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The Journal of Immunology, 2005, 175: 7543-7549.
Copyright © 2005 by The American Association of Immunologists

Molecular and Cellular Basis of the Retrovirus Resistance in I/LnJ Mice1

Laure K. Case, Alexandra Purdy and Tatyana V. Golovkina2

The Jackson Laboratory, Bar Harbor, ME 04609


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Previously, we showed that IFN-{gamma} elicited by mouse mammary tumor virus (MMTV) infection in I/LnJ mice stimulated production of virus-neutralizing Abs, mostly of the IgG2a isotype. These Abs coated virions secreted by infected I/LnJ cells, and thus completely prevented virus transmission to offspring. However, the mechanism of virus neutralization by isotype-specific Abs remained unknown. Ab coating is capable of blocking virus infection by interfering with receptor-virus binding, by virus opsonization, by complement activation, and via Fc{gamma}R-mediated effector mechanisms. The aim of the studies described in this work was to uncover the cellular basis of anti-virus Ab production, to evaluate the importance of the IgG2a subclass of IgGs in virus neutralization, and to investigate which of the blocking mechanisms plays a role in virus neutralization. We showed that I/LnJ-derived bone marrow cells, specifically IFN-{gamma}-producing CD4+ T cells, were key cells conferring resistance to MMTV infection in susceptible mice upon transfer. We also established that a unique bias in the subclass selection toward the IgG2a isotype in infected I/LnJ mice was not due to their potent neutralizing ability, as anti-virus Abs of other isotypes were also able to neutralize the virus, but were a product of virally induced IFN-{gamma}. Finally, we demonstrated that F(ab')2 of anti-MMTV IgGs neutralized the virus as efficiently as total IgGs, suggesting that Ab-mediated interference with viral entry is the sole factor inhibiting virus replication in I/LnJ mice. We propose and discuss possible mechanisms by which infected I/LnJ mice eradicate retrovirus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Exogenous mouse mammary tumor virus (MMTV)3 is a B-type retrovirus that is transmitted through the milk of infected female mice to nursing newborns (1). Cells of the immune system, particularly B cells, are the first targets of this virus (2). MMTV gains access to B cells by traveling though M cells located in the follicular-associated epithelium of Peyer’s patches in the gastrointestinal tract (3). The infected B cells express and present virally encoded superantigen (SAg) in the context of MHC class II molecules to T cells, leading to the stimulation and consequent proliferation of SAg-cognate T cells bearing a particular V{beta}+ chain of TCR (4) and, in turn, proliferation of bystander cells (5, 6). Because MMTV, like most other retroviruses, infects only activated cells (7), these events result in viral amplification and subsequent virus transport to the mammary glands, where it causes mammary tumors (8). Subsequently, activated SAg-cognate T cells are deleted from the immune repertoire, and deletion of these cells can be used as a readout for MMTV infection (9).

In contrast to exogenous MMTVs, the majority of endogenous stably integrated MMTV proviruses (Mtvs) do not produce infectious viral particles because of mutations in their transcriptional regulatory or coding regions (10). However, all endogenous Mtvs express functional SAgs, which stimulate deletion of the V{beta}+ T cell subset during formation of the immune repertoire (4, 11).

There are three documented mechanisms of resistance to MMTV infection in mice. These mechanisms are related to: deletion of SAg-cognate T cells caused by Mtvs (6, 12), failure to efficiently present viral SAgs in the context of certain MHC haplotype molecules (13, 14, 15), and production of uninfectious virions resulting from secretion of virus-neutralizing Abs (16). Whereas numerous strains of mice exhibit resistance to MMTV infection due to expression of particular MHC genes or endogenous Mtvs, only I/LnJ mice are capable of eradicating the virus due to secretion of virus-neutralizing Abs. This resistance mechanism is recessive, because F1 crosses between susceptible C3H/HeN (or BALB/cJ) and resistant I/LnJ mice are susceptible to MMTV and fail to produce virus-neutralizing Abs (16). The neutralizing Abs, predominately of the IgG2a isotype, recognize major virion proteins, including surface glycoproteins (gp52EnvSU), transmembrane proteins (gp36EnvTM), and internal structural proteins (p27GagCA). The production of anti-MMTV Abs is dependent on IFN-{gamma}, because I/LnJ IFN-{gamma}-deficient mice failed to produce anti-virus Abs of any isotype (16). In this study, we investigated the molecular and cellular basis of the virus resistance in I/LnJ mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

