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The Journal of Immunology, 2006, 176: 1148-1153.
Copyright © 2006 by The American Association of Immunologists

Polyoma Virus-Like Particles Elicit Polarized Cytokine Responses in APCs from Tumor-Susceptible and -Resistant Mice1

Palanivel Velupillai*, Robert L. Garcea{dagger} and Thomas L. Benjamin2,*

* Department of Pathology, Harvard Medical School, Boston MA 02115; and {dagger} Section of Pediatric Oncology, University of Colorado School of Medicine, Denver, CO 80262


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
PERA/Ei (PE) mice are highly susceptible to tumor induction by polyoma virus, whereas C57BR/cdj (BR) mice are highly resistant. PE mice respond to viral infection with a type 2 (IL-10) and BR mice with a type 1 (IL-12) cytokine response, underlining the importance of a sustained T cell response for effective antitumor immunity. PE and BR mice showed comparable Ab responses to the virus, indicating that a Th1 response is fully compatible with strong humoral immunity. Tumor susceptibility is dominant, and a type 2 response prevails in F1 mice derived from these strains. In this study, we show that the different cytokine responses of virus-infected hosts are recapitulated in vitro by exposure of APCs from uninfected PE, BR, and F1 animals to the virus. Importantly, virus-like particles formed from recombinant VP1, the major viral capsid protein, elicited the same host-specific cytokine responses as infectious virus. Assembly of VP1 pentamers into capsid shells is required because unassembled VP1 pentamers were ineffective. Binding of virus-like particles to sialic acid is required because pretreatment of APCs with neuraminidase prevented the response. Expression of TLR2 and TLR4 differed among different subpopulations of APCs and also between resistant and susceptible mice. Evidence is presented indicating that these TLRs play a role in mediating the host-specific cytokine responses to the virus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The mouse polyoma virus (Py)3 is a small nonenveloped icosahedral particle with a ~5.2-kb DNA genome. Its outer shell is formed by the linking together of 72 capsomeres, each one a pentamer of the major capsid protein VP1. The VP1 pentamer is able to bind up to five sialic acid molecules (1) linked either to glycolipids or glycoproteins. Specific gangliosides expressed on plasma membranes of a wide variety of cells mediate infection by binding and transporting the virus to the endoplasmic reticulum (2). The virus undergoes steps of disassembly in the endoplasmic reticulum before exit and entry into the nucleus.

Py is highly oncogenic in its natural host under experimental conditions. When inoculated as newborns, mice of susceptible strains rapidly develop a variety of solid tumors (3, 4). Tumor induction depends on expression of the three viral T (tumor) Ags. These early viral gene products act to disrupt signal transduction pathways that regulate cell growth and survival. The subsequent expression of capsid proteins then results in a lytic infection with production of progeny virus and cell death. When separated from the rest of the virus growth cycle, however, continued expression of the T Ags leads to cell transformation in culture and tumor induction in the animal.

Py tumors are highly immunogenic, leading to rejection in most immunocompetent hosts. The T Ags of Py and the closely related primate polyoma virus SV40 comprise the classically defined "tumor-specific transplantation Ags" of these viruses. Rejection is based primarily on cytolytic CD8+ T cells specific for MHC-restricted T Ag-derived peptides (5, 6, 7, 8, 9, 10). Interestingly, in certain crosses between tumor-susceptible and tumor-resistant mice of the same H2 haplotype, susceptibility is found to be dominant (5, 11, 12). Mechanisms that prevent the development or maintenance of tumor immunity must therefore be operating in these susceptible hosts.

Several distinct mechanisms underlie susceptibility in different mouse strains. Among the most highly susceptible hosts are H2k strains that carry an endogenous mouse mammary tumor virus (MMTV). Expression of the superantigen Mtv-7 Sag in these strains during late prenatal and early postnatal life leads to deletion of V{beta}6+ CTL precursors needed for rejection of Py tumors in H2k mice (5). C57BR/cdj (BR) mice, which are H2k but lack Mtv-7, mount V{beta}6+CD8+ T cell responses and develop no tumors (12). A different mechanism is based not on the absence of virus-specific CD8+ T cells but on a failure of these cells to develop effector functions due to expression of the inhibitory NK receptor CD94/NKG2A (13). A third genetically distinct basis of susceptibility operates at the level of innate rather than adaptive immunity (14).

