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Exposure in vivo of murine L5178Y lymphoma cells to cytoreductive triazene derivatives leads to the generation of immunogenic variant lines expressing new transplantation Ags recognized by CTL. In one such clonal variant (clone D), at least one subset of T cell neoepitopes are provided by proteins previously shown by serology to be products of endogenous retroviral env sequences. We report here on characterization of PCR-amplified gp70 env genes in clone D. Relative to known gp70 sequences in parental cells and in current databases, one gp70 sequence presented four distinct nucleotide changes, two of which were apparently unique to clone D DNA and cDNA upon differential hybridization analysis. Transfection experiments with the entire gp70 gene or subgenic fragments encompassing a single putative mutation showed that products of the mutated env gene or fragments may confer immunogenicity in vivo and susceptibility in vitro to lysis by clone D-primed, H-2Kd- or H-2Ld-restricted CTL. By skin test assay of mice primed with either clone D or three mutated synthetic peptides, evidence was obtained that amino acid substitutions at the relevant positions of the gp70 protein may produce immunogenic T cell epitopes and that these epitopes are expressed in vivo by clone D. These data point to the role of mutated retroviral tumor peptides as rejection Ags in histocompatible hosts.

The occurrence of aberrant, retrovirus-related proteins is a common finding in immunogenic clones of the triazene-xenogenized L5178Y lymphoma line (L5178Y/DTIC). In clone D-cells, newly expressed 80 kDa antigens related to xenotropic murine leukemia virus env gene products induce specific humoral and cell-mediated responses and possess biologic activity in vivo. To further clarify the relationship between immunogenic properties of clone D and retroviral gene expression, tumor cells were treated in vitro with antisense oligonucleotides complementary to xenotropic and/or polytropic env sequences of murine leukemia virus. The cells were then assayed for expression of antigens recognized by humoral and cell-mediated responses with specificity for clone D. The results demonstrated that inhibition of env mRNA translation adversely affected the expression of immunogenic determinants in the xenogenized tumor cells.

Highly immunogenic tumor variants are generated by in vitro or in vivo treatment of L5178Y murine lymphoma cells with triazene derivatives. Most of these variants express new transplantation antigens which are not present on the original L5178Y tumor cells. In this study, a polyclonal syngeneic antiserum raised to one such variant (L5178Y/DTIC) was employed in immunoprecipitation studies of cell surface and metabolically labeled L5178Y/DTIC cells. One- and two-dimensional electrophoretic analyses of the immunoprecipitates detected a surface antigen of approximately 80 kDa. Additionally, a 45-kDa component was detected in the lysate of [35S]methionine-labeled cells. Anti-xenotropic MuLV gp70 serum precipitated material whose electrophoretic pattern was similar to that of the 80-kDa surface antigen. Sequential immunoprecipitation analysis revealed that the molecules reactive with the variant-specific antiserum were removed by the anti-xenotropic gp70 antibodies, whereas immunodepletion was only partial when the cell extract was first treated with the variant-specific antibodies. After Western blotting, the 80- and 45-kDa antigens precipitated by the variant-specific antibodies were injected intrasplenically into recipient mice. Only the animals sensitized with the 80-kDa antigen developed specific immunity to L5178Y/DTIC cells in that they displayed an increased frequency in CTL precursors (CTLp) to the variant cells. Sera from mice sensitized to the 80-kDa protein specifically inhibited the development of a primary CTL response to L5178Y/DTIC cells.

Xenogenized variant cells (L5178Y/DTIC) of a murine lymphoma line confer a high degree of specific protection against subsequent challenge of mice with parental L5178Y cells. In an attempt to better define the effect of DTIC-induced determinants on parental antigen recognition and the mechanisms involved in this protection, we evaluated the frequency of anti-parental tumor cytotoxic T lymphocyte precursors following priming of mice with xenogenized cells in vivo. In addition, we tested the effect of host sensitization with the immunogenic, retrovirus-related proteins that are precipitated from the surface of L5178Y/DTIC cells by means of specific antibody. Our results indicated that the novel determinants induced by DTIC treatment on L5178Y cells do not act as helper determinants for the generation of tumor-specific cytotoxic responses. Therefore, increased frequency of tumor-specific cytotoxic lymphocytes does not seem to be a major mechanism of anti-parental tumor immunity induced by xenogenized variant cells.

Treatment of murine lymphomas with triazene derivatives may lead to the appearance of novel drug-mediated tumor antigens, a phenomenon known as chemical xenogenization. Such antigens, which are capable of eliciting specific transplantation resistance in histocompatible mice, have been previously detected by in vivo and in vitro cell-mediated immune responses. In the present report we address the question of the humoral antibody response to a chemically xenogenized lymphoma. Histocompatible mice were given several injections of live cells of the xenogenized tumor. Ten days after each immunization, pooled sera from different animals were analyzed for Ab content by means of flow microfluorometry analysis and CEL-ISA assay. The results reveal that antibodies of both IgG and IgM classes capable of binding the xenogenized tumor can already be detected after one single sensitization. However, the Ab titer gradually increases through subsequent immunizations, reaching a peak level after 3-4 injections at a time when most of the humoral response is made up of antibodies of IgG class. The specificity of the anti-xenogenized tumor hyperimmune sera was subsequently investigated by its reaction with the parental, non-xenogenized line and with normal tissue cells of the same or allogeneic haplotypes. The data obtained point out that cross-reactivity with the parental line could be completely removed by absorption of the hyperimmune sera on parental cells, which removed most of the IgM antibodies. Moreover, the presence of an excess of anti-parental antibodies on the xenogenized tumor cells does not prevent the subsequent binding of the hyperimmune absorbed serum, thus indicating that the novel determinant(s) recognized on xenogenized cells are not spatially related to those shared with the original parental tumor. In addition, the hyperimmune absorbed serum does not cross-react with normal hemopoietic or lymphoid cells of the same (H-2d) or allogeneic H-2b and H-2k haplotypes. Furthermore, no alien histocompatibility antigens of H-2b or H-2k haplotypes could be detected on the xenogenized tumor cell surface. Taken together, these data provide evidence that chemical xenogenization of a murine lymphoma leads to the appearance of novel determinant(s) detectable by specific antibodies.

