Genetic association of the humoral and cellular immune responses of rats to moloney sarcomas

Brown Norway (BN), Lewis (Le), F¹ hybrids of Le × BN (LBN) and parent-to-LBN backcross rats were tested for cellular and humoral responses to a BN Moloney sarcoma. Regardless of AgB phenotype, BN backcrosses produced low levels of cell-mediated cytotoxicity (CMC) that were comparable to those of BN parents. Le backcrosses developed high levels of CMC similar to those produced in Le parents. An inverse relationship between levels of CMC and serum antibodies (cytotoxic for tumor cells and anti-p30 of MuLV) was observed; BN parents and backcrosses produced high levels of serum antibodies whereas levels in Le parents and backcrosses were low. LBN hybrids developed relatively high levels of CMC and serum antibodies. An additional finding was that the CMC response in Le parents and back-crosses was directed primarily against tumor-associated antigens rather than histocompatibility antigens expressed on the tumor cells. The results suggest that humoral and cellular responses to Moloney sarcoma in rats are not determined solely by the major AgB histocompatibility locus but do have a genetic association. This genetic association was detected with a ⁵¹Cr release assay which detects T-cells, suggesting that select populations of effector T-cells may be genetically regulated.     

Alloantigen expression of a rat Moloney sarcoma.

Cells of a brown Norway (BN) rat Moloney sarcoma (MST) failed to express certain BN alloantigen specificities and bound only about 30-50% the amount of labeled alloantibody bound by normal BN spleen cells. MST cells lacked antigen specificities shared by BN and WF rats, but expressed some of the antigen shared by BN and AUG rats. Further loss of alloantigens that occurred with prolonged in vitro culture was associated with reduced virulence of MST cells for syngeneic hosts and with increased expression of tumor-associated antigens. The LEW rats, which are resistant to MST cells, might have rejected the tumor on the basis of factors other than Ag-B antigens.     

Cell-mediated reactivity to antigens shared by Moloney-virus-induced lymphomas (LSTRA) and certain 3-methylcholanthrene-induced mouse sarcomas.

Spleen cells (SC) both from BALB/c mice whose primary Moloney sarcoma virus (MSV)-induced sarcomas had spontaneously regressed and from normal, untreated BALB/c mice, were co-cultivated for 5 days with mitomycin-C-treated LSTRA cells; LSTRA is a BALB/c Moloney lymphoma which shares cell surface antigens with MSV-indiced sarcomas. These SC, referred to as CMR and CU cells, respectively, were shown to be cytotoxic to LSTRA cells in 3 h 51Cr-release assays; CMR cells showed, in most cases, the greatest lytic activity against LSTRA targets. The same SC were also reactive, in 20-h microcytotoxicity and 51Crassays, against target cells from a variety of transplanted sarcomas indiced by 3-methylcholanthrene (MCA) in Balb/c mice. The highest reactivity was seen when CMR or CU cells were tested against target cells from sarcoma lines that expressed an NB-ecotropic MuLV cross-reacting serologically with Moloney virus. Reactivity against isotope-labelled tumor cells expressing MuLV-associated cell surface antigens could be competititively inhibited by adding unlabelled tumor cells expressing such antigens. Finally, Winn assays were performed in which CMR cells strongly inhibited the outgrowth of cells from three sarcoma lines that express the NB-ecotropic MuLV. There was less but significant inhibition of cells from some other MCA sarcomas, either negative for the expression of MuLV-associated antigens or expressing the N-ecotropic endogenous BALB/c MuLV. CU cells enhanced tumor outgrowth in Winn assays at least as often as they inhibited it.     

Secondary in vitro generation of cytolytic T-lymphocytes (CTL's) in the murine sarcoma virus system. Virus-specific CTL induction across the H-2 barrier.

Following in vitro stimulation of murine sarcoma virus Moloney isolate (M-MuSV)-immune spleen cells with syngeneic antigenically related Moloney leukemia cells, highly efficient cytotoxic T-lymphocytes (CTL's) were generated. The cytotoxic effect was directed only against H-2-compatible target cells bearing M-MuSV tumor-associated antigens (TAA). However, in a cold target competition assay a weak but detectable capacity to block CTL activity was also obtained when allogeneic Moloney leukemia cells were added. Moreover, when M-MuSV-immune spleen cells from mice inoculated with virus 14 days previously were stimulated by allogeneic Moloney leukemia cells, a strong cytotoxic effect toward syngeneic and allogeneic tumor cells bearing M-MuSV TAA was elicited.     

