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Neutrophils discriminate between G-CSF producing and non-producing colon carcinoma cells: correlation with tumor inhibition
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IMMUNITY TO 816 MELANOMA IN MICE IMMUNIZED WITH IL-Z SECPJZTING ALLCGENEIC FIBBOBLASTS MELANOMA-ASSOCIATED EXPRESSING ANTIGENS. E.P. Cohen, T.S. Kim and S.J. Russell University of Illinois at Chicago, Chicago, IL 60615 and Chester Beatty Research Laboratories, London, SW36JB, England. 816 melanoma cells form tumor-associated determinants that can act a* targets Of Cell mediated immunity. Nevertheless, the cells proliferate without apparent inhibition in syngeneic, immunocompetent C57BL/6 mice. One possibility is that the melanoma-associated determinants are insufficientlv antioenic. A cellular immunoaen was constructed &at st&lated immunity to the melanoma by cotransfecting genomic DNA from B16 cells along with a plasmid (~TK) carry& a selectable marker into ti(TK-) c&s, a thymidine kinase-deficient allogeneic cell line. After initial selection in HAT, clones of transfected cells expressing tumor-associated antigens were identified by antibody-mediated erythrocyte-rosetting &I situ and individually recovered. Afterwards, the melanoma antigenpositive cells were infected with a retroviral construct (pZip-SV-IL-Z) that carries a promoter, a selectable marker and the gene for human IL-Z. Flow cytometric analysis confirmed the expression of melanoma-associated antigens. IL2 secretion was measured by thymidine incorporation into CTLL2 cells. Southern blotting indicated the presence of a single IL-2 gene in a clone of IL-Z-secreting transfected LM cells. After immunization, the immunogenic properties of the constructs, determined by '&-release from labelled 816 cells, exceeded those of inactivated El6 cells in C57BL/6 mice. The results indicate that the co-ex"ression of melanoma-associated antigens along with allogeneic antigens on a common cell carrier forming IL-2 stimulates heightened iranunity to a weakly antigen& tumor.
MECHANISMS OF REJECTION OF CONSTI~UTIVE IL-1 PRODUCING FIBROSARCOMAS. Amos Douvdevam. Noa Shimoni, Mahmoud tluishel. Slmaa Seql and Ron N Aote. Dewtment of Miaabidogy and Immunology, Faculty of Health’ Sciences, Ben-Gwion University of the Negev. Beer- Sheva, Israel. We have awseed the in vivo gcwth patterns of constitutive IL-1 alpha pducing f&r-comas. A striking curelation was observed between the ccnstitutive eqression of IL-1 alpha and tuma rejection, while IL-1 nonproducing fibrowcomas gew propssively and killed the mice. From the gowth patterns of the -comas in syngeneic and allogeneic mice it appears that constitutive IL-1 alpha expesion enhances the immunoserjcity of weak tumcu cell antigens, resulting in the development of antiitumw immune responses lea&g its era&ation. Indeed, enhanced CTL activity was detected in the spleens of mice of mice with regeasing IL-1 poducing fibrosrwcomas and it is possibly medated through the development of tumcf specific helper T cell activity. Futhermae, an infiltrate of lymphocytes was observed in IL-1 producing fibfoswcomas, which ultimately replaced the tumds mass. In ad&ion, IL-1 notqfoducing fikoswcomas induced in vitro to express IL-1 by treatment with cytokines/LPS. also failed to develop tumors or regewed following initial gowth. The possibility that a cytokine cascade, initiated by endogwous IL-1 expression. leads to tuma rejection was suggested by showing that IL-1 podudng lines also express IL-6 and serxete CSF as well as prostaglandns. These fin&p may serve as the basis of application of novel immunotherapy protocols, aimed to induce IL-1 by the malignant cells w to apply it locally it in the vicinity of the tumor.
