Immunoprevention of Mammary Carcinoma in HER-2/neu Transgenic Mice Is IFN-γ and B Cell Dependent

Immunohistochemistry of mammary tissue with invasive lobular carcinoma in untreated NeuT, vaccinated NeuT-IFN-γ^−/−, and vaccinated NeuT-μMT mice, and with atypical hyperplasia in vaccinated NeuT mice. Immunostaining with anti-p185^neu Ab shows that neoplastic cells of mammary carcinomas developed in untreated NeuT (A) and vaccinated NeuT-IFN-γ^−/− (C) and NeuT-μMT (D) mice express p185^neu in the cytoplasm and on the membrane. The expression of the p185^neu is associated with a marked positivity of PCNA (E, G, H). The mammary glands of vaccinated NeuT mice were mainly composed of ductules lined by a single layer of epithelial cells without or with a faint p185^neu expression mainly confined in the epithelial cell cytoplasm (B); the epithelial proliferation rate was low as assessed by PCNA immunostaining (F). A scanty microvessel network supplies the rare foci of mammary hyperplasia found in vaccinated NeuT mice (J). On the contrary, untreated NeuT (I) and vaccinated NeuT-IFN-γ^−/− (K) mice developed well-vascularized carcinomas. A reduction in microvessel density was observed in vaccinated NeuT-μMT mice (L). A–L, ×400.

Ab response in vaccinated NeuT-μMT mice. A, Sera were collected from NeuT or NeuT-μMT mice after three monthly courses of vaccination and tested at 1/65 dilution against Neu/H-2^q cells by indirect immunofluorescence followed by cytofluorometric analysis. Each point represents the mean fluorescence intensity (MFI) of one serum. B, Mean Ab levels induced by vaccination of NeuT-μMT mice compared with parental NeuT and μMT mice. Each bar represents the mean ± SEM of 6–23 individual mice scored as Ab-positive (MFI, >8). The percentage of Ab-positive mice in each vaccinated group is reported above the hatched bars.

Immunostaining with anti-B220 mAb of lymph node (A), spleen (B), Peyer patch (C), and intestinal lymphoid follicle (D) from NeuT-μMT mice. Positive B cells are scarce in lymph node and spleen, whereas they are well represented in intestinal lymphoid follicle and numerous in Peyer patch. A, ×200; B–D, ×400.

Immunoprevention of mammary carcinoma in NeuT-μMT mice is Ab dependent. Vaccinated NeuT-μMT mice shown in Fig. 1 were stratified according to Ab production in response to vaccination (see Fig. 3). Tumor-free survival curve of vaccinated, Ab-positive mice was significantly different from that of vaccinated, Ab-negative mice (p < 0.01 by the Mantel-Haenszel test).

Whole mounts of inguinal mammary glands from one untreated (A) and two vaccinated NeuT-μMT mice, one Ab deficient (B), the other Ab proficient (C) at the age of 16 wk. The black oval in the center of each picture is the inguinal lymph node. In A and B numerous and large atypical hyperplastic foci (arrows) and a few established mammary carcinomas (arrowheads) were present, whereas a dramatic reduction in diffusion and dimension of hyperplastic foci and the absence of carcinoma were evident in C.

Release of IFN-γ by NeuT-μMT spleen cells. Spleens were obtained from untreated mice or from mice vaccinated with Neu/H-2^q cells plus IL-12. IFN-γ release by spleen cells was assessed by ELISA after 6 days in vitro in the presence or absence of stimulator vaccine cells.

Ab response of vaccinated NeuT-μMT mice analyzed by immunoprecipitation and Western blot. A, Immunoreactivity of sera against native p185^neu protein. Lysates of Neu/H-2^q and Neu^neg/H-2^q cells (here referred to as Neu and Neu^neg, respectively) were immunoprecipitated with pooled sera obtained from four vaccinated Ab-proficient (Ab+) or -deficient (Ab−) NeuT-μMT mice or from untreated NeuT-μMT mice (No vax). Immunoprecipitation with Ab4 anti-rat neu mAb (α-neu Ab) or with sera from vaccinated NeuT mice (NeuT/Vax) are shown for comparison. The C18 anti-p185^neu rabbit Ab was used for Western blot. B, Immunoreactivity of mice sera against cryptic epitopes of p185^neu protein. p185^neu was immunoprecipitated with Ab4 mAb, denatured, and blotted using sera as above or C18 Ab (α-neu Ab). A HRP-linked goat anti-mouse IgG Ab was used for detection.

Subclasses of Abs induced by vaccination in NeuT, NeuT-IFN-γ^−/−, and NeuT-μMT mice. The results of NeuT-IFN-γ^−/− mice were previously published (12 ) and are reported here for comparison. Cytofluorometric analysis of serum binding to Neu/H-2^q vaccine cells with secondary anti-mouse isotype Abs is reported. IgA and IgE Abs were absent from all sera (data not shown). Each bar represents the mean ± SEM of sera (1/65) from three to nine mice at the 17th week of age bled after the third course of vaccination.

Complement-mediated cytotoxicity (A) and ADCC (B) with sera of mice vaccinated with Neu/H-2^q cells plus IL-12. Each point or bar represents the mean ± SEM of sera from three to four mice bled after the third course of vaccination (17 wk of age). ∗, Significantly different from NeuT mice (p < 0.01, Student’s t test).