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and B Cell Dependent1
* Cancer Research Section, Department of Experimental Pathology, University of Bologna, Bologna, Italy;
Istituti Ortopedici Rizzoli, Bologna, Italy;
Aging Research Center, G. D’Annunzio University Foundation, Chieti, Italy; and
Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy
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production (IFN-–/–) or with B cell-deficient mice (µMT). Vaccination did not protect NeuT-IFN-–/– mice, thus confirming a central role of IFN-. The block of Ab production in NeuT-µMT mice was incomplete. About one third of NeuT-µMT mice failed to produce Abs and displayed a rapid tumor onset. By contrast, those NeuT-µMT mice that responded to the vaccine with a robust production of anti-p185neu Ab displayed a markedly delayed tumor onset. In these NeuT-µMT mice, the vaccine induced a lower level of IgG2a and IgG3 and a higher level of IgG2b than in NeuT mice. Moreover, NeuT-µMT mice failed to produce anti-MHC class I Abs in response to allogeneic H-2q molecules present in the cell vaccine. These findings show that inhibition of HER-2/neu carcinogenesis depends on cytokines and specific Abs, and that a highly effective vaccine can rescue Ab production even in B cell-deficient mice. |
Introduction |
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and B cell responses. |
Materials and Methods |
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Transgenic BALB/c (H-2d) female mice, designated here as NeuT mice, overexpressing the transforming activated rat HER-2/neu oncogene under control of the mouse mammary tumor virus promoter (12) were bred under specific pathogen-free conditions by Charles River (Calco, Italy). The establishment of IFN- gene knockout HER-2/neu transgenic mice (NeuT-IFN-–/– mice) has been described previously (12). To obtain B cell-deficient HER-2/neu transgenic mice, one female µMT mouse (knockout for the Ig µ-chain gene) on BALB/c genetic background, a kind gift from Dr. T. Blankenstein (Max-Dellbruck Center for Molecular Medicine, Berlin, Germany), was crossed with one NeuT male mouse. Heterozygous knockout/transgenic F1 male mice were backcrossed with female µMT to obtain mice homozygous for the µ-chain knockout allele and heterozygous for the HER-2/neu transgene, designated here as NeuT-µMT. The level of B220+ B cells was routinely monitored by flow cytometry using mAb RA3-6B2 (BD Pharmingen, San Diego, CA). Individually tagged virgin females used in the experiments were treated according to protocols approved by Institutional Review Boards. Mammary pads were inspected weekly, and tumor masses were measured with calipers in two perpendicular diameters. Progressively growing masses of >3 mm in mean diameter were regarded as tumors. Growth was monitored until all 10 mammary glands displayed a tumor or until a tumor exceeded a mean diameter of 1.5 cm, at which time mice were sacrificed for humane reasons.
Cells
TT12 and N202.1E cell clones were derived from mammary carcinomas of FVB-neuN #202 mice (H-2q), transgenic for the rat HER-2/neu protooncogene (14). TT12 cells (referred to as Neu/H-2q) expressed high levels of p185neu; N202.1E cells (referred to as Neuneg/H-2q) lacked p185neu. Cells were cultured in Dulbecco’s modified minimal essential medium supplemented with 20% FBS (Invitrogen, Milan, Italy) at 37°C in a humidified 5% CO2 atmosphere. In vaccine experiments, cells were treated with 40 µg/ml mitomycin C (Sigma-Aldrich, Milan, Italy) to block cell proliferation. Surface expression of p185neu and class I H-2q molecules was assessed by flow cytometry using mAbs 7.16.4 (p185neu; Oncogene Research Products, Cambridge, MA), KH114 (H-2Kq; BD Pharmingen), 34-7-23S (H-2Dq; Cedarlane, Hornby, Ontario, Canada), and 28-14-8S (H-2D/Lq; BD Pharmingen).