All mice used in this study were bred and maintained at the animal facility of The Jackson Laboratory. I/LnJ, I/LnJ IFN-{gamma} knockout (KO), C3.JK-H2j H2-T18b/Sn (C3.JK), and BALB/cJ mice were purchased from The Jackson Laboratory. C3H/HeN MMTV-free mice were originally purchased from the National Cancer Institute, Frederick Cancer Research Facility. MMTV(LA) viruses (17) were passed on to BALB/cJ mice (BALB/cLA mice). I/LnJ mice were crossed to B6.129S2-Igh-6tm1Cgn mice (18) for 10 repetitive generations, and heterozygous N10 mice were intercrossed to generate I/LnJ mice with a targeted mutation of Igh-6 (I/LnJ B cell-deficient mice). Similarly, I/LnJ mice were crossed to B6.129S2-Cd4tm1Mak (19) and to B6.129S2-Cd8atm1Mak mice (20) for 10 generations to generate I/LnJ mice with targeted mutations of CD4 and CD8, respectively. I/LnJ IFN-{gamma} KO mice were described (16). The studies have been reviewed and approved by the Animal Care and Use Committee at The Jackson Laboratory.

Flow cytometry

Mononuclear PBLs were stained with FITC-coupled mAbs against the V{beta}14+ TCR chain (BD Biosciences). Anti-CD4 Abs coupled to PE (Invitrogen Life Technologies) were used in the second dimension. Leukocytes were recovered from heparinized blood samples by centrifugation through a Ficoll-Hypaque cushion. PBLs were analyzed using a FACScan (BD Biosciences) flow cytometer and the CellQuest software program.

Preparation of purified F(ab')2

Serum samples were collected from 10 MMTV-infected and 10 uninfected I/LnJ mice. The samples were then subjected to ammonium sulfate precipitation, followed by fractionation using a 2.5 x 95-cm ACA22 gel filtration column run at 0.2 ml/min to separate IgMs. Only IgM-negative fractions as determined by ELISA were combined. F(ab')2 were obtained by pepsin (Sigma-Aldrich) digestion, as recommended by the manufacturer. In brief, 15 mg of pepsin-bound agarose was added to 150 mg of each IgG sample in 5 ml of sodium acetate buffer (0.2 M (pH 4.0)). After 24-h incubation at 37°C, an additional 12 mg of pepsin-agarose was supplemented to the reaction and incubation was continued for another 24 h. The reaction was stopped by addition of Tris-HCl buffer (2 M (pH 9.0)) to neutral pH. Digestion products were dialyzed against PBS overnight at +4°C. F(ab')2 and Fc fragments were separated using protein A-agarose beads (Sigma-Aldrich). The concentrations of IgGs and F(ab')2 were measured by Bradford method. To verify the efficiency of digestion and purification, samples were run on a 10% nonreducing SDS-polyacrylamide gel and blotted to nitrocellulose (Bio-Rad). Blots were incubated with anti-mouse IgG (H + L) Abs (Bio-Rad) coupled to HRP and detected using Western blot detection reagents (Amersham Biosciences).

ELISA

MMTV(LA) virions were isolated from the milk of infected BALB/cLA mice, as described (21). Isolated virions were resuspended in PBS with 0.2% Triton X-100 and bound to the plate at 0.5 µg/ml in BBS buffer overnight at +4°C. Nonspecific binding was blocked by 1% BSA for 2 h at 37°C. Total IgGs and F(ab')2 from MMTV-infected and uninfected I/LnJ sera were added at 1 x 10–1 and incubated for 1 h at +4°C. Secondary Abs, against mouse Igs or against mouse Fc fragments conjugated with alkaline phosphatase (AP) diluted in ELISA buffer (PBS, 0.05% Tween 20, and 0.05% sodium azide), were used at the second step. Background obtained with the IgG and F(ab')2 isolated from uninfected I/LnJ sera was subtracted. ELISA to detect anti-virus Abs in chimeric mice was performed, as described (16).

