Gradual Elimination of Retroviruses in YBR/Ei Mice
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病菌学杂志 2006年第5期
The Jackson Laboratory, 600 Main Street, Bar Harbor Maine 04609
ABSTRACT
Mouse mammary tumor virus (MMTV), a well-characterized retrovirus that causes mammary tumors in susceptible mice, is commonly used to investigate virus-host interactions. We have shown that YBR/Ei mice demonstrate a novel, dominant mechanism of resistance to MMTV infection and MMTV-induced mammary tumors. MMTV can both establish infection in YBR/Ei mice and be transmitted by YBR/Ei mice as an infectious virus. However, virus production is severely attenuated, resulting in gradual clearance of infection in successive generations. Our transfer experiments showed that T cells generated in MMTV-infected resistant mice were required to restrict MMTV replication in susceptible mice. These results emphasize the importance of inducing T-cell responses for effective protection against retroviral infections.
INTRODUCTION
The ability to resist retroviruses is advantageous for the infected host because unlimited retroviral replication can be highly mutagenic and may result in numerous diseases. Studies conducted to identify cellular factors that put boundaries on retroviral transmission have resulted in identification and characterization of genes capable of hindering retroviral replication (14). We are using mouse mammary tumor virus (MMTV) to study the genetics of resistance to retroviral infection and to virally induced tumors. MMTV can be transmitted either as an endogenous virus through the germ line or as an exogenous virus (9, 32). Endogenous MMTVs (Mtvs) are commonly found in laboratory mouse strains, but most Mtvs do not produce infectious viral particles due to transcriptional regulatory or coding region mutations (9). Exogenous virus is transmitted via milk and is acquired by newborn pups when they are fostered on viremic mothers. The primary targets of the virus are cells of the immune system, especially antigen-presenting cells such as B cells (5). Infected antigen-presenting cells present the virally encoded superantigen (SAg) by major histocompatibility (MHC) class II molecules to T cells bearing a particular chain of T-cell receptor. In response to SAg presentation, SAg-cognate T cells proliferate, generating a pool of activated infection-competent cells (reviewed in reference 1). Activated SAg-cognate T cells subsequently undergo deletion, and this deletion of SAg-cognate T cells is routinely used as a readout to monitor virus infection (27, 29). Virus propagation in vivo requires SAg activity, because viruses that lack functional SAgs are not infectious (20).
Tumor induction by MMTV is a result of proviral integration in the proximity of a cellular proto-oncogene followed by activation of its expression (33). In addition, virally encoded genes are also essential for virus potency to cause mammary tumors (26, 37a). Proviral expression is regulated by sequences within the long terminal repeats (LTR) that recognize glucocorticoid receptor/steroid hormone complexes present during pregnancy (39). Multiple pregnancies are therefore associated with increased virion production and augmented numbers of infected mammary gland cells within the host. This, in turn, results in an increase in the number of viral reinfections/reintegrations and an increase in the probability of proviral integration next to cellular proto-oncogenes.
Inbred mouse strains have been employed to identify the genetic factors that influence susceptibility to MMTV-induced mammary tumors. Three mechanisms that restrict MMTV-induced tumorigenesis have previously been described: resistance based on MHC haplotype (30, 36), resistance due to the presence of endogenous Mtvs (19, 25), and resistance due to the production of virus-neutralizing antibodies (37).
MHC class II polymorphic membrane glycoproteins are required for presenting degraded, endocytosed foreign antigens to CD4+ T cells. There are two isotypic class II heterodimeric proteins of the mouse, A A (I-A) and E E (I-E), which differ in their efficiency of presenting MMTV SAgs. Inbred strains of b, f, q, or s MHC haplotype do not express I-E molecules due to mutations in the E or E genes (4, 10). Since I-E molecules present viral SAgs more efficiently than I-A molecules, mouse strains that lack the I-E molecule (such as C57BL/6J mice) are relatively resistant to MMTV infection and MMTV-induced tumors (38).
In contrast to exogenous MMTV, endogenous Mtvs present in germ line cells cause negative selection of SAg-cognate T cells during formation of the immune repertoire (1). There are more than 40 different endogenous Mtvs encoding distinct SAgs, and the presence of a particular endogenous Mtv in the mouse genome is indicated by the absence of specific T cells bearing the V chain that interacts with the SAg of this Mtv (12). Mice that inherit Mtvs are resistant to exogenous MMTVs bearing SAgs of the same T-cell specificity (19, 25) and do not succumb to mammary tumors (17).
Mice from the I/LnJ strain inherit a mechanism of resistance to MMTV-induced mammary tumors that is unrelated to the MHC haplotype or endogenous Mtvs (37). This mechanism is recessive and is dependent on production of antivirus neutralizing antibodies (37). Even though I/LnJ mice become infected with MMTV, they secrete antibody-coated virions into the milk and therefore prevent infection of their progeny (37). Since reinfection/reintegration is also ablated in infected I/LnJ mice, they resist MMTV-induced mammary tumors (37).
It has long been recognized that the Y mouse strain is resistant to tumors induced by MMTV (2, 3). Here we show that MMTV is capable of establishing infection in YBR/Ei mice, descendants of the Y strain, and that YBR/Ei mice transmit infectious virus. However, YBR/Ei mice produce the virus at severely attenuated titers and eliminate the virus in successive generations. This paper describes some of the first insights into the retrovirus resistance mechanism inherited by YBR/Ei mice.
MATERIALS AND METHODS
Mice. All mice used in this study were bred and maintained at the animal facility of The Jackson Laboratory, Bar Harbor, Maine. YBR/Ei, BALB/cJ, C3H/HeJ, C3.JK, I/LnJ, NZB/ByJ, RIII/SJ, C57BL/6, and CBySmn.CB17-Prkdcscid/J mice were purchased from The Jackson Laboratory. MMTV(C3H)-infected and uninfected C3H/HeN mice were purchased from the National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD. MMTV(LA) (34) was passed on BALB/cJ mice [BALB/c(LA) mice]. MMTV(C3H)-infected BALB/cJ mice were produced via fostering on MMTV(C3H)-infected C3H/HeN females. The studies have been reviewed and approved by the Animal Care and Use Committee at The Jackson Laboratory.
Mammary gland tumorigenesis. YBR/Ei, C3H/HeN, BALB/cJ, (Y x C)F1 and (Y x B)F1 mice were infected either with MMTV(C3H) by fostering on viremic C3H/HeN females or with MMTV(LA) by fostering on viremic BALB/cLA females. Mammary gland tumor incidence in MMTV(C3H)- or MMTV(LA)-infected mice was monitored by weekly palpation. At least 30 mice were used per group.
Antibodies and fluorescence-activated cell sorting (FACS) analysis. Fluorescein isothiocyanate-coupled monoclonal antibodies against the V14, V2, and V6 T-cell receptor chains (Sigma-Aldrich, St. Louis, MO) and phycoerythrin-coupled anti-mouse CD4 (clone H129.19) antibodies (Invitrogen, Carlsbad, CA) were used to stain mononuclear peripheral blood lymphocytes. Leukocytes were recovered from a heparinized blood sample by centrifugation through a Ficoll-Hypaque cushion. Peripheral blood lymphocytes were analyzed using FACScan (Becton Dickinson, Mountain View, CA) equipment and CellQuest software.
