Immune Rejection of Metastases Arising From Intraocular Tumors in Mice

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1 Immune Rejection of Metastases Arising From Intraocular Tumors in Mice Jerry Y. Niederkorn,* Terrence L. Knisely.t and Elizabeth Moyhew* The role of the immune response in the elimination of spontaneous metastases arising from intraocular tumors was examined in a syngeneic intraocular murine tumor model. P91 mastocytoma (DBA/ 2 origin) expresses strong tumor-specific transplantation antigens and grows transiently in the eyes of syngeneic hosts before undergoing spontaneous rejection. An organ culture technique was used to detect spontaneous metastases in the lungs, spleens, brains, and thymuses of intraocular tumor-bearing mice. Metastatic tumor cells were detected in all organs of immunodeficient mice (i.e., athymic, nude, or x-irradiated DBA/2 mice) within 14 days of intraocular transplantation, and grew progressively thereafter. By contrast, metastatic tumors were rejected in 100% of the immunocompetent DBA/2 mice examined on day 15. Timed enucleation experiments demonstrated that the immune rejection of disseminated tumor cells occurred within hr of their arrival at the various organs. The immune rejection of spontaneous metastases could be adoptively transferred to immunodeficient tumor-bearing mice using spleen cells, but not immune serum, from intraocular tumor-bearing immunocompetent donors. Selective cell depletion experiments revealed that the immune spleen cell effecting immunity was an Lyt 1+, 2+ T cell. The results indicate that the immune rejection of the spontaneous metastases arising from primary intraocular tumors is a T cell-dependent, radiosensitive process that rapidly eliminates metastases within the lungs, brain, thymus, and the spleen of the immunocompetent host. Invest Ophthalmol Vis Sci 27: , 1986 Despite advances in surgical techniques and the development of new therapeutic modalities for treating primary neoplasms, most deaths in cancer patients are due to metastases.' Even early detection and successful management of the primary neoplasm are often insufficient to protect the host from metastatic disease. Enucleation is a time-honored method for treating uveal melanoma, yet 30-50% of the patients treated by this procedure die with liver metastases within 5 yr following surgery. 2 Although more recent studies report a lower mortality rate (i.e., 16%) for patients subjected to enucleation, metastatic melanoma remains to be the leading cause of death in individuals diagnosed with intraocular melanoma. 3 Recent animal studies have suggested that the immune system may be important in the control of metastases arising from intraocular melanomas 4 and From the *Department of Ophthalmology and fgraduate Studies Program in Immunology, University of Texas Health Science Center at Dallas, Dallas, Texas. Supported in part by NIH grants CA30276 and CA09082, an unrestricted grant from the Research to Prevent Blindness, Inc., NY, and a grant from the Bruton Trust Foundation. Dr. Niederkorn is a Research to Prevent Blindness-Olga Keith Wiess Scholar. Submitted for publication: August 19, Reprint requests: Dr. Jerry Y. Niederkorn, Department of Ophthalmology, University of Texas. Health Science Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX mastocytomas. 5 ' 6 Interest in employing immunotherapy for eradicating metastases has been kindled by promising findings using biologic response modifiers, such as recombinant interleukin 2 (IL-2), 7 interferon inducers, 8 and lymphokine activated killer (LAK) cells 910 in mouse tumor models. Other studies have demonstrated that adoptively transferred immune T lymphocytes were able to eliminate disseminated tumors in mice." Thus, immunotherapeutic strategies may hold promise for the treatment of metastatic disease. In the present study, we examined the role of the immune system in controlling spontaneous metastases arising from primary intraocular tumors. By employing an inbred mouse model, it was possible to characterize precisely the immune processes that influence the rejection of spontaneous metastases. Experimental Animals Materials and Methods Adult female DBA/2 (//-2 41 ) mice were purchased from Jackson Laboratories (Bar Harbor, ME) and used as experimental subjects between 3-5 months of age. Female athymic, nude (nu/nu) BALB/c (//-2 d ) mice were purchased from Life Sciences, Inc. (St. Petersburg, FL). The present investigations conform to the ARVO Resolution on the Use of Animals in Research. All 1355

