Development of tumor cell resistance to syngeneic cell-mediated cytotoxicity during growth of ascitic mastocytoma P 815 Y ( tumor-associated antigens / tumor escape )

The immune reactivity to tumor cells within a progressively growing tumor mass in the syngeneic host has been analyzed by studying the cell-mediated cytolytic response of DBA/2 mice to the ascitic mastocytoma P815Y. Peritoneal cells from P815Y tumor-bearing hosts were fractionated by velocity sedimentation at unit gravity. Cell-mediated cytotoxicity of fractionated and unfractionated cells was measured by 51Cr-release from tumor target cells. The cell separation procedure revealed significant levels of specific cell-mediated cytotoxicity to P815Y within peritoneal cell populations at 8-16 days after tumor cell inoculation. Tumor cells purified from the peritoneal cell populations of mice injected with 103 tumor cells 10 days previously were as susceptible to syngeneic and allogeneic cell-mediated cytotoxicity as P815Y grown in vitro. However, tumor cells obtained from mice 16 days after tumor inoculation were resistant to cytolysis by syngeneic, but not allogeneic, effector cells. In addition, day 16 tumor cells did not inhibit syngeneic cell-mediated cytotoxicity against P815Y grown in vitro. Immunoglobulin was not detected on day 16 tumor cells and no circulating antibody to P815Y was found in the ascitic fluid of day 16 tumor-bearing mice. These results indicate that tumor cells may escape immune attack by loss of expression of cell surface tumor-associated antigens in the absence of circulating antibody against tumor. Recent studies in humans and in animal model systems have demonstrated that many tumor-bearing hosts mount both cell-mediated and humoral immune responses to their tumors (1-3). Despite such ongoing antitumor immune responses, many syngeneic tumors are capable of continued growth leading to death of the host. The mechanisms by which tumor cells escape antitumor immune responses are thought to be complex, but may include: (a) antigenic modulation of tumor-associated antigens (4-6); (b) inhibition of cell-mediated immunity by serum blocking factors (7, 8); (c) specific and nonspecific immunosuppression of the hosts' immune responsiveness (9-11); (d) stimulation of tumor growth by the immune response (12); (e) location of the tumor in an immunologically "privileged site" (13); (f) lack of immunogenic determinants on the tumor cell membrane; and (g) tumor growth that proceeds more rapidly than does the induction and action of the antitumor immune response. Most previous studies of cell-mediated immunity to tumorassociated antigens have analyzed the immune capacities of cell populations obtained from various lymphoid organs or peripheral blood. However, the immune reactivities of the cells obtained from these areas, which may be distant from the tumor mass, may not accurately reflect events occurring within or surrounding the tumor (14). In addition, tumor cells maintained in tissue culture are usually used as target cells for assays of cell-mediated cytotoxicity (CMC) in vitro and for the presence of antibodies against tumors. It is possible that tumor cells in tissue culture may lose and/or acquire some characteristics, and therefore, may no longer be representative of tumor cells in the tumor-bearing host (15). To assess more accurately the effect of antitumor immune responses on tumor cells in situ, it is desirable to analyze the cellular and humoral immune responses to the tumor cells occurring within a progressively growing tumor mass. MATERIALS AND METHODS Mice. Female DBA/2, C57BL/10 (B10), C57BL/6 (B6), and B10.D2 mice were obtained from the Jackson Laboratories, Bar Harbor, Me. , and the Institute for Cancer Research, Philadelphia, Pa. , and used at 8 weeks of age. Tumors. The P815Y (DBA/2, H-2d) mastocytoma, L1210 (DBA/2, H-2d) leukemia, and EL4 (B6, H-2b) lymphoma were maintained as ascites tumors in the syngeneic strain and cultured in vitro. Tumor-bearing mice used in this study were DBA/2 mice inoculated intraperitoneally (i.p.) with 103 viable P815Y cells grown in tissue culture and used at the fourth to tenth cell culture passage. Cell Separation Procedures. Peritoneal cells were harvested from normal and P815Y tumor-bearing DBA/2 mice by repeated flushing of the peritoneal cavity with phosphate-buffered saline, washed once with Fischer's modified LI medium (Schwartz BioResearch, Orangeburg, N.Y.), and counted by hemocytometer. Peritoneal cells were fractionated on the basis of cell size by unit gravity velocity sedimentation in a Sta-Put cell separation apparatus (Johns Scientific, Toronto, Ont.) as described by Miller and Phillips (16). Forty to 150 X 106 peritoneal cells in 20 ml of Li medium were loaded into the apparatus followed by a 1500-ml linear gradient of 5-25% fetal bovine serum in Li medium. After sedimentation for 3 hr at 40, 50-ml fractions were collected and cell concentrations determined in a Coulter Counter (Coulter Electronics, Hialeah, Fla.). In Vitro Induction of CMC. Cytotoxic lymphocytes reactive primarily against H-2d transplantation antigens present on P815Y were induced by culturing normal B6 spleen cells with mitomycin C-treated P815Y cells in Marbrook culture vessels as described (17). Assay for CMC. CMC against tumor target cells was measured by a 4-hr 51Cr-release assay as described in detail elsewhere (18). P815Y tumor target cells were obtained from cultures at the fourth to tenth cell culture passage. Cytotoxicity is expressed as percent specific lysis and is equal to experimental release minus spontaneous release divided by hypotonic release minus spontaneous release. All determinations were performed in triplicate, and the SEM rarely exceeded 5% of the mean. Assay for Cell Surface Immunoglobulin. Cell surface immunoglobulin was detected by the binding of fluoresceinconjugated rabbit antibody against mouse gamma globulin 329 Abbreviations: CMC, cell-mediated cytotoxicity; B6, C57BL/6; B1O, C57BL/10; i.p., intraperitoneally; TAA, tumor-associated antigens. 330 Immunology: Biddison and Palmer