Vaccination and IL-12 treatment
Starting at the sixth week of age, mice received successive 3-wk courses of four twice-weekly i.p. vaccinations with 2 x 106 allogeneic Neu/H-2q mammary carcinoma cells in 0.4 ml of PBS, followed by five daily i.p. administrations of recombinant mouse IL-12 (kindly provided by Dr. S. Wolf, Genetics Institute, Andover, MA) in the third week. In the first course, the five injections were of 50 ng of IL-12 in 0.2 ml of PBS supplemented with 0.01% mouse serum albumin (MSA3; Sigma-Aldrich); the subsequent injections were of 100 ng of IL-12. After 1 wk of rest, the course was repeated until mice were sacrificed or were 1 year old. Hereafter, mice receiving this combined treatment are referred to as vaccinated mice. Control mice received either mock vaccination or MSA administration. Their tumor progression mirrored that of untreated mice.
Morphologic and immunohistochemical analysis
Tissue samples were processed as described previously for histologic evaluation or for immunohistochemistry (15). The following Abs were used: anti-endothelial cells (anti-CD31, clone mEC-13.324; provided by A. Vecchi, Istituto M. Negri, Milan, Italy); anti-p185neu (C-18; Santa Cruz Biotechnology, Santa Cruz, CA), anti-proliferating cell nuclear Ag (PCNA; Ylem, Milan, Italy), and anti-CD45/B220 (BD Pharmingen).
Whole-mount preparation of mammary glands
Whole-mount preparations were performed as reported by Medina (16). Briefly, the skin of euthanized mice was fixed overnight in 10% buffered formalin. The mammary fat pads were scored into quarters and gently scraped from the skin. The quarters were immersed in acetone overnight and then rehydrated and stained in ferric hematoxylin (Sigma-Aldrich), dehydrated in increasing concentrations of alcohol, cleared with histo-lemon, and stored in methylsalicylate (Sigma-Aldrich). Digital pictures were taken with a Nikon Coolpix 995 (Nital, Turin, Italy) mounted on a stereoscopic microscope (MZ6; Leica Microsystems, Milan, Italy).
IFN- production by spleen cells
Spleens were collected from vaccinated and control mice, and single-cell suspensions were prepared, washed in PBS, and resuspended in RPMI 1640 supplemented with 10% FBS. Spleen cells (5 x 105 cells/ml) were then restimulated with mitomycin C-treated target cells (5 x 104 cells/ml) for 6 days at 37°C in RPMI 1640 with 10% FBS containing 10 U/ml recombinant mouse IL-2 (PeproTech, Rocky Hill, NJ). Supernatants were assayed for IFN- production by ELISA purchased from Endogen (Woburn, MA).
Ab response
Mice were routinely bled from a lateral tail vein, and sera were stored frozen at –80°C. Analysis of Ab content of sera diluted 1/65 was performed by indirect immunofluorescence followed by flow cytometry. Neu/H-2q and Neuneg/H-2q cells were used as targets for Ab binding. Total Ig binding was evaluated using a FITC-conjugated goat anti-mouse IgG (H+L) chains secondary Ab (Euroclone, Milan, Italy). For Ig subclass analysis, the following secondary FITC-conjugated mAbs were purchased from BD Pharmingen: anti-mouse IgG1 clone A85-1, anti-mouse IgG2a clone R19-15, anti-mouse IgG2b clone R12-3, anti-mouse IgG3 clone R40-82, anti-mouse IgM clone R6-60.2, anti-mouse IgA clone C10-3, and anti-mouse IgE clone R35-72.
Complement-mediated and Ab-dependent cellular cytotoxicity (ADCC)
Complement-mediated cytotoxicity test was performed as described previously (12). The ADCC test was performed according to Sung et al. (17). Neu-positive H-2d mammary tumor cells (12) were plated at 104 cells/well in 96-multiwell plates and allowed to attach overnight. Cultures were incubated for 2 h on ice with sera from experimental mice at different dilutions. After washing, spleen cells from normal BALB/c mice were added at 50:1 E:T ratio and plates were incubated at 37°C overnight. Nonadherent cells were carefully washed out, 20 µl of WST-1 solution (Roche Diagnostics, Milan, Italy) was added to each well, and absorbance at 450 nm was read after an additional 4-h incubation.