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NEUTROPHILS DISCRIMINATE BETWEEN G-CSF PRODUCING AND NON-PRODUCING COLON CARCINOMA CELLS: CORRELATION WITH TUMOR INHIBITION. Mario P. Colombo”. Luciano Lombardi’. Anto nella Stoouacciaro*. Cecilia Melani”. Gioreio Parmiani’. “Istituto Nazionale Tumori. Milan0 and *Diuartimento di Bionatoloaia _ I Umana, Universitl di Roma; Italy. We have demonstrated that mtroviral mediated G-CSF transduction in the murine colon adenocarcinoma C-26 inhibits tumorigenicity of this tumor in syngeneic BALBlc mice. However, tumors were formed after injection of a mixture of infected and uninfected C-26 cells. Infiltrating neutrophilic granulocytes were found 5 but not 10 days after the mixture injection and the growing tumors resulted to be formed by uninfected C-26 cells. We challenged the hypothesis that neutrophils recognize and, in some way, selectively eliminate the G-CSF producing cells only. To this aim we infected the original C-26 cells with a retroviral vector carrying the Escherichia cdi 1ucZ gene and repeated the mixed tumor transplantation assay by injecting p-gal negative, G-CSF producing and b-gal positive, GCSF non-producing C-26 cells. Histological and elecaon microscopy analysis of samples collected 5 days after mixture injection and treated with x-gal revealed the presence of neuaophils all around G-CSG producing, pgal negative tumor cells. A cell-cell contact occurred only between neutrophils and G-CSF producing tumor cells. These results indicate that neutrophils can discriminate between G-CSF producing and no-producing cells and that neutrophils infiltrated the tumors as long as G-CSF producing cells were present.
THERAPEUTIC EFFICACY OF CYCLOPHOSPHAMIDE (0') AND TNFa THERAPY IN A MURINE TUMOR HODEL: POSSIBLE RULE OF SPECIFIC THYMIC CTL ACTIVITY. J. Ehrke. C. Krawczyk, D. Maccubbln and E. Mihich. GCDC. Roswell Park Cancer Inst., Buffalo, NY 14263 Combination treatment protocols consisting of CY and recombinant mouse TNFa were tested for therapeutic effect in C57BL16 mice bearing S.C. implants of EL4 lymphoma. CY was administered 1.~. at a dose of 150 or 250 mglkg and was given once (Day 2 or Day 12) or twice (Day 2 and 12) after tumor. TNF was given i.v. in various doses and schedules. A majority (50) of the 57 protocols tested resulted fn one or more long-term survivors (20-100%); however, only 61% of "cured" mice had developed long-term ianunlty to EL4 (capable of rejecting a rechallenge with EL4). 72% of immune survivors occurred in the groups which received CY on Day 12 only. A comparison of TNF (any schedule) plus CY given on Day 2. Day 12 or Days 2 and 12 revealed that there were 30%. 6896 and 53% long term survivors, respectively and of these, 15%. 96% and 19%. were innnune. Thymus cells frcxn ilrmune survivor animals were tested for specific CTL activity agalnst EL4 tumor cells. Fresh cells were only marginally lytic, however. after 4 days in culture with x-irradiated EL4 cells, they exhibited significant levels of specific killing. This response cannot be generated with thymus cells from naive or tumor bearing animals before or during treatment. These results indicate that long term antitumor imnity In "cured" mice is associated with a long lasting specific antitumor response in the thymus. KA13038, CAO9072, CAZ4538, USPHS)
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MACROPHAGE COLONY STIMULATING FACTOR ENHANCES HOST DEFENSES AGAINST BACTERIA BOTH IN VIVO AND IN VITRO. A. Cross. D. Hoover and M. Garnick. WRAIR, Washington, DC 20307 and Genetics Institute, Cambridge, MA 02149 We previously demonstrated an important role for tumor necrosis factor and interleukin 1 in host defenses against invasive E. coli (EC): the administration of these recombinant cytokines to C3H/HeJ mice (HeJs) that are blocked in their ability to respond to endotoxin significantly enhanced survival from lethal infection (J Exp Med 169:2021, 1989). Since M-CSF stimulates macrophage development and function we assessed its ability to also protect HeJs from lethal infection. Compared to saline-treated controls, significantly more mice survived an EC challenge of 3 LD,,s following pretreatment with M-CSF given subcutaneously (SQ) at 2-4 ng/gm each day X3 (29/35 treated survived v. 5/20 controls, plOOOX reduction In viable EC compared to unprimed PM in an opsonophagocytic assay. Killing was inhibited by treatment of primed PMs with the inhibitors, monomethyl-L-arginine (MMLA) and arginase. Larginine restored EC kill to MMLA-treated primed PMs. We conclude that M-CSF, perhaps via the nitric oxide pathway, enhances host. defenses against EC. Its potential for treatment of invasive bacterial infection deserves further investigation.
