T-cell subsets, IFN-gamma production and efferent specificity in anti-parental tumor immunity induced by mouse sensitization with xenogenized variant cells

In addition to previous evidence for a role of L3T4+ T cells in the protective anti-parental tumor immunity induced by xenogenized variant cells of a murine lymphoma (L5178Y/DTIC), we have investigated the possible participation in this effect of L5178Y tumor-specific lymphocytes of the Lyt-2+ T cell subset. Spleen cells from L5178Y/DTIC tumor-immunized mice produced high levels of IFN-gamma in vitro in response to parental antigens, and this activity was only abolished by treating the responder population with anti-Thy-1.2 antibody or a combination of anti-L3T4 and anti-Lyt-2.2 monoclonal antibodies (MAbs) plus complement. Positively selected L3T4+ and Lyt-2+ cells also produced IFN-gamma in vitro, provided accessory cells (plastic-adherent and Thy-1- Ia- splenocytes, respectively) were added to the lymphocyte-tumor cell cocultures. The production of IFN-gamma by purified L3T4+ and Lyt-2+ cells was inhibited by addition of the respective anti-class-II and anti-class-I H-2 antibody to the cultures. Administration of anti-IFN-gamma MAb in vivo significantly impaired the resistance of L5178Y/DTIC-immune mice to challenge with parental cells, as manifested by survival criteria and increased tumor-cell proliferation in the spleens of antibody-treated mice. Although anti-parental tumor protection in vivo and T-cell activation in vitro for IFN-gamma production were strictly antigen-specific, bystander tumor inhibition was observed when antigenically irrelevant cells were inoculated with the L5178Y lymphoma. These results suggest that both L3T4+ and Lyt-2+ T cells play a role in the protective anti-parental tumor immunity induced by xenogenized cells, and that their activity may involve IFN-gamma-mediated stimulation of non-specific tumoricidal mechanisms.     

Induction of tumor suppression and delayed-type footpad reaction by transfer of lymphocytes sensitized to a xenogenized tumor variant

Splenocytes immune to a highly immunogenic ("xenogenized", L5178Y/DTIC) variant of a murine lymphoma exert anti-parental-tumor activity in a systemic adoptive transfer system, the effect being apparently associated with the Lyt-2- fraction of the lymphocyte population. During investigation of the mechanisms of this protection, we found that the L5178Y/DTIC tumor-immune lymphocytes exhibited an appreciable anti-L5178Y delayed-type hypersensitivity (DTH) response. Enrichment of those lymphocytes in L3T4+ cells significantly enhanced the protective effect as well as the DTH reaction, whereas the use of an anti-L3T4 but not anti-Lyt-2 reagent blocked both activities. In vitro, lymphocyte proliferation against L5178Y cells occurred and was apparently associated with the Lyt-2- fraction of a population of L5178Y/DTIC immune splenocytes.     

Antigenic changes of L5178Y lymphoma after treatment with 5-(3,3-dimethyl-1-triazeno) imidazole-4-carboxamide in vivo.

Immunologic alteration of the L5178Y lymphoma was obtained in vivo after treatment with 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DIC). A single dose of 1,3-bis(2-chlorethyl)-1-nitrosourea (BCNU) "CURED" MICE CHALLENGED WITH L5178Y cells that had been treated with DIC (L5178Y/DIC) for four transplant generations; BCNU did not cure mice bearing the parent tumor. The L5178Y/DIC, treated in vivo for five transplant generations, id not grow in syngeneic mice. L5178Y/OIC cell growth and incidences of death were similar to those of parent cells when inoculated into heavily immunosuppressed mice. Adoptive transfer of lymphocytes from spleens of mice sensitized to the drug-altered tumor specifically protected immunosuppressed mice bearing the L5178Y/DIC tumor. Little protection was afforded by lymphocytes immune to the parent L5178Y tumor, whereas nonimmune lymphocytes or lymphocytes immune against unrelated tumors were completely ineffective. Anti-L5178Y/DIC lymphocytes did not cure mice challenged with the parent L5178Y tumor. Irradiated (400 R) mice previously sensitized to L5178Y/DIC cells rejected 10(2)-10(7) inocula of L5178Y/DIC cells and died when the parent L5178Y was used for challenge. It was concluded that antigeni( alterations of L5178Y cells occurred in (BALB/ctcr X DBA/2Cr)F1 mice after treatment with DIC in vivo.     