2 1356 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / September Vol. 27 surgical procedures were performed using ketamine hydrochloride anesthesia. 12 Tumor Cells A mutagenized tumor clone, designated P91, was isolated from P815 mastocytoma cells (DBA/2 origin) treated in vitro with the mutagen N-nitroso-N-methylnitroguanidine 13 and was a gift from Dr. Thierry Boon (Ludwig Institute for Cancer Research; Brussels, Belgium). The P91 tumor line is a highly immunogenic variant of P815 mastocytoma and undergoes spontaneous immune rejection following intraperitoneal, 14 subcutaneous, 15 or intracameral 15 inoculation in the syngeneic DBA/2 host. P91 tumor inocula 100 times greater than those used in the present study are promptly rejected following intraperitoneal transplantation in syngeneic DBA/2 recipients. 14 Boon et al 14 have shown that P91 expresses distinct new tumor antigens not found on the parental P815 tumor cells, as well as tumor antigens common to P815 mastocytoma. DBA/2 hosts that reject P91 tumors are immune to re-challenge with either P91 or the parental P815 tumor. 14 However, attempts to immunize DBA/2 hosts with P815 and protect against re-challenge with P91 invariably fail. 14 P91 tumor cells do not induce in vitro blastogenic responses in lymphocytes from normal DBA/2 hosts (unpublished findings). DBA/2 histocompatibility antigens appear to be expressed on P91 tumor cells. We have routinely used P91 tumor cells to immunize allogeneic BALB/c (//-2 d ) and C57BL/6 (H-2 b ) hosts against minor and major histocompatibility antigens respectively. Sera from such hosts contain antibodies that lyse DBA/2 lymph node cells in the presence of complement (unpublished findings). P91 tumor cells were cultured in suspension in Falcon 75-cm 2 tissue culture flasks (Falcon Plastics, Oxnard, CA) with Dulbecco's modified Eagle's minimum essential medium (MEM, Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (FCS, Gibco), L-glutamine, and gentamycin (0.05 mg/ml, Schering Corp., Kenilworth, NJ) Anterior Chamber Tumor Inoculations A modified quantitative technique for inoculating a precise number of tumor cells into the anterior segment of the mouse eyes has been described before. 12 Mice were deeply anesthetized with 66 mg/kg of ketamine hydrochloride (Vetalar, Parke, Davis and Co., Detroit, MI) given intramuscularly. P91 tumor cells (10 5 cells/ 5 fi\) were injected intracamerally (IC) into panels of DBA/2 or nude BALB/c recipients. The eyes were examined two to three times per week and the tumor growth scored according to the percent of the anterior chamber occupied by tumor. 12 Detection of Spontaneous Metastases Viable tumor cells were detected at extraocular sites by a culture technique described previously. 6 Selected organs were removed aseptically and monocellular suspensions were prepared by gently forcing the tissues through sterile, stainless steel wire screen (60-mesh). The single-cell suspensions were washed once in Hanks balanced salt solution (HBSS), resuspended in MEM, inoculated into 75-cm 2 tissue culture flasks containing 30 ml of complete MEM, and cultured at 37 C in the presence of 5% CO 2. Cultures were passaged every 5 days by transferring 2 ml of the tissue culture cells into flasks containing fresh tissue culture medium. Organ cultures were scored positive based on the morphology of the dominant cell type (i.e., mastocytoma cells) and the continuous growth of the tumor cells in the respective cultures. Cell cultures failing to demonstrate tumor cell growth after 30 days were scored negative. By co-culturing known numbers of tumor cells with normal organ cell suspensions from tumor-free control mice, it can be shown that this technique can detect as few as 100 tumor cells per organ culture (data not shown). Adoptive Transfer of Cell Suspensions s were collected aseptically from intraocular tumor-bearing DBA/2 mice 15 days after initial tumor inoculation. Monocellular spleen suspensions were prepared by pressing whole spleens through stainless steel screens. cells were washed in HBSS and