ELISA. Virions were purified from 100 μl of MMTV(C3H)-infected C3H/HeN, YBR/Ei, and (Y x C)F1 mouse milk, resuspended in 100 μl of phosphate-buffered saline, and bound to plastic at a dilution of 1 x 10–2 in borate-buffered saline. Nonspecific binding was blocked with 1% bovine serum albumin for 2 h at 37°C. Mouse anti-MMTV gp52SU-specific monoclonal antibodies (37) were used at the second step. The reaction was developed with goat anti-mouse immunoglobulins coupled to alkaline phosphatase (AP). To test for antivirus antibodies in mouse sera, virions isolated from MMTV(C3H)-infected C3H/HeN milk were bound to plastic and incubated with serum samples diluted 1 x 10–2 in phosphate-buffered saline containing 0.05% Tween 20 and 0.05% sodium azide buffer prior to incubation with secondary antibodies. The background obtained with the secondary antibody alone was subtracted. To test for the presence of virus-coating antibodies, purified anti-gp52 monoclonal antibodies of either the immunoglobulin G1 (IgG1) or the IgM isotype (6, 37) were bound to plastic at 3 μg/ml, followed by incubation with virions (at 5 μg/ml of viral proteins) collected from milk of MMTV(C3H)-infected YBR/Ei or I/LnJ mice as described previously (37). The enzyme-linked immunosorbent assay (ELISA) was developed with isotype-specific anti-mouse specific immunoglobulins coupled to AP.
RNase T1 protection assays. RNase T1 protection assays were performed as previously described (18) with probes specific for MMTV(C3H) (21) or MMTV(LA) (22) viral transcripts. Total RNA isolated from milk (5 μg) and lactating mammary glands (40 μg) was analyzed.
Splenocyte transfer experiments. Splenocytes were isolated from MMTV(C3H)-infected and uninfected BALB/cJ and (Y x B)F1 mice. Four different mixtures of splenocytes were injected interperitoneally into 4- to 5-week-old recipient CBySmn.CB17-Prkdcscid/J mice bearing the same MHC haplotype as YBR/Ei mice (H-2d). All recipient groups received 2.5 x 106 splenocytes isolated from MMTV(C3H)-infected BALB/cJ mice mixed with 2.5 x 106 splenocytes isolated from either uninfected BALB/cJ mice, uninfected (Y x B)F1 mice, or MMTV(C3H)-infected (Y x B)F1 mice. In some experiments, T cells were subtracted from infected (Y x B)F1 splenocytes prior to mixing with infected BALB/cJ splenocytes. Negative selection was performed using anti-CD90 (Thy1.2) antibodies (Miltenyi Biotec, Auburn, CA). Recipient mice were mated, and RNA isolated from the milk or from the lactating mammary glands was subjected to RNase T1 protection assay with the MMTV(C3H)-specific probe.
Semiquantitative PCR and footpad injection. High-molecular-weight DNA was isolated from spleens of MMTV(C3H)-infected mice at 8 weeks of age and was resuspended at 0.1 mg/ml. Both endogenous and exogenous proviruses were amplified under semiquantitative conditions (20) using oligonucleotide primers specific to the LTR region of MMTV, i.e., 5'-GAAGATCTTCCCGAGAGTGTCCTACAC-3' and 5'-GAAGATCTTAATGTTCTATTAGTCCAGCCACTG-3'. Since the primers amplify both endogenous and exogenous newly integrated proviruses, PCR products were digested with MfeI (New England Biolabs, Inc., Beverly, MA), which cuts only proviral DNA of exogenous MMTV(C3H) but not endogenous Mtvs (20). Digested products were run on a 1.5% agarose gel, hybridized with the LTR-specific probe, and subjected to Southern blot analysis as previously described (20). Results of experiments were quantified with a PhosphorImager.
For footpad injections, 50 μl of skim milk collected from MMTV(C3H)-infected C3H/HeN females was injected into footpads of YBR/Ei, BALB/cJ, and (Y x B)F1 mice. Mice were sacrificed 4 days after injection, and DNA isolated from draining popliteal lymph nodes was subjected to PCR followed by Southern blot analysis as described above.
RESULTS
YBR/Ei mice become MMTV(C3H) infected but show reduced numbers of integrated proviruses in lymphoid tissue. In the early 1940s, the laboratory of Andervont showed that mammary tumor incidence is very low in both MMTV-infected and uninfected female mice of the Y strain and that this resistance is dominantly inherited (2, 3). To confirm that YBR/Ei mice, descendants of the Y strain, are also resistant to MMTV-induced mammary tumors, females from this strain along with C3H/HeN, BALB/cJ, (Y x C)F1, and (Y x B)F1 females were infected with MMTV(C3H) via fostering and monitored for mammary tumors. Whereas nearly all susceptible C3H/HeN and BALB/cJ females developed tumors by 300 days of age, YBR/Ei, (Y x C)F1, and (Y x B)F1 mice did not develop mammary tumors within this period of time. Mammary tumors contained newly integrated exogenous proviruses as determined by MMTV-specific Southern blot analysis (20, 36) (data not shown). Therefore, YBR/Ei mice resist MMTV-induced mammary tumors in a dominant fashion (Fig. 1).
YBR/Ei mice have an MHC class II H-2d haplotype. This haplotype expresses the I-E molecule, which efficiently presents viral SAgs to cognate CD4+ T cells (31, 36). Unlike those from uninfected YBR/Ei mice, SAg-cognate T cells from MMTV(C3H)-infected YBR/Ei mice responded to viral SAg as these cells were gradually deleted, indicating that mice became infected (Fig. 2). To determine if YBR/Ei mice were capable of transmitting infectious virus, MMTV(C3H)-infected YBR/Ei females were bred and used as foster mothers to susceptible C3H/HeN pups. Whereas uninfected C3H/HeN mice had 7.4% ± 0.3% (n = 5) of CD4+/V14+ T cells among CD4+ T cells, 7-week-old C3H/HeN littermates fostered on viremic YBR/Ei mice had 3.8% ± 1.5% (n = 8) of these cells in the periphery. Furthermore, all C3H/HeN females fostered by infected YBR/Ei mothers developed mammary tumors by 280 days (data not shown). These data indicate that MMTV(C3H)-infected YBR/Ei mice transmitted infectious, tumor-causing virus.
To compare virus loads in primary lymphoid cells of MMTV(C3H)-infected YBR/Ei, C3H/HeN, and (Y x C)F1 mice, genomic DNA isolated from their spleens was subjected to MMTV-specific semiquantitative PCR. MMTV(C3H)-infected YBR/Ei mice had approximately 2.5 times fewer integrated proviruses in lymphoid cells than did infected C3H/HeN mice (Fig. 3, top panel). Like infected YBR/Ei mice, most (Y x C)F1 mice showed diminished viral load, suggesting that the dominant mechanism inherited by YBR/Ei mice controls viral amplification.
