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The immunogenic Friend-Moloney-Rauscher (FMR) virus-induced tumors have been used extensively to clarify the cellular and molecular mechanisms responsible for tumor rejection and to develop immunotherapeutic strategies. We characterize here the trimolecular complex MHC class I-antigenic determinant-T cell receptor involved in the induction of a protective CTL response against the RMA thymoma. This complex is mainly composed by the D(b) molecule interacting with a Rauscher virus antigen (Ag) determinant and the Vbeta5+ T cell receptor. We also show that the chemically induced EL-4 thymoma acquires the susceptibility to recognition by anti-RMA CTLs and the ability to elicit a protective anti-RMA CTL response only upon infection by a virus of the FMR family and that RMA and FMR virus infected EL-4 cells share tumor-associated Ag. The data strongly support the hypothesis that the high immunogenicity of virus-induced or infected tumors is determined by the expression of immunodominant virus-encoded Ag. The demonstration of a different outcome in the immune responses elicited in the presence or in the absence of viral Ag further open the contention of the molecular requirements for immunogenicity and should stimulate a more careful revision of unexpected cross-reactivity among tumors.

We developed a syngeneic mouse IgG2a monoclonal antibody (MAb) A9D41 directed against the Friend leukemia virus envelope gp70 antigen present on the cell surface membranes of virus producer 3C18 Friend leukemia cells (FLC). A9D41 showed a marked antitumor activity in DBA/2 mice given injections of gp70 positive 3C18 FLC, but it was ineffective in mice given injections of gp70 negative 745 FLC or unrelated tumor cells. A9D41 was particularly effective in inhibiting the development of 3C18 FLC liver and spleen metastases. MAb was also effective as adjuvant therapy in inhibiting visceral metastases after excision of an established s.c. FLC tumor, and combined therapy of A9D41 with mouse interferon alpha/beta was more effective than MAb or interferon alpha/beta alone. The immune system of the host played a decisive role in the antimetastatic action of A9D41. Thus, although MAb was cytotoxic for 3C18 FLC in vitro in the presence of rabbit complement, the F(ab')2 fragment was ineffective in vivo, and the antitumor effect of MAb was abolished in mice treated with an antibody to CD4 and diminished in natural killer cell-deficient beige and athymic nude mice. MAb-treated mice surviving injection of FLC developed an immune response to 3C18 FLC.

Tumors may contain immunogenic antigens that are only recognizable in the context of class I, and not of class II, MHC molecules. Therefore, methods were developed to analyze the capacity of Lyt-2+ T-cells to respond to a syngeneic tumor in the absence of a contribution by L3T4+ T-cells. Conditions were defined in which purified Lyt-2+ T-cell populations, as well as L3T4+ T-cell populations, isolated from immune B6 spleen cells, could be induced to proliferate specifically in response to FBL, a retrovirally induced syngeneic tumor, without the addition of exogenous lymphokines. The purity of the subset responses was documented functionally by selective inhibition of the proliferative response of only the appropriate subset following addition of anti-Kb/Db or anti-I-Ab. The antigen and accessory cell (AC) requirements for triggering immune Lyt-2+ and L3T4+ T-cell populations were examined. The response of L3T4+ populations was predominantly specific for retrovirus envelope gp70, whereas Lyt-2+ populations predominantly recognized tumor antigens other than gp70, consistent with the hypothesis that some tumor antigens may be preferentially recognized by only class I- or class II-restricted T-cells. The FBL-stimulated proliferative response of each T-cell subset was dependent upon the presence of syngeneic AC. However, exogenous interleukin 1 was able to replace AC during the response of Lyt-2+ populations, whereas L3T4+ populations required AC also to biochemically process tumor-derived antigen and present it in the context of class II MHC molecules. The results suggest that under some conditions only the presence of AC or interleukin 1 may be limiting for the induction of antitumor responses by Lyt-2+ populations. These studies analyzed the ability to trigger purified Lyt-2+ T-cells in vitro following in vivo priming to tumor, and it remained possible that L3T4+ T-cells made an essential contribution during in vivo priming. Therefore, L3T4(+)-deficient mice were primed with FBL in vivo, and the Lyt-2+ T-cell response was assessed. Although priming was clearly less efficient in the absence of L3T4+ T-cells, Lyt-2+ T-cells from L3T4(+)-deficient mice proliferated and became cytolytically active following stimulation with FBL. Thus, under appropriate conditions, Lyt-2+ T-cells can generate an effective antitumor response in the absence of L3T4+ T-cells or exogenous lymphokines.