resuspended for treatment with the various monoclonal antibodies. Following antibody treatment, spleen cell suspensions were transferred intravenously to intraocular tumor-bearing recipients. Each mouse received the equivalent of 1 X 10 8 treated or untreated spleen cells within 4 hr of enucleation. Negative Selection Experiments cells to be used in adoptive transfer studies were subjected to negative selection in which the cell suspensions were exposed to antibodies able to react with specific T cell surface determinants in the presence of complement (C). Cells bearing the appropriate markers were lysed, and the remainder were used for infusions. The following antibodies were employed: anti-thy 1.2 (New England Nuclear Corp., Boston, MA) diluted 1:1000; anti-lyt 1.1 (New England Nuclear Corp.) diluted 1:1,000; and anti-lyt 2.1 (New England Nuclear Corp.) diluted 1:400. Cell suspensions were incubated with equal volumes of the respective antibody dilutions at 4 C for 1 hr, washed in HBSS, and incubated for 1 hr at 37 C with a 1:20 dilution of rabbit complement (Cedarlane Laboratories Ltd.,

3 No. 9 IMMUNE REJECTION OF METASTASES/Niederkorn er ol Table 1. Detection of metastatic P91 mastocytoma cells in peripheral organs following intracameral transplantation* No. of positive cultures^ Organ Day /5 5/5 5/5 2/5 1/5 3/5 2/5 1/5 2/4 1/5 N.D4 * Normal DBA/2 mice received 1 X 10 5 P91 cells inoculated into the left eye on day 0. Mice were sacrificed on the indicated days and organs were cultured in vitro as described in Materials and Methods. t Number of positive P91 cultures/number of organs cultured. X = not done. Hicksville, N.Y.). The cells were washed in HBSS two additional times, resuspended in HBSS, and the remaining cells (starting concentration = 1 X 10 8 cells per mouse) injected intravenously into the various recipients. Enucleation Tumor-containing globes were excised from mice under metofane anesthesia. Metofane inhalation was selected for anesthesia since its duration time is brief, it allows the experimental animals to recover rapidly, and unlike ether, it does not carry the risk of explosion. Simple extirpation with curved iris scissors was accomplished and was followed by application of pressure to the socket to achieve hemostasis. Care was taken not to puncture the globe in order to prevent local seeding of tumor cells. Gamma Irradiation of Mice Mice were subjected to sublethal, whole-body gamma irradiation (500 R) at a rate of 109 rad/min, in a Gammacell (Atomic Energy of Canada, Ltd., Ottawa, Canada) containing a 137 Cs source. This dose of gamma irradiation is known to render mice lymphopenic and unable to reject tumor grafts. 16 Immune Serum Sera from P91 tumor-bearing DBA/2 mice contain specific antibodies directed against the tumor-specific transplantation antigens found on the cell membrane of these mastocytoma cells. 15 The peak antibody titers are found on day 14 following intraocular tumor inoculation. 15 Accordingly, serum was collected from intraocular tumor-bearing mice on day 14 and used in lieu of spleen cells in the adoptive transfer of immunity studies described above. One donor-equivalent of immune serum (i.e., the total volume of serum routinely recovered from a normal mouse = 0.5 ml) was injected intravenously (0.25 ml) and intraperitoneally (0.25 ml) within 2-4 hr after enucleation of the respective recipients. Statistics The Fisher Exact Probability test was employed for analyzing the statistical significance of the various data. A probability value less than 0.05 was considered significant. Results Spontaneous Metastasis of Intraocular Tumors The first series of experiments was designed to determine if intraocular P91 tumors disseminate to distant bodysites and, if so, are these metastases rejected in the normal syngeneic host. Panels of DBA/2 mice received a P91 tumor cell inoculum of 1 X 10 5 cells delivered into the anterior chamber of the left eye on day 0. Organs were removed and cultured for the detection of viable metastases at several time points thereafter. Repeated attempts to detect metastases in livers of intraocular tumor-bearing hosts failed. Suspecting that toxic products and enzymes were liberated from liver cell suspensions, we tested the viability of tumor cells co-cultured with normal liver cell suspensions. When