Immunoprecipitation and Western blot analysis
Neu/H-2q cells and Neuneg/H-2q cells were lysed for 30 min on ice with 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS, 10% glycerol, 150 mM NaCl plus phosphatase and protease inhibitors, 1 mM PMSF, 1 µg/ml aprotinin, 50 mM NaF, and 10 mM sodium pyrophosphate (all reagents were purchased from Sigma-Aldrich). Cell lysates were normalized at a protein concentration of 4 mg/ml with lysis buffer and diluted 1/3 with TBS (50 mM Tris-HCl (pH 7.5) plus 150 mM NaCl) to decrease detergent concentration. Cell lysates (500 µg of total protein each sample) were immunoprecipitated with 3 µg/ml anti-rat neu mAb 7.16.4 (Ab-4; Oncogene Research Products) or with 20 µl of sera from untreated or vaccinated mice for 2 h at 4°C. Immunoprecipitated proteins were eluted following overnight incubation with protein A-agarose (Santa Cruz Biotechnology), washed twice in TBS, and denatured at 100°C for 10 min in 2x Laemmli sample buffer (Bio-Rad, Hercules, CA) containing SDS and 5% 2-ME (Sigma-Aldrich). Proteins were then separated on 7.5% SDS-PAGE gels (Ready Gel; Bio-Rad) and transferred onto polyvinylidene fluoride membranes (Bio-Rad). After blocking with PBS containing 0.1% Tween 20 and 5% nonfat dry milk, membranes were incubated overnight at 4°C with anti-neu rabbit polyclonal Ab diluted 1/200 in blocking buffer (C18; Santa Cruz Biotechnology). Finally, the presence of immunoprecipitated HER-2/neu proteins was detected by 1-h incubation at room temperature with HRP-linked goat anti-rabbit Ab (Santa Cruz Biotechnology; diluted 1/1000 in blocking buffer), followed by a colorimetric reaction (Opti-4CN Substrate kit; Bio-Rad).
To determine whether vaccination-induced Abs recognize cryptic HER-2/neu epitopes, Neu/H-2q and Neuneg/H-2q cell lysates were immunoprecipitated with Ab-4 anti-rat neu mAb, denatured under reducing conditions, and blotted as described above. Membranes were then blocked with PBS containing 0.1% Tween 20 and 5% BSA and incubated with mice sera diluted 1/250 in PBS containing 0.1% Tween 20 and 2% BSA overnight at 4°C. Bound Abs were detected by HRP-labeled goat anti-mouse IgG Ab (Santa Cruz Biotechnology; diluted 1/1000), followed by the colorimetric reaction.
To study the effect of serum Abs on the phosphorylation status of p185neu, Neu/H-2q cells were treated for 1 h at 37°C with sera from untreated and vaccinated mice, diluted 1/10 in DMEM without serum. Cell lysates were immunoprecipitated with anti-rat neu Ab-4 mAb as described above. Aliquots of immunoprecipitated proteins were analyzed in parallel by Western blotting using C18 rabbit anti-neu polyclonal Ab to detect HER-2/neu receptor expression level and 1.5 µg/ml biotinylated antiphosphotyrosine Ab (4G10; Upstate, Lake Placid, NY) to detect the phosphorylated HER-2/neu receptor.
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or B cells
HER-2/neu transgenic mice, referred to as NeuT mice, develop mammary carcinomas in all 10 mammary glands by the age of 32 wk. We found that a combination of MHC allogeneic tumor cells expressing p185neu and systemic rIL-12 was the minimal vaccine that could arrest in all mice mammary carcinogenesis and reduce tumor incidence by 90–100% (12). The major responses elicited by the combined treatment in transgenic mice were an abundant production of IFN- by T cells, in particular by CD8+ lymphocytes, and a strong Ab response directed against p185neu. To analyze their importance in the protection from mammary carcinoma afforded by vaccination, NeuT transgenic mice were crossed with knockout mice deficient in IFN- release (NeuT-IFN-–/–) or in B cell immunity (NeuT-µMT).