Effects of horse and fetal calf serum on the expression of tumor-associated antigen and tumorigenicity of L5178Y leukemia/lymphoma cells

A tumor antigen (TA) associated with murine leukemia-lymphoma L5178Y cells has been identified by the enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF) techniques. The antigen was present in both non-solubilized and 0.5% NP-40 solubilized membrane extracts. Rabbit anti-L5178Y lymphoma serum (RALS), extensively absorbed with normal mouse tissues, identified TA in extracts of L5178Y lymphoma and L5178Y leukemia cells grown in horse serum (L5178Y/HS), but not in extracts of L5178Y cells grown in fetal calf serum (L5178Y/FCS). Similarly, absorbed rabbit anti-L5178Y/HS serum specifically reacted with extracts of lymphoma and L5178Y/HS but not with L5178Y/FCS cells. Membrane IIF showed positive reactivity in 88% of lymphoma and 73% of L5178Y/HS cells, whereas splenic lymphocytes and L5178Y/FCS cells were negative. Goat anti-AKR virus serum reacted with soluble extracts of lymphoma, L5178Y/HS, and L5178Y/FCS as well as with normal DBA/2 tissues in the ELISA. However, goat anti-AKR virus serum did not block the reactivity of RALS to lymphoma in the blocking ELISA (BELISA). Expression of TA, but not murine leukemia viral antigen(s), was correlated with the in-vivo tumorigenicity of the L5178Y cells. The antigenic activity of lymphoma extract was reduced by incubation for 1 h at 56 and 65 degrees C, by trypsin digestion, and by exposure to pH 2.8 or 11.0 for 1 h. The antigen, sequentially purified by gel filtration and Lentil-lectin affinity chromatography, was a glycoprotein, with a molecular weight of approx. 64,000 daltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.     

Murine natural anti-tumor antibodies. I. Rapid in vivo binding of natural antibody by tumor cells in syngeneic mice.

A competitive radioimmunoassay (RIA) for the detection of cell-bound antibody was used to study the in vivo acquisition of immunoglobulin (Ig) by tumor cells. Several tumor lines acquired Ig rapidly between 3 and 18 h after intraperitoneal implantation into normal syngeneic mice and this Ig was recovered by elution with basic or acid buffers. The Ig eluted from the L5178Y lymphoma showed higher binding to the L5178Y than to thymocytes, bone-marrow cells, 1509a sarcoma and P-815-X2 mastocytoma. In addition, binding of the eluates to the L5178Y was specifically inhibited by L5178Y cells or by solubilized membrane antigens of the L5178Y. The in vivo acquisition of Ig by the L5178Y could also be blocked by the IV and IP injections of tumor antigen although both L5178Y and 1509a solubilized membrane antigens were effective. Some of the Ig acquired by the tumor cells was found to be complement-fixing antibody since normal rabbit complement lysed 80% of L5178Y cells obtained from the peritoneal cavity of syngeneic mice 18 h after implantation, but did not lyse in vitro L5178Y cells. The in vivo binding of the complement-fixing antibodies was also inhibited by tumor antigens in the same way as the acquisition of Ig detected by RIA. It was shown that the acquisition of Ig during the first 18h of IP growth was a T-independent phenomenon because tumor cells acquire as much Ig in AT X BM mice as in sham-thymectomized controls. In a study with 11 different clones derived from the L5178Y lymphoma, a high correlation (r = 0.75, p less than 0.005) was found between the amount of Ig acquired after in vivo implantation and the amount of Ig bound to the cells after in vitro incubation with normal syngeneic serum. It is suggested that the rapid in vivo acquisition of Ig was due to the in vivo binding of natural antibodies to tumor cells.     

Humoral response against murine lymphoma cells xenogenized by drug treatment in vivo

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.     

Systemic adoptive immunotherapy of a highly immunogenic murine lymphoma growing in the brain

Systemic adoptive immunotherapy with specifically immune lymphocytes was successfully performed in immunodepressed mice challenged intracerebrally with L5178Y/DTIC lymphoma cells, a tumor subline rendered highly immunogenic through in vivo exposure to 5-(3,3-dimethyl-1-triazenyl)-IH-imidazole-4-carboxamide (DTIC). The systemic immunotherapy relied on the infusion of the in vitro-generated primary cytotoxic T-lymphocytes (CTL), the anti-tumor activity of which was evaluated by measuring tumor-cell proliferation in the brain in terms of radiolabel uptake and by recording survival times of recipient leukemic mice. The protection afforded by CTL appeared to be specific and was largely conditioned by dose and time of CTL administration. The levels of protection correlated well with the in vitro killing of L5178Y/DTIC tumor cells.     