MMTV(C3H)-infected YBR/Ei females secrete virus at attenuated titers and eliminate infectious virus across successive generations. To confirm that viral infection progresses to the mammary gland, RNA isolated from lactating mammary glands of MMTV(C3H)-infected YBR/Ei and C3H/HeN mice was subjected to RNaseT1 protection analysis. Although MMTV-specific transcripts were detected in mammary glands of infected YBR/Ei mice, their quantity was decreased compared to that in susceptible C3H/HeN mice (Fig. 3, bottom panel). Since proviral expression is under the control of hormones that govern pregnancy and lactation, analysis of virus-specific transcripts in mammary glands of mice that had undergone a second pregnancy was performed. In contrast to infected C3H/HeN mice, MMTV(C3H)-infected YBR/Ei mice did not show a significant increase in virus load after a second pregnancy (Fig. 3, bottom panel).
To compare virus titers secreted into the milk by MMTV(C3H)-infected susceptible C3H/HeN and resistant YBR/Ei mice, milk RNA was subjected to RNase T1 protection analysis, whereas virion proteins were subjected to virus-specific ELISA. Even though virus was detected in the milk of MMTV(C3H)-infected YBR/Ei mice, virus titers produced by these mice were severely reduced compared to virus titers produced by MMTV-infected C3H/HeN mice (Fig. 4).
To test whether attenuation of virus production resulted in loss of virus in infected YBR/Ei mouse pedigrees, we monitored the virus fate over successive generations of MMTV(C3H)-infected YBR/Ei mice. First, we compared percentages of SAg-cognate T cells in MMTV(C3H)-infected YBR/Ei mice across different generations (generation 1 [G1] through G4). While all MMTV(C3H)-infected YBR/Ei G1 mice showed deletion of SAg-cognate T cells (Fig. 2 and Fig. 5, top left panel), some G2 mice showed no deletion of SAg-cognate T cells, suggesting that MMTV(C3H) was eliminated in these G2 animals (Fig. 5, top left panel). Percentages of SAg-cognate T cells in almost all G3 animals and in all G4 animals were similar to percentages of these cells in uninfected mice, suggesting that the virus was lost in G4 mice (Fig. 5, top right panel). To confirm this, milk of subsequent generations of MMTV(C3H)-infected YBR/Ei mice was collected and the virus titer was assayed. While different lineages of mice eliminated virus at distinct generations (Fig. 5, bottom left panel), virus was eliminated in 100% of mice by G4 (Fig. 5, bottom right panel). MMTV(C3H) is thus gradually eliminated in infected YBR/Ei pedigrees.
YBR/Ei mice are also resistant to a different MMTV variant, MMTV(LA). YBR/Ei mice were infected with MMTV(LA) to determine whether viral resistance in YBR/Ei mice pertains to MMTV variants other than MMTV(C3H). Exogenous MMTV(LA) is carried by BALB/cJ mice and is produced at higher viral titers than exogenous MMTV(C3H) (22, 35). MMTV(LA) consists of three exogenous viruses, i.e., BALB2, BALB14, and BALBLA, with V2-, V14-, and V6/V8.1-specific SAgs, respectively (34). YBR/Ei mice were infected with MMTV(LA) via fostering, and infection was monitored by determining percentages of SAg-cognate T cells in the periphery of infected mice, by analyzing virus production into the milk, and by monitoring infected females for mammary tumors. MMTV(LA)-infected YBR/Ei mice were also bred to produce subsequent generations of infected mouse pedigrees. SAg-cognate CD4+ and CD8+ T cells were deleted in all MMTV(LA)-infected G1 and G2 YBR/Ei mice (Table 1 [shown for CD4+ T cells only]). However, mice from MMTV(LA)-infected YBR/Ei pedigrees showed deletion of SAg-cognate T cells at later generations than mice from MMTV(C3H)-infected YBR/Ei pedigrees (Table 1 and Fig. 5, top panels). Similarly, the number of mice producing virus into the milk gradually decreased over successive generations of MMTV(LA)-infected YBR/Ei mice (Fig. 6, top panels). Importantly, mice from later generations were more resistant to mammary tumors than mice from earlier generations (Fig. 6, bottom panel). Thus, in addition to MMTV(C3H), YBR/Ei mice also attenuate another MMTV variant, MMTV(LA). However, it takes more generations to eliminate this high-titer MMTV(LA) than to eliminate low-titer MMTV(C3H).
The mechanism of resistance inherited by YBR/Ei mice is unrelated to endogenous Mtvs, production of virus-neutralizing antibodies, or affected expression of integrated proviruses. Uninfected YBR/Ei mice have 9.5% ± 0.3% of CD4+/V14+ cells, 10.2% ± 0.5% of CD4+/V 6+ cells, and 7.5% ± 0.4% of CD4+/V2+ cells (n = 6) among CD4+ T cells, and they have 6.5% ± 0.2% of CD8+/V14+ cells, 8.8% ± 0.8% of CD8+/V6+ cells, and 7.7% ± 0.2% of CD8+/V2+ cells among CD8+ T cells. Furthermore and as mentioned above, SAg-cognate T cells in infected YBR/Ei mice responded to viral SAgs as they underwent gradual deletion (Fig. 2 and Table 1). This suggests that SAg-cognate T cells utilized by MMTV(C3H) and MMTV(LA) viruses are not negatively selected due to the presence of endogenous Mtvs in YBR/Ei mice and are not anergized.
Previously we showed that MMTV-infected I/LnJ mice produce virus-neutralizing antibodies which coat virions secreted by infected I/LnJ cells and completely prevent virus transmission to offspring (37). Even though we concluded that MMTV-infected YBR/Ei mice transmitted infectious virions, since susceptible C3H/HeN mice fostered on their milk became MMTV infected (see above), it was still possible that YBR/Ei mice were capable of raising a weak but virus-neutralizing immune response. To determine if YBR/Ei mice produce antivirus antibodies, serum samples were collected from MMTV(C3H)-infected YBR/Ei mice and tested for the ability to bind to viral proteins. Similarly, viral particles were purified from the milk of MMTV(C3H)-infected G1 YBR/Ei mice and tested for the presence of virion-associated antivirus antibodies. Unlike MMTV(C3H)-infected I/LnJ mice, infected YBR/Ei mice did not produce antivirus antibodies (Fig. 7, top panel) and did not secrete virions coated with antivirus antibodies (Fig. 7, bottom panel). These data, together with the fact that MMTV-infected YBR/Ei mice transmit infectious virions to susceptible mice, eliminated the possibility that a virus-neutralizing immune response underlies the resistance mechanism inherited by YBR/Ei mice.