EXPANSION OF CD4+ AND CD8+ TUMOR-INFILTRATING LYMPHOCYTES (TILs) IN SITU FOLLOWING ADOPTIVE Il’lflUNOTHERAPY IN A MURINE TUMOR MODEL: CYTOKINE GENE EXPRESSION. llobar M. The Jacksoa Laborstay. Bar Barba. ME 04609.
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IMMUNITY TO 816 MELANOMA IN MICE IMMUNIZED WITH IL-Z SECPJZTING ALLCGENEIC FIBBOBLASTS MELANOMA-ASSOCIATED EXPRESSING ANTIGENS. E.P. Cohen, T.S. Kim and S.J. Russell University of Illinois at Chicago, Chicago, IL 60615 and Chester Beatty Research Laboratories, London, SW36JB, England. 816 melanoma cells form tumor-associated determinants that can act a* targets Of Cell mediated immunity. Nevertheless, the cells proliferate without apparent inhibition in syngeneic, immunocompetent C57BL/6 mice. One possibility is that the melanoma-associated determinants are insufficientlv antioenic. A cellular immunoaen was constructed &at st&lated immunity to the melanoma by cotransfecting genomic DNA from B16 cells along with a plasmid (~TK) carry& a selectable marker into ti(TK-) c&s, a thymidine kinase-deficient allogeneic cell line. After initial selection in HAT, clones of transfected cells expressing tumor-associated antigens were identified by antibody-mediated erythrocyte-rosetting &I situ and individually recovered. Afterwards, the melanoma antigenpositive cells were infected with a retroviral construct (pZip-SV-IL-Z) that carries a promoter, a selectable marker and the gene for human IL-Z. Flow cytometric analysis confirmed the expression of melanoma-associated antigens. IL2 secretion was measured by thymidine incorporation into CTLL2 cells. Southern blotting indicated the presence of a single IL-2 gene in a clone of IL-Z-secreting transfected LM cells. After immunization, the immunogenic properties of the constructs, determined by '&-release from labelled 816 cells, exceeded those of inactivated El6 cells in C57BL/6 mice. The results indicate that the co-ex"ression of melanoma-associated antigens along with allogeneic antigens on a common cell carrier forming IL-2 stimulates heightened iranunity to a weakly antigen& tumor.
MECHANISMS OF REJECTION OF CONSTI~UTIVE IL-1 PRODUCING FIBROSARCOMAS. Amos Douvdevam. Noa Shimoni, Mahmoud tluishel. Slmaa Seql and Ron N Aote. Dewtment of Miaabidogy and Immunology, Faculty of Health’ Sciences, Ben-Gwion University of the Negev. Beer- Sheva, Israel. We have awseed the in vivo gcwth patterns of constitutive IL-1 alpha pducing f&r-comas. A striking curelation was observed between the ccnstitutive eqression of IL-1 alpha and tuma rejection, while IL-1 nonproducing fibrowcomas gew propssively and killed the mice. From the gowth patterns of the -comas in syngeneic and allogeneic mice it appears that constitutive IL-1 alpha expesion enhances the immunoserjcity of weak tumcu cell antigens, resulting in the development of antiitumw immune responses lea&g its era&ation. Indeed, enhanced CTL activity was detected in the spleens of mice of mice with regeasing IL-1 poducing fibrosrwcomas and it is possibly medated through the development of tumcf specific helper T cell activity. Futhermae, an infiltrate of lymphocytes was observed in IL-1 producing fibfoswcomas, which ultimately replaced the tumds mass. In ad&ion, IL-1 notqfoducing fikoswcomas induced in vitro to express IL-1 by treatment with cytokines/LPS. also failed to develop tumors or regewed following initial gowth. The possibility that a cytokine cascade, initiated by endogwous IL-1 expression. leads to tuma rejection was suggested by showing that IL-1 podudng lines also express IL-6 and serxete CSF as well as prostaglandns. These fin&p may serve as the basis of application of novel immunotherapy protocols, aimed to induce IL-1 by the malignant cells w to apply it locally it in the vicinity of the tumor.