Immunosensitivity and histocompatibility antigens in drug-altered leukemic cells.

New antigenic properties of experimental lymphomas have been reported previously following in vivo treatment with antitumor agents. 5-(3,3-Dimethyl-1-triazeno)imidazole-4-carboxamide (DIC) induced new antigenic characteristics on L1210 and L5178Y lymphomas, that were previously investigated in studies in animals compatible with the original untreated parental tumors. Here the L1210/DIC and L5178Y/DIC susceptibility to the cytotoxic effects of allogeneic and xenogeneic lymphocytes and sera obtained from animals sensitized to DBA/2 histocompatibility antigens were studied. The original and the DIC tumors showed the same sensitivity to anti-DBA/2 cellular and humoral cytotoxicity. The immune response electied in allogeneic mice by the original and DIC sublines was evaluated by in vitro cell-mediated and humoral cytotoxic assay. Beyond the immune response to histocompatibility antigens, a specific, anti-DIC-antigen immunoreaction was not found. Inhibition assay of the cell-mediated cytotoxicity and absorption of the humoral cytotoxicity demonstrated that DIC-induced antigens are not reciprocally related in cell-surface concentration to the natural DBA/2 histocompatibility antigens associated with tumor cells of DIC lines. An experiment was conducted in which specific activity against the DIC-treated L5178Y/DIC cells was observed with anti-L5178Y/DIC rabbit immune serum absorbed with the parental L5178Y lymphoma. This finding provides additional support to previous studies indicating that treatment with DIC induced new antigens on the lymphoma cells.     

Inhibition of murine lymphoma growth by adoptive transfer of lymphocytes sensitized to a xenogenized tumor variant

Lymphocytes immune to a highly immunogenic ("xenogenized") variant of a murine lymphoma--which were shown to exert anti-xenogenized tumor activity in a previously described model of tumor immunotherapy--were tested in the present study for possible suppressive effects on the growth of an i.c. graft of the original lymphoma. Remarkable tumor-inhibitory effects followed the i.v. infusion of splenic lymphocytes sensitized or restimulated in vitro, or derived from animals immunized in vivo with the xenogenized tumor cells. The pattern of inhibition of the parental cells was apparently similar to that previously described for the xenogenized variant, but preliminary evidence suggests that the underlying mechanisms may be different.     

Immunogenic tumor variants induced by drug treatment of the L5178Y lymphoma: search for serologically defined antigens at the clonal level.

Highly immunogenic tumor variants are generated by in vitro or in vivo treatment of the murine L5178Y lymphoma line with triazene derivatives. Most of these variants express new transplantation- and antibody-defined antigens that previous studies have shown to be closely related. One such 80-kDa protein on the surface of clone-D cells was found to be related to xenotropic MuLV gp70 molecules. To investigate the possible relevance of clone-D data to general properties of immunogenic variants in this tumor model system, polyclonal syngeneic antisera raised to a panel of immunogenic clones (including clone D) of the drug-treated L5178Y lymphoma line were employed in the immunoprecipitation of cell-surface and intrinsically labeled variant cells. In all clones, 1- and 2-dimensional electrophoretic analysis of the immunoprecipitates detected an antigen of approximately 80 kDa, and 35S-labeled 80-kDa molecules could be cross-precipitated from all clones by the panel of clone-specific antisera. In addition, 45- and 30-kDa components were also found in metabolically labeled variant cells. While the surface 80-kDa component was reactive with anti-xenotropic gp70 antibodies, the 30-kDa molecule was removed by anti-gag p30 antibody in sequential immunoprecipitation experiments. These data suggest that expression of aberrant, retrovirus-related proteins is a common finding in immunogenic cells of the drug-treated L5178Y lymphoma line.     