Retroviral LTR sequences regulate transcription of viral genes by providing signals for cellular transcriptional control machinery. Since LTR sequences of endogenous and exogenous MMTVs share homology, host factors that modulate endogenous proviral expression may also direct expression of exogenous integrated proviruses. To determine if attenuated virus production in YBR/Ei mice correlates with decreased expression of endogenous proviruses, expression of Mtv17 (inherited by YBR/Ei mice) (Fig. 8) in YBR/Ei and other Mtv17-bearing mice was compared. Since endogenous Mtv17 is known to be highly expressed in mammary glands (23), decreased expression of Mtv17 in the mammary glands of YBR/Ei mice could indicate that the resistance to MMTV infection in YBR/Ei mice functions by downmodulating proviral expression. Mtv17 was found to be equally expressed in mammary glands of YBR/Ei mice and of other Mtv17-bearing mice, indicating that the retrovirus resistance mechanism is not due to decreased expression of integrated proviruses (Fig. 9).
Thus, attenuation of virus production in YBR/Ei mice is unrelated to endogenous Mtvs, production of virus-neutralizing antibodies, or affected expression of integrated proviruses.
YBR/Ei T cells restrict virus replication. To determine whether the virus resistance mechanism in YBR/Ei mice operates at the cellular or organismal level, viral loads in susceptible and resistant mice were compared during a short course of viral infection. MMTV(C3H) was injected into the footpads of resistant YBR/Ei and (Y x B)F1 mice and susceptible BALB/cJ and C3H/HeN mice. Mice were sacrificed 4 days after injection, and viral integrations in DNA isolated from draining popliteal lymph node cells were quantified by semiquantitative PCR. No difference in viral load between resistant and susceptible mice was observed at the initial stages of infection (Fig. 10).
The results described above indicate that virus restriction in YBR/Ei mice is controlled at the organismal level. Therefore, we next sought to determine whether the resistance mechanism is related to the function of the immune system. Accordingly, we monitored virus infection in susceptible immunodeficient CBySmn.CB17-Prkdcscid/J mice reconstituted with splenocytes from MMTV(C3H)-infected syngeneic BALB/cJ mice. The donor cells were mixed with spleen cells from either infected or uninfected resistant (Y x B)F1 mice prior to transfer. We reasoned that if YBR/Ei mice suppress viral infection via an adaptive immune mechanism, then recipient mice receiving "virus-educated" (Y x B)F1 splenocytes from infected mice should be infected to a lesser degree than recipient mice receiving splenocytes from uninfected (Y x B)F1 mice. Reconstituted recipient mice were bred, and titers of virus produced into their milk were compared. As expected, recipient control mice injected with mixed splenocytes from infected BALB/cJ and uninfected (Y x B)F1 mice produced high virus titers in the milk, while recipient mice reconstituted with mixed splenocytes from infected BALB/cJ and infected (Y x B)F1 mice produced much less virus into the milk (Fig. 11, top panel). Furthermore, removal of T cells from infected (Y x B)F1 splenocytes completely abolished the ability of the remaining cells to inhibit virus replication upon transfer (Fig. 11, bottom panel). These results indicate that the immune system, and specifically T cells, is capable of controlling virus replication in YBR/Ei mice.
DISCUSSION
We have shown that YBR/Ei mice exhibit a novel strategy to restrict MMTV infection and resist MMTV-induced tumors. Like susceptible BALB/cJ and C3H/He mice, YBR/Ei mice transmit infectious tumor-causing virus. However, unlike susceptible mice, YBR/Ei mice inhibit virus production as evidenced by the decreased number of newly integrated proviruses in lymphoid tissues and by reduced titers of virus secreted into the milk. The viral load was reduced progressively with successive passages through YBR/Ei mice and became uniformly negative. As a result, two different MMTV variants, MMTV(C3H) and MMTV(LA), were eliminated after consecutive passages through YBR/Ei mice. However, the number of virus passages across generations required for viral clearance was dependent on the initial input virus titer. It took 3 to 4 generations longer to clear a high-titer MMTV(LA) than a low-titer MMTV(C3H).
An antiviral immune response is directed by innate and adaptive immune systems. The innate immune response is a rapid, nonspecific response to infection that provides an early line of defense and also controls the highly specific, adaptive response that takes longer to develop. Several host factors that restrict retroviral replication through innate mechanisms have been described previously. These include mechanisms controlled by Fv1, Ref1, Fv4, APOBEC3G, TRIM5, Lv2, and ZAP (for reviews, see references 15 and 16). These factors control/block early events in retroviral infection. However, since resistant YBR/Ei mice are infected to the same degree as susceptible BALB/cJ mice at the initial stages of infection, the resistance mechanism inherited by these mice is not solely due to innate mechanisms. Instead, it appears that retrovirus resistance in YBR/Ei mice is influenced by a slower-developing, virus-specific adaptive immune response. This conclusion is supported by our experimental data showing that "infection-educated" but not naive T cells were capable of conferring virus resistance to susceptible mice upon transfer. Thus, an immune T-cell-mediated antivirus response is generated to clear infection in YBR/Ei mice. However, this response is not robust, because it takes generations to eliminate virus from infected mouse pedigrees. Interestingly, the number of generations required to get rid off the virus also varies between distinct mouse pedigrees. This is reflected by noticeable variations in the number of integrants in infected YBE/Ei G1 mice. Thus, natural variation between individual mice is to be expected, especially since the mechanism of such reduction is immune mediated.
The control of Friend murine leukemia virus (F-MuLV) infection by recovery from Friend virus 3 (Rfv3) is one model of retroviral resistance that similarly features adaptive immunity of the host. Even though mice carrying the dominant resistant allele of Rfv3 (C57BL/6J mice) become infected with F-MuLV, they completely eradicate the virus and infected cells within 60 days of infection (7). While the mechanism by which Rfv3 confers resistance to the virus is unknown, it has been shown that anti-F-MuLV neutralizing antibodies, CD4+ T cells, and CD8+ T cells are all required for protective immunity against F-MuLV (8, 11, 24). The resistance mechanism inherited by YBR/Ei mice is clearly distinct from Rfv3-mediated restriction because MMTV-infected YBR/Ei mice secrete infectious virions into the milk, retain infected cells throughout the life of the animal, and do not secrete virus-neutralizing antibodies. Furthermore, T cells, which seem to play an important role in restricting MMTV in YBR/Ei mice, fail to rid the YBR/Ei system of infected cells. This suggests that the resistance mechanism in YBR/Ei mice may be unrelated to virus-specific cytotoxic T cells. One possibility is that CD4+ T cells in infected YBR/Ei mice secrete immunomodulating cytokines, such as gamma interferon (28), and thus influence antiviral effects elicited by other cell types.
The ability of the immune system of YBR/Ei mice to clear retroviral infection in the absence of virus-neutralizing antibodies makes this model an important subject of study. Subsequently, identifying the genetic basis and molecular mechanism that governs restriction of MMTV infection in YBR/Ei mice may prove to be valuable for better understanding retroviral resistance in humans.
ACKNOWLEDGMENTS
We thank Alexander Chervonsky for helpful discussion, Martha Buzzell for mouse care assistance, and Pat Cherry for administrative service.
