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Mice infected intracerebrally (ic) or intraperitoneally (ip) with Pseudomonas aeruginosa were treated subcutaneously with graded doses of five antibiotics all of which displayed anti-pseudomonal activity in vitro. The analysis of MIC, ED50 in the ip infection model (ED50-ip) and that in the ic system (ED50-ic) for each agent showed: gentamicin, the most active agent in vitro, was also active in vivo, with an ED50-ic nearly six-fold higher than ED50-ip; ceftazidime, considerably active both in vitro and in vivo, showed comparable values of ED50 in the two systems; ceftriaxone, also active in vitro, showed limited activity in vivo, its two ED50 values being similar; aztreonam, moderately active both in vitro and in vivo, showed comparable activity in the two infection models; carbenicillin, the least active agent in vitro, gave poor results in vivo, with an ED50-ic 2.6-fold higher than ED50-ip.

The ability of mouse brain to provide T-dependent antilymphoma graft responses was studied in a model in which graded numbers of tumor cells were implanted intracerebrally into recipient hosts. By using survival criteria, it was possible to demonstrate the occurrence of both primary and secondary responses against tumor-associated histocompatibility antigens in allogeneic models as well as tumor-associated transplantation antigens in histocompatible recipients. The patterns of this intracerebral graft resistance did not differ significantly from those of peripheral T-dependent immune reactivity. The findings are discussed with regard to the concept of the brain as an immunologically privileged site.

Equitoxic doses of 5-(3-3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC) and aryl-triazene derivatives (compounds all capable of inducing a marked increase in murine tumor cell immunogenicity) were studied for their effects on the host immune system. At different times after drug exposure the animals were tested for allograft responses, competence in producing lymphocytes active in lethal graft-versus-host disease, delayed-type hypersensitivity, humoral antibody production, and mitogen responsiveness. While some of the aryl-triazenes tested (DM-COOK DM-NO2) showed a pattern of immunodepression similar to that of DTIC, others were less (MIC, MM-COOK, MM-Cl) or far less (DM-Cl, MM-NO2) active than DTIC in impairing host immunocompetence, although all retained or even augmented their ability to induce chemical xenogenization.

A chemically induced lymphosarcoma line (LSBM-1) of C57BL/10 (H-2b) origin lacks detectable TATA and is scarcely immunogenic for H-2-incompatible congenic recipients. New antigenic specificities, defined as drug-mediated tumor antigen(s) (DMTA) were found in a subline (LBD-1) obtained by in vivo treatment of LSBM-1 with the anti-neoplastic agent dimethyl-1-triazeno-imidazole-carboxamide (DTIC) over several transplant generations. It was concluded that the presence of detectable TATA is not a prerequisite for the induction of new antigenic specificities (DMTA).

The relationship between induction of novel immunogenicity by xenogenizing chemicals and DNA-methylating activity in murine tumors was investigated at the clonal level in L1210Ha cells treated with 5-azacytidine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 1-(p-chlorophenyl)-3,3-dimethyltriazene (DM-Cl). Cells were exposed to the drugs in vitro, cloned by limiting dilution, and assayed for transplantation immunogenicity and 5-methylcytosine content. The results showed that 0% (0/29, 5-azacytidine), 6.8% (2/29, MNNG) and 87.5% (28/32, DM-Cl) of the resulting clones were highly immunogenic, as judged by their tumorigenicity in intact compared to immunodepressed hosts. Frequency distribution analysis of the 5-methylcytosine content of drug-treated and parental clones showed that the methylation pattern was not significantly modified by tumor exposure to either 5-azacytidine or MNNG, and the two immunogenic clones induced by MNNG had methylcytosine levels very close to the 50th percentile value. In contrast, the extent of DNA methylation was increased in the cells treated with DM-Cl, but no obvious association was found between methylation status and immunogenicity of the drug-treated clones. In four 5-azacytidine-treated clones that displayed little or no immunogenicity, additional rounds of drug exposure led to progressive DNA demethylation, but failed, as a rule, to enhance tumor cell immunogenicity. Taken together, the present data indicate that, at least for the examined tumor, immunogenic variants are generated by mutagen treatment at high (MNNG) or very high (DM-Cl) frequencies under conditions in which hypomethylation-induced antigen amplification is unlikely.

To determine whether antigen-presenting ability might be involved in the superior immunogenicity of chemically xenogenized tumors over that of parental cells, we tested a murine lymphoma line xenogenized by a triazene derivative for expression of Ia antigens, ability to present soluble antigen in vitro, and production of factor(s) active in a mouse thymocyte assay. Results showed that Ia antigens, absent on nonimmunogenic parental L5178Y cells, were expressed on a xenogenized, highly immunogenic tumor variant (clone D), as detected by immunofluorescence. While the ability of parental cells to stimulate lymphocyte proliferation in vitro was lost on removal of Ia+ cells from the responder population, considerable augmentation of reactivity was observed upon depletion of Ia+ cells from the population of splenocytes responding to the xenogenized cells. Under these conditions, stimulation was blocked by anti-Ia antibodies, or an anti-L3T4 reagent or antibodies to the novel antigenic determinants induced by xenogenization. In addition, no stimulating activity was observed following exposure of clone D cells to glutaraldehyde or lysosomotropic agents such as chloroquine and ammonia. When the ability of clone D cells to present ovalbumin in vitro was assayed, it was found that the xenogenized cells could present the soluble antigen to specifically primed lymphocytes. Moreover, clone D cells could substitute for splenic adherent cells in the proliferative reaction of splenocytes to concanavalin A. Finally, when the supernate from clone D-cell culture pulsed with phorbol myristic acetate was tested in a mouse thymocyte assay, considerable IL-1-like activity was disclosed.

Novel drug-mediated tumor antigens (DMTA) have been detected in chemically induced L5178Y lymphoma of DBA/2 origin, following treatment of tumor-bearing hosts with 5-(3,3-dimethyl-1-triazenyl)-1H-imidazole-4-carboxamide (DTIC). Studies were conducted on the susceptibility of the DTIC-treated subline, maintained in tissue culture, to NK-mediated cytolysis in vitro. The results of the experiments showed that DTIC-treated lymphoma cells are less susceptible to NK cell lysis than the parental line (i.e. L5178Y); the DTIC-treated lymphoma, maintained in vivo as ascitic form, behaved as the corresponding in vitro line, thus suggesting that NK-resistant phenotype was not dependent upon propagation conditions. The intrinsic susceptibility to cell-mediated lysis of the DTIC-treated tumor was unchanged, as demonstrated by lysis produced by alloimmune cytotoxic cells or by natural cytotoxic mesenteric lymph node effectors. "Cold" competition experiments and target binding assay performed with L5178Y/tc and its DTIC-treated subline showed that DTIC-altered cells are less efficient than the parental line as "cold" competitor cells for NK-mediated lysis and bind less efficiently than L5178Y tumor to NK lymphocytes. These data suggested that the NK resistance of the DTIC-treated lymphoma may result from a failure to bind to effector cells, as a consequence of a profound rearrangement of the cell surface produced by DTIC treatment of cancer cells.