fresh P91 tumor cells were co-cultured with liver cell suspensions from normal donor mice, the admixed tumor cells perished rapidly (data not shown). Moreover, several maneuvers designed to augment P91 tumor growth in normal liver cell cocultures likewise failed; exhaustive washing of cell suspensions in HBSS, filtration over nylon wool columns, and density gradient centrifugation did not permit tumor cell survival following mixture with normal liver cell suspensions. Thus, it was not possible to use this assay system for detecting viable metastases in liver cell suspensions. By contrast, metastases could readily be cultured in cell suspensions from the spleen, lung, thymus, and brain. The results in Table 1 show that the lungs and spleen were the primary organs for the development of metastases; viable tumor cells were

4 1358 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / September 1986 Vol. 27 Table 2. Detection of metastatic P91 mastocytoma cells in normal and immunocompromised hosts* No. of positive cultures^ Host Day Normal DBA/2 X-irradiated DBA/2* Balb/c nude^: 5/5 1/5 5/5 9/9 8/9 1/1 8/10 7/7 1/1 5/5 4/4 2/5 4/4 * Panels of mice received 1 X 10 3 P91 cells inoculated into the left eye on day 0. On the specified days, organs were cultured from normal DBA/2, X- irradiated DBA/2 (500R, day -1) and BALB/c nude mice. t Number of positive P91 cultures/number of organs cultured. X The majority of these animals died from progressive tumor growth by day 21. isolated in 100% of these organs on day 7. However, by day 15 metastases could no longer be detected in these or any other organs examined. Thus, intraocular P91 tumors disseminate rapidly to the spleen and lungs but are rejected in the syngeneic host. Rejection of Spontaneous Metastases is an Immune Process The rapid disappearance of pulmonary and splenic metastases suggested that the syngeneic host was able to reject the peripheral tumors by an immune process. To test this possibility, panels of immunocompetent and immunologically impaired hosts were examined for their ability to reject spontaneous metastases. Table 3. Evidence that spontaneous metastases originate and disseminate from the primary intraocular tumor* Host Normal DBA/2 X-irradiated DBA/2 BALB/c nude Organ No. of positive cultures^ Days following enucleation 1 1/4 1/4 1/4 N.D4 2 1/11 0/11 0/11 0/11 7/8 7/9 0/9 3/9 6/6 5/6 1/6 6 4/4 3/3 * On day 8 post-intracameral inoculation of 1 X 10 s P91 tumor cells, the tumor-bearing eye was enucleated. On the specified days following enucleation, organs were cultured in vitro as described in Materials and Methods. t Number of positive P91 cultures/number of organs cultured. X = Not done. Athymic nude mice, lymphopenic, gamma-irradiated (500 R) DBA/2 mice, and normal DBA/2 mice received intraocular inoculations of P91 tumor cells on day 0. Mice were killed and their spleens and lungs examined for the presence of viable metastases on days 7, 14, and 21. The data shown in Table 2 indicate that nude mice and lymphopenic DBA/2 mice were unable to reject their splenic and pulmonary metastases, and ultimately died from progressive tumor growth between days 21 and 28 post-tumor inoculation. By contrast, immunocompetent DBA/2 mice were essentially free of metastases by day 14. Thus, the rejection of metastases from intraocular P91 tumors is a T cell-dependent, radiosensitive immune process. Tumor Metastasis to the and s is a Continuous "Seeding Process" Results from the previous experiments indicated that tumor cells disseminated to the spleen by day 7. The continued presence of tumor cells on days raises the question of whether the splenic metastases found on day 14 were the same tumor cell population detected earlier. Conversely, it may be reasoned that the primary tumor continuously seeds tumor emboli to the spleen where the tumor cells are rejected. In the latter case, metastases detected on day 14 might represent recent arrivals rather than tumor cells persisting from day 7. To distinguish between these possibilities, panels of tumor-bearing mice were subjected to enucleation on day 8 and their organs examined for metastases 1, 2, and 6 days later. The results in Table 3 emphasize the rapid rejection of metastases following the removal of the primary intraocular tumor. Within 24 hr after