Production of mAbs

Splenocytes were isolated from 2-wk-old I/LnJ mice fostered on viremic BALB/cLA females and fused with the AG8 myeloma cell line, according to a standard protocol. Selected clones were screened by ELISA for Ab production. MMTV(LA) virions and proteins contained within the same density fraction isolated from the milk of uninfected BALB/cJ mice were used for this screen. Hybridomas interacting with MMTV virions, but not with milk proteins, were scored as positive. Of 4420 hybridomas screened, 4 were found to be reactive against virion proteins by ELISA and/or immunoprecipitation. The isotype of mAbs was determined using Abs against different isotypes coupled to AP at the second step of ELISA.

Transmission electron microscopy and immunogold labeling

Mm5MT cells (MMTV(C3H)-induced mammary gland tumor cell line; American Type Culture Collection) were stained with mAbs, according to a published protocol (22). Goat anti-mouse IgGs conjugated to 15-nm colloidal gold particles (Electron Microscopy Sciences) were used as secondary Abs.

Virus neutralization assays

IgGs, F(ab')2, and mAbs produced by MMTV-infected I/LnJ mice were tested for their ability to neutralize virus in two types of assays. First, 90 µl of IgGs and F(ab')2 resuspended in PBS at 166 mg/ml was incubated with 50 µl of purified MMTV(LA) (corresponding to the content of one newborn stomach) for 2 h at room temperature and injected into a footpad of BALB/cJ mice. Four days after injection, cells isolated from the draining popliteal lymph node were analyzed by FACS for the percentage of V{beta}14+ T cells among CD4+ T cells. Alternatively, 200 µl of supernatants derived from hybridoma clones was incubated with 50 µl of MMTV(LA) virions for 2 h at room temperature, followed by i.p. injection into BALB/cJ mice. Five weeks after injection, mice were bled and peripheral T cells were analyzed by FACS for the percentage of V{beta}14+ T cells among CD4+ T cells.

Bone marrow chimeras

Bone marrow was isolated from the femora, tibias, and humeri of I/LnJ mice and (C3.JK x I/LnJ)F1 hybrid mice, and 5 x 106 cells in 0.5 ml of PBS were injected i.v. into 8- to 10-wk-old recipient mice that had been irradiated at 900 rad. To determine the percentage of chimerism, mice were bled and PBLs were stained with Abs against the CD5-1 and CD5-1.1 variants of CD5 (BD Pharmingen). I/LnJ mice have the CD5-1 allele of the gene, whereas C3H/He mice have the CD5-1.1 allele. The percentage of I/LnJ bone marrow cells was determined by the percentage of CD5-1+/CD5-1.1– cells of the total CD5+ cells.

Splenocyte transfer

To produce chimeric mice containing different subsets of mature progenitors of the bone marrow cells, splenic cells from uninfected I/LnJ mice were subjected to negative selection (see below) and the residual cells (5 x 106) were i.v. injected into susceptible IFN-{gamma}-deficient I/LnJ mice irradiated at 700 rad before transfer. We also used splenocytes obtained from I/LnJ mice deficient in B, CD4+, or CD8+ T cells for our transfer experiments. Mice were bled 3 mo after injection and assayed by ELISA for anti-MMTV Ab production.

Splenocyte purification by negative selection

Splenocytes were incubated with anti-CD11c (N418) expressed on dendritic cells (DCs), anti-CD11b expressed on macrophages (M{phi}) (M1/70.15.11.5), anti-MHC class II Ia-specific (M5/114.15.2), and anti-NK cell (DX5) Abs bound to magnetic beads (Miltenyi Biotec). Cells attached to microbeads were run through depletion columns, as described by the manufacturer (Miltenyi Biotec). The remaining cells (>95% pure as determined by FACS) were injected into recipient mice.

Assay for IFN-{gamma}

Thioglycolate-elicited peritoneal exudate cells (PEC) (1.8 x 106) were incubated with purified MMTV virions in 24-well plates for 60 h, as described (23). IFN-{gamma} concentration was measured in the culture’s supernatants by an ELISA kit, according to the manufacturer’s protocol (BD Biosciences). The same density fraction isolated from an equal amount of virus-free BALB/cJ milk was used as control.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Cells of I/LnJ bone marrow origin are capable of conveying the ability to produce anti-MMTV-neutralizing Abs