Although the combined treatment protected almost all NeuT mice, none of the NeuT-IFN-–/– or NeuT-µMT were protected (Fig. 1). Differences between NeuT-µMT and NeuT-IFN-–/– were apparent in tumor latency and in the number of carcinomas per mouse (tumor multiplicity), which were significantly (p < 0.01 at least) reduced by vaccinations in B cell-deficient mice, but not in mice lacking IFN-.
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- or B cell-deficient micePathological studies revealed that by the third week of age the mammary glands of NeuT mice developed foci of atypical hyperplasia, which subsequently extended to all mammary glands. Foci of carcinoma in situ, first apparent around the 15th week of age, evolved to invasive carcinomas by the 20th week of age and were present in all the glands 10–12 wk later.
In NeuT mice vaccinated with Neu/H-2q cells plus IL-12, the mammary glands showed the development of a limited number of atypical hyperplastic foci often surrounded by reactive cells. These hyperplastic foci decreased with time so that, at 45–50 wk of age, the mammary glands were free of hyperplastic foci and tumors. In NeuT-IFN-–/– and NeuT-µMT mice treated with IL-12 or Neu/H-2q cells plus IL-12, the invasive lobular carcinomas that eventually developed in mammary glands were histologically very similar to those of untreated NeuT mice.
Importantly, in NeuT mice, the combined treatment resulted in a scarce expression of p185neu, the HER-2/neu gene product that, if present, was mainly confined to the cytoplasm of mammary duct epithelial cells, which rarely stained for PCNA, a marker of tumor cell proliferation (Fig. 2, B and F). By contrast, a marked cytoplasmic and membrane p185neu expression, similar to that found in hyperplastic and neoplastic lesions of untreated mice (Fig. 2A), was displayed by neoplastic cells of carcinomas developed in both NeuT-µMT and NeuT-IFN-–/– mice (C and D). Expression of p185neu was associated with a marked expression of PCNA (Fig. 2, E, G, and H).
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0.005) decrease in the number of microvessels supplying hyperplastic or neoplastic lesions in vaccinated NeuT and NeuT-µMT mice when compared with that of untreated NeuT mice (vessel count in vaccinated NeuT mice, 6.1 ± 1.7; in vaccinated NeuT-µMT, 9.8 ± 1.2; in vaccinated NeuT-IFN-–/–, 11.7 ± 1.9; in untreated NeuT mice, 14.4 ± 2.2). A similar reduction in microvessel density was observed in NeuT and NeuT-µMT mice treated with IL-12 only (9.0 ± 1.8 and 9.2 ± 1.5, respectively). Vaccination elicits Ab production in B cell-deficient NeuT-µMT mice
It has been reported that B cells from µMT mice, in particular on a BALB/c genetic background, can bypass the IgM defect and undergo class switch leading to Ig secretion (18, 19, 20, 21). We found that only one third of NeuT-µMT mice receiving the vaccine was truly unresponsive, whereas the remaining two thirds of mice produced abundant high-titer Abs against the vaccine (Fig. 3A). We then compared the serum Ab levels of Ab-proficient NeuT-µMT mice with those of the two parental mouse lines, NeuT and µMT (Fig. 3B). The frequency of Ab production in response to the vaccine was 100% both in NeuT and µMT mice, and the levels of serum Abs were also similar. It could be concluded that an effective vaccine elicited very high levels of Abs even in B cell-deficient µMT mice. Only when the two genetic lesions were combined in the same mouse (i.e., in NeuT-µMT mice), we observed a lack of anti-vaccine Abs production in about one third of mice and reduced Ab levels in the remaining two thirds. This may be attributed to the fact that HER-2/neu expression makes NeuT-µMT mice tolerant to HER-2/neu gene product p185neu (9).