enucleation, 81 % (13/16) of the organs from immunocompetent mice were free of demonstrable metastases. By 48 hr, only 1 of 44 organs was positive for tumor cells. By contrast, removal of tumor-containing eyes in immunologically compromised hosts failed to alter the incidence of metastases; tumor cells were isolated in 100% of the spleens and lungs of gamma-irradiated DBA/2 mice and athymic, nude mice 6 days after enucleation (Table 3). Thus, the rapid elimination of metastases following enucleation of the tumor-containing eyes of immunocompetent hosts indicates that the metastases detected in hosts not subjected to enucleation are due to a continuous seeding process, and not to the tumor cells persisting in the target organ. Immune Rejection of Spontaneous Metastases is a T Cell-Mediated Process The previous results indicated that rejection of the spontaneous metastases was a T cell-dependent, radio-

5 No. 9 IMMUNE REJECTION OF METASTASES/Niederkorn er ol Table 4. Adoptive transfer of immune spleen cells and immune serum to immunosuppressed hosts bearing metastases arising from intraocular tumors* Cell or serum transfer No. ofpositive cultures^ None (control) Normal spleen cells Immune spleen cells Normal serum Immune serum 16/17(94%) 8/12(67%) (P = 0.07)J 1/12 (8%) (P< ) 4/4 (100%) (P = 0.8) 5/5 (100%) (P= 1.0) 16/16(100%) 11/13(85%) (P=0.19) 3/12(25%) (P< ) 4/4 (100%) (P=l.0) 4/5 (80%) (P = 0.23) 6/7 (86%) 3/13(23%) {P = 0.0\) 0/12(0%) (P< ) 4/4 (100%) (/>=1.0) 2/5 (40%) (P = 0A) 11/17(65%) 5/13(38%) (P = 0.1) 0/12 (0%) (P< 0.001) 2/4 (50%) (P = 0.36) 3/5 (60%) (P = 0.38) * DBA/2 mice were x-irradiated (500 R) 1 day prior to receiving 1 X 10 s P91 cells in the left eye. On day 8, the tumor-bearing eye was enucleated and the animals infused intravenously with either 1 X 10 8 spleen cells or 0.5 ml of serum from normal DBA/2 mice or from donor mice bearing 14-day intraocular tumors. On day 14, the organs were cultured in vitro for the detection of metastases. t Number of positive P91 organ cultures/number of organs cultured. Percentage of organs positive for metastases is in parenthesis. t Probability value determined by Fisher Exact Probability test comparing each experimental group with control group (line 1). sensitive process. Adoptive transfer experiments were employed to identify the T cell-dependent mechanism^) responsible for metastatic tumor rejection. Immune serum and spleen cells were collected from immunocompetent intraocular tumor-bearing DBA/2 hosts on day 15 post-tumor inoculation. For the adoptive transfer studies, one donor equivalent of either serum (=0.5 ml) or spleen cells (1 X 10 8 ) was transferred intravenously. Additional experiments involved the depletion of specific T cell subsets by lysis with monoclonal antibody in the presence of complement. In each case, the initial cell suspension contained 1 X 10 8 spleen cells. The cells remaining after lysis with the respective antibody were transferred and considered to be a splenic equivalent for that category of immune cell. Recipient mice consisted of x-irradiated (500 R; day 1) DBA/2 mice that received intraocular P91 tumor inocula on day 0 and were subjected to enucleation on day 8. Either spleen cells or sera were infused intravenously within 2-4 hr after enucleation. Mice were killed 6 days later, and the organs examined for metastases. The results in Table 4 show that untreated hosts and hosts that received normal spleen cells were impaired in their ability to reject metastases. Likewise, neither immune serum nor normal serum exerted a protective effect; these mice did not differ significantly (P> 0.05) from untreated controls. However, protection was noted in hosts that received immune spleen cells. The number of positive tumor cultures in immune spleen cell recipients was significantly (P < ) reduced for each category of organ tested (Table 4). The immune spleen cells that produced immunity against pulmonary and splenic metastases were T lymphocytes, since the protection could be ablated by pretreatment of the cells with anti-thy 1.2 antibody in the presence of complement (Table 5). Further negative selection experiments indicated that this T cell population expressed the Lyt 1.1 surface determinants, since pretreatment with this antibody almost completely removed the protective effect