Vaccines prepared from purified viral envelope complexes are effective against certain animal model tumors induced by exogenous retroviruses. Related viruses have recently been isolated from humans and obviously cause adult T-cell leukemia and the acquired immunodeficiency syndrome. Knowledge accumulated in experiments with subunit vaccines against animal retroviruses could help to develop immunopreventive regimens against human retroviruses.

The influence of cyclophosphamide (CY) on Friend virus leukemogenesis was studied in SJL/J, C57BL/10J, and C57BL/10J X SJL/J F1 (hereafter called B10SJF1) mice. All three differ in their susceptibility to the viral oncogenic effect. Immunosuppressive doses of CY, which by themselves produced no cancer, were followed 2 days later by injection of Friend leukemia virus. The virus doses were the same as used previously. Although in other experiments preinjection of various chemical carcinogens augmented leukemogenesis by Friend leukemia virus in SJL/J mice, in the present study, pretreatment by CY had no such effect. In contrast, CY increased Friend erythroleukemia incidence from 15 to 100% in B10SJF1 mice and from 0 to 85% in C57BL/10J mice. The disease in C57BL/10J mice had a 190-day incubation period, which is approximately 5 times that in the SJL/J and B10SJF1 mice. During this latent period, the C57BL/10J mice harbored infectious Friend leukemia virus in their plasma.

Autogenous immune sera from several strains of mice have been examined for type-, group-, or interspecies-specific reactivities against leukemia virus envelope antigens and virus-induced cell surface proteins. The natural antibody of these test sera react with gp69/71, gp43, and p15 structural components on murine leukemia viruses including AKR, Friend, Rauscher, Moloney, and xenotropic BALB:virus-2. Furthermore, comparable radioimmune titration curves are obtained when these viruses are used in radioimmune precipitation assays. Competition experiments, however, suggest that natural immune sera are predominantly type specific and only weakly cross-react with the Rauscher or Friend virus. Natural immune sera react with the virion envelope but not with the virus-induced cell surface antigen. With respect to the biological activity of autogenous immune sera, there appears to be an inconsistency between the spectrum of virus-precipitating antibody and virus-neutralizing antibody. Although normal mouse serum readily neutralizes xenotropic viruses (BALB:virus-2), only weak neutralization of the ecotropic viruses can be achieved in vitro. Although there is a lack of direct evidence to indicate that autogenous immunity to murine leukemia virus is involved in the control of virus-mediated neoplasia, several empirical correlations point in this direction.

A strong transplantation resistance to a fibrosarcoma (KMT-17) is induced in syngeneic Wistar King Aptekman/Hok rats after a single s.c. immunization with Friend virus-infected (xenogenized) viable KMT-17 cells. The resistance induced by the repeated immunizations with irradiated Friend virus-infected KMT-17 cells, however, was unexpectedly weaker when compared with that induced by irradiated KMT-17 cells. The immunogenicity of tumor-associated antigen on xenogenized Friend virus-infected KMT-17 cells was correlated with their shortened survival time in the peritoneal cavity after i.p. inoculation, especially in rats preimmunized with xenogenized tumor cells. Furthermore, xenogenized tumor cells producing a medium amount of virus-associated antigen (VAA) but not those producing a large amont of VAA showed an augmented immunogenicity even in normal rats. On the other hand, the tumor-associated antigen immunogenicity was augmented by immunization with xenogenized tumor cells which expressed a relatively small amount of VAA in rats presensitized with VAA. These findings indicate that the immunogenicity of xenogenized tumor cells is augmented by the middle-grade immune responses to VAA produced on xenogenized tumor cells.