Previously, we showed that F1 progeny of a cross between susceptible MHC-congenic C3.JK and resistant I/LnJ mice were susceptible to MMTV infection and MMTV-induced mammary tumors (24). We therefore used these F1 mice in bone marrow transfer experiments to determine the cellular basis of the I/LnJ resistance. Adult MMTV-infected (C3.JK x I/LnJ)F1 mice were lethally irradiated at 900 rad and injected with bone marrow isolated from I/LnJ mice. All recipient (I/LnJ x C3.JK)F1 mice became chimeric and MMTV infected as they demonstrated deletion of SAg-cognate T cells (Table I). The chimeric mice were tested for their ability to produce anti-MMTV Abs 70 days after transfer. All I/LnJ to F1 chimeras showed production of Abs against MMTV in their sera (Table I and Fig. 1). These results show that cells of I/LnJ bone marrow origin are sufficient to convey the ability to produce neutralizing Abs in susceptible mice.


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  		Table I. Ab production in bone marrow chimeras

 


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  		FIGURE 1. Bone marrow cells from virus-resistant I/LnJ mice are solely responsible for conveying ability to produce anti-virus Abs to susceptible mice. Serum samples from MMTV(LA)-infected bone marrow chimeras, as well as from MMTV(C3H)-infected I/LnJ, C3.JK, and uninfected I/LnJ mice were tested for reactivity against MMTV virion proteins by ELISA. Anti-mouse IgG2a Abs coupled to AP were used at the second step. Sera were diluted 10–3. Background obtained with the secondary Ab alone was subtracted.

 
CD4+ Th1 T cells are required for the production of virus-neutralizing Abs by MMTV-infected I/LnJ mice

Once we established that I/LnJ-derived bone marrow cells were solely responsible for the production of virus-neutralizing Abs, we sought to elucidate which cellular subset was necessary and sufficient for the production of anti-MMTV Abs. To address this question, we transferred splenic cells from uninfected I/LnJ mice into MMTV-infected susceptible syngeneic mice. For these experiments, we choose to use I/LnJ IFN-{gamma}-deficient mice as recipients to eliminate reactivity against minor histocompatibility Ags that we observed with (I/LnJ x C3.JK)F1 recipient mice in our pilot experiments. Splenocytes were isolated from uninfected I/LnJ mice and subjected to negative selection by MACS or were isolated from B cell-, CD4+ T cell-, and CD8+ T cell-deficient I/LnJ mice, as described in Materials and Methods. The remaining cells, and cells isolated from mice with targeted mutations of different genes, were injected into MMTV-infected 4-wk-old I/LnJ IFN-{gamma}-deficient mice that were irradiated at 700 rad. I/LnJ IFN-{gamma}-deficient mice replenished with splenocytes derived from uninfected I/LnJ mice were used as controls. Three months after injection, recipient mice were assayed for MMTV infection, for chimerism, and for Ab production.

The efficiency of conferring anti-MMTV Ab production to recipient mice was ~50%, because only one-half of the control mice produced anti-MMTV Abs (Table II). Whereas recipient mice produced anti-MMTV Abs after receiving lymphocytes that lacked B cells, DCs, M{phi}, all MHC class II-positive cells, NK cells, and CD8+ T cells, mice that received lymphocytes lacking CD4+ T cells did not (Table II). Importantly, these mice did not produce Abs of any isotype (Table II). Therefore, IFN-{gamma}-producing CD4+ Th1 T cells are necessary and sufficient for conferring the ability to produce anti-MMTV Abs to susceptible IFN-{gamma}-deficient I/LnJ mice.


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  		Table II. Anti-virus Ab production in MMTV-infected chimeric I/LnJ IFN-{gamma}-deficient recipient micea

 
Naturally occurring anti-virus Abs of distinct isotypes neutralize the virus with similar efficiency

Mice with disruption of the IFN-{gamma} and IFN-{gamma} receptor genes are highly sensitive to viral infections despite normal cytotoxic and Th cell responses (25, 26, 27). Because both IFN-{gamma} receptor and IFN-{gamma} KO mice demonstrate decreased baseline IgG2a levels (27, 28), it was suggested that this subset of IgGs plays a major role in protection against viruses. In fact, IgG2a Abs have been shown to display functional characteristics different from IgG1 Igs (29, 30, 31) and to predominate in responses against various viruses, including retroviruses (32, 33). MMTV-infected I/LnJ mice also mount an IgG2a-specific anti-virus immune response that is dependent on IFN-{gamma} (16).