of the immune spleen cells for all four categories of organs tested (Table 5). The immune spleen cells also expressed the Lyt 2.1 surface marker. Pretreatment of immune cells with anti-lyt 2.1 antibody in the presence of complement resulted in significant reduction in immunity to pulmonary and splenic metastases (P < 0.04 and P < respectively). By contrast, pretreatment of immune cells with rabbit complement alone had an insignificant effect on the capacity of immune spleen cells to confer protection against spontaneous metastases in any of the organ categories tested. Collectively, the results of the adoptive transfer experiments, shown in Table 5, indicate that the immune rejection of spontaneous metastases is mediated by a T cell population that expresses surface determinants characteristic of cytotoxic T lymphocytes (i.e., Lytl+, 2+). Discussion The development of spontaneous metastasis is a complex process involving several sequential steps in which malignant cells are released from the primary tumor and disseminate to distant sites where they proliferate to form new tumor foci.' A successful metastatic cell must be able to detach from the primary tumor, invade host tissues and gain entrance into the circulation, survive in the circulation, arrest at an organ capillary bed, extravasate, and grow in the new environment. Although many experimental studies of metastases in animal models have used intravenously injected tumor cells, such investigations may be misleading since this protocol circumvents the early steps in the metastatic process. However, the present study

6 1360 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / September 1986 Vol. 27 Table 5. Phenotype of immune cells conveying protection against metastases arising from intraocular P91 tumors* Treatment of immune spleen cell suspensionx No. positive culture^ None Conly anti-thy C anti-lyt 1.1 +C anti-lyt C 1/12(8%) 4/14(29%) (P = 0.18) 7/11 (64%) (P = 0.008) 9/13(69%) (P = 0.002) 10/18(56%) (P = 0.009) 3/12(25%) 8/15(53%) (P=0.1) 8/9 (89%) (P = 0.005) 10/13(77%) (P = 0.01) 11/18(61%) (P = 0.04) 0/12(0%) 0/14(0%) (/>= 1.0) 3/10(30%) (P = 0.07) 9/13(69%) (P = ) 3/19(16%) (P = 0.21) 0/12(0%) 0/15(0%) (P= 1.0) 3/10(30%) (P = 0.07) 6/12(50%) (P = 0.006) 4/18(22%) (/ > = 0.11) * DBA/2 mice were x-irradiated (500 R) one day prior to receiving 1 X 10 5 P91 cells in the left eye. On day 8, the tumor-bearing eye was enucleated and the animals infused intravenously with the equivalent of 1 X 10 8 immune spleen cells. Immune spleen cell suspensions were treated with monoclonal antibodies and rabbit complement before intravenous injection as described in Materials and Methods. On day 14, the organs were cultured in vitro for the detection of metastases. t Number of positive P91 cultures/number of organs cultured. Percentage of organs positive for metastases is indicated in parenthesis. t Line 1 (immune spleen cells; no treatment) represents data also shown in Table 4 (line 3). Probability value determined by Fisher Exact Probability Test. Each experimental group was compared with the control group (Line 1 = immune spleen cells; no treatment). examined spontaneous metastases that were produced through the normal metastatic process. An additional advantage of the present intraocular tumor model is the ability to detect minute numbers of peripheral metastases in selected organs. This feature permits experiments designed to monitor not only the organ distribution of metastases, but also precise determination of the time required for immune rejection of extraocular tumor foci. We are aware that mastocytomas such as P91 do not normally occur in the eye, and that their metastatic behavior may, therefore, differ significantly from other tumors (e.g., melanoma) that are known to occur in the eye. Nonetheless, several fundamental observations are worth noting. The data indicate that intraocular P91 tumors are capable of rapid metastasis to distant organs and, in the absence of a normal immune system, form progressively growing secondary tumors. In the immunocompetent host, the majority of these spontaneous metastases are rejected within 24 hr. The presence of a primary intraocular tumor produces continuous seeding of tumor emboli to peripheral organs. This continuous seeding process comes to an abrupt end with either the surgical removal of the primary tumor or its spontaneous rejection. The immune rejection of spontaneous metastases was found to be T cell-dependent and radiosensitive. The observation that metastatic tumor rejection was absent in athymic nude mice suggests that natural killer (NK) cells are not required for this process, since nude mice have normal or slightly elevated NK cell-mediated immunity The early disappearance of P91 metastases coincides with the acquisition of systemic tumor-specific cytotoxic T lymphocyte (CTL), antibody, and delayed-type hypersensitivity (DTH) immune responses. 