We have previously shown that mice simultaneously infected with the murine mammary tumor virus (MuMTV) and with certain slow murine leukemia viruses (MLV) have an increased resistance to the pathological effects of both agents. Here we report that milk-transmitted MuMTV also delays the development of the acute erythroleukemia induced by Friend leukemia virus (FLV), and retards, or prevents in some cases, the development of long-term lymphomas caused by its helper component. This is confirmed by the observation that the average life span of MuMTV-carrying mice infected with FLV or its helper component is prolonged by over 30% as compared to that of MuMTV-free animals infected with the same agents and by the finding that the replication of Friend viruses is reduced in mice neonatally exposed to MuMTV. Since the antibody responses of mice to MuMTV and to FLV were not cross-reactive, we searched for antiviral activity in the tissues of mice exposed to MuMTV, FLV, or the helper component of FLV. Low levels of interferon-alpha/beta were consistently detected in spleen extracts from mice infected with all these agents but could not be demonstrated in the spleens of uninfected BALB/c mice; thus, the chronic production of endogenous interferon is likely to play a major role in the reciprocal interference in vivo between retroviruses belonging to different genera.

The expression of the major glycoprotein, gp71, of murine leukemia virus was studied on the surfaces of a variety of normal murine cell lines with a monospecific rabbit antiserum raised against purified Friend murine leukemia virus gp71. Using viable cell membrane immunofluorescence, most established and early passage normal murine cell lines were significantly reactive with the antiserum, irrespective of neoplastic transformation, strain genotype, or whether they were of embryonic or adult tissue origin. The only murine cells tested not detectably expressing gp71 determinants were BALB/3T3 lines. Although some Friend gp71 interspecies reactivity was discernible on normal murine cells, the principal reactivity was shown to be group specific. Fresh thymocytes from BALB/cJ (GIX-), C57BL/6J (GIX-), and 129/J (GIX+) mouse strains were also reactive with Friend gp71 antiserum, and this activity, as well as that of an antiserum prepared against purified AKR gp71, were also group specific. An activity discriminating GIX+ from GIX- thymocytes was not observed with either Friend or AKR gp71 antisera.

Previous studies have demonstrated that spleen cells from DBA/2 mice protected against challenge with a leukemogenic dose of Friend leukemia virus (FLV) by passive administration of xenogeneic antiviral or anti-FLV Mr 71,000 viral envelope glycoprotein antisera can adoptively transfer antiviral resistance to unimmunized irradiated syngeneic recipients. In addition, elimination of T-cells by treatment with anti-Thy 1.2 antibodies plus complement had no effect on the ability of spleen cells from serum-protected mice to adoptively transfer antiviral resistance. We now show that similar depletion of B-cells with rabbit anti-mouse immunoglobulin G plus complement or macrophages by adherence to Sephadex G-10 columns also leaves intact the protective capacity of spleen cells from serum-protected mice. That these results reflect the ability of more than one spleen cell population to transfer antiviral resistance rather than the activity of a non-T, non-B, nonmacrophage cell compartment is supported by the finding that purified splenic T- or B-cells alone from serum-protected DBA/2 mice can adoptively transfer antiviral resistance. Given the previously reported effects of sublethal irradiation on FLV leukemogenesis which could potentially complicate the interpretation of adoptive transfer experiments carried out in this system, analogous studies were performed using a Winn-type assay in which putative effector cells were preincubated with virus before inoculation of the mixture in unirradiated mice. These Winn assay experiments yielded identical results in that serum-protected spleen cells again prevented viral leukemogenesis, and the separate elimination of T-cells, B-cells, or macrophages had no effect on their protective activity. In addition, mixed transfer of serum-protected and normal spleen cells also protected irradiated mice against FLV challenge, providing further evidence that this adoptive protection truly reflects the presence of virus-specific effector cells in the spleens of serum-protected mice and not an inability of these spleen cells to replace radiation-sensitive viral target cells in recipient animals, since these should be supplied by the normal spleen cells in the transferred mixture.

