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The absence of endogenous mouse mammary tumor viruses (MMTVs) in the congenic mouse strain, BALB/Mtv-null, restricts the early steps of exogenous C3H MMTV infection, preventing the superantigen (Sag) response and mammary tumorigenesis. Here we demonstrate that BALB/Mtv-null mice also resist tumor induction by FM MMTV, which encodes a stronger Sag compared to C3H MMTV. In contrast to infections with C3H MMTV, Mtv-null mice show FM-MMTV Sag-specific responses comparable to those observed in susceptible BALB/c mice. Neither virus shows significant replication in the spleen or mammary gland. Thus, Mtv-null mice restrict MMTV replication and mammary tumorigenesis even after a robust Sag response.
Mouse mammary tumor virus (MMTV) is an oncogenic retrovirus that causes mammary carcinoma in mice after a long latency (6 to 9 months) (16). MMTV is transmitted exogenously through the milk of infected female mice to the progeny during the first 2 weeks of life, whereas the stably integrated endogenous proviruses (Mtvs) found in most laboratory mice are inherited through the germ line according to Mendelian rules (13). A majority of the endogenous proviruses are defective for replication and mammary tumorigenesis due to mutations (7, 17). However, most of these proviruses encode functional superantigens (Sags), which are expressed at the surface of antigen-presenting cells in association with major histocompatibility complex class II molecules (4, 6, 11). Sag presentation induces peripheral or intrathymic clonal deletion of most Sag-reactive T cells, resulting in an altered mouse T-cell repertoire (3, 18). Strong Sags, e.g., Mtv7, induce proliferation of cognate T cells prior to clonal deletion (20), whereas weak Sags, e.g., C3H or GR Sag, fail to induce proliferation of reactive T cells or expression of interleukin-2 receptors (15). C3H MMTV infection also has been shown to induce expression of endogenous Mtv sag gene expression (21).
Our previous work demonstrated that endogenous Mtvs play a critical role in determining susceptibility to multiple pathogens in BALB/c mice (5). BALB/c mice harbor endogenous Mtv6, Mtv8, and Mtv9 on chromosomes 16, 6, and 12, respectively (13), and are susceptible to tumorigenesis by exogenous C3H MMTV, as well as the T-cell-tropic MMTV variant, type B leukemogenic virus (TBLV). BALB/c-congenic mice lacking all endogenous Mtvs (BALB/Mtv-null) are resistant to infection and tumorigenesis by exogenous C3H MMTV (5). These mice also are resistant to TBLV, which lacks a functional sag gene (2). Interestingly, BALB/Mtv-null mice demonstrate reduced replication and mortality resulting from infection by the gram-negative bacterium, Vibrio cholerae. Genetic experiments showed that the presence of any single Mtv provirus reconstituted susceptibility to V. cholerae within 48 h, suggesting that endogenous Mtvs manipulate innate immunity (5). Since Sag is the only protein known to be encoded by all of the endogenous Mtvs of BALB/c mice, we proposed that endogenous sag genes may control immunity to multiple pathogens. In the present study, we attempted to determine whether the strong exogenous Sag encoded by the FM-MMTV strain could overcome the restriction to MMTV replication in Mtv-null animals.
We previously demonstrated that BALB/Mtv-null mice develop only a 10% incidence of mammary carcinomas after milk-borne infection or after intraperitoneal (i.p.) inoculation with the C3H-MMTV-based infectious clone, HYB-MTV (5, 19). C3H infection in the BALB/Mtv-null mice is impaired at an early stage since peripheral deletion of C3H Sag-reactive (CD4+ T-cell-receptor [TCR] Vβ14+) T cells is not observed (5). These results suggest that endogenous Mtv-encoded Sags are required for successful infection leading to a Sag response and tumorigenesis by C3H MMTV. The C3H MMTV strain encodes a weak Sag that causes a slow deletion of reactive T cells and causes weak cell proliferation in mixed lymphocyte reactions (1, 8).
