gel and immunoblotted for pSTAT1 (Y701). Lysates of the entire tumor were separated on a 15% SDS-PAGE gel and immunoblotted for IFN-γ. β-Actin was used as a loading control. A representative blot from one of three experiments is shown.

FIGURE 8.

Deficient IFN-γ signaling limits STAT1 phosphorylation within irradiated tumors. Western blot analysis of pSTAT1 and total STAT1 in B16/OVA (lanes 1–4) and B16/OVA/DNM tumors (lanes 5 and 6) grown in WT (lanes 1, 2, 5, and 6) or IFN-γ−/− (lanes 3 and 4) mice that received 0 Gy (lanes 1, 3, and 5) or 15 Gy (lanes 2, 4, and 6) of radiation. Whole cell lysates of tumor after removal of the CD45+ population were run on an 8% SDS-PAGE gel and immunoblotted for pSTAT1 (Y701). Lysates of the entire tumor were separated on a 15% SDS-PAGE gel and immunoblotted for IFN-γ. β-Actin was used as a loading control. A representative blot from one of three experiments is shown.

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Tumor Ags are capable of eliciting specific T cell activation in draining lymph nodes but the homing and retention of these effector cells into the tumor is very limited. This mechanism of tumor evasion is often attributed to the physiology of the tumor microenvironment, which can restrict both the infiltration and activation of CTL (30). Lack of associated inflammation and the presence of an immunosuppressive network within the tumor are two important barriers to effective and long-term immune control of tumor growth (3). Combining radiotherapy with immunotherapy may offer a means to overcome the impairment of T cell infiltration into the tumor (5). Localized irradiation of the tumor results in alterations to the microenvironment that are beneficial for immune function by generating inflammation and altering the balance between stimulatory and suppressive cytokines (3). Although much is known about the tumor microenvironment affecting responses to radiation, such as hypoxia, (31), there is much less information on the resulting changes to the tumor microenvironment following irradiation and with respect to its impact on endogenous immune responses and immunotherapies.

In this study, we have evaluated the role of IFN-γ produced following localized irradiation in conditioning the tumor microenvironment for effective T cell infiltration. Extravasation of effector T cells into tumors from the vasculature is the initial step in immune-mediated antitumor responses and is thought to depend on the presence of an activated vessel phenotype (32). The expression of adhesion molecules on vasculature is a critical component of lymphocyte trafficking into tissues (33, 34). We have observed a dependence on inflammatory cytokine IFN-γ for the up-regulation of VCAM-1 on vasculature in irradiated tumors. The deficient VCAM-1 expression 7 days after tumor irradiation in the IFN-γ−/− mice may also involve IL-17. In a recent study by Schnyder et al. (35), IL-17 was shown to reduce the levels of VCAM-1 on a variety of in vitro cytokine-activated human cell lines. Because IL-17 levels are slightly elevated in IFN-γ−/− mice, this provides a rational explanation for the altered VCAM-1 expression that we have observed in the deficient mice (36). Because many tumors are not permissive to T cell infiltration, treatments that activate the vasculature through increases in IFN-γ production may prove beneficial for T cell trafficking and retention within the tumor.

The multistep process of lymphocyte trafficking involves not only adhesion molecules, but also chemokines that serve to attract effector T cells to the tumor site (37). Most tumors have a complex chemokine network which can influence immune responses and the composition of lymphocyte infiltration (38). Our data would suggest that localized irradiation benefits the antitumor immune response through up-regulation of inflammatory chemokine levels. IFN-γ-inducible chemokines, MIG and IP-10, in all irradiated tumors were found at slightly increased levels over untreated tumors. These two chemokines, along with the increased expression of MIP-1α, serve to attract T cells that express the receptors CXCR3 and CCR5 into inflamed tissues, including the irradiated tumors in our experiments (39). The tumor cells themselves appear to contribute to the expression of both MIG and IP-10, as evidenced by the decrease in both chemokine levels in tumors that were incapable of responding to IFN-γ. Production of MIG and IP-10 appears to be a common feature in many tumor cells, as we and others have reported this trait in other tumor lines in vitro (14, 24). In addition to recruiting IFN-γ-producing T cells, MIG and IP-10 also have antiangiogenic properties (40). Interestingly, these two properties of the IFN-γ-inducible chemokines do not appear advantageous for the tumor to evade immune responses and sustain angiogenesis for the growing mass of cells. The significance of tumor cell production of these chemokines warrants additional study. Whatever their cellular origin, accumulation of MIG and IP-10 within the tumor microenvironment may contribute to successful treatment outcomes through two distinct mechanisms, the recruitment of activated CTL and the inhibition of angiogenesis (41, 42). Production of these chemokines by immune cells alone may not be sufficient for tumor control. However, therapies that seek to enhance the expression of MIG and IP-10 by the tumor cells themselves may prove to be important for control of tumor growth (43).