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NEUTROPHILS DISCRIMINATE BETWEEN G-CSF PRODUCING AND NON-PRODUCING COLON CARCINOMA CELLS: CORRELATION WITH TUMOR INHIBITION. Mario P. Colombo”. Luciano Lombardi’. Anto nella Stoouacciaro*. Cecilia Melani”. Gioreio Parmiani’. “Istituto Nazionale Tumori. Milan0 and *Diuartimento di Bionatoloaia _ I Umana, Universitl di Roma; Italy. We have demonstrated that mtroviral mediated G-CSF transduction in the murine colon adenocarcinoma C-26 inhibits tumorigenicity of this tumor in syngeneic BALBlc mice. However, tumors were formed after injection of a mixture of infected and uninfected C-26 cells. Infiltrating neutrophilic granulocytes were found 5 but not 10 days after the mixture injection and the growing tumors resulted to be formed by uninfected C-26 cells. We challenged the hypothesis that neutrophils recognize and, in some way, selectively eliminate the G-CSF producing cells only. To this aim we infected the original C-26 cells with a retroviral vector carrying the Escherichia cdi 1ucZ gene and repeated the mixed tumor transplantation assay by injecting p-gal negative, G-CSF producing and b-gal positive, GCSF non-producing C-26 cells. Histological and elecaon microscopy analysis of samples collected 5 days after mixture injection and treated with x-gal revealed the presence of neuaophils all around G-CSG producing, pgal negative tumor cells. A cell-cell contact occurred only between neutrophils and G-CSF producing tumor cells. These results indicate that neutrophils can discriminate between G-CSF producing and no-producing cells and that neutrophils infiltrated the tumors as long as G-CSF producing cells were present.
THERAPEUTIC EFFICACY OF CYCLOPHOSPHAMIDE (0') AND TNFa THERAPY IN A MURINE TUMOR HODEL: POSSIBLE RULE OF SPECIFIC THYMIC CTL ACTIVITY. J. Ehrke. C. Krawczyk, D. Maccubbln and E. Mihich. GCDC. Roswell Park Cancer Inst., Buffalo, NY 14263 Combination treatment protocols consisting of CY and recombinant mouse TNFa were tested for therapeutic effect in C57BL16 mice bearing S.C. implants of EL4 lymphoma. CY was administered 1.~. at a dose of 150 or 250 mglkg and was given once (Day 2 or Day 12) or twice (Day 2 and 12) after tumor. TNF was given i.v. in various doses and schedules. A majority (50) of the 57 protocols tested resulted fn one or more long-term survivors (20-100%); however, only 61% of "cured" mice had developed long-term ianunlty to EL4 (capable of rejecting a rechallenge with EL4). 72% of immune survivors occurred in the groups which received CY on Day 12 only. A comparison of TNF (any schedule) plus CY given on Day 2. Day 12 or Days 2 and 12 revealed that there were 30%. 6896 and 53% long term survivors, respectively and of these, 15%. 96% and 19%. were innnune. Thymus cells frcxn ilrmune survivor animals were tested for specific CTL activity agalnst EL4 tumor cells. Fresh cells were only marginally lytic, however. after 4 days in culture with x-irradiated EL4 cells, they exhibited significant levels of specific killing. This response cannot be generated with thymus cells from naive or tumor bearing animals before or during treatment. These results indicate that long term antitumor imnity In "cured" mice is associated with a long lasting specific antitumor response in the thymus. KA13038, CAO9072, CAZ4538, USPHS)
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MACROPHAGE COLONY STIMULATING FACTOR ENHANCES HOST DEFENSES AGAINST BACTERIA BOTH IN VIVO AND IN VITRO. A. Cross. D. Hoover and M. Garnick. WRAIR, Washington, DC 20307 and Genetics Institute, Cambridge, MA 02149 We previously demonstrated an important role for tumor necrosis factor and interleukin 1 in host defenses against invasive E. coli (EC): the administration of these recombinant cytokines to C3H/HeJ mice (HeJs) that are blocked in their ability to respond to endotoxin significantly enhanced survival from lethal infection (J Exp Med 169:2021, 1989). Since M-CSF stimulates macrophage development and function we assessed its ability to also protect HeJs from lethal infection. Compared to saline-treated controls, significantly more mice survived an EC challenge of 3 LD,,s following pretreatment with M-CSF given subcutaneously (SQ) at 2-4 ng/gm each day X3 (29/35 treated survived v. 5/20 controls, p
EXPANSION OF CD4+ AND CD8+ TUMOR-INFILTRATING LYMPHOCYTES (TILs) IN SITU FOLLOWING ADOPTIVE Il’lflUNOTHERAPY IN A MURINE TUMOR MODEL: CYTOKINE GENE EXPRESSION. llobar M. The Jacksoa Laborstay. Bar Barba. ME 04609.