Differences in innate immune responses to Py have been documented between certain susceptible "wild-derived" inbred strains such as PERA/Ei (PE) and the classical inbred BR strain, which is resistant (12, 14). These strains are identical in their H2k haplotype and in being free of interfering endogenous superantigen(s). They differ, however, in their cytokine responses to Py infection, PE mice responding with a type 2 and BR mice with a type 1 response. The susceptibility of PE is dominant (or semidominant) in crosses with BR. Adherent cells from spleens of infected PE and F1 mice when examined ex vivo produce high levels of IL-10. The CTL response in these mice is weak and transient. In contrast, adherent spleen cells from infected BR mice produce IL-12. These mice sustain an effective CTL response through induction of IFN-{gamma} (14). Administration of rIL-12 to PE mice before infection results in a lower incidence and a significant delay in tumor development. The same treatment of F1 animals confers complete protection against tumor development (14).

In the present study, we exposed APCs from uninfected PE, BR, and F1 mice in vitro to infectious virus and to well-characterized subviral assemblies of the major viral capsid protein VP1. These interactions lead to the same polarized cytokine responses as previously documented in virus-infected hosts. We show that different subpopulations of APCs are involved in production of the signature cytokines depending on the host donor. We propose that differential expression of TLR2 and TLR4 in these APC subpopulations is responsible, at least in part, for the host-specific cytokine responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mouse strains

BR and PE mice were purchased from The Jackson Laboratory. All mice were bred and maintained in our virus Ab-free barrier facility before use in the experiments.

mAbs and conjugates

mAbs to cell surface markers CD11b, CD11c, B220, CD3, CD40, and IAk were purchased from BD Pharmingen. The anti-TLR2 and TLR4 were obtained from eBiosciences.

FACS

Total splenic leukocytes were obtained following lysis of erythrocytes in ammonium chloride solution (0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM Na2EDTA). Peritoneal exudate cells (PEC) were harvested from peritoneal cavity by injecting 5 ml of ice-cold PBS containing heparin sulfate. Cells (1 x 106) were stained in PBS containing 2% FCS and 0.01% sodium azide. Cells were first incubated with purified rat anti-mouse CD16/CD32 mAb and then with fluorochrome-labeled mAbs or isotype control Igs (BD Pharmingen). Cells were incubated for 1 h at 4°C, followed by three washes in buffer, and fixed in PBS containing 1% paraformaldehyde. Flow cytometry was performed on a Becton Dickinson FACScan using CellQuest software (BD Biosciences).

Generation of virus-like particles (VLPs) and VP1 pentamers

Py VLPs were purified from Sf9 cells after infection with a recombinant baculovirus-expressing VP1 (15, 16). Pentamers of a VP1 mutant truncated at the C terminus were purified as described (17) and further adsorbed against polymyxin B Sepharose to remove any residual endotoxin. Both protein preparations had equivalent trace endotoxin levels (≤125 IU/ml) measured by the limulus assay (Sigma-Aldrich).

Neuraminidase treatment of splenocytes

Total splenocytes were harvested and suspended in PBS. Cells were incubated with 1 U/ml neuraminidase (Sigma-Aldrich) at 37°C for 1 h and washed in RPMI 1640 containing FCS. Treated cells were infected with the virus, stimulated with VLP, or anti-CD40.

Generation of cell-culture supernatants

Subsets of APCs (5 x 106) from different mouse strains were either infected with virus or stimulated with VLP. Cells were cultured in complete IMDM for 36 h, and then the supernatants were harvested for cytokine analysis.

Detection of cytokines

A two-site capture ELISA was performed to detect cytokines in the cell-culture supernatants. Pairs of matched rat mAbs specific to murine IL-10 and IL-12 (p70) and the relevant recombinant proteins as standards were all purchased from BD Pharmingen. Polystyrene microtiter plates (Dynex) were coated with 2 µg/ml (50 µl/well) murine cytokine-specific purified mAbs. The plates were incubated overnight at 4°C, washed in PBS containing 0.05% Tween 20 (Sigma-Aldrich), and blocked with PBS containing 10% FCS. The culture supernatants were then added to wells and incubated overnight at 4°C for capture of cytokines. The wells were washed followed by addition of 1 µg/ml (100 µl/well) biotinylated mAbs and allowed to stand at room temperature for 2 h. Avidin-peroxidase (Sigma-Aldrich) was used for detection in the presence of tetramethylbenzidine as substrate (Kirkegaard and Perry Laboratories). Absorbance was measured at 450 nm in an ELISA microplate reader (Dynatech). Serial dilutions of recombinant cytokine proteins were included to construct a standard curve for quantitating the amount of cytokine in the culture supernatants.