Genetic control of immune responses to Moloney sarcomas in rats: role of non-RT-1 background genes

Bone marrow chimeras, athymic nude rats and a congeneic strain were utilized to verify and further examine non-RT-1 linked background genes that influence immune responses of BN and LEW rats to Moloney sarcomas. In transplants that did not involve RT-1 incompatibility, infusion of high-responder bone marrow into a lethally irradiated low-responder recipient, or low-responder bone marrow into a high-responder recipient, would restore a high antibody response to the gp70 antigen of MuLV. Such transplants did not restore a high response to the p30 antigen. Athymic nude rats did not exhibit a significant response to either p30 or gp70 while euthymic littermates exhibited a significant response to both antigens. Growth of Moloney sarcomas as well as antibody and cellular responses to antigens expressed by such tumors were measured in LEW-IN rats which carry the RT-1 of BN and the background of LEW. For each of these parameters, LEW-IN resembled LEW more closely than BN.     

Cell-mediated cytotoxicity to moloney sarcoma in syngeneic and allogeneic rats

Cell-mediated cytotoxicity in the primary immune response to Moloney sarcoma tumor (MST) in allogeneic and syngeneic rats was found to be predominantly T-cell-dependent. A minor non-T-cell cytotoxic activity may also have been detected. CMC was presumably directed against tumor and viral related antigens in the syngeneic host and primarily against alloantigens in the allogeneic host. CMC was more vigorous in the allogeneic recipient than in the syngeneic host. This may be due to differences in quantities or immunogenicities of the various antigens involved. Two peaks of T-cells in effector populations were observed during a 20-day post-inoculation period. The first peak corresponded to peak T-cell-mediated cytotoxicity on day 8 and the second peak occurred on days 13 or 14 when CMC was minimal or undetectable.     

Augmented immunogenicity of tumor cell membranes produced by infection with influenza virus as compared to Moloney sarcoma virus.

The tumor-associated transplantation antigens (TATA) of crude membrane extracts from SV40-transformed BALB/3T3 tumor cells lytically infected with influenza virus were markedly more immunogenic than were extracts from uninfected cells measured either by the ability to induce heightened resistance to tumorgraft challenge or by heightened lymphocyte-mediated cytotoxicity against tumor cells in vitro. When intact tumor cells (as opposed to membrane extracts) were productively infected with Moloney sarcoma virus, they were made so immunogenic that they would only grow in X-irradiated syngeneic animals. Yet crude membrane extracts from the Moloney sarcoma virus-infected tumor cells showed no increase in TATA activity analogous to that seen after infection with influenza virus. Thus, influenza virus augmentation of tumor membrane TATA may operate by a different mechanism than does the oncornavirus augmentation of intact tumor cell TATA reported by others. It appears that Moloney sarcoma virus and possibly other oncornaviruses cannot be used to augment the TATA activity of tumor cell membranes in the same way that other surface-budding viruses can.     

Inhibition of cell-mediated cytotoxicity against tumor-associated antigens by suppressor cells from tumor-bearing mice.

Spleen cells from mice bearing primary tumours induced by Moloney strain of murine sarcoma virus (M-MuSV) strongly inhibited the in vitro generation of specific secondary cell-mediated cytotoxic response of spleen cells from M-MusV regressor mice. These suppressor cells were resistant to treatment with anti-theta serum and complement or to X-irradiation. It appeared that suppressor cells may have had a role in limiting the host's immune response against tumor growth.     

Specificity of serum from rodents immune to Moloney C-type virus-induced tumours.

When cells are infected by C-type viruses such as Moloney sarcoma/leukaemia virus, new antigens appear on the cell membrane. Mice and rats will respond immunologically to the antigen(s). It was uncertain whether the antigens were related to the viral structural proteins or to non-virion, tumour-specific surface antigens (TSSA) or both. In an 125I-antiglobulin binding assay, Moloney virus completely blocked the binding of mouse and rat sera to virus shedding target cells, thus suggesting that mice and rats recognise only viral proteins. Mice responded to type-specific and rats mainly to group-specific determinants on the virus. Individual Moloney viral proteins were prepared using the guanidine HCl method and were used to block the binding of the rat anti-Moloney immune serum to Moloney virus shedding target cells. By this method, it was demonstrated that the rat serum contains specificities for the viral proteins gp70 and p30, but it was not possible to detect any antibodies directed towards non-virion or TSSA-like molecules.     