To determine whether a stronger Sag could overcome the requirement for endogenous Mtv Sag, we inoculated BALB/Mtv-null and BALB/c mice in the footpad with mammary tumor extracts containing FM-MMTV virions. Similar to MMTV(SW), FM MMTV encodes a strong Sag that induces a three- to fourfold enlargement of draining lymph nodes and a substantial increase in the number of cognate (TCR Vβ8.2+) T cells, as well as B cells in the lymph nodes, after subcutaneous (s.c.) virus inoculation (12, 22). In contrast, the weak C3H-MMTV Sag causes only a marginal increase in reactive TCR Vβ14-expressing T cells and B cells in the draining lymph nodes (12). Single-cell suspensions were prepared from the draining popliteal lymph nodes harvested from FM-MMTV-treated or -untreated mice at 4 days postinoculation. The cells were surface stained with antibodies against CD4 and TCR Vβ8. Interestingly, the BALB/Mtv-null mice responded to the FM-MMTV Sag at levels similar to those observed in BALB/c mice. A twofold increase was observed in the proportion of CD4+ TCR Vβ8+ T cells in the draining lymph nodes of FM-MMTV-infected BALB/Mtv-null and BALB/c mice compared to uninfected mice (Fig. (Fig.1).1). Thus, FM MMTV stimulates the initial stages of viral replication to give a similar Sag response in both mouse strains, suggesting that endogenous Mtvs are not required for the initial infection by exogenous MMTVs encoding strong Sags.
Since C3H MMTV generates only a negligible increase in reactive TCR Vβ14-expressing T cells in the draining lymph nodes of BALB/c mice (8, 12), a long-term infection assay was performed to compare infectivity of the two viral strains. BALB/c and BALB/Mtv-null mice were injected s.c. and i.p. with mammary tumor extracts containing FM MMTV. Peripheral blood lymphocytes were prepared from age-matched uninfected mice and FM-MMTV-infected mice at 1.5 and 6 months postinfection. These cells were dually stained with antibodies against CD4 and either TCR Vβ8 or Vβ3 and analyzed by flow cytometry for Sag-mediated peripheral deletion of cognate CD4+ Vβ8+ T cells. As observed in the footpad assay, the magnitude and kinetics of the FM-MMTV Sag response in Mtv-null mice were very similar to those observed in the BALB/c strain. Infected BALB/c and Mtv-null mice demonstrated ca. 50% peripheral deletion of FM-MMTV Sag-reactive CD4+ Vβ8+ T cells within 1.5 months postinfection (Fig. (Fig.2A).2A). The deletion of FM-MMTV Sag-reactive T cells was maintained in the Mtv-null mice even at 6 months postinfection (Fig. (Fig.2B).2B). No deletion was observed in the Sag nonreactive CD4+ Vβ3+ T-cell subset, confirming the specificity of the response to FM-MMTV Sag (Fig. (Fig.2B).2B). This result suggests that an MMTV strain encoding a strong Sag can overcome the need for endogenous Mtvs in establishing the initial infection, leading to a robust response to the exogenous Sag. This observation contrasts with our previous results using C3H MMTV, which encodes a weak Sag and is restricted at an earlier stage in the BALB/Mtv-null mice, preventing a Sag response.
BALB/c mice inoculated with FM MMTV develop a 100% incidence of mammary carcinomas within ~8 months of infection (Table (Table1).1). Unexpectedly, despite a strong FM-MMTV Sag response, Mtv-null mice remained highly resistant to FM-MMTV-induced mammary tumors. Reactivity to FM-MMTV Sag also did not facilitate TBLV-induced lymphomagenesis in Mtv-null mice coinfected with FM MMTV and the TBLV molecular clone (HYB-TBLV) (14) expressed from stably transfected Jurkat T cells (Table (Table1).1). These mice demonstrated peripheral deletion of FM-MMTV Sag-reactive CD4+ Vβ8+ T cells (data not shown) but failed to develop TBLV-induced lymphomas. The tumorigenicity of HYB-TBLV was verified by infection of adult BALB/c mice, which developed a 75% incidence of TBLV-induced thymic T-cell lymphomas within 4 months (Table (Table11).
To confirm that infection was restricted in the Mtv-null mice, RNA was extracted as previously described (5) from multiple organs of BALB/c and Mtv-null animals infected with FM MMTV and subjected to reverse transcription-PCR (RT-PCR) (Fig. (Fig.2C).2C). Mammary glands (lane 1), spleens (lanes 7, 18, and 19), and salivary glands (lanes 22 and 23) from FM-MMTV-infected BALB/c animals all showed the 402-bp fragment expected from transcription of integrated FM-MMTV proviruses, a finding consistent with the spread of the viral infection. However, Mtv-null mice lacked detectable FM-MMTV transcription in these same tissues (lanes 3, 4, 5, 9, 10, 11, 20, 21, 24, and 25) even after multiple pregnancies and lactations, which should amplify virus expression from hormone response elements in the LTR. In addition, mammary tumors arising in FM-MMTV BALB/c mice had easily detectable viral transcripts (lanes 14 to 16), whereas the single mammary tumor arising in FM-MMTV-infected Mtv-null mice did not (lane 17). The quality of all cDNA preparations was verified using primers specific for glyceraldehyde-3-phosphate dehydrogenase (gapdh). Together, these results suggest that Sag-specific deletion in FM-MMTV-infected Mtv-null mice does not lead to significant infection of B and T cells or other cell types.