Cell fractionation of APCs and FACS

Total splenocytes and PECs were separated into subpopulations and stained using fluorochrome-labeled Abs. Cells were plated onto a polystyrene plastic dish and incubated at 37°C for 2 h. The adherent cells (macrophages and dendritic cells) were left overnight for further incubation. At the end of this incubation, the detached floating cells (dendritic cells) were harvested. The adherent cells (macrophages) were harvested using a cell scraper. Plastic nonadherent cells were further plated on a anti-CD3 Ab-coated plastic surface and incubated for further 2 h. The nonadherent cells (B cells) were harvested and used in the additional experiments. These different lymphocyte populations were immunostained using cell surface-specific mAbs. Dendritic cells were 70–77% positive for CD11c, macrophages were 82–87% for CD11b, and B cells were 85–90% for B220. Levels of cross-contamination were as follows: B cells contained 2–4% CD11b- and CD11c-positive cells; macrophages contained 3–5% of B220- and CD11c-positive cells; and dendritic cells contained 4–6% of B220- and CD11b-positive cells. CD3-positive cells were present at <1% in all APC subpopulations.

Virus neutralization

Serum samples were serially (2-fold) diluted and preincubated with the virus (8 hemagglutination units final concentration) in a V-bottom 96-well plate and incubated at 37°C for 60 min. An equal volume of a 0.4% (vol/vol) suspension of guinea pig erythrocytes (Colorado Serum Company) was added to each well, and the plates were incubated at 4°C overnight. The hemagglutination inhibition (HAI) titers were determined as the reciprocal of the highest dilution of serum giving complete agglutination.

N.B.

Work on mice described in this report has been conducted under protocols reviewed and approved by the Institutional Animal Care and Use Committee of Harvard Medical School.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Py infections of PE and BR mice elicit comparable antiviral humoral responses

It was shown previously that infection of newborn PE and BR mice with Py elicits opposite innate immune responses, a type 2 response in PE and a type 1 response in BR (14). As expected, the type 1 response of BR mice driven by IL-12 and IFN-{gamma} gives rise to a sustained cytolytic T cell response directed at T Ag determinants and to effective immune surveillance against Py-induced tumors (12, 14). The type 2 (IL-10) response, which prevails in PE and F1 mice, in contrast, might be expected to favor a stronger humoral response to the virus itself. This was tested by comparing the Ab titers in sera of infected mice at weekly intervals beginning at 2 wk postinoculation. Virus-neutralizing Ab titers were determined by HAI. Results showed sharply rising titers from 2 to 5 wk in all infected mice, with no indication of differences between parental and F1 animals in either the time course or magnitude of the response (Table I). Thus, the critical difference in the outcome of innate immune responses to Py in these hosts is the interfacing of the type 1 response with adaptive responses and development of CTLs specific for T Ag determinants and not with humoral immunity to the virus.


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  		Table I. Virus-neutralizing Ab titers in sera of Py-infected micea

 
Py VLPs elicit host-specific cytokine responses from APCs in vitro

The host-specific cytokine responses that determine susceptibility vs resistance to tumor induction in the intact animal were elicited in vitro by exposure of APCs from spleen to infectious virus (Fig. 1, top panels). B cells and macrophages from PE and F1 mice were the major IL-10 responders, whereas B cells and dendritic cells from BR mice were the major IL-12 producers. Importantly, the same host-specific cytokine responses were elicited using VLPs instead of infectious virus (Fig. 1, middle panels). This was found in all instances except for macrophages from PE and F1 animals, which responded with IL-10 only to complete virus and not to VLPs. Macrophages as well as dendritic cells were successfully infected by virus as indicated by synthesis of the viral T Ags and development of characteristic cytopathic effects (results not shown). The VLPs, purified from recombinant baculovirus-infected insect cells, consist of empty shells of VP1, the major viral capsid protein, and are devoid of both minor capsid proteins VP2 and VP3 as well as of the viral DNA (15, 16). VLPs elicited the host-specific cytokines as efficiently as infectious virus based on their hemagglutination titers. These results demonstrate that induction of cytokine responses by the virus does not depend on expression of T Ags or other viral protein but only on interaction between the APCs and the outer capsid shell of the virus.



