Immune response to a syngeneic mammary adenocarcinoma. III. Development of memory and suppressor functions modulating cellular cytotoxicity.

Measurement of the development of cytolytic activity by mammary tumor primed or unprimed syngeneic spleen cells on in vitro monolayers of the 13762 rat mammary tumor operationally defined several subpopulations of lymphoid cells involved in the cytotoxic response. In vitro sensitization of cells from Fischer 344 animals injected 2 to 10 days earlier with 2 x 10(7) viable tumor cells always resulted in a higher and earlier lytic response than cells from non-inoculated animals. Adoptive transfer of the same in vivo primed cells for 5 days in irradiated syngeneic hosts removed any cytotoxic cells originally present but subsequent in vitro sensitization still resulted in a higher and earlier cytolytic response. We defined such cells as "memory" cells for cytotoxicity. Memory cells were radiosensitive and specific for the immunizing target cell. In contrast to cells from animals inoculated for 3 to 10 days, cells obtained 11 and 12 days after immunization had a lower response than unprimed cells on vitro sensitization. The anamnestic response could be restored either by culturing 12-day primed cells in vitro for 2 days without antigen or by adoptive transfer for 5 days into irradiated syngeneic rats. This suggests that another population of cells is present in spleen and suppresses the conversion of memory to cytotoxic cells. A more direct measurement of suppressor cell function was obtained by coincubating tumor-primed and unprimed cells on monolayers during in vitro sensitization. Cells from animals bearing tumors for 5 to 10 days always caused an increase in the response of the mixed lymphocyte groups, whereas 11- to 13-day tumor primed cells always caused a marked decrease in the cytolytic response. These results suggest the following interpretation of the kinetics of cell-mediated cytotoxicity to syngeneic tumor inoculation. Cytotoxic cells appear about 6 days after immunization, reach peak levels 2 days later, and then decrease rapidly. Memory cells are generated at a faster rate, reach peak levels before maximum cytolytic activity, but are then functionally inhibited from converting into differentiated cytotoxic cells by a new population of suppressor cells which reach peak activity about 12 days after immunization.     

An intradermal assay for quantification and kinetics studies of tumor angiogenesis in mice.

A method is reported for the study of early phases of neovascularization in syngeneic murine tumors and human tumor xenografts in nude mice. Using this method, the effect of irradiation of tumor cells or tumor bed on tumor angiogenesis was studied. Tumor cells were injected intradermally in the abdominal skin flap, which was reopened at 2-day intervals to quantify newly formed blood vessels at the site of tumor cell injection. Both tumor cell injection and blood vessel counting were performed under a dissecting microscope. Using three syngeneic murine tumors and two clones of a human colonic adenocarcinoma, it was observed that new blood vessels started appearing within a few days after tumor cell injection and that this event preceded measurable tumor growth. The number of blood vessels increased exponentially for several days but then their further growth slowed. The extent of angiogenesis depended on the tumor type and the number of tumor cells injected. The exposure of the skin flap to ionizing radiation prior to tumor cell injection reduced neovascularization. We further observed that heavily irradiated tumor cells retained their ability to induce angiogenic response and that lymphoid cells (peritoneal exudate and spleen cells) could also elicit an angiogenic response, although it is weaker than the response elicited by tumor cells. Thus this method is suitable for quantification and kinetics of early phases of tumor angiogenesis in individual mice bearing transplants of syngeneic tumors or human tumor xenografts, and it can be useful for investigating various regulators of tumor angiogenesis.     