T cell-mediated proliferative and cytotoxic responses occur in vitro to syngeneic tumor cells antigenically altered by mutagen treatment. One such xenogenized variant of the murine L5178Y lymphoma elicits IgG antibodies reactive with determinants on variant cells that are not expressed at detectable levels on parental or normal cells of the same H-2d haplotype and are also unrelated to public specificities of H-2b or H-2k histocompatibility antigens. In the present study we investigated the effect of those antibodies on development of cell-mediated responses in vitro to the xenogenized cells used for induction of the humoral response. The proliferative reaction, generation of cytolytic activity and target cell lysis were all inhibited by the anti-xenogenized tumor immune serum, whereas the corresponding reactions to the parental cells by syngeneic or allogeneic effector lymphocytes were not. In order to investigate the possible H-2 association of T cell-mediated responses to xenogenized cells, we also examined the effect on those reactions of antibodies specific for Class I or Class II products of the H-2d complex. The results obtained suggested a role for I-Ad molecules in the T cell proliferative response to the xenogenized cells, and also indicated a preferential association of the cytotoxic response with H-2Kd determinants.

n vivo exposure of tumor-bearing mice to the antineoplastic agent 5-(3,3-dimethyl-1-triazenyl)-1H-imidazole-4-carboxamide (DTIC) results in increased immunogenicity of tumor cells, an event often referred to as "chemical xenogenization" (CX). To verify the hypothesis of DTIC-induced somatic mutation(s) as the major mechanism underlying CX, studies were performed to dissociate CX from the onset of drug resistance, which was invoked in the past as an event leading to selection of preexisting immunogenic clones. Therefore, experiments were done with a DTIC-susceptible tumor line treated with DTIC and quinacrine dihydrochloride (Q), an antimutagenic compound, according to selected experimental schedules. At different transplant generations, the CX and the onset of drug resistance were evaluated. The results show that a) Q does not prevent the onset of DTIC resistance, b) DTIC-resistant clones arising after treatment with DTIC plus Q are not immunogenic, and c) CX is selectively antagonized by Q. The present data confirm that DTIC-induced immunogenicity is not the result of a selection mechanism mediated by the drug and give further support to the hypothesis that the molecular mechanism of CX may be related to somatic mutation(s).

Drug-mediated tumor antigens (DMTA), which appear after in vivo treatment of murine lymphomas with 5-(3,3'-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC), are shown to elicit strong primary cell-mediated responses in vitro. Specific cytotoxic response is obtained using CD2F1 (H-2d/H-2d) spleen cells cocultured in vitro for 5 days with inactivated histocompatible L5178Y/DTIC lymphoma cells in a one-way mixed lymphocyte tumor culture. Sensitized lymphoid cells are markedly cytotoxic for L5178Y/DTIC targets and are minimally or not active against the parental lymphoma. Marginal or no cross-reactivity is found between L5178Y/DTIC lymphoma and other untreated or DTIC-treated tumor or normal cells. Cytotoxic activity is T dependent, since it is abrogated by treatment in vitro with anti-serum and complement and is not elicited by spleen cells of athymic (nude) donors.

Previous work has shown that murine lymphoma cells antigenically altered ("xenogenized") by drug treatment elicit strong in vivo resistance to the original cells. Moreover, splenocytes immune to a drug-treated variant (L5178Y/DTIC) of a murine lymphoma exert anti-parental tumor activity in an adoptive transfer system, an effect mediated by L3T4+ lymphocytes and associated with the detection of an anti-L5178Y delayed-type hypersensitivity (DTH) response. We now report that the in vivo activity of the tumor-immune L3T4+ lymphocytes is a radio-sensitive (2,500 rad in vitro) phenomenon that requires collaboration with radio-resistant, silica-sensitive syngeneic cells in the host, and is inhibited by treatment of recipient mice with monoclonal antibodies (MAbs) to the L3T4 antigen or murine interferon-gamma (IFN-gamma). In vitro, the tumor-immune L3T4+ lymphocytes produce interleukin 2 (IL-2), lymphotoxin (LT) and IFN-gamma activities on incubation with L5178Y cells and spleen-adherent cells. These results suggest that the mechanisms of anti-parental tumor protection by xenogenized cells involve specific induction of a DTH response mediated by the "inflammatory" (THI) subset of L3T4+ T lymphocytes and IFN-gamma activated macrophages.

An antigenically altered DTIC-treated tumor subline was tested for sensitivity to the cytotoxic activity of murine Natural Killer (NK) cells in comparison to that of the original (parental) tumor. Direct lysis assays and "cold" inhibition procedures showed that the DTIC-subline is consistently less susceptible to lysis that the parental tumor.

In an attempt to elucidate the cellular mechanisms responsible for the therapeutic activity of systemic immunotherapy in an adoptive transfer system, lymphoma cells were implanted i.c. It was found that, upon peripheral injection of cytotoxic T-lymphocytes with specificity for the tumor, the cells reached and infiltrated the diseased brain but did not accumulate selectively in the malignant graft. In order to accomplish significant tumor inhibition, the infused lymphocytes, largely expressing the Lyt-1+2+ phenotype, apparently cooperated with radioresistant phagocytic cells present in histocompatible hosts and athymic mice.