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  		FIGURE 1. VLPs elicit host-specific cytokine responses. Five million cells (macrophages, dendritic cells (DC), or B cells) from PE, BR, and F1 spleens were either infected with polyoma virus, stimulated with VLP, or C-terminal truncated VP1 pentamers in 24-well tissue culture plate. The cells were incubated for 36 h at 37°C, and the supernatants were harvested. The cytokines were measured by double sandwich ELISA. Vertical bar represents the mean ± SE of cytokine concentration of three animals in each group.

 
The interaction of VP1 with APCs requires partially or fully assembled virus shells, because free pentamers of VP1 tested at the same protein concentration as used for VLPs were 10- to 20-fold less efficient in eliciting the expected cytokine responses (Fig. 1, bottom panels). These VP1 pentamers fail to assemble into shells due to a deletion of 63 aas at the C terminus that are involved in interpentamer contacts. They nevertheless retain the ability to bind to cells via sialic acid-containing receptors (17).

PECs show host-specific cytokine response

PECs are likely the first cells to encounter virus following i.p. inoculation, a route of inoculation of newborns used in studies of tumor induction (12). B cells and macrophages from peritoneal exudates differ in their lineages and functional properties from those in spleen (18). We therefore prepared B cells and macrophages from PECs of naive PE, BR, and F1 mice and exposed them to VLPs. PE and F1 animals again showed an IL-10 response from B cells, whereas BR mice gave an IL-12 response mainly from macrophages (Fig. 2). These results confirm and extend those in Fig. 1 by demonstrating the ability of VLPs to elicit host-specific cytokine responses and the emergence of different subpopulations of APCs from the two strains as the primary responders.



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  		FIGURE 2. Induction by VLPs of cytokines in PECs from susceptible and resistant mice. Macrophages and B cells (one million) from peritoneal cavity of PE, BR, and F1 mice were stimulated with VLP in 48-well tissue culture plate. The cells were incubated for 36 h at 37°C, and the supernatants were harvested. The cytokines were measured by double sandwich ELISA. The cytokine concentrations were shown as mean ± SE of three animals in each group.

 
Selective up-regulation of MHC class II molecules in APC subpopulations by VLPs

Previous experiments using splenocytes from virus-infected hosts showed up-regulation of B7.1 in BR and of B7.2 in PE and F1 animals (14). In addition to these costimulatory molecules, mature APCs are expected to show up-regulation of MHC class II molecules (19). We found that exposure of APCs in vitro to VLPs led to up-regulation of class II in the same subpopulations of APCs as produce the cytokines. A roughly 2-fold induction of class II molecules (IAk) was found in B cells from PE and F1 mice and in dendritic cells from BR mice following exposure to VLPs (Table II), consistent with the results in Figs. 1 and 2.


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  		Table II. Expression of class II in VLP-stimulated spleen cellsa

 
Pretreatment of APCs with neuraminidase prevents VLP-induced cytokine release

The identity of virus receptors on APCs that mediate cytokine responses to Py and Py-VLPs is unknown. Conventional receptors for the virus are known to contain sialic acid in an {alpha}-2,3 linkage to carriers, which may be either glycolipids (gangliosides) or possibly sialoglycoproteins, which remain to be identified. TLRs on APCs may conceivably bind VLPs either by virtue of carbohydrate side chains bearing sialic acid or through protein sequences involved in recognition of pathogen-associated molecular patterns independently of sialic acid. To investigate these possibilities, we determined the effect of removal of cell surface sialic acids on the ability of APCs to respond. Pretreatment of APCs from parental as well as F1 mice with neuraminidase completely blocked their ability to secrete the expected cytokine in response to infectious virus or VLPs (Table III). Such treatment had no effect on IL-12 secretion by APCs stimulated with anti-CD40, indicating the importance of VLP binding through specific sialic acid linkages.