To determine whether anti-virus Abs have to be of the IgG2a isotype to neutralize infection, we examined anti-virus Abs of other isotypes for their virus-neutralizing ability. Accordingly, we produced B cell hybridomas from splenocytes of I/LnJ mice infected with MMTV as neonates. The clones were screened for reactivity against MMTV and for isotype specificity. The supernatants containing mAbs produced by selected clones were then subjected to immunoprecipitation to determine which viral proteins the mAbs were reactive against. Four Abs were selected: one IgG1-specific mAb reactive against gp36TM (9G6), one IgG2b-specific anti-gp52SU mAb (3E11), one IgM-specific mAb reactive against gp52SU (1D5), and one IgM-specific mAb (5E9) whose Ag specificity we were unable to determine, probably due to a very low affinity binding (Fig. 2, A–C).



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  		FIGURE 2. Neutralizing ability of Abs is not dependent on the IgG2a isotype. A, B cell hybridomas made from spleens of MMTV(LA)-infected I/LnJ mice produce Env-specific mAbs. Virion proteins were immonoprecipitated with distinct mAbs, separated on 10% acrylamide gel, transferred to nitrocellulose, and incubated with mAbs against gp36TM and gp52SU, followed by incubation with goat anti-mouse polyvalent Abs coupled to HRP. Unlike other mAb, 5E9 did not show reactivity against both Env and Gag (data not shown) in Western blot. B, 5E9 mAb recognizes a viral protein in ELISA. Virions purified from the milk of MMTV(LA)-infected BALB/cJ mice were bound to plastic and incubated with mAbs 5E9. The reaction was developed with goat anti-mouse polyvalent Igs coupled to AP. Reactivity of the Abs against virus-free milk was subtracted. C, Electron micrograph showing reactivity of 3E11 mAb on Mm5MT cells. Arrow indicates budding virion-containing gold particles. D, Anti-gp52SU, but not anti-gp36TM Abs, exhibit neutralizing activity. Purified MMTV(LA) virions incubated with different mAbs were injected directly into the mammary glands of previously uninfected BALB/cJ mice. Uninfected control BALB/cJ mice have 11.8 ± 0.5% of peripheral CD4+V{beta}6+ T cells (n = 10). The deletion of SAg-cognate T cells measured 5 wk after infection was used as indicator of virus infectivity. The mAb 514 of the IgM isotype that recognizes gp70SU of MCF and polytropic murine leukemia virus (34 ) and medium with 5% FCS were used as controls. I/LnJ+, total serum from MMTV-infected I/LnJ mice. Four to five recipient BALB/cJ mice were used per group.

 
Abs produced by MMTV-infected I/LnJ mice were tested for their ability to neutralize virus. For that reason, mAb supernatants were used in an in vitro neutralization assay. IgM-specific mAb 514 recognizing gp70SU of murine leukemia virus (MuLV) (34), as well as plain medium, were used as controls. Purified MMTV(LA) virions incubated with different mAbs were injected directly into the mammary glands of previously uninfected BALB/cJ mice. All successfully infected mice showed deletion of SAg-cognate T cells, which starts ~4 wk after infection (9, 35). Therefore, infected mice were bled and percentages of SAg-cognate T cells were measured in the periphery 5 wk after infection. Uninfected control BALB/cJ mice have 11.8 ± 0.5% of peripheral CD4+V{beta}6+ T cells (n = 10). BALB/cJ mice injected with virus preincubated with mAbs 9G6, 514, or plain medium became infected with MMTV, showing a diminution in the percentage of these SAg-cognate T cells (Fig. 2D). However, BALB/cJ mice injected with MMTV virions preincubated with mAbs 3E11, 5E9, and 1D5 remained virus free, because they either did not show (5E9, 311E), or showed a nonsignificant deletion (1D5), of SAg-cognate T cells (Fig. 2D). Thus, MMTV pretreatment with anti-gp52SU mAbs of the IgM and IgG2b isotype in vitro results in virus neutralization. These data indicate that Abs produced by MMTV-infected I/LnJ mice do not need to be of the IgG2a isotype to be neutralizing. Instead, the class switch is most likely due to virally elicited production of IFN-{gamma}.