Anti-tumoral activity of L and D isomers of aspartic acid beta-hydroxamate on L5178Y leukemia

D and L isomers of aspartic acid beta-hydroxamate (respectively DAH and LAH) were compared for their in vitro and in vivo activity against the murine leukemia L5178Y and their tolerance in vivo in DBA/2 mice. DAH and LAH displayed comparable cytotoxic activity against L5178Y leukemia in vitro. Death of leukemia cells was observed at concentrations above 1.2 mM for both DAH and LAH. High concentrations of L-asparagine partially reversed the growth-inhibitory effects of DAH and LAH on L5178Y cells for concentrations of DAH and LAH lower than 0.6 mM. Intraperitoneal administration of DAH and LAH to mice showed that the LD10, LD50 and LD90 of DAH was 3- to 4-fold greater for DAH than for LAH. DAH was able to eradicate L5178Y tumors in mice without inducing toxic deaths, whereas LAH at comparable doses killed all the animals treated.     

Formation of 1-beta-D-arabinofuranosylcytosine diphosphate choline in neoplastic and normal cells.

1-beta-D-Arabinofuranosylcytosine diphosphate choline was formed from 1-beta-D-arabinofuranosylcytosine (ara-C) during incubation in vitro of peripheral myeloblasts from patients with acute myelogenous leukemia and cultured cells (nonleukemic human lymphocytes, mouse lymphoma L5178Y, and HeLa); as well, 1-beta-D-arabinofuranosylcytosine diphosphate choline was formed in vivo in mouse leukemia L1210 cells and mouse liver. 3-Deazauridine enhanced the anabolism of ara-C in nonleukemic lymphocytes in vitro and leukemia L1210 cells in vivo but did not influence ara-C anabolism in the other cell types. In acute myelogenous leukemia myeloblasts incubated in vitro with ara-C, concentrations of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate were maximal after 8 hr of incubation and formation of the latter preceded that of 1-beta-D-arabinofuranosylcytosine diphosphate choline.     

A new type of antimetastatic peptide derived from fibronectin.

PURPOSE: We found previously that fibronectin (FN) has a cryptic functional site (YTIYVIAL sequence within the 14th type III repeat) opposing cell adhesion to extracellular matrix. A 22-mer FN peptide containing this site, termed FNIII14, inhibits beta1 integrin-mediated adhesion without binding to integrins. The present study shows that FNIII14 has the potential to prevent lymphoma cell metastasis. EXPERIMENTAL DESIGN: Antimetastatic effect of FNIII14 has been evaluated through in vitro or in vivo experiments. RESULTS: FNIII14 inhibited the integrin alpha4beta1-mediated B lymphoma Ramos cell adhesion to VCAM-1 on venule endothelial cells, as well as to FN. Murine T lymphoma L5178Y-ML25 cells, which are known to metastasize to liver and spleen, preferentially adhered to vitronectin (VN) and migrated toward VN concentration gradients. FNIII14 abrogated both the integrin alphavbeta3-mediated adhesion and migration of L5178Y-ML25 cells. Inhibition of the alphavbeta3mediated L5178Y-ML25 cell adhesion by FNIII14 was reversed by phenylarsine oxide, a protein tyrosine phosphatase inhibitor. In addition, FNIII14 abrogated the VN-stimulated tyrosine phosphorylation of intracellular signaling proteins, including focal adhesion kinase (p125(FAK)) and paxillin, suggesting that such a diversity of FNIII14 effects might be because of the negative regulation of p125(FAK) and paxillin tyrosine phosphorylation, which has been involved in adhesion signals transduced by different integrins. The in vivo experiment using a murine metastasis model showed that FNIII14 would inhibit liver and spleen metastases of L5178Y-ML25 cells at a dose much lower than that of RGDS. CONCLUSIONS: FNIII14 might be applicable as a new type of antimetastatic agent distinct from integrin-binding peptides.     