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The bimodal distribution of Ab responses among vaccinated NeuT-µMT mice prompted us to re-evaluate the survival curves shown in Fig. 1. A clear difference in survival became evident when vaccinated NeuT-µMT mice were stratified according to their Ab response (Fig. 5). Mice that did not produce Igs in response to vaccination were prone to early onset of mammary carcinoma with a slight delay in comparison to untreated mice. On the contrary, tumor onset was significantly delayed in vaccinated NeuT-µMT mice able to mount a sizeable Ab response (Fig. 5). The analysis of whole-mount preparation of mammary glands before the onset of macroscopic carcinomas (16-wk-old mice) showed the presence of diffuse atypical hyperplasia and occasional in situ carcinomas in both NeuT and Ab-deficient NeuT-µMT mice, whereas in the mammary glands of Ab-proficient NeuT-µMT mice, hyperplastic nodules were rare and in situ carcinomas were absent (Fig. 6).
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production by NeuT-µMT lymphocytes
The survival curves and tumor multiplicities of Ab-deficient NeuT-µMT clearly showed a residual protection from mammary carcinoma that was completely absent in NeuT-IFN-–/– mice (compare Figs. 1 and 5). IFN-, besides its well-known activities on B and T cells, can directly inhibit the growth of preneoplastic and neoplastic mammary cells (12); moreover, it can hamper tumor growth through the induction of the antiangiogenic chemokines monokine induced by IFN- and IFN--inducible protein 10. We found that IFN- release by spleen cells of vaccinated NeuT-µMT mice was clearly detectable in vitro, although at a level lower than that of vaccinated NeuT mice (Fig. 7). After in vitro restimulation with the vaccine, splenocytes from both NeuT and NeuT-µMT mice released comparable amount of IFN-. The residual protection from mammary carcinoma produced by vaccination in Ab-negative NeuT-µMT mice provides an estimate of the relative importance of non-B cell-mediated effects of IL-12 and IFN- in this system.
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Mammary carcinogenesis was significantly delayed by vaccination in Ab-proficient NeuT-µMT mice; however, all mice eventually succumbed to progressive tumors, unlike NeuT mice that remained tumor free for >1 year. This suggests that the Ab response of B cell-deficient NeuT-µMT mice was lacking in comparison with that of NeuT mice. We have already shown that anti-vaccine Ab level of NeuT-µMT mice was quantitatively inferior to that of NeuT mice (Fig. 3). Western blot analysis of Ab specificity showed that sera from Ab-proficient NeuT-µMT mice specifically recognized and immunoprecipitated p185neu molecules from HER-2/neu-positive transgenic cell lysates; however, a strong quantitative difference was evident in comparison to sera from NeuT mice (Fig. 8A). Sera from Ab-proficient NeuT-µMT mice showed a marginal activity against denatured p185neu, and sera from Ab-deficient mice were again negative (Fig. 8B), thus indicating the absence of Abs against cryptic determinants. We never observed a significant difference between sera of untreated and vaccinated mice when tested on cell lysates of p185neu-negative cells.
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–/–, and NeuT-µMT mice in response to vaccination (Fig. 9). We reported previously (12) that the absence of tumor prevention in NeuT-IFN-–/– mice was associated with the lack of IgG2a, IgG2b, and IgG3 subclasses, whereas IgG1 production was similar to that of NeuT mice. The isotype spectrum of NeuT-µMT mice was clearly distinct from that of NeuT-IFN-–/–; however, it showed some subtle differences in comparison with NeuT mice. In particular, both IgG2a and IgG2b were produced by NeuT-µMT mice, but the IgG2a:IgG2b ratio was reversed in comparison with NeuT mice. IgG3 production was low in vaccinated NeuT mice, but was completely absent in NeuT-µMT mice.
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–/– (Fig. 10A). Only sera from immunocompetent NeuT mice determined significant levels of ADCC (Fig. 10B).
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Discussion |
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or B cells were not protected from mammary carcinoma by a highly effective vaccination combining IL-12 and MHC allogeneic cells expressing the p185neu Ag.