Not surprisingly, milk-borne transmission of FM MMTV from infected Mtv-null females to their offspring was not observed, as demonstrated by the lack of Sag-specific T-cell deletion (Fig. (Fig.3)3) or mammary tumor development (Table (Table1).1). In contrast, the progeny of FM-MMTV-infected BALB/c females developed a 100% incidence of mammary tumors, indicating successful milk-borne transmission in susceptible mice (Table (Table1).1). Our previous experiments revealed a strong correlation between the extent of Sag-mediated deletion of cognate T cells and MMTV tumorigenicity (5). However, the BALB/Mtv-null mice appear to restrict FM MMTV after a Sag-mediated T-cell response, thus preventing viral tumorigenesis, as well as milk-borne transmission.
Our results suggest that the absence of endogenous Mtvs creates different blocks to the replication and tumorigenicity of exogenous MMTV in BALB/Mtv-null mice. If the exogenous MMTV encodes a weak Sag, e.g., C3H MMTV, endogenous Sags are essential during the early stages of infection, leading to exogenous Sag presentation and cognate T-cell deletion (5). C3H MMTV infection is known to cause a rapid induction of endogenous Mtv Sag expression in gut-associated lymphoid tissue, spleen, and thymus as early as 24 h postinfection in BALB/c mice (21). In contrast, the endogenous Mtv Sag expression remains low to undetectable in infected C57BL/6 mice, which are relatively resistant to C3H MMTV. The increased endogenous Mtv Sag expression could be an indirect effect of cellular activation after exogenous MMTV infection (21). In addition, increased amounts of endogenous Sag might be indicative of their critical role in establishing infection by C3H MMTV in BALB/c mice.
Exogenous viral strains expressing strong Sags, such as FM MMTV, also can establish the first stages of infection (presumably the infection of dendritic cells) (9) in the absence of endogenous Mtvs. BALB/Mtv-null mice show a robust response to FM-MMTV Sag, indicating that the initial steps of viral infection and Sag expression occur to the same extent as in the BALB/c mice. The ability of FM-MMTV Sag to overcome the requirement for endogenous Mtvs in establishing a robust viral infection might be due to its property of inducing proliferation of cognate T cells prior to clonal deletion. However, this T-cell response does not lead to significant MMTV replication in B or T cells or the spread of virus to the mammary gland. On the other hand, C3H-MMTV Sag, which lacks the ability to induce cognate T-cell proliferation, may depend on endogenous Mtvs to achieve viral amplification in the earliest stages of infection that would eventually lead to Sag-specific T-cell deletion and virus-induced mammary tumorigenesis. Nevertheless, we cannot rule out that FM and C3H MMTV have essentially the same block to infection. Both viruses may cause a low-level infection and presentation of their respective Sag proteins in the same cell type, but only the stronger Sag may function.
Despite the establishment of infection and a robust FM-MMTV Sag response, the Mtv-null animals remain highly resistant to FM-MMTV-induced mammary tumorigenesis (Table (Table1),1), lack detectable proviral transcription in mammary glands (Fig. (Fig.2C),2C), and also fail to transmit the virus in their milk (Fig. (Fig.3).3). This observation suggests that the absence of endogenous Mtvs results in a later block to replication of viruses encoding strong Sags. Our experiments indicate that MMTV infection is not established in B and T cells, which results in the lack of transmission to the mammary epithelial cells. Transfer of infected splenocytes from C3H MMTV-infected BALB/c mice into BALB/Mtv-null mice also failed to establish MMTV infection and tumorigenesis in the Mtv-null recipients (data not shown). The restriction of MMTV-induced tumorigenesis may also result from a cell-mediated immune response against MMTV-infected cells. Our previous results show that, unlike BALB/c mice, Mtv-null animals completely restrict the outgrowth of BALB/c B-cell lymphomas (A20) stably expressing HYB-MTV, while A20 cells lacking HYB-MTV readily form tumors in both mouse strains (5). Another possibility is that endogenous Mtv-encoded Sags activate NK cells and cytokines that are critical for the growth of exogenous MMTV-induced tumors (10), which is consistent with our previous hypothesis that endogenous Mtvs alter innate immunity (5). In summary, our results demonstrate that Mtvs play a crucial role at early stages during the infection cycle of exogenous MMTVs leading to tumorigenesis and milk-borne viral transmission.
We acknowledge helpful suggestions from the Dudley lab.
This study was supported by National Institutes of Health grant R01 CA116813.
Published ahead of print on 15 October 2008.