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ABSTRACT
Mouse mammary tumor virus (MMTV), a well-characterized retrovirus that causes mammary tumors in susceptible mice, is commonly used to investigate virus-host interactions. We have shown that YBR/Ei mice demonstrate a novel, dominant mechanism of resistance to MMTV infection and MMTV-induced mammary tumors. MMTV can both establish infection in YBR/Ei mice and be transmitted by YBR/Ei mice as an infectious virus. However, virus production is severely attenuated, resulting in gradual clearance of infection in successive generations. Our transfer experiments showed that T cells generated in MMTV-infected resistant mice were required to restrict MMTV replication in susceptible mice. These results emphasize the importance of inducing T-cell responses for effective protection against retroviral infections.
INTRODUCTION
The ability to resist retroviruses is advantageous for the infected host because unlimited retroviral replication can be highly mutagenic and may result in numerous diseases. Studies conducted to identify cellular factors that put boundaries on retroviral transmission have resulted in identification and characterization of genes capable of hindering retroviral replication (14). We are using mouse mammary tumor virus (MMTV) to study the genetics of resistance to retroviral infection and to virally induced tumors. MMTV can be transmitted either as an endogenous virus through the germ line or as an exogenous virus (9, 32). Endogenous MMTVs (Mtvs) are commonly found in laboratory mouse strains, but most Mtvs do not produce infectious viral particles due to transcriptional regulatory or coding region mutations (9). Exogenous virus is transmitted via milk and is acquired by newborn pups when they are fostered on viremic mothers. The primary targets of the virus are cells of the immune system, especially antigen-presenting cells such as B cells (5). Infected antigen-presenting cells present the virally encoded superantigen (SAg) by major histocompatibility (MHC) class II molecules to T cells bearing a particular chain of T-cell receptor. In response to SAg presentation, SAg-cognate T cells proliferate, generating a pool of activated infection-competent cells (reviewed in reference 1). Activated SAg-cognate T cells subsequently undergo deletion, and this deletion of SAg-cognate T cells is routinely used as a readout to monitor virus infection (27, 29). Virus propagation in vivo requires SAg activity, because viruses that lack functional SAgs are not infectious (20).
Tumor induction by MMTV is a result of proviral integration in the proximity of a cellular proto-oncogene followed by activation of its expression (33). In addition, virally encoded genes are also essential for virus potency to cause mammary tumors (26, 37a). Proviral expression is regulated by sequences within the long terminal repeats (LTR) that recognize glucocorticoid receptor/steroid hormone complexes present during pregnancy (39). Multiple pregnancies are therefore associated with increased virion production and augmented numbers of infected mammary gland cells within the host. This, in turn, results in an increase in the number of viral reinfections/reintegrations and an increase in the probability of proviral integration next to cellular proto-oncogenes.
Inbred mouse strains have been employed to identify the genetic factors that influence susceptibility to MMTV-induced mammary tumors. Three mechanisms that restrict MMTV-induced tumorigenesis have previously been described: resistance based on MHC haplotype (30, 36), resistance due to the presence of endogenous Mtvs (19, 25), and resistance due to the production of virus-neutralizing antibodies (37).
MHC class II polymorphic membrane glycoproteins are required for presenting degraded, endocytosed foreign antigens to CD4+ T cells. There are two isotypic class II heterodimeric proteins of the mouse, A A (I-A) and E E (I-E), which differ in their efficiency of presenting MMTV SAgs. Inbred strains of b, f, q, or s MHC haplotype do not express I-E molecules due to mutations in the E or E genes (4, 10). Since I-E molecules present viral SAgs more efficiently than I-A molecules, mouse strains that lack the I-E molecule (such as C57BL/6J mice) are relatively resistant to MMTV infection and MMTV-induced tumors (38).
In contrast to exogenous MMTV, endogenous Mtvs present in germ line cells cause negative selection of SAg-cognate T cells during formation of the immune repertoire (1). There are more than 40 different endogenous Mtvs encoding distinct SAgs, and the presence of a particular endogenous Mtv in the mouse genome is indicated by the absence of specific T cells bearing the V chain that interacts with the SAg of this Mtv (12). Mice that inherit Mtvs are resistant to exogenous MMTVs bearing SAgs of the same T-cell specificity (19, 25) and do not succumb to mammary tumors (17).
Mice from the I/LnJ strain inherit a mechanism of resistance to MMTV-induced mammary tumors that is unrelated to the MHC haplotype or endogenous Mtvs (37). This mechanism is recessive and is dependent on production of antivirus neutralizing antibodies (37). Even though I/LnJ mice become infected with MMTV, they secrete antibody-coated virions into the milk and therefore prevent infection of their progeny (37). Since reinfection/reintegration is also ablated in infected I/LnJ mice, they resist MMTV-induced mammary tumors (37).
It has long been recognized that the Y mouse strain is resistant to tumors induced by MMTV (2, 3). Here we show that MMTV is capable of establishing infection in YBR/Ei mice, descendants of the Y strain, and that YBR/Ei mice transmit infectious virus. However, YBR/Ei mice produce the virus at severely attenuated titers and eliminate the virus in successive generations. This paper describes some of the first insights into the retrovirus resistance mechanism inherited by YBR/Ei mice.
MATERIALS AND METHODS
Mice. All mice used in this study were bred and maintained at the animal facility of The Jackson Laboratory, Bar Harbor, Maine. YBR/Ei, BALB/cJ, C3H/HeJ, C3.JK, I/LnJ, NZB/ByJ, RIII/SJ, C57BL/6, and CBySmn.CB17-Prkdcscid/J mice were purchased from The Jackson Laboratory. MMTV(C3H)-infected and uninfected C3H/HeN mice were purchased from the National Cancer Institute, Frederick Cancer Research Facility, Frederick, MD. MMTV(LA) (34) was passed on BALB/cJ mice [BALB/c(LA) mice]. MMTV(C3H)-infected BALB/cJ mice were produced via fostering on MMTV(C3H)-infected C3H/HeN females. The studies have been reviewed and approved by the Animal Care and Use Committee at The Jackson Laboratory.
Mammary gland tumorigenesis. YBR/Ei, C3H/HeN, BALB/cJ, (Y x C)F1 and (Y x B)F1 mice were infected either with MMTV(C3H) by fostering on viremic C3H/HeN females or with MMTV(LA) by fostering on viremic BALB/cLA females. Mammary gland tumor incidence in MMTV(C3H)- or MMTV(LA)-infected mice was monitored by weekly palpation. At least 30 mice were used per group.
Antibodies and fluorescence-activated cell sorting (FACS) analysis. Fluorescein isothiocyanate-coupled monoclonal antibodies against the V14, V2, and V6 T-cell receptor chains (Sigma-Aldrich, St. Louis, MO) and phycoerythrin-coupled anti-mouse CD4 (clone H129.19) antibodies (Invitrogen, Carlsbad, CA) were used to stain mononuclear peripheral blood lymphocytes. Leukocytes were recovered from a heparinized blood sample by centrifugation through a Ficoll-Hypaque cushion. Peripheral blood lymphocytes were analyzed using FACScan (Becton Dickinson, Mountain View, CA) equipment and CellQuest software.