The levels of serum thymic factor(s) (STF), of Thy-1.2 positivity of splenocytes [as measured by their azathioprine (AZ) sensitivity], and of Thy-1.2-positive "spontaneous" spleen rosette-forming cells (SSRFCs), as well as the presence of infectious virus in the thymus, were assessed as a function of time after virus inoculation in susceptible DBA/2, partially resistant BALB/c, and fully resistant C57BL/6 mice given the polycythemia- or anemia-inducing strain of Friend leukemia virus (FLV-P and FLV-A, respectively). As early as Days 2 to 3, the levels of STF and of AZ sensitivity of splenocytes were profoundly decreased in DBA/2 mice, and, to a lesser extent, in BALB/c mice given FLV-P; however, SSRFCs/spleen were increased in both mouse strains. Conclusive evidence of infectious FLV-P was obtained in the thymuses of DBA/2 mice soon after infection. In mice of the same strains infected with FLV-A, STF levels were similarly decreased, but AZ sensitivity of splenocytes was unaffected, and SSRFCs were decreased. Evidence of early FLV-A infection in the thymus of DBA/2 mice was likewise obtained. In C57BL/6 mice given FLV-A, STF levels, AZ sensitivity of splenocytes, and SSRFC showed changes similar to, but of lower magnitude than, those in BALB/c mice. On the other hand, in C57BL/6 mice given FLV-P, the decrease in STF and AZ sensitivity was almost as pronounced as in susceptible DBA/2 mice in the face of complete absence of infectious virus or viral markers in the thymuses. The observed changes are ascribed to virus infection in view of the following: (a) good temporal correlation between these changes and virus infection; (b) absence of any change in mice given heat-inactivated viruses or spleen homogenate of normal DBA/2 mouse spleen; (c) overall good correlation between mouse genotype and genetic (Fv-1 and Fv-2) restrictions of virus infection on one hand and the magnitude of the observed changes on the other. In particular, the decrease in STF and SSRFC levels is ascribed to the replication-competent (Friend-murine leukemia virus) component of Friend leukemia virus complex, whereas the decrease in AZ sensitivity of splenocytes and the increase of SSRFCs are ascribed to the defective spleen focus-forming virus component of the complex. All changes described so far were transient, since they were not detectable beyond 42 days after virus inoculation in overtly leukemic animals. The observed derangements of thymus-derived immune functions may play an important cofactor role during the onset of leukemia in mice genetically permissive to Friend leukemia virus replication and transformation, but they do not seem relevant to the maintenance of leukemia.

Neonatal injections of Friend virus (FV) or Gross virus (GV) into rats produced immunological tolerance to the virus-induced tumors. The inoculation of specific immune lymphoid cells into the FV-induced tolerant rats brought about the runting syndrome, whereas the GV-induced tolerance was completely abrogated by the same procedure. To investigate the mechanism of the runting syndrome, rats were made doubly tolerant by neonatal injections of a mixture of FV and GV. The adoptive transfer of lymphoid cells from rats immunized with the FV-induced lymphomas into the doubly tolerant rats produced the runting syndrome. On the other hand, adoptive transfer of lymphoid cells from rats immunized with GV lymphomas into the doubly tolerant rats did not produce the runting syndrome and broke down the GV-induced tolerance but not the FV-induced tolerance. By the use of a complement-dependent cytotoxicity test, FV-infected cells were homogeneously detected in thymus, spleen, and bone marrow of the doubly tolerant rats, whereas GV-infected cells were detected only in the thymus. Studies with a rosette formation test as a rat thymus marker showed that none of the FV lymphomas formed rosettes with guinea pig erythrocytes, whereas GV lymphomas formed rosettes. These results suggest that FV and GV have different target cells for infection and transformation and that the development of the runting syndrome is closely associated with infection of bone marrow and spleen cells with FV.