Evidence for cytostatic T cell activity in the effector mechanism against syngeneic TMT mammary tumor cells in mice

The effector mechanism of immune spleen cells against syngeneic TMT mammary tumor cells was analyzed in vitro. C3H/He mice were first inoculated with TMT tumor cells, and then the tumors were x-irradiated with 2000 rad 1 wk after the inoculation. Spleen cells from these treated mice inhibited the growth of tumor cells in vitro when assessed by (3H)-TdR incorporation by tumor cells (cytostatic activity). The same spleen cells did not have any cytotoxic activity on TMT tumor cells detected by a 51Cr-release assay. The cytostatic activity was mediated by Lyt-1+23- T cells. The purified T cells alone could not inhibit the growth of tumor cells, but accessory cells were required for the induction of cytostatic T cell activity. The accessory cells were Ia-positive, macrophage-like adherent cells. Furthermore, both T cells and macrophages were also required for the inhibition of tumor growth even after the spleen cells were activated in vitro. These results suggest T cells and macrophages play an important role in the effector mechanism against TMT mammary tumor cells. The mechanism of cytostasis by T cells and macrophages was discussed from the standpoint of the cellular interaction.     

Kinetics of the reactivity of subpopulations of spleen cells of mice bearing virus-induced mammary tumors to syngeneic antigenic extracts in vitro

Summary This study was designed to investigate the nature of lymphocyte reactivity to soluble tumor antigens with respect to the kinetics of the reactivity, the responding cell type, and the role of accessory cells, within a syngeneic system. BALB/c mice were inoculated with 1×10⁶ viable cells of sygeneic MTV-induced mammary tumors. Assessment of proliferative activity of spleen cells of these animals by DNA synthesis (³H-thymidine incorporation in vitro) indicated a biphasic response to stimulation by 200 g of a syngeneic perchloric acid (PCA)-soluble extract (AMMT) of the tumor over a 25-day period, with peak activities at days 13 and 19 post inoculation. The response was predominantly T-cell-mediated. Splenic macrophage population rose from less than 2% of total spleen cells by day 25 without any appreciable change in the T or B cell population. Depletion of spleen cells of macrophages abolished the first peak activity (at day 13) but significantly enhanced the second (at day 19). Reconstitution of the depleted cells with macrophages prepared from peritoneal exudates of tumor-bearing or normal mice restored the responses to undepleted values, thus indicating an accessory role for macrophages in these responses. These results provide new data which should contribute to a better understanding of the tumor-host relationship.     

In vitro generation of secondary cell-mediated cytotoxic response against a syngeneic Gross virus-induced lymphoma in rats.

Cell-mediated cytotoxic responses against a syngeneic Gross virus-induced lymphoma, (C58NT)D, in W/Fu rats were generated in vitro by using mixed lymphocyte-tumor cell cultures. The source of responding cells was either spleens from normal rats or spleens from rats carrying or having rejected (C58NT)D tumors. Mitomycin C-treated (C58NT)D tumor cells were used as stimulating cells. The secondary anti-tumor cytotoxic response occurred more rapidly and reached higher levels than the primary response, and it was antigen specific. T cells, but nor B cells or macrophages, were essential for both the induction and the effector phases of the secondary anti-tumor responses. These data suggest that specific memory T cells persist for long periods of time in the lymphoid organs of (C58NT)D immune rats, which can rapidly become cytotoxic upon re-exposure to antigen.     

Augmentation of immune response to syngeneic fibrosarcoma T241 with in vivo protection

Summary Lewis T241 fibrosarcoma, a syngeneic tumor in C57 BL/6J mice, was found to be poorly immunogenic. When tumor-bearing animals (TBA) were challenged with tumor cells either concomitantly or after excision of a growing tumor no protection was observed. In vivo (Winn) neutralization assays also showed a lack of tumor immunogenicity. However, in vitro studies showed that a significant proliferative response could be elicited from the spleen cells of TBA when these cells were cultured with either mitomycin-C-treated tumor cells or KCl tumor extract. Similarly, macrophage migration inhibition factor (MIF) was produced by TBA spleen cells upon incubation with KCl tumor extract, but no cell-mediated cytotoxicity to T241 target cells was observed with various lymphoid cell populations at any stage of tumor growth. Immunization of syngeneic animals with Vibrio cholerae neuraminidase(VCN)-treated, irradiated tumor cells alone or admixed with Freund's complete adjuvant (FCA) resulted in decreased tumor growth and fewer pulmonary metastases following challenge with 10⁶ tumor cells. No complete tumor rejection was observed. In contrast, 13 of 16 animals immunized with irradiated tumor cells admixed with FCA rejected 10⁵ tumor cells. Animals that grew tumors had significantly reduced tumor growths and pulmonary metastases. Lymph node and peritoneal exudate cells (PEC) of immunized animals showed significant cytotoxicity to T241 cells.     

