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Supplementary Figure S3

Multiple studies have associated the transcription factor IRF1 with tumor-suppressive activities. Here, we report an opposite tumor cell–intrinsic function of IRF1 in promoting tumor growth. IRF1-deficient tumor cells showed reduced tumor growth in MC38 and CT26 colon carcinoma and B16 melanoma mouse models. This reduction in tumor growth was dependent on host CD8+ T cells. Detailed profiling of tumor-infiltrating leukocytes did not show changes in the various T-cell and myeloid cell populations. However, CD8+ T cells that had infiltrated IRF1-deficieint tumors in vivo exhibited enhanced cytotoxicity. IRF1-deficient tumor cells lost the ability to upregulate PD-L1 expression in vitro and in vivo and were more susceptible to T-cell–mediated killing. Induced expression of PD-L1 in IRF1-deficient tumor cells restored tumor growth. These results indicate differential activity of IRF1 in tumor escape.

Supplementary Figure S2

Supplementary Figure S1

Supplementary Figure legends

Mouse Strains from Jackson Laboratories used in experimentation.

Ingenuity Canonical Pathways.

P815 tumor cell line authentication.

IFNγ resistance in E.G7-OVA and P815 tumors.

Ocular immune privilege (IP) limits the immune surveillance of intraocular tumors as certain immunogenic tumor cell lines (P815, E.G7-OVA) that are rejected when transplanted in the skin grow progressively when placed in the anterior chamber of the eye. As splenectomy (SPLNX) is known to terminate ocular IP, we characterized the immune mechanisms responsible for rejection of intraocular tumors in SPLNX mice as a first step toward identifying how to restore tumoricidal activity within the eye. CD8+ T cells, IFNγ, and FasL, but not perforin, or TNFα were required for the elimination of intraocular E.G7-OVA tumors that culminated in destruction of the eye (ocular phthisis). IFNγ and FasL did not target tumor cells directly as the majority of SPLNX IFNγR1−/− mice and Fas-defective lpr mice failed to eliminate intraocular E.G7-OVA tumors that expressed Fas and IFNγR1. Bone marrow chimeras revealed that IFNγR1 and Fas expression on immune cells was most critical for rejection, and SPLNX increased the frequency of activated macrophages (Mφ) within intraocular tumors in an IFNγ- and Fas/FasL-dependent manner, suggesting an immune cell target of IFNγ and Fas. As depletion of Mφs limited CD8 T cell–mediated rejection of intraocular tumors in SPLNX mice, our data support a model in which IFNγ- and Fas/FasL-dependent activation of intratumoral Mφs by CD8+ T cells promotes severe intraocular inflammation that indirectly eliminates intraocular tumors by inducing phthisis, and suggests that immunosuppressive mechanisms that maintain ocular IP interfere with the interaction between CD8+ T cells and Mφs to limit the immunosurveillance of intraocular tumors. Cancer Immunol Res; 2(12); 1175–85. ©2014 AACR.

IFNγR1 and Fas/FasL interactions are required for rejection of intraocular P815 tumors in splenectomized mice.

Characterization of myeloid cells in Luc-E.G7 tumor bearing eyes.