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  		Table III. Pretreatment of APCs with neuraminidase prevents VLP-induced cytokine synthesisa

 
Evidence for involvement of TLR2 and TLR4 in host-specific cytokine responses

The ability of intact virus and VLPs to stimulate cytokine responses and the inability of VP1 pentamers to do the same (Fig. 1) are consistent with a role of TLRs, which are known to recognize highly repetitive motifs in structural components of various pathogens (20, 21). Results in Figs. 1 and 2 further suggested the possibility that the inherent differences in host responses to the virus may be determined in part by differential expression of TLRs in different subpopulations of APCs. As a first step in investigating the possible involvement of TLRs, we determined the levels of expression of TLR2 and TLR4 in APC subpopulations from uninfected PE, BR, and F1 mice. We found that TLR4 was expressed at higher levels in B cells from PE and F1 mice, whereas TLR2 was preferentially expressed in macrophages and dendritic cells from BR mice (Table IV). These results correlated well with those in Figs. 1 and 2 and also Table II, which showed that B cells from PE and F1 are the major IL-10 responders to VLPs, whereas B cells and dendritic cells from BR are the major producers of IL-12.


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  		Table IV. TLR2 and TLR4 expression in APCs from resistant and susceptible micea

 
To test whether TLR2 and TLR4 play functional roles in the host-specific cytokine responses to Py, total spleen cells from PE, BR, and F1 mice were pretreated with anti-TLR2 or anti-TLR4 Ab and then exposed to VLPs (Table V). Anti-TLR4 strongly suppressed IL-10 secretion by splenocytes from PE and F1 mice. A weaker but significant inhibition of the IL-12 response in splenocytes from BR was seen with anti-TLR2. These results support a role for these TLRs in specific APC subpopulations in mediating the host-specific cytokine responses to the virus.


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  		Table V. Effect of anti-TLR2 and anti-TLR4 on VLP-induced cytokine productiona

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The encounter between viral structural proteins and cells of the innate immune system are critical early events in virus-host interactions and has been well documented in a number of different virus systems (22, 23, 24). These interactions may either subvert or promote effective immune defenses against viral infection (23). Innate immune responses in mice to the oncogenic polyoma virus differ among inbred strains and are linked to dramatically different outcomes. BR mice respond with a type 1 cytokine response leading to effective T cell immunity against the viral T Ags and to protection against tumor development (5, 6, 14). A type 2 response develops in infected PE and (PE x BR) F1 mice, which fail to mount a sustained T cell response and develop multiple tumors (14). In this study, we have shown that Py infection of naive APCs from PE and BR mice in vitro elicits the same responses as in these virus-infected hosts. Thus, spleen-derived B cells and dendritic cells from resistant BR mice respond by secretion of IL-12, whereas B cells and, to a lesser extent, macrophages from susceptible PE mice produce IL-10. Although humoral responses to the virus might be expected to be stronger following the type 2 response in PE, this outcome is not supported by the data that show equally vigorous Ab responses in PE and BR mice.

IL-12 and IL-10 are among the key cytokines that set the stage for generating Th1 or Th2 responses. MMTV glycoprotein alone activates B cells (25). MMTV infection of susceptible C3H/HeN mice leads to secretion by B cells of IL-10, a strong suppressor of Th1 cytokines (26). As shown in the present study, B cells from PE mice respond to Py VLPs by secretion of IL-10. B1 cells in the peritoneum are an important source of IL-10 (27), and these cells may be a major source of this cytokine when the virus is introduced i.p. in PE mice (14). IL-10 is an important regulator of MHC class II molecules, often down-regulating expression in macrophages and dendritic cells but up-regulating expression in B cells (28). B cells from PE and F1 mice indeed show increased class II expression when exposed to VLPs. These cells may present processed viral Ag to naive T cells, leading further to a Th2 response.