ELISA. Virions were purified from 100 μl of MMTV(C3H)-infected C3H/HeN, YBR/Ei, and (Y x C)F1 mouse milk, resuspended in 100 μl of phosphate-buffered saline, and bound to plastic at a dilution of 1 x 10–2 in borate-buffered saline. Nonspecific binding was blocked with 1% bovine serum albumin for 2 h at 37°C. Mouse anti-MMTV gp52SU-specific monoclonal antibodies (37) were used at the second step. The reaction was developed with goat anti-mouse immunoglobulins coupled to alkaline phosphatase (AP). To test for antivirus antibodies in mouse sera, virions isolated from MMTV(C3H)-infected C3H/HeN milk were bound to plastic and incubated with serum samples diluted 1 x 10–2 in phosphate-buffered saline containing 0.05% Tween 20 and 0.05% sodium azide buffer prior to incubation with secondary antibodies. The background obtained with the secondary antibody alone was subtracted. To test for the presence of virus-coating antibodies, purified anti-gp52 monoclonal antibodies of either the immunoglobulin G1 (IgG1) or the IgM isotype (6, 37) were bound to plastic at 3 μg/ml, followed by incubation with virions (at 5 μg/ml of viral proteins) collected from milk of MMTV(C3H)-infected YBR/Ei or I/LnJ mice as described previously (37). The enzyme-linked immunosorbent assay (ELISA) was developed with isotype-specific anti-mouse specific immunoglobulins coupled to AP.
RNase T1 protection assays. RNase T1 protection assays were performed as previously described (18) with probes specific for MMTV(C3H) (21) or MMTV(LA) (22) viral transcripts. Total RNA isolated from milk (5 μg) and lactating mammary glands (40 μg) was analyzed.
Splenocyte transfer experiments. Splenocytes were isolated from MMTV(C3H)-infected and uninfected BALB/cJ and (Y x B)F1 mice. Four different mixtures of splenocytes were injected interperitoneally into 4- to 5-week-old recipient CBySmn.CB17-Prkdcscid/J mice bearing the same MHC haplotype as YBR/Ei mice (H-2d). All recipient groups received 2.5 x 106 splenocytes isolated from MMTV(C3H)-infected BALB/cJ mice mixed with 2.5 x 106 splenocytes isolated from either uninfected BALB/cJ mice, uninfected (Y x B)F1 mice, or MMTV(C3H)-infected (Y x B)F1 mice. In some experiments, T cells were subtracted from infected (Y x B)F1 splenocytes prior to mixing with infected BALB/cJ splenocytes. Negative selection was performed using anti-CD90 (Thy1.2) antibodies (Miltenyi Biotec, Auburn, CA). Recipient mice were mated, and RNA isolated from the milk or from the lactating mammary glands was subjected to RNase T1 protection assay with the MMTV(C3H)-specific probe.
Semiquantitative PCR and footpad injection. High-molecular-weight DNA was isolated from spleens of MMTV(C3H)-infected mice at 8 weeks of age and was resuspended at 0.1 mg/ml. Both endogenous and exogenous proviruses were amplified under semiquantitative conditions (20) using oligonucleotide primers specific to the LTR region of MMTV, i.e., 5'-GAAGATCTTCCCGAGAGTGTCCTACAC-3' and 5'-GAAGATCTTAATGTTCTATTAGTCCAGCCACTG-3'. Since the primers amplify both endogenous and exogenous newly integrated proviruses, PCR products were digested with MfeI (New England Biolabs, Inc., Beverly, MA), which cuts only proviral DNA of exogenous MMTV(C3H) but not endogenous Mtvs (20). Digested products were run on a 1.5% agarose gel, hybridized with the LTR-specific probe, and subjected to Southern blot analysis as previously described (20). Results of experiments were quantified with a PhosphorImager.
For footpad injections, 50 μl of skim milk collected from MMTV(C3H)-infected C3H/HeN females was injected into footpads of YBR/Ei, BALB/cJ, and (Y x B)F1 mice. Mice were sacrificed 4 days after injection, and DNA isolated from draining popliteal lymph nodes was subjected to PCR followed by Southern blot analysis as described above.
RESULTS
YBR/Ei mice become MMTV(C3H) infected but show reduced numbers of integrated proviruses in lymphoid tissue. In the early 1940s, the laboratory of Andervont showed that mammary tumor incidence is very low in both MMTV-infected and uninfected female mice of the Y strain and that this resistance is dominantly inherited (2, 3). To confirm that YBR/Ei mice, descendants of the Y strain, are also resistant to MMTV-induced mammary tumors, females from this strain along with C3H/HeN, BALB/cJ, (Y x C)F1, and (Y x B)F1 females were infected with MMTV(C3H) via fostering and monitored for mammary tumors. Whereas nearly all susceptible C3H/HeN and BALB/cJ females developed tumors by 300 days of age, YBR/Ei, (Y x C)F1, and (Y x B)F1 mice did not develop mammary tumors within this period of time. Mammary tumors contained newly integrated exogenous proviruses as determined by MMTV-specific Southern blot analysis (20, 36) (data not shown). Therefore, YBR/Ei mice resist MMTV-induced mammary tumors in a dominant fashion (Fig. 1).
YBR/Ei mice have an MHC class II H-2d haplotype. This haplotype expresses the I-E molecule, which efficiently presents viral SAgs to cognate CD4+ T cells (31, 36). Unlike those from uninfected YBR/Ei mice, SAg-cognate T cells from MMTV(C3H)-infected YBR/Ei mice responded to viral SAg as these cells were gradually deleted, indicating that mice became infected (Fig. 2). To determine if YBR/Ei mice were capable of transmitting infectious virus, MMTV(C3H)-infected YBR/Ei females were bred and used as foster mothers to susceptible C3H/HeN pups. Whereas uninfected C3H/HeN mice had 7.4% ± 0.3% (n = 5) of CD4+/V14+ T cells among CD4+ T cells, 7-week-old C3H/HeN littermates fostered on viremic YBR/Ei mice had 3.8% ± 1.5% (n = 8) of these cells in the periphery. Furthermore, all C3H/HeN females fostered by infected YBR/Ei mothers developed mammary tumors by 280 days (data not shown). These data indicate that MMTV(C3H)-infected YBR/Ei mice transmitted infectious, tumor-causing virus.
To compare virus loads in primary lymphoid cells of MMTV(C3H)-infected YBR/Ei, C3H/HeN, and (Y x C)F1 mice, genomic DNA isolated from their spleens was subjected to MMTV-specific semiquantitative PCR. MMTV(C3H)-infected YBR/Ei mice had approximately 2.5 times fewer integrated proviruses in lymphoid cells than did infected C3H/HeN mice (Fig. 3, top panel). Like infected YBR/Ei mice, most (Y x C)F1 mice showed diminished viral load, suggesting that the dominant mechanism inherited by YBR/Ei mice controls viral amplification.