Activity of tumor-associated lymphoid cells at short intervals after administration of irradiated syngeneic and allogeneic tumor cells.

After a single intraperitoneal injection of irradiated tumor cells, host cells capable of responding against syngeneic tumors were detected in peritoneal exudates of mice. Although irradiation of the injected tumor prevented its overgrowth, it did not significantly alter the antigenicity of the tumor. Immunologic activities of tumor-associated host cells in the peritoneal cavity were continuously monitored, starting 48 hr after tumor administration. In vitro cell-mediated lysis of syngeneic tumors appeared as early as 3 days after irradiated tumor administration. In addition, peritoneal exudate cells from inoculated mice were capable of adoptively transferring immunity. Purification of these peritoneal exudate cells on nylon wool columns yielded a nonadherent Ig-negative lymphocyte fraction whose cytolysis was tumor-specific and T cell-associated. The macrophage-free lymphocyte fraction exhibited a higher in vitro activity against tumors than unpurified peritoneal exudates. This tumor-host system allowed the study of cells which directly interact with the tumor cells in vivo, starting shortly after tumor administration. The results reported in this paper show that tumor-associated lymphoid cells capable of mounting anti-tumor response in vivo and in vitro can be demonstrated as early as 3 days after tumor inoculation.     

Cell-mediated immune response to syngeneic UV induced tumors: I. The presence of tumor associated macrophages and their possible role in the in Vitro generation of cytotoxic lymphocytes

A primary in vitro sensitization system employing a chromium release assay was utilized to investigate reactivity of murine spleen cells toward syngeneic ultraviolet (uv) light induced fibrosarcomas. These tumors are immunologically rejected in vivo when implanted into normal syngeneic mice but grow progressivly when implanted into syngeneic mice that had previously been irradiated with subcarcinogenic levels of uv light. Following appropriate sensitization, spleen cells from both normal and uv irradiated mice are capable of developing cytotoxic lymphocytes in vitro against the uv induced tumors. It was subsequently discovered that in situ uv induced tumors all contained macrophages of host origin that became demonstrable only after enzymatic dissociation of the tumor tissue. These macrophages were immunologically active in vitro as their presence in the stimulator cell population was necessary to achieve an optimum anti-tumor cytotoxic response following in vitro sensitization. Anti-tumor reactivity generated by mixing spleen cells and tumor cells in the absence of tumor derived macrophages could be greatly enhanced by the addition of normal syngeneic peritoneal macrophages. When in vitro anti-tumor reactivity of spleen cells from normal and uv treated mice was compared under these conditions we again found no significant difference in the magnitude of the responses. In addition, the cytotoxic cells generated in response to uv induced tumors appeared to be highly cross reactive with respect to their killing potential. Cross reactive killing was observed between all uv induced tumors tested as well as with a syngeneic benz[a]pyrene (BP) induced fibrosarcoma. No cytotoxicity was observed against normal syngeneic PEC's even through these cells were shown to be susceptible to lysis by anti-H-2^k effector cells. It was concluded that: (a) A significant number of host-derived macrophages are present in uv tumor tissue. (b) These macrophages are important for the in vitro generation of tumor specific cytotoxicity. (c) Spleen cells from uv treated mice are capable of recognizing and responding against uv tumor associated antigens in vitro. Cytotoxic effector cells generated in response to uv induced tumors appear to have specificity for tumor associated antigens (TAA) present on all uv tumors tested as well as a syngeneic BP induced tumor. The relationship between in vivo and in vitro reactivity against uv tumors is discussed.     