In vitro studies of cell-mediated immunity to Moloney murine leukemia virus and Moloney leukemia-associated surface antigens.

Cell-mediated immunity to Moloney murine leukemia virus (M-MuLV) and to tumor-associated surface antigens of leukemia cells induced by the virus was studied with an in vitro migration inhibition factor assay. Spleen cells of C57BL/6N mice at Day 14 following inoculation with Moloney murine sarcoma virus, produced migration inhibition factor in response to M-MuLV. The Moloney murine sarcoma virus-immune spleen cells, however, did not respond to other murine type C viruses, to AKR and Rauscher viruses, or to murine mammary tumor virus. The immune spleen cells also responded specifically to purified glycoprotein with molecular weights of 69,000 and 71,000 and proteins with molecular weights of 30,000 and 12,000, but not to protein with a molecular weight of 10,000, of the homologous M-MuLV. Migration inhibition factor production was also observed in response to soluble 3 M KCl extracts of leukemia cells, MBL-2, induced by M-MuLV. Similarly, the immune spleen cells responded to membrane fractions purified from the MBL-2 cells. Comparable membrane fractions prepared from a Gross virus-induced leukemia, E male G2, and a radiation-induced leukemia, RL male 1, were not active. The tumor-associated surface antigens of MBL-2 membranes could be solubilized by the detergent, Nonident P-40. Thus, C57BL/6N mice inoculated with Moloney murine sarcoma virus developed cell-mediated immunity to envelope and some internal antigens of M-MuLV and also to tumor-associated surface antigens of a tumor induced by this leukemia virus.     

Genetic control of immune responses to Moloney leukemia virus in rats.

When BN and LEW rats were immunized with untreated or with inactivated Moloney murine leukemia virus (M-MuLV), BN rats produced high antibody responses to the p15, p30, and gp70 antigens of the virus, whereas LEW rats were low responders to these antigens. BN rats also exhibited a high response and LEW rats a low response when the two strains were immunized with purified p30. Studies of (LEW X BN)F1 and backcross rats suggested that factors associated with AgB exerted major influences on responses to antigens of M-MuLV and that other factors were also important. When other rat strains representing 5 AgB alleles were tested, some were high and some were low responders to M-MuLV, and responses to p15, p30, and gp70 were not always parallel. Since M-MuLV replication was greater in cells of BN rats than in cells of LEW rats, replication of M-MuLV may have influenced the levels of responses to some viral antigens. Control of virus replication appeared to be due to cell mechanisms rather than to the environment of the host.     

The effect of alloimmunization on recurrence in an experimental colon cancer model

Primary bowel tumors were induced in Wistar/Furth (W/Fu) rats by 16 weekly injections of 1,2-dimethylhydrazine (DMH). After curative resection, 70% to 75% of control rats who receive no further treatment developed local or regional recurrence within 22 weeks. Rats immunized with three weekly subcutaneous inoculations of 1 X 10(6) irradiated (10,000 rad) Brown Norwegian (BN) X W/Fu F1 or Buffalo (Bu) tumor cells (both containing colon cancer tissue-type specific antigens [TSA] but differing from the W/Fu at Ag-B [histocompatibility] loci) developed recurrent tumor at a rate not significantly different from untreated or concurrent Ag-B antigen matched non-TSA treated controls. By 22 weeks after tumor resection, 63% of rats immunized with BN X W/Fu F1 colon adenocarcinoma and 52% of those immunized with Bu adenocarcinoma had recurred. Sixty-one percent and 44% were the respective recurrence rates for rats immunized with strain matched renal cell tissue. These data show nonspecific protection against tumor recurrence because of alloimmunization but clearly demonstrate the subordination of any beneficial colon cancer TSA immunotherapeutic effect by contained histocompatibility antigens. The problem of such nonspecific immune stimulation by alloantigen overriding an expected specific effect by TSA and possible alternative methods of immunization to prevent this phenomenon are discussed.     