IFN- release was the most conspicuous T cell response present in vaccinated HER-2/neu transgenic mice, in the absence of detectable CTL (12). The complete disappearance of protection from mammary carcinoma in transgenic mice lacking IFN- confirmed the pivotal role of this cytokine. Morphological data indicated that IFN- production by reactive cells was fundamental to elicit an efficient antitumor response (12). Its absence, in fact, resulted in a scarce intratumoral recruitment of reactive cells, which were also unable to produce proinflammatory cytokines and chemokines (data not shown). Inhibition of tumor neoangiogenesis, mainly induced by IL-12-elicited IFN-, contributed to the effectiveness of the combined treatment, because the vascular network supplying mammary hyperplasia in NeuT or carcinomas in NeuT-µMT mice was reduced in comparison with untreated NeuT and treated NeuT-IFN-–/– mice.
However, from a heuristic point of view, the pleiotropic action of IFN-, which is simultaneously a regulatory molecule in most antitumor immune responses and a direct effector acting on tumor cells themselves, did not allow a clear dissection of further immune mechanisms.
We found that different immune treatments were able to delay mammary carcinogenesis (25), but the jump from delay to effective prevention of carcinoma was invariably accompanied by the appearance of anti-p185neu Abs (6, 12), thus indicating a fundamental role for the B cell response in combination with T cell-derived cytokines (8, 22, 26). Abs directed against membrane oncoproteins like p185neu may play multiple antitumor roles that include block of mitogenic signal transduction through inhibition of receptor dimerization and induction of internalization and recycling along with immune-mediated functions like complement-mediated cytotoxicity and ADCC (8, 12, 22, 23, 24, 27, 28).
To formally demonstrate the role of Abs in cancer prevention, we vaccinated B cell-deficient, HER-2/neu transgenic NeuT-µMT mice. To our surprise, two thirds of NeuT-µMT mice responded to vaccination with a copious production of specific Abs of various Ig subclasses. This result is in general agreement with recent findings that µMT mice of BALB/c background are capable of mounting "natural" and Ag-induced Ab responses (20, 21). In µMT mice, Abs are produced by plasma cells deriving from B cells that completed development either via surface expression of IgD or possibly through premature switching to IgG1 or other isotypes. It has been reported that immunization with either T cell-dependent or -independent Ags induces Ab responses only in about one third of µMT mice (20), whereas our vaccine was able to induce specific Abs in 100% of µMT mice. This finding points to the great potency of this combination of IL-12, allogeneic MHC, and p185neu.
At variance with µMT mice, NeuT-µMT mice were not uniformly able to mount Ab responses to our vaccine. HER-2/neu transgenic mice are tolerant to the p185neu protein (9); thus, the difference in the proportion of vaccine responses between µMT and NeuT-µMT mice may result from the immunological tolerance of the latter. This dichotomous behavior of NeuT-µMT mice in response to our vaccine was exploited to investigate long-term prevention of mammary carcinoma in the absence of Ab response. Without protective Abs, the preventive effect of the vaccine vanished, whereas a high Ab response went along with a delayed onset of mammary carcinomas.
The mean latency of tumors in vaccinated, Ab-deficient NeuT-µMT mice was about 1 month longer than in untreated mice. A similar delay (3 wk) was induced by the administration of IL-12 alone, without allogeneic cells. These data can be compared with the complete inefficiency of vaccination in NeuT-IFN-–/– mice, allowing us to dissect immune-mediated from direct antitumor effects of IFN-. It could be concluded that the major contribution of IFN- in the prevention of mammary carcinoma was its activity of immune mediator, in particular for what concerns the induction of a switch to Th1-type Igs. On the contrary, direct antitumor activities, such as the induction of antiangiogenic chemokines (monokine induced by IFN- and IFN--inducible protein 10), or the inhibition of preneoplastic cell proliferation, in the absence of Abs, produced only a short delay in tumor appearance.