Effect of tumor cell culture media and sera from tumor hosts on spreading,phagocytosis, and antitumor cytotoxicity of C. parvum-activated murine macrophages

Summary We investigated whether the media of tumor cell cultures and sera from tumor-bearing hosts exert inhibitory effects upon macrophage spreading, phagocytosis, and cytotoxicity. Peritoneal macrophages from normal and Corynebacterium parvum-treated C3Hf/Bu mice were incubated in media from a syngeneic mammary carcinoma, allogeneic Ehrlich ascites carcinoma, and human malignant melanoma or cervical carcinoma cell cultures, or in the serum of hosts bearing these tumors. Such media and sera inhibited the ability of both normal and C. parvum-activated macrophages to spread on glass surfaces and to ingest latex particles. In contrast, they did not interfere with the in vitro destruction of tumorigenic L929 cells by C. parvum-activated macrophages. Media of murine embryo fibroblasts and a human benign tumor and sera from healthy mice or humans did not, however, inhibit either of the macrophage functions tested.     

B7-H3 enhances tumor immunity in vivo by costimulating rapid clonal expansion of antigen-specific CD8+ cytolytic T cells

B7-H3 is a B7 family molecule with T cell costimulatory function in vitro. The in vivo role of B7-H3 in the stimulation of tumor immunity is unclear. We report here that expression of B7-H3 by transfection of the mouse P815 tumor line enhances its immunogenicity, leading to the regression of tumors and amplification of a tumor-specific CD8+ CTL response in syngeneic mice. Tumor cells engineered to express B7-H3 elicit a rapid clonal expansion of P1A tumor Ag-specific CD8+ CTL in lymphoid organs in vivo and acquire the ability to directly stimulate T cell growth, division, and development of cytolytic activity in vitro. Our results thus establish a role for B7-H3 in the costimulation of T cell immune responses in vivo.     

Morphological and functional characteristics of cells infiltrating and destroying tumor multicellular spheroids in vivo.

EMT6 mammary sarcoma cells were grown in vitro as multicellular spheroids to model for the heterogeneity of microenvironments and structural changes which develop in many tumors, including micrometastases. Spheroids of 700-900 micron diameter were implanted into and recovered at different times from the peritoneal cavities of sensitized or nonsensitized allogeneic and syngeneic mice. The colony forming efficiency of spheroid tumor cells recovered at 24 and 48 h from sensitized allogeneic mice was markedly decreased as compared with those from nonsensitized allogeneic or syngeneic animals. These recovered spheroids were extensively infiltrated by both lymphocytes and macrophages, which ultrastructurally had very close membrane associations with tumor cells. Host cells recovered from spheroids exhibited cytotoxic activity in an in vitro 51Cr release assay. Thus, multicellular spheroids in vivo provide a unique experimental model to study the functional capacity of host cells within a spheroical tumor. Although lacking the stroma and the vasculature of in vivo solid tumors, this model does have many similarities to in vivo tumors and is thus suitable for studying the tumor cell-host cell interactions within the tumor microenvironment. In addition, the system offers the potential for quantitative study of the effects of treatment modalities on tumor cell-host cell interactions.     

Chronic bacterial osteomyelitis suppression of tumor growth requires innate immune responses

Clinical studies over the past several years have reported that metastasis-free survival times in humans and dogs with osteosarcoma are significantly increased in patients that develop chronic bacterial osteomyelitis at their surgical site. However, the immunological mechanism by which osteomyelitis may suppress tumor growth has not been investigated. Therefore, we used a mouse model of osteomyelitis to assess the effects of bone infection on innate immunity and tumor growth. A chronic Staphylococcal osteomyelitis model was established in C3H mice and the effects of infection on tumor growth of syngeneic DLM8 osteosarcoma were assessed. The effects of infection on tumor angiogenesis and innate immunity, including NK cell and monocyte responses, were assessed. We found that osteomyelitis significantly inhibited the growth of tumors in mice, and that the effect was independent of the infecting bacterial type, tumor type, or mouse strain. Depletion of NK cells or monocytes reversed the antitumor activity elicited by infection. Moreover, infected mice had a significant increase in circulating monocytes and numbers of tumor associated macrophages. Infection suppressed tumor angiogenesis but did not affect the numbers of circulating endothelial cells. Therefore, we concluded that chronic localized bacterial infection could elicit significant systemic antitumor activity dependent on NK cells and macrophages.     