Murine liver metastasis model using L5178Y-ML lymphoma and the effect of antitumor agents on the metastasis

A reproducible tumor model for liver metastasis has been developed from murine L5178Y lymphoma line by sequential cycles of subcutaneous inoculation of liver tumor cells, that were originally generated in livers of female (BALB/c x DBA/2)F1 mice by injecting the parental cells into the tail vein. This variant (L5178Y-ML) metastasized predominantly to the liver after intravenous or subcutaneous injection. The livers of the animals killed 9 days after intravenous implantation of 5 x 10(5) tumor cells were about 3 times the weight of control livers. All tumor-bearing mice died 10 to 12 days after inoculation. Subcutaneous implantation of L5178Y-ML in the side flank of mice induced metastatic nodules spontaneously in the livers. The tumor cells proliferated more in livers than in the implanted sites, compared with the parental L5178Y cells. The effects of 5-fluorouracil, mitomycin C, cis-platinum and doxorubicin on the liver metastasis of L5178Y-ML were examined at subtoxic doses; 5-fluorouracil was the most effective in both inhibiting the tumor growth in livers and prolonging the survival period of mice. This model provides a useful tool for the experimental therapy of hepatic tumors in mice.     

Induction of high-grade tumor-specific immunity in a host using a cytotoxic T-lymphocyte clone specific for a stable tumor antigen on murine leukemia L1210

T-cell clone K4L, the cell surface phenotypes of which were Thy-1+, Lyt-1-, Lyt-2+, and L3T4-, was established from the spleen cells of a murine leukemia L1210-immune mouse. Clone K4L was specific for antigen B on L1210, and this antigen was different from antigen A for which the previously reported T-cell clone K7L was specific. K4L possessed cytotoxicity and tumor growth-inhibitory activity against both L1210 and antigen A loss variant, L1210-K7L-, but not against syngeneic tumor P388 or L5178Y. Previously we showed that antigen A was lost frequently for generation of antigen loss variants. In contrast, antigen B was barely found to be lost. When mice were inoculated with L1210 plus a moderate dose of K4L, the tumor grew after initial suppression but this newly emerging tumor was K4L sensitive and was ultimately rejected. The mice initially given L1210 plus K4L attained a high-grade tumor-specific immunity for rejecting the subsequently challenged high-dose (10(7) cells) L1210. This immunity did not involve any bystander antitumor activity against the third party P388 lymphoma that was injected together with L1210 but accompanied the increase in the L1210-specific cytotoxic T-lymphocyte activity. Evidence was provided that the live L1210, the outgrowth of which was inhibited by K4L, induced an effective immune response of radiation-sensitive host lymphocytes including L3T4+ helper T-cells. Taken together, our results show a novel strategy for inducing high-grade host-dependent antitumor immunity by use of a cytotoxic T-lymphocyte clone specific for a stable tumor-specific transplantation antigen.     

CD47 agonist peptide PKHB1 induces immunogenic cell death in T‐cell acute lymphoblastic leukemia cells

T‐cell acute lymphoblastic leukemia (T‐ALL) has a poor prognosis derived from its genetic heterogeneity, which translates to a high chemoresistance. Recently, our workgroup designed thrombospondin‐1‐derived CD47 agonist peptides and demonstrated their ability to induce cell death in chronic lymphocytic leukemia. Encouraged by these promising results, we evaluated cell death induced by PKHB1 (the first‐described serum‐stable CD47‐agonist peptide) on CEM and MOLT‐4 human cell lines (T‐ALL) and on one T‐murine tumor lymphoblast cell‐line (L5178Y‐R), also assessing caspase and calcium dependency and mitochondrial membrane potential. Additionally, we evaluated selectivity for cancer cell lines by analyzing cell death and viability of human and murine non‐tumor cells after CD47 activation. In vivo, we determined that PKHB1‐treatment in mice bearing the L5178Y‐R cell line increased leukocyte cell count in peripheral blood and lymphoid organs while recruiting leukocytes to the tumor site. To analyze whether CD47 activation induced immunogenic cell death (ICD), we evaluated damage‐associated molecular patterns (DAMP) exposure (calreticulin, CRT) and release (ATP, heat shock proteins 70 and 90, high‐mobility group box 1, CRT). Furthermore, we gave prophylactic antitumor vaccination, determining immunological memory. Our data indicate that PKHB1 induces caspase‐independent and calcium‐dependent cell death in leukemic cells while sparing non‐tumor murine and human cells. Moreover, our results show that PKHB1 can induce ICD in leukemic cells as it induces CRT exposure and DAMP release in vitro, and prophylactic vaccinations inhibit tumor establishment in vivo. Together, our results improve the knowledge of CD47 agonist peptides potential as therapeutic tools to treat leukemia.     

A human/mouse hybrid model for the study of human genetic factors influencing tumor cell growth.