The possible involvement of host macrophages in the passive serum therapy of Friend leukemia virus (FLV)-induced disease has been examined with the use of agents inhibiting normal macrophage functions, including silica and a tumor-produced macrophage chemotaxis inhibitor. Under conditions in which macrophage chemotaxis inhibitor. Under conditions in which macrophage functions are at least transiently abrogated by these agents, no effect was seen on the anti-fLV protection afforded by the passive administration of chimpanzee anti-FLV antiserum to infected DBA/2 mice, as monitored by the development of virus-induced splenomegaly and the level of infectious virus. The macrophage inhibitors also did not influence the appearance of the host antiviral humoral immune response which normally accompanies serum protection. These results suggest that the normal functioning of host macrophages do not play a central role in the passive serum therapy protective mechanism leading to resistance to FLV infection.

Cell-mediated immune reactions appear to play an important role in resistance against growth of leukemia cells in mice. Possible mechanisms for in vivo protection in two tumor systems are discussed. These tumor models, which are a Friend leukemia virus-induced transplantable tumor, FBL-3, and primary murine sarcoma virus (MSV) -induced tumors, are strongly antigenic; under some conditions, tumors regress completely. In mice with regressing FBL-3 tumors, cell-mediated cytotoxicity was measured by release of [125I]iododeoxyuridine. The response was biphasic, with an initial peak at 10 days and a 2nd peak after 30 days. A boost in reactivity could be elicited by later challenge with tumor cells. All of the reactivity was dependent on T-cells, being eliminated by treatment with anti-theta plus complement. The specificity of the reactions was not completely defined, but it was consistent with Friend type-specific antigen plus broader, common antigens. In mice with regressing MSV tumors, strong cell-mediated cytotoxicity, measured mainly by release of 51Cr, was seen against RBL-5, a Rauscher virus-induced leukemia. A single peak of response occurred at about 14 days after virus inoculation. Upon later challenge with RBL-5 cells, a vigorous and rapid secondary response was elicited, mainly in the region of tumor challenge. This cytotoxic reactivity and in vivo resistance to leukemia.lso was completely dependent on T-cells. In addition, macrophage-mediated inhibition of leukemia cell growth in vitro was seen in this system at the time of peak tumor development. The 51Cr release cytotoxicity was specific and directed primarily against an antigen, MEV-SA1, associated with mouse endogenous C-type viruses. The macrophage-induced growth inhibition appeared to be nonspecific. In both the FBL-3 and MSV tumor systems, protection against tumor growth could be adoptively transferred by immune lymphoid cells. In addition to induction of cell-mediated immunity by tumor cell or virus inoculation, cell-mediated cytotoxic reactivity was found to occur naturally in most young mice. This natural killer activity was quite distinct from the experimentally elicited reactions, being mediated by N-cells, a subpopulation of lymphoid cells with no clearly identifiable cell surface markers. The natural cytotoxicity was also directed against antigenic specificities different from those recognized by the MSV-immune cells. The central issue in all of these studies has been to determine the relationships between the in vitro-detected cell-mediated reactivity and in vivo resistance to leukemia.

The specificity of a single rabbit antiserum pool raised against the purified major glycoprotein, gp71, of Friend murine leukemia virus was determined for a variety of virus-producing mouse, feline, and gibbon ape cell lines by viable cell membrane immunofluorescence absorption. Among murine cells examined, Friend gp71 type specificity was shared only with Rauscher virus-producing cells, and a group specificity was present for all the murine leukemia virus-producing cells tested. Friend and Rauscher murine leukemia virus-infected cells shared interspecies cross-reactivity with feline leukemia and gibbon ape lymphoma virus-producing cells. However, Moloney, Gross, and other virus-producing murine cells shared some, but not all, of these gp71 interspecies determinants with the feline and primate cells. Immunoferritin electron microscopy localized these gp71 antigenic determinants on both virus and cell membranes.