Immunotherapy with autologous tumor cells engineered to secrete Tag7/PGRP, an innate immunity recognition molecule

BACKGROUND: Recent studies indicate that the innate component of immune defense plays an important role in the establishment of antigen-specific immune response. We have previously isolated a novel mouse gene tag7/PGRP that was shown to be involved in the innate component of the immune system, and its insect homologue is an upstream mediator of Toll signaling in Drosophila. METHODS: Transiently or stably genetically modified mouse tumor cell lines expressing Tag7 were used. Tumor growth rate and animal survival were analyzed. Possible effector cells involved in tumor suppression were detected immunohistochemically. RESULTS: Transfection of mammary gland adenocarcinoma cells with the tag7 cDNA did not alter their growth rate in vitro but diminished their tumorogenicity in vivo in syngeneic and immunodeficient animals. Increased incidence of apoptosis was registered in the modified tumors. Transient expression of Tag7 by mouse melanoma M3 cells elicited protective immunity against parental tumor cells. Immunohistochemical analysis revealed that tumors after immunization with the genetically modified cells were infiltrated with Mac1(+) cells, B220(+) cells, and NK cells. Using nude mice we observed rejection of modified cells, but did not detect memory formation. CONCLUSIONS: We can conclude that secretion of the Tag7 protein by genetically modified cells can induce mobilization of antigen-presenting cells and innate effectors. Memory mechanisms are mediated by T cell response. For the first time our results demonstrate that local secretion of Tag7-the molecule involved in innate immunity-may play an important role in the induction of effective antitumor response in mice.     

Evidence for the involvement of cytolytic macrophages in rejection of SV40-induced tumors

The potential role of cytolytic macrophages in in vivo resistance to tumors induced by simian virus 40 (SV40) was evaluated in two experimental systems. First, a cell line produced by sequential in vivo passage of SV40-transformed fibroblasts through syngeneic C3H/HeJ mice was found to develop both increased neoplastic character and resistance to macrophage-mediated lysis, suggesting in vivo selection pressure against the macrophage-sensitive phenotype. In the second approach, SV40-transformed cells from C3H.OL mice, a strain that fails to produce SV40-specific cytolytic T lymphocytes (CTL), were cloned, and the cloned cells were tested for susceptibility to macrophage cytolysis in vitro. Two clones SV-COL-E8 and SV-COL-F5, which represent the extremes of macrophage susceptibility and resistance, respectively, were tested for progressive growth in syngeneic C3H.OL recipients. Progression in vivo was found to correlate with resistance to macrophage cytolysis in vitro. Other in vitro measures of the neoplastic phenotype, cell division rate and anchorage-independent growth, did not predict the relative abilities of clones E8 and F5 to form tumors. Likewise, the cells were indistinguishable in their sensitivity to cytolysis by allogeneic CTL and by natural killer cells. Finally, the presence of activated macrophages in the peritoneum of mice rejecting a challenge of syngeneic SV40-transformed cells was confirmed in both CTL responder and nonresponder strains. These studies suggest that cytolytic macrophages are indeed generated during rejection of SV40-induced mouse tumors and that, in the absence of an effective anti-SV40 CTL response, resistance of the transformed cell to macrophage-mediated cytolysis can be a determining factor in in vivo tumor growth.     