Studies on murine sarcoma virus: II. Detection of group-specific antigens by immunofluorescence

The group-specific (GS) antigen of murine tumor viruses was demonstrated by immunofluorescence in mouse cells recently infected by mouse sarcoma virus, strain Moloney (MSV-M), with the serum of rats carrying long-transplanted MSV-M tumors. GS antigen was detected 15 h post-infection and was also present in various mouse and rat cell lines chronically infected with murine tumor viruses. The antigen was strictly localized in the cytoplasm of infected cells and was also found in mouse and rat cells chronically infected by members of the two major subgroups of murine tumor viruses. Further, the sera employed were shown to contain exclusively GS antibodies and the tumors used for immunization were found by several techniques to be free of virus envelope (V) antigens after a given number of passages in vivo. V antigens were visible only at the cell membrane and the time course of appearance of both GS and V antigens in recently infected cells was parallel. In contrast, GS antigen was not observed in two hamster tumor lines transformed by MSV-M.     

Studies on cellular immunity and its serum mediated inhibition in Moloney-virus-induced mouse sarcomas

A colony-inhibition technique was used to demonstrate lymph-node cell (LNC) mediated immune reactions against tumor-specific transplantation antigens of primary, Moloney sarcoma virus induced mouse sarcomas. Lymph-node cells from mice in which Moloney sarcomas had regressed spontaneously (regressors), as well as LNC from mice carrying progressively growing tumors (progressors), induced by virus inoculation at an age of 14 days or older, reduced the plating efficiency of Moloney sarcoma target cells. Serum from mice with progressively growing Moloney sarcomas, but not from mice with spontaneous mammary carcinomas or methylcholanthrene-induced sarcomas, abrogated the inhibitory effect of regressor LNC on Moloney sarcoma cells. Additional evidence for the specificity of the serum effect was obtained in experiments showing that the protective effect of progressor sera could be specifically removed by absorption with Moloney sarcoma cells. Regressor sera gave no protection against target cell inhibition by regressor LNC. It is suggested that progressor sera contain antibodies which mediate an efferent form of immunological enhancement.     

Immunotherapy of murine sarcoma by adoptive transfer of resident peritoneal macrophages proliferating in culture

Normal resident peritoneal macrophages from BALB/c mice were continuously grown and expanded in vitro as non tumorigenic cells on a confluent layer of mesothelial cells. These peritoneal macrophages expanded in vitro (EPM) were very cytotoxic against EMT6 sarcoma, Abelson myeloma, EL4, and L929S cells in culture. This tumoricidal effect was fully expressed without further activation with bacterial lipopolysaccharides (LPS). In vivo, adoptive transfer of one million EPM to BALB/c mice bearing subcutaneous EMT6 sarcoma caused regression of the solid tumor. In contrast, macrophages produced by 10 days' culture of bone marrow stem cells, or freshly isolated from the peritoneal cavity of BALB/c mice, were not cytotoxic in vitro or in vivo. Local injection in the vicinity of the tumor as well as intravenous transplantation of EPM effectively inhibited tumor growth. This antitumoral effect was further enhanced by intraperitoneal injection of 2 micrograms LPS to the tumor bearing mice.     

Murine sarcoma virus (MSV)-induced tumors in mice. I. Distribution of MSV-immune cytolytic T lymphocytes in vivo.

Quantitative studies using a 51Cr release assay were performed to analyse the time-course of appearance and specificity of cytolytic cells in C57Bl/6 mice inoculated with Moloney murine sarcoma virus (MSV). Various lymphoid organs were studied including spleen, mesenteric lymph nodes (MLN), lymph nodes regional to the MSV-induced tumor (RLN), and peripheral blood. Furthermore, the kinetics of appearance of cytolytic cells within the MSV-induced tumor was determined. In agreement with previous studied, it was found that cytolytic T lymphocytes (CTL) specific for MSV-associated antigens were present among the cells extracted from the tumor and from the lymphoid organs. However, differences in the kinetics of CTL activity could be documented: lymphoid cells from spleen, RLN and peripheral blood showed peak activity at the time of maximum tumor diameter, while intratumoral cells showed peak activity at the onset of tumor regression. MLN cells showed cytolytic activity only when tumor regression had begun. Naturally cytotoxic cell populations of thymus-independent origin, present in normal control mice, were also detected in MSV-infected mice.     