Somatic cell hybridization (fusion) was carried out between cells of the murine lymphoma L-5178Y(r) and peripheral human lymphocytes. The L-5178Y(r) line is deficient in hypoxanthine-guanine phosphorybosyl transferase (HGPRT). Using a selective medium system, it was possible to isolate hybrid cell clones having the complete complement of murine chromosomes as well as the human X chromosome on which the gene for HGPRT is located. This model system may be used to study the effect of the human X chromosome on the phenotype of the murine lymphoma.     

Murine Liver Metastasis Model Using L5178Y-ML Lymphoma and the Effect of Antitumor Agents on the Metastasis

A reproducible tumor model for liver metastasis has been developed from murine L5178Y lymphoma line by sequential cycles of subcutaneous inoculation of liver tumor cells, that were originally generated in livers of female (BALB/c × DBA/2)F₁ mice by injecting the parental cells into the tail vein. This variant (L5178Y-ML) metastasized predominantly to the liver after intravenous or subcutaneous injection. The livers of the animals killed 9 days after intravenous implantation of 5 × 10⁵ tumor cells were about 3 times the weight of control livers. All tumor-bearing mice died 10 to 12 days after inoculation. Subcutaneous implantation of L5178Y-ML in the side flank of mice induced metastatic nodules spontaneously in the livers. The tumor cells proliferated more in livers than in the implanted sites, compared with the parental L5178Y cells. The effects of 5-fluorouracil, mitomycin C, cis -platinum and doxorubicin on the liver metastasis of L5178Y-ML were examined at subtoxic doses; 5-fluorouracil was the most effective in both inhibiting the tumor growth in livers and prolonging the survival period of mice. This model provides a useful tool for the experimental therapy of hepatic tumors in mice.     

Immune regulation of the L5178Y murine tumor-dormant state: induction of cell- and soluble factor-mediated inhibition of tumor cell growth by tumor necrosis factor and gamma-interferon

Small concentrations of recombinant murine tumor necrosis factor (rMuTNF) synergized with recombinant murine gamma-interferon (rMuIFN-gamma) to inhibit tumor cell growth in peritoneal cell (PC) cultures from tumor-dormant mice. A soluble inhibitor of tumor cell growth was produced in the rMuTNF-treated PC cultures, but there was no synergistic enhancement of production of this inhibitor by the addition of rMuIFN-gamma. Treatment of both plastic-adherent PC and nonadherent PC cultures which contained L5178Y cells with rMuTNF + rMuIFN-gamma resulted in inhibition of tumor cell growth. However, in the absence of L5178Y cells, rMuTNF + rMuIFN-gamma induced antitumor cytotoxic activity in the plastic-adherent but not in the plastic nonadherent PC. These results indicate that the antitumor activity of rMuTNF + rMuIFN-gamma in PC from tumor-dormant mice involves both cell- and soluble factor-mediated mechanisms.     

Characterization of L5178Y cell phenotypes isolated during progression of the tumor-dormant state in DBA2 mice

During the course of the L5178Y tumor-dormant state in DBA/2 mice, there is continual selection of a tumor cell subpopulation ("emergent" phenotype) from the uncloned original L5178Y population used to initiate the tumor-dormant state. In vivo and in vitro experiments show that the emergent-phenotype tumor cells are less capable than "original"-phenotype cells, which constitute the majority of the L5178Y cell inoculum, of restimulating cytolytic T-lymphocyte (CTL) activity in tumor-dormant mice and are less susceptible to lysis by those CTL. Both original- and emergent-phenotype tumor cells are capable of eliciting an immune CTL response in naive mice, but again emergent-phenotype cells are poorly lysed by this response. As a consequence of these characteristics, emergent-phenotype cells rapidly form ascitic tumors when used as a challenge in L5178Y cell-immunized mice and cannot establish a tumor-dormant state. Results presented here and in previous publications indicate that CTLs are the major host cells involved in the selection of emergent-phenotype L5178Y cells during the course of the tumor-dormant state. Heterogeneity of the tumor cell challenge inoculum is important in establishing the L5178Y tumor-dormant state. The state, once established, is maintained by an immune CTL response which is continuously being restimulated by the strongly immunogenic original-phenotype L5178Y cells. The tumor-dormant state terminates when the less immunogenic and more immunoresistant emergent-phenotype tumor cells predominate and escape the waning immune response.