Murine mammary carcinoma exosomes promote tumor growth by suppression of NK cell function

Many tumor cells shed specialized membrane vesicles known as exosomes. In this study, we show that pretreatment of mice with exosomes produced by TS/A or 4T.1 murine mammary tumor cells resulted in accelerated growth of implanted tumor cells in both syngeneic BALB/c mice and nude mice. As implanted TS/A tumor cells grew more rapidly in mice that had been depleted of NK cells, we analyzed the effects of the tumor-derived exosomes on NK cells. The tumor-derived exosomes inhibit NK cell cytotoxic activity ex vivo and in vitro as demonstrated by chromium release assays. The treatment of mice with TS/A tumor exosomes also led to a reduction in the percentages of NK cells, as determined by FACS analysis, in the lungs and spleens. Key features of NK cell activity were inhibited, including release of perforin but not granzyme B, as well as the expression of cyclin D3 and activation of the Jak3-mediated pathways. Human tumor cell lines also were found to produce exosomes that were capable of inhibiting IL-2-stimulated NK cell proliferation. Exosomes produced by dendritic cells or B cells did not. The presentation of tumor Ags by exosomes is under consideration as a cancer vaccine strategy; however, we found that pretreatment of mice with tumor exosomes blunted the protective effect of syngeneic dendritic cells pulsed ex vivo with tumor exosomes. We propose that tumor exosomes contribute to the growth of tumors by blocking IL-2-mediated activation of NK cells and their cytotoxic response to tumor cells.     

Lysis of fresh murine mammary tumor cells by syngeneic natural killer cells and lymphokine-activated killer cells

Summary We have compared the ability of natural killer (NK) cells from two substrains of C3H mice that differ with respect to their susceptibility to the development of mammary adenocarcinomas to lyse fresh syngeneic mammary tumor cells. Single cell suspensions of mammary tumors from retired breeder females were used as targets in 22-h ⁵¹Cr-release cytotoxicity assays with syngeneic NK cells. Tumor cell suspensions were prepared by enzymatic digestion of finely minced tissue followed by centrifugation through a discontinuous Percoll gradient. Effector cells were prepared by passing spleen cells over nylon wool followed by centrifugation through Percoll fraction 7. Syngeneic NK cells had significant levels of lysis against 5/8 tumors studied. NK cells from low risk animals (C3Heb/FeJ) consistently demonstrated greater cytotoxicity against tumor cell preparations than did effectors from the high tumor substrain (C3H/OuJ). Study of cytocentrifuge preparations stained with Wright-Giemsa revealed that the two substrains were identical with respect to the number of azurophilic granules present in the cytoplasm of their NK cells. We have also shown that lymphokine-activated killer (LAK) cells can be generated from splenocytes in C3H mice. While LAK cells from both substrains were capable of lysing fresh syngeneic mammary tumor cells in vitro, LAK cells from the animals at high risk for the formation of mammary adenocarcinomas had greater cytotoxicity against tumor cell suspensions than LAK cells from the low tumor substrain.     

Effector mechanisms of cytolytically activated macrophages. I. Secretion of neutral proteases and effect of protease inhibitors

Peritoneal macrophages from C56BL/6J mice, when activated by bacillus Calmette-Guérin, lysed syngeneic MCA-I sarcoma targets but not syngeneic embryo fibroblasts. Inflammatory macrophages elicited by concanavalin A (Con A) did not appreciably lyse either target. The activated macrophages secreted more neutral proteases into the extracellular compartment, both absolutely and relative to intracellular content, than did the Con A inflammatory macrophages. Bovine pancreatic trypsin inhibitor (BPTI) (750 KIU/ml) and diisopropylfluorophosphate (2 x 10(-3) M) inhibited cytolysis of neoplastic targets by the activated macrophages. The BPTI had to be present during the 48-hr macrophage-tumor cell interaction to reduce cytolysis; pretreatment of either the macrophages or the targets by the BPTI did not reduce cytolysis. The inhibitors, at the concentrations found to inhibit cytolysis, were not toxic to the macrophages as judged by morphology, by the ability of the macrophages to incorporate leucine into protein, and by the potential for cytolytic activation of the macrophages in vitro. It is suggested that neutral serine protease(s) secreted by activated macrophages participate in the cytolytic destruction of neoplastic cells.     