The WKA/Mk rat tumors induced by Friend virus complex, Rauscher virus complex, and their associated lymphatic leukemia viruses were investigated for their antigenic relationhips with transplantation experiments and cytotoxicity tests. It was found that Friend lymphatic leukemia virus-induced tumors lacked part of the tumor-associated transplantation antigens (TATA's) on Friend virus complex-induced tumors, and the former did not express the type-specific (Friend) TATA for the latter not shared by Rauscher virus complex-induced tumors, which was previously reported by the authors. In contrast, the antigenic differences between TATA's of Rauscher virus complex-induced tumors and those of Rauscher lymphatic leukemia virus-induced tumors were not clearly demonstrated. Furthermore, these studies indicated that Rauscher lymphatic leukemia virus-induced tumors had a weak type-specific TATA not shared by the tumors induced by Friend lymphatic leukemia virus. These results of transplantation studies were also serologically supported by cytotoxicity tests.

Inhibition or enhancement of Friend leukemia virus disease could be produced by treatment of mice with the immunopotentiator, pyran copolymer. The result depended on the route of inoculation of the drug. Prophylactic administration of the drug i.p. retarded splenomegaly, reduced splenic foci, and increased survival time of mice infected with Friend leukemia virus. Conversely, when the same dose and regimen of pyran was administered i.v., splenomegaly was enhanced, splenic foci were increased, and survival time was decreased. Histopathological examination of the spleens of mice revealed that i.p. pyran administration caused a marked increase in the splenic marginal zone with some increase in erythropoiesis in the red pulp, while i.v. pyran administration did not markedly change the splenic marginal zone but caused an early and sustained increase in erythropoiesis in the red pulp.

The purpose of this study was to develop an animal system of protective immunity against oncornaviruses and to test whether such immunization had an inhibitory effect upon chemical sarcomagenesis. Several murine sarcoma virus (MSV) pseudotypes were used as immunogens and tested against themselves, against other pseudotypes, against leukemogenesis by their helper viruses, and against sarcomagenesis by 3-methylcholanthrene. Five MSV pseudotypes were obtained by rescuing complete MSV from MSV-genome carrier, nonproducer hamster tumor cells, using five different leukemia viruses as helpers. The immunogenic properties of these pseudotypes could be specified on the basis of the following observations. 1) They all induced sarcomas in newborn mice and regressing sarcoma nodules in young adult mice. After regression, most mice remained free of neoplastic disease, but some developed sarcoma or leukemia relapses. 2) They had an individual host range pattern, usually determined by the helper virus, as tested by inoculation of a constant virus dose in BALB/c, C57BL/Ka, and Swiss mice. 3) They were all immunogenic, in the sense that the first virus inoculation prevented sarcoma induction by a second challenge, either viral or cellular. 4) They were cross-reactive in vivo, one pseudotype immunizing against another, in the combinations tested. 5) They were able to immunize against leukemogenesis induced by their helper viruses. This was shown by prevention of leukemic deaths by Rauscher and Friend viruses, by a slight prolongation of survival after challenge with the Precerutti-Law leukemia virus, and by inhibition of splenomegaly by Moloney leukemia virus. In a second stage of the study, we investigated whether immunization with any of the MSV psuedotypes had an inhibitory effect upon sarcomagenesis induced by near-threshold doses of 3-methylcholanthrene. The incidence of these sarcomas was essentially the same in virus-immunized and control mice. It was concluded that immunizing procedures able to prevent sarcomagenesis when the inducer is a virus did not have any consistent preventive effect when the inducer was a chemical.