MMTV virions stimulate IFN-{gamma} secretion by I/LnJ PEC

Experiments described in the previous section suggested that infection per se must be a key to production of IFN-{gamma} in I/LnJ mice. To test this hypothesis, we incubated PEC of I/LnJ mice with intact MMTV virions isolated from the milk of MMTV(LA)-infected BALB/cJ mice in vitro. Culture supernatants were assayed for IFN-{gamma} production 60 h later (Fig. 3). IFN-{gamma} secretion was induced in response to MMTV virions, but not against the virus-like fraction isolated from MMTV-free milk (Fig. 3). Thus, MMTV virions trigger a signaling cascade that leads to secretion of IFN-{gamma}.



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  		FIGURE 3. PECs from I/LnJ mice produce IFN-{gamma} in response to MMTV virions. IFN-{gamma} concentration detected in supernatants of thioglycolate-elicited cells from the peritoneal cavity of I/LnJ mice after incubation with MMTV virions (left) or with the same density fraction isolated from an equal amount of virus-free BALB/cJ milk (right). Thioglycolate-elicited cells collected from the peritoneal cavity consist mostly of M{phi}, but also contain cells of other origin, including CD4+ T cells (~5%).

 
Ability of anti-MMTV Abs to neutralize the virus is solely due to virion coating

We have shown previously that MMTV-infected I/LnJ mice produce uninfectious viruses that are coated with anti-virus Abs (16). We hypothesized that these Abs, specifically anti-Env Abs, control virus infectivity because they prevent virus binding to the plasma membrane receptors expressed on susceptible cells. However, in addition to direct interference with viral entry, Abs may also cause virus eradication by opsonization, by Ab-dependent cellular cytotoxicity, and via complement activation (36, 37). This led us to question which of these mechanisms operates in infected I/LnJ mice. Because all the mechanisms, except for virus coating/interference with viral entry, are initiated by the Fc fragment of IgGs, we reasoned that we would be able to address this question by comparing the neutralizing capacity of total IgGs with F(ab')2. Accordingly, sera were collected from MMTV-infected I/LnJ and uninfected mice, and IgGs were subjected to digestion using pepsin after separation of IgMs by size fractionation. Pepsin digestion results in the cleavage of disulfide bonds and the release of the Fc portion of the Ab from the F(ab')2 fraction. Protein A-bound agarose was used to remove Fc fragments, as well as any undigested IgGs (Fig. 4, A and B).



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  		FIGURE 4. Virus-neutralizing capacity of Abs produced by infected I/LnJ mice is solely due to their ability to coat virions. A, F(ab')2 of anti-virus IgGs bind to MMTV Ags. ELISA was conducted with MMTV(LA) virion proteins bound to plates and the IgG or the F(ab')2 fraction of Igs isolated from MMTV(LA)-infected and uninfected I/LnJ mice. The ELISA was developed with either goat anti-mouse polyvalent Igs or goat anti-mouse Fc-specific Igs coupled to AP. B, Western blot analysis of the IgGs. IgGs (untreated, digested with pepsin before and after incubation with protein A agarose) were separated on 10% nonreducing acrylamide gel, blotted to nitrocellulose, and incubated with anti-mouse polyvalent Abs, which recognized both H and L chains coupled to HRP. *, Nonspecific band. C, F(ab')2 of IgGs exhibit virus-neutralizing capacity. Four days after footpad injection of MMTV(LA) virions preincubated with total IgGs or F(ab')2 of IgGs isolated from MMTV-infected or uninfected I/LnJ mice, cells derived from popliteal lymph nodes were double stained with anti-CD4 PE and anti-V{beta}6 (or anti-V{beta}14) FITC-labeled mAbs and analyzed by FACS. Uninfected BALB/cJ mice have 11.4 ± 0.4% and 9.2 ± 0.8% of peripheral CD4+V{beta}6+ and CD4+V{beta}14+, respectively (n = 5). BALB/cJ mice infected with virus preincubated with total IgGs or F(ab')2 of IgGs isolated from uninfected I/LnJ mice showed an increase of SAg-cognate T cells, suggesting that the virus was infectious. On the contrary, BALB/cJ mice infected with virus preincubated with total IgGs or F(ab')2 of IgGs isolated from MMTV(LA)-infected I/LnJ mice did not demonstrate elevation of SAg-cognate T cells, suggesting that the virus was neutralized.