MMTV(C3H)-infected YBR/Ei females secrete virus at attenuated titers and eliminate infectious virus across successive generations. To confirm that viral infection progresses to the mammary gland, RNA isolated from lactating mammary glands of MMTV(C3H)-infected YBR/Ei and C3H/HeN mice was subjected to RNaseT1 protection analysis. Although MMTV-specific transcripts were detected in mammary glands of infected YBR/Ei mice, their quantity was decreased compared to that in susceptible C3H/HeN mice (Fig. 3, bottom panel). Since proviral expression is under the control of hormones that govern pregnancy and lactation, analysis of virus-specific transcripts in mammary glands of mice that had undergone a second pregnancy was performed. In contrast to infected C3H/HeN mice, MMTV(C3H)-infected YBR/Ei mice did not show a significant increase in virus load after a second pregnancy (Fig. 3, bottom panel).
To compare virus titers secreted into the milk by MMTV(C3H)-infected susceptible C3H/HeN and resistant YBR/Ei mice, milk RNA was subjected to RNase T1 protection analysis, whereas virion proteins were subjected to virus-specific ELISA. Even though virus was detected in the milk of MMTV(C3H)-infected YBR/Ei mice, virus titers produced by these mice were severely reduced compared to virus titers produced by MMTV-infected C3H/HeN mice (Fig. 4).
To test whether attenuation of virus production resulted in loss of virus in infected YBR/Ei mouse pedigrees, we monitored the virus fate over successive generations of MMTV(C3H)-infected YBR/Ei mice. First, we compared percentages of SAg-cognate T cells in MMTV(C3H)-infected YBR/Ei mice across different generations (generation 1 [G1] through G4). While all MMTV(C3H)-infected YBR/Ei G1 mice showed deletion of SAg-cognate T cells (Fig. 2 and Fig. 5, top left panel), some G2 mice showed no deletion of SAg-cognate T cells, suggesting that MMTV(C3H) was eliminated in these G2 animals (Fig. 5, top left panel). Percentages of SAg-cognate T cells in almost all G3 animals and in all G4 animals were similar to percentages of these cells in uninfected mice, suggesting that the virus was lost in G4 mice (Fig. 5, top right panel). To confirm this, milk of subsequent generations of MMTV(C3H)-infected YBR/Ei mice was collected and the virus titer was assayed. While different lineages of mice eliminated virus at distinct generations (Fig. 5, bottom left panel), virus was eliminated in 100% of mice by G4 (Fig. 5, bottom right panel). MMTV(C3H) is thus gradually eliminated in infected YBR/Ei pedigrees.
YBR/Ei mice are also resistant to a different MMTV variant, MMTV(LA). YBR/Ei mice were infected with MMTV(LA) to determine whether viral resistance in YBR/Ei mice pertains to MMTV variants other than MMTV(C3H). Exogenous MMTV(LA) is carried by BALB/cJ mice and is produced at higher viral titers than exogenous MMTV(C3H) (22, 35). MMTV(LA) consists of three exogenous viruses, i.e., BALB2, BALB14, and BALBLA, with V2-, V14-, and V6/V8.1-specific SAgs, respectively (34). YBR/Ei mice were infected with MMTV(LA) via fostering, and infection was monitored by determining percentages of SAg-cognate T cells in the periphery of infected mice, by analyzing virus production into the milk, and by monitoring infected females for mammary tumors. MMTV(LA)-infected YBR/Ei mice were also bred to produce subsequent generations of infected mouse pedigrees. SAg-cognate CD4+ and CD8+ T cells were deleted in all MMTV(LA)-infected G1 and G2 YBR/Ei mice (Table 1 [shown for CD4+ T cells only]). However, mice from MMTV(LA)-infected YBR/Ei pedigrees showed deletion of SAg-cognate T cells at later generations than mice from MMTV(C3H)-infected YBR/Ei pedigrees (Table 1 and Fig. 5, top panels). Similarly, the number of mice producing virus into the milk gradually decreased over successive generations of MMTV(LA)-infected YBR/Ei mice (Fig. 6, top panels). Importantly, mice from later generations were more resistant to mammary tumors than mice from earlier generations (Fig. 6, bottom panel). Thus, in addition to MMTV(C3H), YBR/Ei mice also attenuate another MMTV variant, MMTV(LA). However, it takes more generations to eliminate this high-titer MMTV(LA) than to eliminate low-titer MMTV(C3H).
The mechanism of resistance inherited by YBR/Ei mice is unrelated to endogenous Mtvs, production of virus-neutralizing antibodies, or affected expression of integrated proviruses. Uninfected YBR/Ei mice have 9.5% ± 0.3% of CD4+/V14+ cells, 10.2% ± 0.5% of CD4+/V 6+ cells, and 7.5% ± 0.4% of CD4+/V2+ cells (n = 6) among CD4+ T cells, and they have 6.5% ± 0.2% of CD8+/V14+ cells, 8.8% ± 0.8% of CD8+/V6+ cells, and 7.7% ± 0.2% of CD8+/V2+ cells among CD8+ T cells. Furthermore and as mentioned above, SAg-cognate T cells in infected YBR/Ei mice responded to viral SAgs as they underwent gradual deletion (Fig. 2 and Table 1). This suggests that SAg-cognate T cells utilized by MMTV(C3H) and MMTV(LA) viruses are not negatively selected due to the presence of endogenous Mtvs in YBR/Ei mice and are not anergized.
Previously we showed that MMTV-infected I/LnJ mice produce virus-neutralizing antibodies which coat virions secreted by infected I/LnJ cells and completely prevent virus transmission to offspring (37). Even though we concluded that MMTV-infected YBR/Ei mice transmitted infectious virions, since susceptible C3H/HeN mice fostered on their milk became MMTV infected (see above), it was still possible that YBR/Ei mice were capable of raising a weak but virus-neutralizing immune response. To determine if YBR/Ei mice produce antivirus antibodies, serum samples were collected from MMTV(C3H)-infected YBR/Ei mice and tested for the ability to bind to viral proteins. Similarly, viral particles were purified from the milk of MMTV(C3H)-infected G1 YBR/Ei mice and tested for the presence of virion-associated antivirus antibodies. Unlike MMTV(C3H)-infected I/LnJ mice, infected YBR/Ei mice did not produce antivirus antibodies (Fig. 7, top panel) and did not secrete virions coated with antivirus antibodies (Fig. 7, bottom panel). These data, together with the fact that MMTV-infected YBR/Ei mice transmit infectious virions to susceptible mice, eliminated the possibility that a virus-neutralizing immune response underlies the resistance mechanism inherited by YBR/Ei mice.
