Demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice

Abstract

This study demonstrates that endogenously produced interferon gamma (IFN-gamma) forms the basis of a tumor surveillance system that controls development of both chemically induced and spontaneously arising tumors in mice. Compared with wild-type mice, mice lacking sensitivity to either IFN-gamma (i.e., IFN-gamma receptor-deficient mice) or all IFN family members (i.e., Stat1-deficient mice) developed tumors more rapidly and with greater frequency when challenged with different doses of the chemical carcinogen methylcholanthrene. In addition, IFN-gamma-insensitive mice developed tumors more rapidly than wild-type mice when bred onto a background deficient in the p53 tumor-suppressor gene. IFN-gamma-insensitive p53(-/-) mice also developed a broader spectrum of tumors compared with mice lacking p53 alone. Using tumor cells derived from methylcholanthrene-treated IFN-gamma-insensitive mice, we found IFN-gamma's actions to be mediated at least partly through its direct effects on the tumor cell leading to enhanced tumor cell immunogenicity. The importance and generality of this system is evidenced by the finding that certain types of human tumors become selectively unresponsive to IFN-gamma. Thus, IFN-gamma forms the basis of an extrinsic tumor-suppressor mechanism in immunocompetent hosts.

Figure 1

IFN-γ-insensitive mice demonstrate an increased susceptibility to development of spontaneous and chemically induced tumors. (A) Groups of 15–20 129/Sv/Ev wild-type mice (▴) and syngeneic IFN-γR^−/− mice (•) were injected with a single dose of methylcholanthrene, and tumor development was quantitated for 130 days. (B) Groups of wild-type, IFN-γR^−/−, and Stat1^−/− mice were injected with MCA, and tumor development was monitored for 165 days. Values represent the composite of four independent experiments. (C) Spontaneous tumor development in IFN-γR^−/− × p53^−/− (•) and 129/Sv/Ev × p53^−/− mice (▴). The difference in average tumor development times between 129/Sv/Ev × p53^−/− (18.5 weeks) and IFN-γR^−/− × p53^−/− (13.7 weeks) is statistically significant by the Wilcoxon rank sum test (P = 0.001).

Figure 2

RAD-gR tumors demonstrate equivalent growth kinetics in IFN-γR^−/− and 129/Sv/Ev mice. Three representative IFN-γR^−/− tumor lines were injected subcutaneously into naive IFN-γR^−/− (•) and 129/Sv/Ev (▴) mice at the indicated doses. Tumor growth kinetics were monitored by measuring the diameter of the tumor masses and are represented as an average ± SE of four to five mice per group.

Figure 3

Growth of reconstituted and unreconstituted RAD-gR.28 cells in wild-type and immunodeficient mice. (A) Rejection of reconstituted RAD-gR.28.mgR in 129/Sv/Ev mice. IFN-γ-insensitive RAD-gR.neo (▴) or RAD-gR.mgRΔIC (•) tumor cells and IFN-γ-sensitive RAD-gR.28.mgR (▪) cells were injected subcutaneously at a dose of 10⁶ cells per animal into 129/Sv/Ev mice. RAD-gR.28.mgR cells were also injected subcutaneously into 129/Sv/Ev mice that had been pretreated on days −1, +2, and +5 with i.p. injections of 250 μg of a neutralizing mAb to murine IFN-γ (⧫) or saline (data not shown). (B) RAD-gR.28.mgR is resistant to the antiproliferative actions of IFN-γ. Triplicate cultures of RAD-gR.28.neo and RAD-gR.28.mgR cells were incubated for 24 hr with different combinations of IFN-γ (5,000 units/ml), IFN-α (1,000 units/ml), TNFα (10 ng/ml), IL-1 (10 ng/ml), and IL-12 (50 units/ml), and proliferation was assessed by monitoring ³H-labeled thymidine incorporation. Values are expressed as the percentage of incorporation compared with untreated cells. (C) RAG1^−/− mice cannot reject reconstituted, IFN-γ-sensitive RAD-gR.28.mgR cells. RAD-gR.28.mgR cells were injected subcutaneously at a dose of 10⁶ cells per mouse into either 129/Sv/Ev (▴) or RAG1^−/− (▪) mice. Values represent the average ± SE tumor diameter of four to five mice per group.

Figure 4

IFN-γ responsiveness can be restored to SK-LC-7 by enforced expression of the IFN-γR α chain and to SK-LC-2 and SK-LC-19 by expression of Jak2. The ability of cells to enhance expression of MHC class I proteins is measured after 72 hr of incubation with 1,000 IRU/ml of either HuIFN-γ (Left) or HuIFN-α_A/D (Right) (thick lines) or buffer (thin lines). (A) SK-LC-7 cells transfected with expression plasmids encoding either the HuIFN-γR α chain (Upper) or HuIFN-γR β chain (Lower). (B) SK-LC-2 cells transfected with plasmids encoding either Jak1 (Upper) or Jak2 (Lower). (C) SK-LC-19 cells transfected with plasmids encoding Jak1 (Upper) or Jak2 (Lower).