 
To investigate whether F(ab')2 were as efficient in neutralizing MMTV as total IgGs, we performed a neutralization experiment. Biologically active MMTV(LA) virions were incubated with F(ab')2 or with total IgGs and then injected into the footpads of uninfected BALB/cJ mice. This route of MMTV infection induces a vigorous localized immune reaction during which the numbers of SAg-cognate T cells increase in draining popliteal lymph nodes (38, 39). MMTV(LA) is a natural mixture of three different viruses with V{beta}2-, V{beta}14-, and V{beta}6-specific SAgs, respectively (17, 40). Four days after injection, cells isolated from the draining lymph nodes were analyzed for the percentage of SAg-cognate V{beta}6+ T and V{beta}14+ T cells among CD4+ T cells. A significant increase in the percentage of CD4+V{beta}6+ and CD4+V{beta}14+ T cells was detected in the regional lymph nodes of recipient mice injected with virus incubated with F(ab')2 and total IgGs isolated from sera of uninfected mice (Fig. 4C). In contrast, no T cell-specific stimulation was observed in recipient mice injected with the virus preincubated with F(ab')2 and total IgGs purified from sera of infected mice (Fig. 4C). The results from this neutralization experiment confirmed our hypothesis and indicated that F(ab')2 of IgGs alone are solely responsible for the neutralizing ability of Abs produced by MMTV-infected I/LnJ mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Viruses have the unique ability to evade host immunity by either avoiding or inhibiting immune responses. This ability is necessary because it permits virus persistence and replication to higher titers, thus increasing chances of transmission to a susceptible recipient. MMTV is a highly infectious retrovirus that exploits numerous mechanisms to evade host immune responses in various strains of mice (23, 41, 42). Nevertheless, MMTV is unable to persist in I/LnJ mice and is eradicated in the infected mouse pedigree (16). This mechanism of virus resistance is related to rapid and sustained production of virus-neutralizing Abs, which remain at high levels through the life of the animal (16). The vast majority of anti-virus Abs belong to the IgG2a isotype, and their production requires IFN-{gamma} (16). IgG2a is the major complement-binding and opsonizing Ab and is also highly potent in activating Fc{gamma}R-mediated effector mechanisms. In this study, we demonstrated that the neutralizing ability of anti-virus Abs produced by MMTV-infected I/LnJ mice is not related to the above mechanisms, but is solely due to virus coating, which prevents the attachment of virions to the cell (Fig. 5).



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  		FIGURE 5. Schematic representation of working hypothesis for this study. I/LnJ mice produce neutralizing Abs directed against the SU env gene product, and thus, all cell-free viral particles are coated with Igs affecting recognition of the viral receptor expressed on susceptible cells.

 
Despite the isotypic bias toward IgG2a, rare Abs of other isotypes found in MMTV-infected I/LnJ mice were as efficient as total anti-virus Abs in virus neutralization (Fig. 2). IFN-{gamma} induces a class switch to IgG2a in Ab-producing B cells. Therefore, it is plausible that the biased selection of IgG2a-specific anti-virus Abs is a byproduct of IFN-{gamma} secretion rather than a requirement for virus neutralization. This suggests that infection per se is a key to the production of IFN-{gamma} in I/LnJ mice. In support of this notion, purified MMTV virions stimulated production of IFN-{gamma} by I/LnJ PEC (Fig. 3). Previously, we showed that MMTV uses innate immune TLR4 for its own benefit by stimulating the secretion of immune suppressor cytokine IL-10, which blocks the ongoing anti-virus immune response (23). Thus, it is possible that in addition to IL-10, MMTV-induced TLR4 signaling elicits secretion of IFN-{gamma}. Our preliminary experiments validated this hypothesis, as splenocytes from TLR4-deficient mice were unable to produce IFN-{gamma} in response to MMTV (data not shown).

Cells of bone marrow origin purified from resistant I/LnJ mice were fully proficient in transferring the ability to produce anti-virus Abs in susceptible mice (Table I). These results implicated a subset of bone marrow cell progenitors and also ruled out the stromal cells of the secondary lymphoid organs and follicular DCs in establishing and maintaining resistance against retrovirus. Using susceptible IFN-{gamma}-deficient I/LnJ mice, we identified CD4+ T cells as necessary and sufficient cells for the production of virus-neutralizing Abs. Moreover, our experiments also established that it is IFN-{gamma}-secreting Th1 T cells that are responsible for anti-virus Ab production, because IFN-{gamma}-deficient recipient I/LnJ mice that received IFN-{gamma}-sufficient splenocytes lacking CD4+ T cells failed to make anti-virus Abs (Table II). What are the functions provided by CD4+ T cells that are essential for virus resistance mechanism? First, CD4+ T cells provide immunological help to the B cell response. Clearly, all isotype immune responses in MMTV-infected I/LnJ mice are T cell dependent because susceptible recipient mice that received I/LnJ lymphocytes lacking CD4+ T cells do not produce Abs of any isotype (Table II). Second, IFN-{gamma}-secreting CD4+ T cells also provide direct antiviral activities, particularly during persistent infection. However, it has not been determined whether the latter mechanism operates in I/LnJ mice.