The immune response to a chemically induced fibrosarcoma

Summary A clone of C3H10T 1/2 fibroblasts transformed in vitro with the carcinogen 3-methylcholanthrene readily produced tumors when as few as 10³ cells were injected into immunocompetent adult syngeneic mice. A non-transformed clone of the same parentage did not produce tumors. Because the cell-mediated immune response has an important role in inhibiting the growth of tumors, we have compared the ability of both these transformed and non-transformed fibroblasts to stimulate and to act as targets in cell-mediated cytotoxicity (CMC) assays. This model is unique in that studies of the immune response to tumors rarely have or utilize appropriate normal controls. When both types of irradiated fibroblasts were used as stimulators in vitro, neither syngeneic nor allogeneic effector spleen cells capable of efficiently lysing the tumor fibroblasts were generated. In contrast, the normal fibroblasts could both stimulate and be lysed by allogeneic cytolytic T cells (CTL). However, the tumor fibroblasts could be lysed by allogeneic effector spleen cells that had been sensitized to C3H/He spleen cells. These results suggest that the expression of alloantigenic determinants necessary for stimulating a CMC response may vary substantially among normal cell types. They further indicate that the tumor cells are not resistant to lysis by appropriately stimulated effector cells. Thus, they must express antigenic determinants necessary for immune lysis and they do not inhibit the functional expression of cytolytic cells once generated. Consequently, tumor growth in vivo may be dependent, in part, upon a failure of the syngeneic host's immunocompetent cells to respond appropriately to the tumor cells. Additional data are provided which suggest that this failure is attributable in large part to immunosuppressive properties of the tumor cells.     

Bispecific antibodies retarget murine T cell cytotoxicity against syngeneic breast cancer in vitro and in vivo

Bispecific antibodies with specificity for CD3 and a tumor antigen can redirect cytolytic T cells to kill tumor targets, regardless of their natural specificity. To assess the clinical potential of bispecific antibodies for treatment of human cancers we have, in the present study, adapted a totally syngeneic mouse model to the targeting of mouse T cells against mouse tumors in immunocompetent mice. We show that gp52 of the mouse mammary tumor virus (MTV) can serve as a tumor-specific antigen for redirected cellular cytotoxicity. Chemically crosslinked and genetically engineered bispecific antibodies with specificities for gp52 and murine CD3 -chain induced activated mouse T cells to specifically lyse mouse mammary tumor cells from cultured lines and primary tumors from C3H-MTV⁺ mice. Retargeted T cells also blocked the growth of mammary tumors in vitro as well as their growth in syngeneic mice. These findings identify murine MTV-induced mammary adenocarcinomas as a solid-tumor, animal model for retargetin T cells with bispecific antibodies against syngeneic breast cancer.     

Specific inhibition of cytotoxic memory cells produced against UV-induced tumors in UV-irradiated mice.

Cytotoxic responses of UV-irradiated mice against syngeneic UV-induced tumors were measured by using a 51Cr-release assay to determine if UV treatment induced a specific reduction of cytotoxic activity. The in vivo and in vitro primary responses against syngeneic tumors and allogeneic cells were unaffected, as was the "memory" response (in vivo stimulation, in vitro restimulation) against alloantigens. In contrast, the memory response of UV-treated mice against syngeneic, UV-induced tumors was consistently and significantly depressed. The cytotoxicity generated by tumor cell stimulation in vivo or in vitro was tumor-specific and T cell-dependent. Since the primary response against syngeneic UV-induced tumors produces apparently normal amounts of tumor-specific cytotoxic activity, UV-treated mice may not reject transplanted syngeneic tumors because of too few T effector memory cells. These results imply that, at least in this system, tumor rejection depends mostly on the secondary responses against tumor antigens and that at least one carcinogen can, indirectly, specifically regulate immune responses.