Cell-mediated suppression of tumor immunity has a non-specific component. II. Evidence from cell culture experiments

In vitro studies were performed to search for cells in the thymus of BALB/c mice with MCA-induced sarcomas which could suppress either a secondary immune response to MuLV-associated tumor antigens present on a Moloney leukemia virus-induced lymphoma, LSTRA, or a primary cytotoxic response to C57BL/6 alloantigens. They showed that thymocytes from mice with sarcomas were suppressive in both systems, and that suppression was independent of whether or not the sarcomas expressed MuLV antigen gp70. Furthermore, normal thymocytes, when combined with mitomycin-C-treated cells from any of several different MCA-induced sarcomas, suppressed the in vitro generation of a secondary cytolytic response to LSTRA. We postulate that exposure of mice to certain tumor antigens induces cells which, in the presence of the same tumor antigens which are not detectably present on the tumor cells inducing the response.     

The lymphocyte response to a primary viral neoplasm (MSV) through its entire course in BALB-c mice

BALB/c mice injected with Moloney sarcoma virus (MSV) developed tumors within 5–8 days which usually completely regressed by day 20–25. Maximal tumor size was around day 15. Lymphocytes from these animals were examined for activity against target cells bearing the Moloney leukemia virus (MLV) associated cell surface antigen at 2- to 5-day intervals for 30 days. Significantly cytotoxic lymphocytes were obtained from animals which had developed no palpable tumor as early as 2 days after infection and from animals in which tumors had completely regressed. The least active lymphocytes were from tumor-bearing animals. The cytotoxicity of the active lymphocytes was demonstrated to be antigen-specific by testing also against target cells which were MLV-negative.     

Expression of oncofetal antigens on murine sarcomas characterized for expression of endogenous MuLV.

A rabbit antiserum raised by repeated immunization with BALB/c fetuses obtained at 10-14 days of gestation was used to search for oncofetal antigens (OFA) in murine sarcomas which had previously been characterized for the expression of endogenous murine leukemia virus (MuLV). Iodinated protein A from staphylococcus aureus (IPA) was used to quantitate binding of the antiserum to cultured tumor or fetal cells or to saline extracts of tumors and fetuses. Use of the "antigen" extracts facilitated the assay: the extracts bound to plastic and served as targets for the binding assay, eliminating the need to establish tumors in culture. After absorbtion in vitro and in vivo with adult tissues the rabbit antiserum bound to day 10-14 fetal cells and extract but not to endogenous MuLV (BALB virus 1). The antiserum bound equally well to MuLV-negative and MuLV-positive sublines of MCA-induced sarcomas 1420 and 1414 but not to Moloney sarcoma cells and MCA-induced sarcoma 1386. Thus, the absorbed antiserum detects a class of common cross-reacting antigens which are serologically distinct from MuLV-associated antigens.     

Specific induction of local antitumor effector cells mediated in vivo by the circulating lymphocyte pool

Wistar rats are specifically resistant to the growth of the chemical carcinogen induced syngeneic tumors, Mc7 and Mc107 sarcoma, after being immunized with s.c. implants of irradiated tumor tissue. The central lymph of such immunized rats contains cells able to systemically transfer resistance against tumor growth to normal or irradiated recipient rats. The thoracic duct lymphocytes (TDL) from tumor immune donors are not directly cytotoxic against tumor targets. However recipients of i.v. infused immune TDL develop cytotoxic activity in the peritoneal cavity when challenged with the immunizing tumor at that site. Although the induction of maximum cytotoxicity is tumor specific, peritoneal lavage cells are cytotoxic against both Mc7 and Mc107 tumor targets in the 51Cr release assay. Inhibition of cytotoxicity in the assay by addition of unlabeled Mc7 or Mc107 sarcoma cells to labeled tumor targets suggests that there is both specific and nonspecific activity in these peritoneal lavage cells. Resistance to in vivo tumor growth in adoptively immunized recipients of TDL challenged with both Mc7 and Mc107 is specific for the immunizing tumor. However growth of a mixture of Mc7 and Mc107 sarcoma cells is inhibited in recipients of immune TDL. The results support the notion that mediator lymphocytes circulate in tumor immunized rats in a noncytotoxic state, specifically recognize tumor cells at a challenge site, and mediate induction of effector cells locally. These effectors are at least in part nonspecific in their cytotoxic activity.