The production of virus-neutralizing Abs underlies resistance to Friend MuLV in some strains of inbred mice (43). C57BL/B6J (B6) and C57BL/10J (B10) mice are resistant to Friend MuLV infection, whereas BALB/cJ mice are susceptible (43). Like the anti-MMTV response in I/LnJ mice, the anti-Friend MuLV-neutralizing Ab response in resistant mice is also IFN-{gamma} and CD4+ T cell dependent (44, 45, 46). However, there are numerous differences between the two models. First, the MuLV resistance mechanism inherited by B6/B10 mice is dominant, whereas the MMTV resistance mechanism inherited by I/LnJ mice is recessive (16). Second, IFN-{gamma}-deficient I/LnJ mice are susceptible to MMTV infection, remain infected throughout their lifetimes, and do not produce anti-virus Abs of any isotype (16). On the contrary, MuLV-infected IFN-{gamma}-deficient B6 mice make anti-virus-neutralizing Abs of the IgM isotype and demonstrate a temporal recovery from infection (45). B6 and B10 mice are resistant to MMTV because of the nonpermissive MHC haplotype (47). However, hybrid mice obtained from crosses between B6 or B10 mice and I/LnJ mice are susceptible to MMTV infection and mammary tumors (14) (data not shown). This argues against the same genes conferring resistance mechanisms inherited in B6 and I/LnJ mice.

HIV-infected individuals continuously produce virus-specific Abs throughout most of the clinical course. However, levels of neutralizing Abs found in these individuals tend to be low (48, 49, 50) and neither increase in titer nor suppress virus recovered from other infected people (51). Maintenance of high levels of anti-virus-neutralizing Abs is therefore a challenging goal that would require long-lived memory B cells and/or plasma cells. Various mechanisms have been proposed to achieve this goal, including continuous exposure to Ag, reimmunization, and trapping of Ag/Ab complexes in follicular DCs (52, 53, 54). This has resulted in the development of anti-HIV vaccines that elicit neutralizing Abs somewhat effectively. However, all exhibited a relatively short t1/2 (55). Therefore, the key in preventing or aborting virus infection is to achieve rapid generation of virus-neutralizing Abs and sustain their production at high levels. I/LnJ mice can mount an immune response against retrovirus so efficient that it does not allow retroviruses to evolve and escape immune recognition. Even though the IgG2a isotype of the Abs is not required for efficient virus neutralization, the predominance of this class of Abs after retroviral infection could correspond to the selection of a most competent response to the virus. Therefore, elucidation of the mechanism of the anti-virus-neutralizing Ab production in I/LnJ mice is of fundamental importance, because it will ultimately lead to increased knowledge about anti-virus immune response and variations in susceptibility to viral infections in humans.


   Acknowledgments
 
We are thankful to members of the laboratory and to Dr. Alexander Chervonsky for helpful discussion; Rob Wilpan for chromatography; Lesley Bechtold for electron microscopy; and Sara Williams for the graphics work.


   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 Public Health Service Grant CA89116 and by an award from the American Cancer Society to T.V.G. This work was also supported by a grant (CA34196) from the National Cancer Institute to The Jackson Laboratory. Back

2 Address correspondence and reprint requests to Dr. Tatyana V. Golovkina, at current address, University of Chicago, 920 East 58th Street, CLSC 711A, Chicago, IL 60637. E-mail address: tgolovki{at}bsd.uchicago.edu Back

3 Abbreviations used in this paper: MMTV, mouse mammary tumor virus; AP, alkaline phosphatase; DC, dendritic cell; KO, knockout; M{phi}, macrophage; Mtv, MMTV provirus; MuLV, murine leukemia virus; PEC, peritoneal exudate cell; SAg, superantigen. Back

Received for publication July 6, 2005. Accepted for publication September 23, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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