Induction of host-specific cytokine responses in APCs by Py does not require infection, although infection may well occur. Rather, the responses depend solely on binding, and possibly internalization, of virus particles. Two well-characterized subviral assemblies of the major viral capsid protein VP1 have been studied. VLPs consisting largely of shells of the same size and morphology as full virus particles elicited the same response as infectious virus. Moreover, these VLPs acted with essentially the same efficiency as infectious virus based on protein concentration or hemagglutination titers. Sialic acid, an essential component of receptors for Py on erythrocytes and conventional target cells, was also required on APCs for virus and VLPs to elicit secretion of cytokines. In contrast, pentameric assemblies of a mutant form of VP1 were 10- to 20-fold less efficient than virus or VLPs when tested at equivalent protein concentrations. These "free" pentamers lack sequences at the C terminus of VP1 required for pentamer-pentamer interaction but are unaltered with respect to sialic acid binding. Although pentamers engage a maximum of five sialic acids, capsid shells may easily be envisaged to engage 30 or more of the sugars on the cell surface. The requirement for assembly of pentamers into shells to induce a response suggests a role of TLRs that recognize molecular patterns with highly repetitive structural motifs in pathogen components (20, 21).

Viral structural proteins signal through specific TLRs to activate cells of the innate immune system (23). Thus, the envelope glycoproteins of MMTV and the Moloney leukemia virus activate B cells through TLR4 (22, 23), whereas the hemagglutinin of measles virus and glycoprotein B of human CMV induce type 1 responses through TLR2 and TLR-9, respectively (29, 30). Our results indicate inherent quantitative differences in expression of TLR2 and TLR4 among different subpopulations of APCs in PE and BR mice. Overall, TLR4 is preferentially expressed over TLR2 in APCs from PE and F1 mice, whereas APCs from BR mice preferentially express TLR2. This bias in TLR usage is particularly evident in those subpopulations that produce the host-specific cytokines in response to Py VLPs. Results of experiments using anti-TLR Abs before VLP exposure indicate functional roles for these TLRs in this system.

The full identity of virus/VLP receptors on APCs and the mechanisms of signaling that mediate cytokine release remain largely unknown. The requirement for sialic acid is consistent with a role for gangliosides as primary receptors (2). These sialoglycolipids are present on the outer leaflet of the plasma membranes of most cells. Results are also consistent with a direct interaction of the virus with TLRs, which carry complex N-linked oligosaccharide chains. Recent structural studies of the ectodomain of TLR3 reveal a heavily glycosylated surface as well as a glycosylation-free surface, a feature that may be shared with other TLRs, although ligand binding site(s) remain unknown (31). Following stimulation with bacterial LPS, TLR2 and TLR4 are found to associate with lipid-rich membrane microdomains, which are also enriched in gangliosides (32). Gangliosides have been reported to affect maturation and various functions of APCs (33, 34, 35). It is thus possible that both gangliosides and TLRs are involved in some manner in the binding and signaling by Py VLPs.

Although they harbor no endogenous MMTV superantigens, PE mice nevertheless transmit their susceptibility to tumor induction by Py in a dominant fashion in crosses with BR (12). Roughly 50% of backcross mice ((PE x BR) x BR) inoculated with the virus develop tumors, indicating a single dominant gene (12). F2 mice challenged with Py and analyzed for production of IL-12 and IL-10 likewise gave results in agreement with a single dominant gene, leading to a PE-like (IL-10) response (our unpublished results). Dominance is incomplete in that F1 animals develop fewer tumors per animal than PE (12). F1 animals inoculated with virus show a transient type 1 response that rapidly gives way to type 2. rIL-12 gives only partial protection to PE mice but confers complete protection on F1 animals (14). Along with results presented in this study, the combined evidence makes it highly likely that the gene governing the innate immune response to Py acts in a dosage-dependent manner and underlies the dominant susceptibility to tumor induction by Py in the PE x BR cross. Efforts to map and identify the gene along with further analysis of the VLP-APC interactions should help to define the underlying mechanism.


   Acknowledgments
 
We acknowledge the expert help and assistance of John Carroll in the animal experiments.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the National Cancer Institute RO1 CA-90992 (to T.L.B.) and RO1 CA37667 (to R.L.G.). Back

2 Address correspondence and reprint requests to Dr. Thomas L. Benjamin, Harvard Medical School, Pathology, NRB-939I, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail address: thomas_benjamin{at}hms.harvard.edu Back

3 Abbreviations used in this paper: Py, mouse polyoma virus; MMTV, mouse mammary tumor virus; PEC, peritoneal exudate cell; VLP, virus-like particle; HAI, hemagglutination inhibition. Back

Received for publication August 18, 2005. Accepted for publication October 26, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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