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Combined allogeneic tumor cell vaccination and systemic interleukin 12 prevents mammary carcinogenesis in HER-2/neu transgenic mice.

Nanni P, Nicoletti G, De Giovanni C, Landuzzi L, Di Carlo E, Cavallo F, Pupa SM, Rossi I, Colombo MP, Ricci C, Astolfi A, Musiani P, Forni G, Lollini PL - J. Exp. Med. (2001)

Bottom Line: IL-12 treatment strongly increased the cell vaccine efficacy.Specific anti-HER-2/neu antibodies were produced and a nonpolarized activation of CD4(+) and CD8(+) cells secreting IL-4 and interferon (IFN)-gamma were evident.In conclusion, our data show that an allogeneic HER-2/neu-expressing cell vaccine combined with IL-12 systemic treatment can prevent the onset of genetically determined tumors.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Section, Department of Experimental Pathology, University of Bologna, Italy.

ABSTRACT
Transgenic Balb/c mice expressing the transforming rat HER-2/neu oncogene develop early and multifocal mammary carcinomas. Within the first 5 months of life the tissue-specific expression of HER-2/neu causes a progression in all their 10 mammary glands from atypical hyperplasia to invasive carcinoma. It was previously observed that chronic administration of interleukin (IL)-12 increased tumor latency, but every mouse eventually succumbed to multiple carcinomas. A significant improvement in tumor prevention was sought by administering allogeneic mammary carcinoma cells expressing HER-2/neu combined with systemic IL-12. This treatment reduced tumor incidence by 90% and more than doubled mouse lifetime. For the maximum prevention p185(neu) antigen must be expressed by allogeneic cells. IL-12 treatment strongly increased the cell vaccine efficacy. The mammary glands of mice receiving the combined treatment displayed a markedly reduced epithelial cell proliferation, angiogenesis, and HER-2/neu expression, while the few hyperplastic foci were heavily infiltrated by granulocytes, macrophages, and CD8(+) lymphocytes. Specific anti-HER-2/neu antibodies were produced and a nonpolarized activation of CD4(+) and CD8(+) cells secreting IL-4 and interferon (IFN)-gamma were evident. A central role for IFN-gamma in the preventive effect was proven by the lack of efficacy of vaccination in IFN-gamma gene knockout HER-2/neu transgenic Balb/c mice. A possible requirement for IFN-gamma is related to its effect on antibody production, in particular on IgG2a and IgG2b subclasses, that were not induced in IFN-gamma knockout HER-2/neu mice. In conclusion, our data show that an allogeneic HER-2/neu-expressing cell vaccine combined with IL-12 systemic treatment can prevent the onset of genetically determined tumors.

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Antibody production by Balb-neuT mice. (A) Cytofluorometric analysis of serum binding to Neu/H-2q cells. Each bar represents the mean ± SEM of sera (diluted 1:65) from three mice at the fifteenth week of age bled after the second course of the indicated treatment. (B) Neu/H-2q and Neuneg/H-2q cell extracts were immunoprecipitated by sera from three mice treated with Neu/H-2q cells plus IL-12, PBS as negative control, and anti–rat p185neu monoclonal antibody 7.16.4 as positive control (Neu mAb). Western blot analysis was performed using the antineu polyclonal antibody C18. (C) Complement dependent cytotoxicity against syngeneic Neu/H-2d cells and Balb-neuT lymphocytes, which do not express p185neu (H-2d). The percentage of viable lymphocytes was determined after a 30-min incubation with sera of mice bled after the second course of treatment (diluted 1:10) followed by a 30-min incubation with rabbit low-tox complement (diluted 1:10). Results are expressed as 100 − % viable lymphocytes.
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fig9: Antibody production by Balb-neuT mice. (A) Cytofluorometric analysis of serum binding to Neu/H-2q cells. Each bar represents the mean ± SEM of sera (diluted 1:65) from three mice at the fifteenth week of age bled after the second course of the indicated treatment. (B) Neu/H-2q and Neuneg/H-2q cell extracts were immunoprecipitated by sera from three mice treated with Neu/H-2q cells plus IL-12, PBS as negative control, and anti–rat p185neu monoclonal antibody 7.16.4 as positive control (Neu mAb). Western blot analysis was performed using the antineu polyclonal antibody C18. (C) Complement dependent cytotoxicity against syngeneic Neu/H-2d cells and Balb-neuT lymphocytes, which do not express p185neu (H-2d). The percentage of viable lymphocytes was determined after a 30-min incubation with sera of mice bled after the second course of treatment (diluted 1:10) followed by a 30-min incubation with rabbit low-tox complement (diluted 1:10). Results are expressed as 100 − % viable lymphocytes.