15 Although tumor-specific anti-p91 IgG antibody is capable of lysing P91 tumor cells in vitro, 15 the likelihood that cytolytic antibody was involved in the eradication of metastases in vivo is remote, since passive transfer of immune serum failed to protect immunologically impaired hosts. Moreover, experiments using adoptively transferred immune spleen cell populations indicated that the rejection of spontaneous metastases was mediated by a population of T lymphocytes bearing the Lyt 1.1 and Lyt 2.1 surface determinants, since pretreatment of the immune spleen cells with either anti-lyt 1.1 or Lyt 2.1 antibody in the presence of complement removed most of the protective effect of the immune spleen cells. We conclude that the Lyt 1+, 2+ effector cells are cytotoxic T lymphocytes, since they express the Lyt 2+ surface marker, which is characteristically restricted to the cytotoxic/suppressor T cell subset. 19 Depletion of immune effector cells by pretreatment with anti-lyt 1.1 antibody suggests that the same population of immune cells co-expresses Lyt 1 and Lyt 2 surface determinants. This is not unexpected, since virtually all T lymphocytes, including CTL, express Lyt 1 molecules. 20 ' 21 Thus, the surface phenotype of CTL is more appropriately considered to be Lyt 1+, Since spontaneous metastases are eradicated in the normal immunocompetent DBA/2 mouse within hr of their arrival in the spleen or lungs, one would predict direct cytolysis by CTL as the most likely immune effector mechanism for metastatic tumor destruction. Although we favor the hypothesis that metastases are eliminated by direct cytolysis by CTL, it is possible that the Lyt 1+, 2+ effector cells function indirectly by elaborating a variety of lymphokines that activate

7 No. 9 IMMUNE REJECTION OF METASTASES/Niederkorn er ol second level effector cells, such as tumoricidal macrophages, 22 lymphokine activated killer (LAK) cells, 910 or even radiation-resistant, host-derived CTL. 23 Regardless of the precise in situ effector mechanism of metastatic tumor rejection, it is clear that Lyt 1+, 2+ T-cells play a pivotal role. The present results, along with previous findings, 15 reveal an interesting contrast in the host's deployment of immune effector mechanisms for controlling P91 tumors. We have recently concluded that the rejection of primary intraocular P91 tumors in DBA/2 hosts relies on a DTH-mediated immune process, which culminates in tumor destruction by ischemic necrosis. 15 By contrast, the results reported here suggest that metastases arising from primary intraocular P91 tumors are rejected by CTL. Thus, it appears that the host employs two different immune mechanisms for rejecting metastases and primary intraocular P91 tumors. Based on the present findings, we conclude that the rejection of metastases occurs by the action of Lyt 1+, 2+ CTL that function most effectively in the spleen and the lungs. However, the precise mechanism for metastatic tumor cell destruction in these organs remains to be characterized. Key words: intraocular tumors, spontaneous metastases, immune rejection, immunology, T cells Acknowledgments The excellent technical assistance of Ms. Jessamee Mellon is greatly appreciated. P91 tumor cultures were graciously provided by Dr. Thierry Boon, Ludwig Cancer Institute, Brussels, Belgium. The manuscript was carefully prepared by Ms. Sara Howard. References 1. Poste G and Fidler IJ: The pathogenesis of cancer metastasis. Nature 283:139, Shields JA: Diagnosis and Management of Intraocular Tumors. St. Louis, CV Mosby Co, 1983, pp Gass JDM: Comparison of prognosis after enucleation vs cobalt 60 irradiation of melanomas. 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