Vaccination induced a significant delay of mammary carcinogenesis in Ab-proficient NeuT-µMT mice, but all mice eventually developed tumors. By contrast, NeuT mice were completely protected by vaccination. This suggests that the B cell deficit extant in Ab-proficient NeuT-µMT mice precluded a complete prevention of mammary carcinoma, a fact that could shed further light on the nature of protective Abs. As mentioned above, quantitative differences in Ab titers may play a role in lowering the protection. Even Ab-proficient NeuT-µMT mice had significantly lower levels of anti-p185neu Abs than NeuT mice. Major qualitative differences among NeuT, NeuT-IFN-–/–, and NeuT-µMT were evident in the IgG subclasses elicited by vaccination. An abundant IgG1 response in NeuT-IFN-–/– mice, which were not protected by vaccination, indicates that the 1 chain was not involved in prevention. NeuT-IFN-–/– mice lacked anti-vaccine IgG2a and IgG2b, two Ab subclasses that were produced both by NeuT and by Ab-proficient NeuT-µMT mice. In NeuT mice, the IgG2a:IgG2b ratio was skewed in favor of IgG2a, whereas in NeuT-µMT, the reverse was true. It has been shown that, in the absence of µ-chains, the 2b chain can support B cell development (29, 30); thus, the imbalance found in NeuT-µMT mice could be the consequence of the alternative pathways leading to B cell survival and differentiation in this knockout mouse (20). From the point of view of cancer prevention, the imbalance found in NeuT-µMT suggests that a response skewed to IgG2a might provide a better protection from mammary carcinoma. IgG3 were induced by vaccination in NeuT mice, but not in NeuT-IFN-–/– or in NeuT-µMT mice. Because µMT mice are specifically deficient in IgG3 production (20, 21), the NeuT-µMT model could not be used to assess the relative importance of this isotype in cancer prevention.
An intriguing alteration in the specificity of nonprotective, "bystander" Abs was evident. We found that vaccination induced protective Abs directed against p185neu both in NeuT and in NeuT-µMT mice; thus, we believe that quantitative and isotypic differences discussed above explain the lower degree of cancer prevention obtained in Ab-proficient NeuT-µMT mice. However, the combined vaccine is expected to induce a wide range of Abs recognizing determinants other than p185neu. In particular, the expression of allogeneic MHC molecules by cells in the vaccine should elicit anti H-2q class I Abs. Such Abs are a byproduct devoid of preventive efficacy, because mice receiving IL-12 plus allogeneic cells lacking p185neu are not protected from mammary carcinoma (12). The very low level of anti-class I Abs found in vaccinated, Ab-proficient NeuT-µMT mice indicates a profound alteration in the ability to respond to multiple Ags. A possible explanation is that the selection caused by the p185neu Ag irreversibly shapes the development of B cells in deficient NeuT-µMT mice (31, 32).
In conclusion, we showed that B cell responses regulated by IFN- were a key mechanism mediating prevention of mammary carcinoma in mice vaccinated with allogeneic tumor cells and IL-12. This conclusion is important both to direct further investigation of the B cell-mediated mechanisms at work in the HER-2/neu system (e.g., Ag presentation, cytokine release, complement-mediated cytotoxicity, ADCC, direct down-modulation of p185neu) and to design similar attempts at preventing cancer in humans.
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Acknowledgments |
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Footnotes |
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1 This work was supported by grants from the Italian Association for Cancer Research; the Italian Ministry for Education, University, and Research; and the Universities of Bologna and Torino. A.A. and S.C. are supported by fellowships from the Italian Foundation for Cancer Research. 
2 Address correspondence and reprint requests to Dr. Pier-Luigi Lollini, Sezione di Cancerologia, Viale Filopanti 22, I-40126 Bologna, Italy. E-mail address: pierluigi{at}lollini.dsnet.it
3 Abbreviations used in this paper: MSA, mouse serum albumin; PCNA, proliferating cell nuclear Ag; ADCC, Ab-dependent cellular cytotoxicity.
Received for publication September 18, 2003. Accepted for publication June 7, 2004.