Mentions: High-titer antibodies binding Neu/H-2q cells were detected in sera from mice vaccinated with allogeneic Neu/H-2q cells (Fig. 9 A). Staining of NeuNeg/H-2q cells was much lower than Neu/H-2q cells (mean fluorescence intensity 61 ± 9 vs. 332 ± 11), thus suggesting that sera of H-2d mice vaccinated with Neu/H-2q plus IL-12 contained p185neu-reactive antibodies along with alloreactive anti–H-2q antibodies. Neither untreated control mice nor mice treated with IL-12 alone produced antibody against Neu/H-2q cells (Fig. 9 A). To circumvent the issue of alloreactivity we performed a Western blot analysis (Fig. 9 B), which showed that antibodies of mice vaccinated with Neu/H-2q plus IL-12 specifically recognized p185neu (Fig. 9 B). Only sera from mice receiving cell vaccines resulted cytotoxic against syngeneic Neu/H-2d tumor cells, whereas no lysis was observed against syngeneic p185neu-negative H-2d lymphocytes (Fig. 9 C). The lysis was stronger when Neu/H-2q cell vaccine was combined with IL-12 administration.


Combined allogeneic tumor cell vaccination and systemic interleukin 12 prevents mammary carcinogenesis in HER-2/neu transgenic mice.

Nanni P, Nicoletti G, De Giovanni C, Landuzzi L, Di Carlo E, Cavallo F, Pupa SM, Rossi I, Colombo MP, Ricci C, Astolfi A, Musiani P, Forni G, Lollini PL - J. Exp. Med. (2001)

Antibody production by Balb-neuT mice. (A) Cytofluorometric analysis of serum binding to Neu/H-2q cells. Each bar represents the mean ± SEM of sera (diluted 1:65) from three mice at the fifteenth week of age bled after the second course of the indicated treatment. (B) Neu/H-2q and Neuneg/H-2q cell extracts were immunoprecipitated by sera from three mice treated with Neu/H-2q cells plus IL-12, PBS as negative control, and anti–rat p185neu monoclonal antibody 7.16.4 as positive control (Neu mAb). Western blot analysis was performed using the antineu polyclonal antibody C18. (C) Complement dependent cytotoxicity against syngeneic Neu/H-2d cells and Balb-neuT lymphocytes, which do not express p185neu (H-2d). The percentage of viable lymphocytes was determined after a 30-min incubation with sera of mice bled after the second course of treatment (diluted 1:10) followed by a 30-min incubation with rabbit low-tox complement (diluted 1:10). Results are expressed as 100 − % viable lymphocytes.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195980&req=5

