Research Article Splenectomy Promotes Indirect Elimination of Intraocular Tumors by CD8 þ T Cells That Is Associated with IFNg- and Fas/FasL-Dependent Activation of Intratumoral Macrophages Maxine R. Miller 1 , Jonathan B. Mandell 1 , Kelly M. Beatty 1 , Stephen A.K. Harvey 1 , Michael J. Rizzo 1,2 , Dana M. Previte 1,2 , Stephen H. Thorne 3,4,5 , and Kyle C. McKenna 1,3,5 Abstract Ocular immune privilege (IP) limits the immune surveillance of intraocular tumors as certain immuno- genic 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 þ T cells, IFNg , and FasL, but not perforin, or TNFa were required for the elimination of intraocular E.G7-OVA tumors that culminated in destruction of the eye (ocular phthisis). IFNg and FasL did not target tumor cells directly as the majority of SPLNX IFNg R1 / mice and Fas-defective lpr mice failed to eliminate intraocular E.G7-OVA tumors that expressed Fas and IFNg R1. Bone marrow chimeras revealed that IFNg R1 and Fas expression on immune cells was most critical for rejection, and SPLNX increased the frequency of activated macrophages (Mf) within intraocular tumors in an IFNg - and Fas/FasL-dependent manner, suggesting an immune cell target of IFNg and Fas. As depletion of Mfs limited CD8 T cell–mediated rejection of intraocular tumors in SPLNX mice, our data support a model in which IFNg - and Fas/FasL-dependent activation of intratumoral Mfs by CD8 þ 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 þ T cells and Mfs to limit the immunosurveillance of intraocular tumors. Cancer Immunol Res; 2(12); 1175–85. Ó2014 AACR. Introduction Ocular "immune privilege" (IP) is exemplified by the obser- vations that certain immunogenic tumor cell lines (P815 and E.G7-OVA), which are rejected by host immune responses when transplanted in the skin, grow progressively when placed into the anterior chamber (a.c.) of the eye (1). IP is not immune ignorance, as several studies have shown that intraocular tumor growth primes systemic immune responses to tumor antigens (2–4). Rather, ocular immune responses are very tightly regulated to limit inflammation during pathogen removal so that certain intraocular tissues, which do not regenerate and are essential for vision, are not damaged (5, 6). Ocular IP is maintained by anatomic and biochemical barriers to host immune responses along with the generation of systemic tolerance to antigens encountered within the eye (reviewed in ref. 7). Splenectomy (SPLNX) terminates ocular IP and promotes rejection of immunogenic tumors transplanted in the a.c. of the eye (8). However, the mechanism of tumor elimination has not been defined. Herein, we characterize the requirements for elimination of intraocular tumors in SPLNX mice as a first step toward identifying how to overcome IP and restore tumoricidal activity within intraocular tumors. We demonstrate that CD8 þ T cells, macrophages (Mf), IFNg , and FasL, but not perforin, or TNFa were necessary for intraocular tumor elimination. Although tumors expressed IFNg R1 and Fas, IFNg and FasL did not directly target tumors. Rather, IFNg and Fas/FasL interactions were required for intratumoral Mf activation that was associated with severe ocular inflamma- tion, which indirectly eliminated intraocular tumors by induc- ing complete destruction of the eye (ocular phthisis). Our data suggest that immunosuppressive mechanisms that preserve ocular IP interfere with the complex interplay between CD8 þ 1 Department of Ophthalmology, University of Pittsburgh, Pittsburgh, Penn- sylvania. 2 Graduate Program in Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania. 3 Department of Immunology, University of Pitts- burgh, Pittsburgh, Pennsylvania. 4 Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania. 5 University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania. Note: Supplementary data for this article are available at Cancer Immu- nology Research Online (http://cancerimmunolres.aacrjournals.org/). Corresponding Author: Kyle C. McKenna, Department of Biology, Fran- ciscan University of Steubenville, Sts. Cosmas and Damian Hall, 1235 University Boulevard, Steubenville, OH 43952. Phone: 740-283-6765; Fax: 740-283-6363; E-mail: kmckenna@franciscan.edu doi: 10.1158/2326-6066.CIR-14-0093-T Ó2014 American Association for Cancer Research. Cancer Immunology Research www.aacrjournals.org 1175 on December 8, 2021. © 2014 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from Published OnlineFirst September 23, 2014; DOI: 10.1158/2326-6066.CIR-14-0093-T