In addition to its role as a proinflammatory cytokine, IFN-γ is capable of inducing MHC class I expression on a variety of cell types, including most tumor cells (28). The result of MHC up-regulation on the surface of the tumor cells is recognition by CTL. Irradiation of the tumor mass resulted in increased MHC class I expression on the tumor cells and this correlated with their lytic susceptibility to the TILs that infiltrated the tumors. IFN-γ responsiveness was found to be critically important in order for the tumor cells to up-regulate MHC class I. Other proinflammatory cytokines, such as IFN-α and TNF-α, have been shown to mediate a similar response in tumor cell expression of MHC class I (19, 44); however, IFN-γ is the most likely cytokine to induce these effects in our model as evidenced by the similarities in H-2Kb expression between the untreated and irradiated B16/OVA/DNM tumors (Fig. 5). Although other proinflammatory cytokines are capable of up-regulating MHC class I expression, they do not induce an increase in Ag-processing components quite to the extent as IFN-γ (45, 46). These molecules play an important role in MHC class I presentation and recognition of the tumor cells by the CTL. Treatment options that increase IFN-γ in the tumor microenvironment may therefore be important when CTL immunotherapies are also involved to generate tumor cells that can be recognized and lysed.

In all of the IFN-γ-dependent processes we have examined, intracellular signaling via STAT1 appears to regulate the resulting response to the cytokine (47). Especially important for CTL-mediated control of tumor cells is the expression of MHC class I on the surface of the target. Although many tumor cells express low levels of MHC class I, these are often insufficient for CTL recognition and therefore induced higher expression is necessary to elicit lytic responses by effector cells. IFN-γ is capable of up-regulating not only MHC class I and its associated Ag presentation components, but also caspases that are involved in apoptotic signaling (11, 48, 49). In addition to its role in promoting apoptosis in cells, STAT1 has also been shown to inhibit production of proangiogenic molecules by tumor cells (50). The wide ranging effects of STAT1 activation in tumors suggests that this process can be targeted to potentiate antitumor therapies by negatively influencing processes that promote tumor growth (i.e., angiogenesis and metastasis) and positively directing processes that enhance immune responses (i.e., up-regulation of MHC and caspases) (51, 52). Tumor cells that do not respond to IFN-γ have been found to lack STAT1 activation (53). We have observed an increase in STAT1 activation in irradiated tumors that is partially mediated by IFN-γ. There was a correlation between diminished STAT1 activation and decreased lytic susceptibility to CTL. Immunotherapies might therefore benefit when combined with other treatment modalities that increase STAT1 activation in tumor cells. However, increases in STAT1 are associated with a radioresistant tumor cell phenotype (54). Therefore, it will be necessary to maintain the sensitivity of the tumor cell to immune destruction without affecting the inherent cellular radiosensitivity. Further examination into the role of STAT1 in tumor cells for successful combination treatment is warranted.

In summary, we have examined the mechanism by which IFN-γ promotes effective T cell function within irradiated tumors. Processes that are important for CTL trafficking and recognition of tumor cells were found to be dependent on the presence of the cytokine in the microenvironment and the ability of the tumor cell to respond to IFN-γ. Although untreated tumors contain IFN-γ in the microenvironment, it is at an insufficient level to induce VCAM-1, MHC class I, and chemokine up-regulation. Localized irradiation of the tumor results in an increase in IFN-γ production that alleviates the low levels of these key molecules required for CTL responses. Irradiation also improved the lytic sensitivity of tumor cells to CTL when the targets were capable of responding to IFN-γ in the microenvironment through increases in apoptosis-inducing STAT1 activation. The importance of this cytokine in overall tumor growth was demonstrated by depleting animals of IFN-γ, which resulted in enhanced tumor growth kinetics after irradiation treatment. Therefore, treatment options such as radiotherapy that induce inflammation and accumulation of IFN-γ in the tumor can be successfully combined with immunotherapies due to their ability to condition the microenvironment for T cell infiltration and tumor cell recognition.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health Grant CA28332 and by a grant from the Sally Edelman and Harry Gardner Cancer Research Foundation. A.A.L., S.A.G., and J.P.M. were supported by the National Institutes of Health Training Grant AI07285.

3

Abbreviations used in this paper: IP-10, IFN-γ-inducible protein 10; WT, wild type; TIL, tumor-infiltrating lymphocyte.

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