Retroviral LTR sequences regulate transcription of viral genes by providing signals for cellular transcriptional control machinery. Since LTR sequences of endogenous and exogenous MMTVs share homology, host factors that modulate endogenous proviral expression may also direct expression of exogenous integrated proviruses. To determine if attenuated virus production in YBR/Ei mice correlates with decreased expression of endogenous proviruses, expression of Mtv17 (inherited by YBR/Ei mice) (Fig. 8) in YBR/Ei and other Mtv17-bearing mice was compared. Since endogenous Mtv17 is known to be highly expressed in mammary glands (23), decreased expression of Mtv17 in the mammary glands of YBR/Ei mice could indicate that the resistance to MMTV infection in YBR/Ei mice functions by downmodulating proviral expression. Mtv17 was found to be equally expressed in mammary glands of YBR/Ei mice and of other Mtv17-bearing mice, indicating that the retrovirus resistance mechanism is not due to decreased expression of integrated proviruses (Fig. 9).
Thus, attenuation of virus production in YBR/Ei mice is unrelated to endogenous Mtvs, production of virus-neutralizing antibodies, or affected expression of integrated proviruses.
YBR/Ei T cells restrict virus replication. To determine whether the virus resistance mechanism in YBR/Ei mice operates at the cellular or organismal level, viral loads in susceptible and resistant mice were compared during a short course of viral infection. MMTV(C3H) was injected into the footpads of resistant YBR/Ei and (Y x B)F1 mice and susceptible BALB/cJ and C3H/HeN mice. Mice were sacrificed 4 days after injection, and viral integrations in DNA isolated from draining popliteal lymph node cells were quantified by semiquantitative PCR. No difference in viral load between resistant and susceptible mice was observed at the initial stages of infection (Fig. 10).
The results described above indicate that virus restriction in YBR/Ei mice is controlled at the organismal level. Therefore, we next sought to determine whether the resistance mechanism is related to the function of the immune system. Accordingly, we monitored virus infection in susceptible immunodeficient CBySmn.CB17-Prkdcscid/J mice reconstituted with splenocytes from MMTV(C3H)-infected syngeneic BALB/cJ mice. The donor cells were mixed with spleen cells from either infected or uninfected resistant (Y x B)F1 mice prior to transfer. We reasoned that if YBR/Ei mice suppress viral infection via an adaptive immune mechanism, then recipient mice receiving "virus-educated" (Y x B)F1 splenocytes from infected mice should be infected to a lesser degree than recipient mice receiving splenocytes from uninfected (Y x B)F1 mice. Reconstituted recipient mice were bred, and titers of virus produced into their milk were compared. As expected, recipient control mice injected with mixed splenocytes from infected BALB/cJ and uninfected (Y x B)F1 mice produced high virus titers in the milk, while recipient mice reconstituted with mixed splenocytes from infected BALB/cJ and infected (Y x B)F1 mice produced much less virus into the milk (Fig. 11, top panel). Furthermore, removal of T cells from infected (Y x B)F1 splenocytes completely abolished the ability of the remaining cells to inhibit virus replication upon transfer (Fig. 11, bottom panel). These results indicate that the immune system, and specifically T cells, is capable of controlling virus replication in YBR/Ei mice.
DISCUSSION
We have shown that YBR/Ei mice exhibit a novel strategy to restrict MMTV infection and resist MMTV-induced tumors. Like susceptible BALB/cJ and C3H/He mice, YBR/Ei mice transmit infectious tumor-causing virus. However, unlike susceptible mice, YBR/Ei mice inhibit virus production as evidenced by the decreased number of newly integrated proviruses in lymphoid tissues and by reduced titers of virus secreted into the milk. The viral load was reduced progressively with successive passages through YBR/Ei mice and became uniformly negative. As a result, two different MMTV variants, MMTV(C3H) and MMTV(LA), were eliminated after consecutive passages through YBR/Ei mice. However, the number of virus passages across generations required for viral clearance was dependent on the initial input virus titer. It took 3 to 4 generations longer to clear a high-titer MMTV(LA) than a low-titer MMTV(C3H).
An antiviral immune response is directed by innate and adaptive immune systems. The innate immune response is a rapid, nonspecific response to infection that provides an early line of defense and also controls the highly specific, adaptive response that takes longer to develop. Several host factors that restrict retroviral replication through innate mechanisms have been described previously. These include mechanisms controlled by Fv1, Ref1, Fv4, APOBEC3G, TRIM5, Lv2, and ZAP (for reviews, see references 15 and 16). These factors control/block early events in retroviral infection. However, since resistant YBR/Ei mice are infected to the same degree as susceptible BALB/cJ mice at the initial stages of infection, the resistance mechanism inherited by these mice is not solely due to innate mechanisms. Instead, it appears that retrovirus resistance in YBR/Ei mice is influenced by a slower-developing, virus-specific adaptive immune response. This conclusion is supported by our experimental data showing that "infection-educated" but not naive T cells were capable of conferring virus resistance to susceptible mice upon transfer. Thus, an immune T-cell-mediated antivirus response is generated to clear infection in YBR/Ei mice. However, this response is not robust, because it takes generations to eliminate virus from infected mouse pedigrees. Interestingly, the number of generations required to get rid off the virus also varies between distinct mouse pedigrees. This is reflected by noticeable variations in the number of integrants in infected YBE/Ei G1 mice. Thus, natural variation between individual mice is to be expected, especially since the mechanism of such reduction is immune mediated.
The control of Friend murine leukemia virus (F-MuLV) infection by recovery from Friend virus 3 (Rfv3) is one model of retroviral resistance that similarly features adaptive immunity of the host. Even though mice carrying the dominant resistant allele of Rfv3 (C57BL/6J mice) become infected with F-MuLV, they completely eradicate the virus and infected cells within 60 days of infection (7). While the mechanism by which Rfv3 confers resistance to the virus is unknown, it has been shown that anti-F-MuLV neutralizing antibodies, CD4+ T cells, and CD8+ T cells are all required for protective immunity against F-MuLV (8, 11, 24). The resistance mechanism inherited by YBR/Ei mice is clearly distinct from Rfv3-mediated restriction because MMTV-infected YBR/Ei mice secrete infectious virions into the milk, retain infected cells throughout the life of the animal, and do not secrete virus-neutralizing antibodies. Furthermore, T cells, which seem to play an important role in restricting MMTV in YBR/Ei mice, fail to rid the YBR/Ei system of infected cells. This suggests that the resistance mechanism in YBR/Ei mice may be unrelated to virus-specific cytotoxic T cells. One possibility is that CD4+ T cells in infected YBR/Ei mice secrete immunomodulating cytokines, such as gamma interferon (28), and thus influence antiviral effects elicited by other cell types.
The ability of the immune system of YBR/Ei mice to clear retroviral infection in the absence of virus-neutralizing antibodies makes this model an important subject of study. Subsequently, identifying the genetic basis and molecular mechanism that governs restriction of MMTV infection in YBR/Ei mice may prove to be valuable for better understanding retroviral resistance in humans.
ACKNOWLEDGMENTS
We thank Alexander Chervonsky for helpful discussion, Martha Buzzell for mouse care assistance, and Pat Cherry for administrative service.
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