fig9: Antibody production by Balb-neuT mice. (A) Cytofluorometric analysis of serum binding to Neu/H-2q cells. Each bar represents the mean ± SEM of sera (diluted 1:65) from three mice at the fifteenth week of age bled after the second course of the indicated treatment. (B) Neu/H-2q and Neuneg/H-2q cell extracts were immunoprecipitated by sera from three mice treated with Neu/H-2q cells plus IL-12, PBS as negative control, and anti–rat p185neu monoclonal antibody 7.16.4 as positive control (Neu mAb). Western blot analysis was performed using the antineu polyclonal antibody C18. (C) Complement dependent cytotoxicity against syngeneic Neu/H-2d cells and Balb-neuT lymphocytes, which do not express p185neu (H-2d). The percentage of viable lymphocytes was determined after a 30-min incubation with sera of mice bled after the second course of treatment (diluted 1:10) followed by a 30-min incubation with rabbit low-tox complement (diluted 1:10). Results are expressed as 100 − % viable lymphocytes.
Mentions: High-titer antibodies binding Neu/H-2q cells were detected in sera from mice vaccinated with allogeneic Neu/H-2q cells (Fig. 9 A). Staining of NeuNeg/H-2q cells was much lower than Neu/H-2q cells (mean fluorescence intensity 61 ± 9 vs. 332 ± 11), thus suggesting that sera of H-2d mice vaccinated with Neu/H-2q plus IL-12 contained p185neu-reactive antibodies along with alloreactive anti–H-2q antibodies. Neither untreated control mice nor mice treated with IL-12 alone produced antibody against Neu/H-2q cells (Fig. 9 A). To circumvent the issue of alloreactivity we performed a Western blot analysis (Fig. 9 B), which showed that antibodies of mice vaccinated with Neu/H-2q plus IL-12 specifically recognized p185neu (Fig. 9 B). Only sera from mice receiving cell vaccines resulted cytotoxic against syngeneic Neu/H-2d tumor cells, whereas no lysis was observed against syngeneic p185neu-negative H-2d lymphocytes (Fig. 9 C). The lysis was stronger when Neu/H-2q cell vaccine was combined with IL-12 administration.

Bottom Line: IL-12 treatment strongly increased the cell vaccine efficacy.Specific anti-HER-2/neu antibodies were produced and a nonpolarized activation of CD4(+) and CD8(+) cells secreting IL-4 and interferon (IFN)-gamma were evident.In conclusion, our data show that an allogeneic HER-2/neu-expressing cell vaccine combined with IL-12 systemic treatment can prevent the onset of genetically determined tumors.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Section, Department of Experimental Pathology, University of Bologna, Italy.

ABSTRACT
Transgenic Balb/c mice expressing the transforming rat HER-2/neu oncogene develop early and multifocal mammary carcinomas. Within the first 5 months of life the tissue-specific expression of HER-2/neu causes a progression in all their 10 mammary glands from atypical hyperplasia to invasive carcinoma. It was previously observed that chronic administration of interleukin (IL)-12 increased tumor latency, but every mouse eventually succumbed to multiple carcinomas. A significant improvement in tumor prevention was sought by administering allogeneic mammary carcinoma cells expressing HER-2/neu combined with systemic IL-12. This treatment reduced tumor incidence by 90% and more than doubled mouse lifetime. For the maximum prevention p185(neu) antigen must be expressed by allogeneic cells. IL-12 treatment strongly increased the cell vaccine efficacy. The mammary glands of mice receiving the combined treatment displayed a markedly reduced epithelial cell proliferation, angiogenesis, and HER-2/neu expression, while the few hyperplastic foci were heavily infiltrated by granulocytes, macrophages, and CD8(+) lymphocytes. Specific anti-HER-2/neu antibodies were produced and a nonpolarized activation of CD4(+) and CD8(+) cells secreting IL-4 and interferon (IFN)-gamma were evident. A central role for IFN-gamma in the preventive effect was proven by the lack of efficacy of vaccination in IFN-gamma gene knockout HER-2/neu transgenic Balb/c mice. A possible requirement for IFN-gamma is related to its effect on antibody production, in particular on IgG2a and IgG2b subclasses, that were not induced in IFN-gamma knockout HER-2/neu mice. In conclusion, our data show that an allogeneic HER-2/neu-expressing cell vaccine combined with IL-12 systemic treatment can prevent the onset of genetically determined tumors.

Show MeSH
Related in: MedlinePlus