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In vivo treatment of mouse leukemia L1210 with DTIC can induce new antigens on tumor cells that are not detectable on parental cells and that are transmissible as a genetic character. Moreover, L1210/DTIC is rejected by syngeneic hosts. The aim of this study was to investigate whether DTIC selects pre-existing immunogenic clones rather than inducing ex novo new antigenic determinants and to verify the number of induced antigens. L1210 leukemia was cloned in vitro and 4 clones were treated in vivo with DTIC. All the treated clones displayed antigenic properties since they were rejected by syngeneic hosts. Cytotoxic T lymphocytes (CTL) activated against one DTIC clone could recognize and lyse the relevant target. One of these DTIC-modified clones (L4/DTIC) was recloned and the subclones were tested in vivo and in vitro. Two out of six subclones were rejected by syngeneic hosts. CTL specific against these two clones were able to recognize and lyse all the other clones to different degrees. The degree of susceptibility to lysis did not correlate with the capability to evoke an immune response in vivo. Based on these findings we conclude that DTIC does not select pre-existing clones but modifies the tumor cells antigenically, and that the antigenicity induced by DTIC in a cloned tumor line is due to the presence of common antigens shared to different degrees with treated cells.

Antineoplastic compounds can induce on tumour cells new antigens that undetectable on parental cells and which are transmissible as a genetic character. In this study mouse leukaemia L1210 was cloned in vitro by limiting dilution and one cloned line was recloned in vivo. Four subcloned tumour cell lines (A,D,R,S) were xenogenized in vivo by DTIC treatment (A/DTIC, D/DTIC, R/DTIC, S/DTIC) following a schedule previously described. Up to 10(7) cells of these xenogenized subclones, injected i.p., were rejected by syngeneic hosts, although they grew in immunosuppressed hosts. The DTIC treated subclones were lysed by in vivo-primed, in vitro-restimulated (with the relevant subclone) lymphocytes. The cytotoxic lymphocyte activity was not strictly specific since parental, DTIC-untreated cells were also lysed, although less efficiently. CTL directed against the D/DTIC subclone were cloned by limiting dilution. Ninety-four CTL clones were assayed against L1210 subcloned cells, DTIC-treated and untreated, and against different murine tumours (syngeneic or allogenic). Three specific antigens could be identified in the 51Cr release assay. The DTIC subclones expressed one antigen that was specifically recognized by a set of CTL clones. A number of CTL clones were able to lyse the L1210 subcloned cell exclusively, targetting a tumour-associated antigen that did not appear to be modified in the DTIC-treated subclones. A third antigen was demonstrated in the parental and DTIC treated D subclone. On the basis of these results it was postulated that there was at least one common DTIC-inducible antigen specific and reproducible within an identical cell population. Moreover, DTIC treatment did not modify histocompatibility antigens or TAA pre-existing in L1210 cells. The findings discussed here provide new information about permanent xenogenization of tumour cells, which might be exploited for experimental chemo-immunotherapy of cancer.

The reticulum cell sarcomas (RCS) of SJL/J mice are of particular interest since they readily induce the proliferation of syngeneic T-lymphocytes. Previous cellular studies examined the antigens on the RCS which stimulated this response and suggested that the tumor expressed allogeneic I-region-associated (Ia) antigens normally associated with the E alpha:E beta molecular complex (S. M. Wilbur and B. J. Bonavida, Exp. Med., 153: 501-513, 1981). These particular Ia glycoproteins are not expressed on normal SJL/J cells due to a defect in the E alpha polypeptide synthetic pathway. However, the E beta subunit is synthesized normally by these animals but remains intracellular. The SJL/J-derived RCS may circumvent this defect in E alpha subunit biosynthesis. The aberrant synthesis of this polypeptide is thought to allow membrane presentation of an intact pseudoallogeneic Ia glycoprotein which utilized the normally dormant E beta s polypeptide. In the present study, two monoclonal antibodies directed against the Ia.7 specificity of the E alpha chain (13/18, 14-4-4S) were used to examine more directly the expression of this polypeptide on the tumor. Surprisingly, neither antibody was effective against the RCS in a direct complement-mediated cytolysis assay. Nevertheless, the tumor was found to specifically adsorb lytic activity of both the monoclonal antibodies. In addition, both a cold-cell competition assay and indirect immunofluorescence corroborated the data and indicated that the RCS does express detectable levels of the Ia.7 antigen. Normal spleen cells and lipopolysaccharide B-derived blasts from SJL/J mice were found in all experiments to be devoid of any specific reactivity with these monoclonal antibodies. In addition, continued in vivo passage of transplantable RCS was found to cause down-modulation of the Ia.7 specificities on these tumors. Newer RCS transplantable lines, however, expressed demonstrable levels of this alloantigen in both cellular and serological assays. The observed down-modulation could explain the difficulties encountered in defining this specificity on long-term transplantable RCS. In conclusion, the present serological study corroborates the early cell-binding data. An Ia.7 antigen is shown to be expressed on the RCS, yet this specificity could not be detected on normal SJL/J cells.

Treatment of murine tumors with the anti-tumor agent 5-(3,3 dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC) has resulted in the induction of antigenic specificities not found in parental cells. After the withdrawal of DTIC treatment, the antigenic sublines maintained the new properties indefinitely, as a heritable character although the mechanism of induction and the molecular nature of the new antigens are essentially unknown. Seven clones from the murine immunogenic leukemia L1210/DTIC were selected and studied in some detail. Three of the seven clones showed an increased immunogenicity in vivo since two clones (D5 and D7) were rejected by syngenic mice and one (D6) prolonged the life span for 3 weeks (untreated tumours killed the mice in 7 days). The seven clones and the L1210/DTIC were recognized and lysed by in vivo primed, in vitro stimulated (with L1210/DTIC) lymphocytes. Therefore, the seven clones shared antigens with the L1210-DTIC immunogenic subline. Secondary stimulated lymphocytes to clone D5, and clone D7 were able to lyse D5, D6 and D7 cells, respectively, but were unable to recognize the remaining clones. From in vivo and in vitro studies, all the L1210/DTIC sublines are composed of cells carrying two or more distinct antigens. Each cell clone expressed one set of antigens. It was concluded that only a few antigens expressed in different cells were induced by a DTIC treatment of L1210 leukemia.

The treatment in vivo with anti-tumour drugs can induce an antigenic alteration of L1210 leukemia resulting in the rejection of an inoculum of 10 X 10(6) viable cells in syngeneic mice. As drug-induced antigens appeared in excess of any pre-existing tumour-associated transplantation antigens (TATA), viable altered cells have been used to sensitize syngeneic animals. Experiments showed that viable altered cells elicited stronger anti-TATA reaction than X-inactivated parental cells, as measured by host survival to a challenge of L1210 leukemia. TATA immunogenicity of parental cells has been preminent, in the strain of animals used, to determine the sensitizing effectiveness of 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) cells. Host protection to an inoculum of parental tumours has been more proficient with the DTIC subline derived from highly immunogenic L1210Ha cells than from poorly immunogenic L1210Cr cells. Immunoprophylactic inocula, proper chemotherapeutic treatments and adoptive transfer of immune lymphocytes used in combination exerted a synergic host protection.