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J. T. Garrett, S. Rawale, S. D. Allen, G. Phillips, G. Forni, J. C. Morris, and P. T. P. Kaumaya Novel Engineered Trastuzumab Conformational Epitopes Demonstrate In Vitro and In Vivo Antitumor Properties against HER-2/neu J. Immunol., June 1, 2007; 178(11): 7120 - 7131. [Abstract] [Full Text] [PDF]
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B. J. Czerniecki, G. K. Koski, U. Koldovsky, S. Xu, P. A. Cohen, R. Mick, H. Nisenbaum, T. Pasha, M. Xu, K. R. Fox, et al. Targeting HER-2/neu in Early Breast Cancer Development Using Dendritic Cells with Staged Interleukin-12 Burst Secretion Cancer Res., February 15, 2007; 67(4): 1842 - 1852. [Abstract] [Full Text] [PDF]
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S. Rolla, C. Nicolo, S. Malinarich, M. Orsini, G. Forni, F. Cavallo, and F. Ria Distinct and Non-Overlapping T Cell Receptor Repertoires Expanded by DNA Vaccination in Wild-Type and HER-2 Transgenic BALB/c Mice J. Immunol., December 1, 2006; 177(11): 7626 - 7633. [Abstract] [Full Text] [PDF]
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T. Pannellini, M. Spadaro, E. Di Carlo, E. Ambrosino, M. Iezzi, A. Amici, P. L. Lollini, G. Forni, F. Cavallo, and P. Musiani Timely DNA Vaccine Combined with Systemic IL-12 Prevents Parotid Carcinomas before a Dominant-Negative p53 Makes Their Growth Independent of HER-2/neu Expression. J. Immunol., June 15, 2006; 176(12): 7695 - 7703. [Abstract] [Full Text] [PDF]
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T. P. Moran, M. Collier, K. P. McKinnon, N. L. Davis, R. E. Johnston, and J. S. Serody A Novel Viral System for Generating Antigen-Specific T Cells J. Immunol., September 1, 2005; 175(5): 3431 - 3438. [Abstract] [Full Text] [PDF]
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W. Luo, J. C.-f. Hsu, C.-Y. Tsao, E. Ko, X. Wang, and S. Ferrone Differential Immunogenicity of Two Peptides Isolated by High Molecular Weight-Melanoma-Associated Antigen-Specific Monoclonal Antibodies with Different Affinities J. Immunol., June 1, 2005; 174(11): 7104 - 7110. [Abstract] [Full Text] [PDF]
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L. A. O'Mara, L. A. Norian, D. Kreamalmeyer, J. M. White, and P. M. Allen T Cell-Mediated Delay of Spontaneous Mammary Tumor Onset: Increased Efficacy with In Vivo versus In Vitro Activation J. Immunol., April 15, 2005; 174(8): 4662 - 4669. [Abstract] [Full Text] [PDF]
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J. M. Park, M. Terabe, Y. Sakai, J. Munasinghe, G. Forni, J. C. Morris, and J. A. Berzofsky Early Role of CD4+ Th1 Cells and Antibodies in HER-2 Adenovirus Vaccine Protection against Autochthonous Mammary Carcinomas J. Immunol., April 1, 2005; 174(7): 4228 - 4236. [Abstract] [Full Text] [PDF]
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 A. Astolfi, L. Landuzzi, G. Nicoletti, C. De Giovanni, S. Croci, A. Palladini, S. Ferrini, M. Iezzi, P. Musiani, F. Cavallo, et al. Gene Expression Analysis of Immune-Mediated Arrest of Tumorigenesis in a Transgenic Mouse Model of HER-2/neu-Positive Basal-Like Mammary Carcinoma Am. J. Pathol., April 1, 2005; 166(4): 1205 - 1216. [Abstract] [Full Text] [PDF]
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-neu Ab) or with sera from vaccinated NeuT mice (NeuT/Vax) are shown for comparison. The C18 anti-p185neu rabbit Ab was used for Western blot. B, Immunoreactivity of mice sera against cryptic epitopes of p185neu protein. p185neu was immunoprecipitated with Ab4 mAb, denatured, and blotted using sera as above or C18 Ab (
