INTRODUCTION

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Mouse mammary tumor virus (MMTV) is a replication-competent B-type murine retrovirus and causes mammary adenocarcinomas in some strains of laboratory mice (30). MMTV can be transmitted exogenously through milk and endogenously through a germ line as proviruses (Mtv). Both exogenous MMTV and endogenous Mtv proviruses have an open reading frame (ORF) encoding superantigen (SAG) in the 3' long terminal repeat (LTR); SAG binds to major histocompatibility complex (MHC) class II molecules and leads to stimulation and consequent deletion of mature T cells bearing particular V gene products (1, 2, 10, 21, 22, 27-29, 34). As T-cell recognition of SAG is mediated predominantly by the T-cell receptor (TCR) V domain, SAG can stimulate much higher proportions of T cells than can conventional peptide antigens (3, 19). After B cells are infected with exogenous MMTV, viral SAGs are presented on the cell surface in the context of MHC class II molecules. Through the SAG-MHC class II complex, the infected B cells then induce the proliferation of CD4⁺ T cells bearing specific TCR V chains (11, 24, 44). These T cells lead to the expansion of infected B cells, resulting in amplification of the infection with MMTV (3, 5, 20, 39, 41). On the other hand, SAG expression from inherited provirus usually leads to depletion of immature T cells expressing reactive TCR  chains during intrathymic T-cell development (14). Thus, the characteristic of SAG for strong T-cell stimulation is critical in successful infection of the mammary gland for exogenous MMTV and in skewing the T-cell repertoire via clonal deletion for endogenous MMTV. Since most of the T cells recognizing SAG expressed by MMTV, irrespective of their maturation stage, are finally deleted after stimulation with SAG, a direct role of SAG in tumorigenicity for a mammary tumor seems unlikely. However, there are several lines of evidence showing a link between tumor formation and SAG expression. Reticulum cell sarcoma tumors, which are derived from germinal center B cells, overexpressed SAG mRNA from a novel Mtv provirus and grew in a SAG-specific CD4⁺ T-cell-dependent manner (39). Thus, it is most likely that the development of a reticulum cell sarcoma tumor is dependent on SAG expression on the tumor. A similar paracrine mechanism has been implicated in the generation of human follicular B-cell lymphoma (12, 15). On the other hand, SAGs, especially bacterial SAGs, are often used as immunostimulants for infection and tumor immunity because of their strong T-cell stimulation activity (26). Thus, the direct role of SAG in tumor development remains to be addressed.

In the present study, to investigate whether viral SAG expression is linked to immunogenicity or tumorigenicity in tumor development (23, 37-39), we examined in vivo tumor growth with the BALB/c myeloma cell line FO transfected with a V6-specific SAG gene from the 3' Mtv-50 LTR ORF (31, 32, 43). Transfectants with the viral SAG gene grew more rapidly than did mock transfectants after subcutaneous inoculation in BALB/c mice but not in athymic nude mice. The implications for the role of viral SAG in tumorigenicity are discussed.

	MATERIALS AND METHODS

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Animals. Male BALB/c (H-2^d Mls-1^b) mice, 6 weeks old, were purchased from Charles River Japan Inc. (Hino, Japan). Male BALB/c nu/nu mice, 6 weeks old, were purchased from Japan SLC (Shizuoka, Japan).

Cells and cell cultures. The mouse myeloma cell line FO was obtained from the American Type Culture Collection (Manassas, Va.) and has been previously described (13). The cells were cultured in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% heat-inactivated fetal calf serum (Sigma Chemical Co., St. Louis, Mo.), 100 U of penicillin per ml, 100 µg of streptomycin per ml, and 10 mM HEPES.

Plasmids. The isolation of Mtv-50 LTR ORF cDNA was described previously (31, 32, 43). The Mtv-50 LTR ORF was cloned in the EcoRI site of vector pCR3 (Invitrogen, Carlsbad, Calif.) by PCR, resulting in the expression plasmid pCR3-Mtv-50. The structure of the constructs was confirmed by restriction enzyme mapping and DNA sequence analysis.

Establishment of stable transfectants. Twenty micrograms of expression plasmid pCR3-Mtv-50 was introduced into FO cells by electroporation with Cell-Porator (Gibco BRL, Rockville, Md.), and transfectants were isolated using the antibiotic G418 sulfate (0.4 mg/ml) (Promega, Madison, Wis.). The expression of Mtv in the isolated clones was examined by reverse transcription (RT)-PCR with common Mtv-specific sense and antisense primers. Representative clones, termed M14, M19, M110, M112, and M114, expressing Mtv-50 SAG were used for further analyses. The isolated transfectants were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and 0.4 mg of G418 per ml.

RT-PCR analysis. Total RNA was extracted from transfectants or popliteal lymph node (LN) cells from mice inoculated with Mtv-50 SAG transfectants or mock transfectants by the acid guanidinium thiocyanate-phenol-chloroform (AGPC) assay (36). First-strand cDNA synthesis and RT-PCR were done as described by Saiki et al. (35). First-strand cDNA was synthesized from 2 µg of total RNA with SuperScript II reverse transcriptase (Life Technologies, Gaithersburg, Md.) and 20 pmol of random primer (Life Technologies) in 20 µl of reaction mixture according to the manufacturer's instructions. The synthesized first-strand cDNA (2 µl) was amplified by PCR with 40 pmol of each primer and 2.5 U of recombinant Taq (Takara Shuzo, Osaka, Japan) in a total volume of 100 µl of reaction buffer consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, and specific primers, as follows: common Mtv-specific sense, 5'-AGA CAG GTG GTG GCA ACC A-3' (positions 603 to 621); common Mtv-specific antisense, 5'-AAG TCA GGA AAC CAC TTG T-3' (positions 1061 to 1080); Mtv-50-specific antisense, 5'-AAA AGG GAT CGA AGC CAA-3'; interleukin 5 (IL-5) sense, 5'-CTC TAG TAA GCC CAC TTC TA-3'; IL-5 antisense, 5'-TGA TAC CTG AAT AAC ATC CC-3'; IL-6 sense, 5'-TGG AGT CAC AGA AGG AGT GGC TAA G-3'; IL-6 antisense, 5'-TCT GAC CAC AGT GAG GAA TGT CCA C-3'; -actin sense, 5'-TGG AAT CCT GTG GCA TCC ATG AAA C-3'; and -actin antisense, 5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3'. PCR thermocycles consisted of 1 min at 94°C, 1 min at 54°C, and 30 s at 72°C. Before the first cycle, a denaturation step for 5 min at 94°C was included, and after 35 cycles, the extension was prolonged for 2 min at 72°C. The PCR product was resolved by electrophoresis on a 1.0% agarose gel (Nakalai Tesque, Kyoto, Japan), transferred to a GeneScreen plus filter (NEN Research Products, Boston, Mass.), and hybridized with a ³²P-labeled oligonucleotide probe as follows: common Mtv, 5'-AAC AGG TAC ATG ATT AT-3' (positions 824 to 840); IL-5, 5'-TCT GAT TCA TAC ATA GGA CA-3'; IL-6, 5'-TAG AAA TTC TTC AAG GAT T-3'; and -actin, 5'-TTC TGC ATC CTG TCA GCA AT-3'. After the membranes were incubated for 16 h at 60°C in 1 M NaCl-10% dextran sulfate-100 mg of heat-denatured salmon sperm DNA per ml, they were washed for 30 min in 2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-1% sodium dodecyl sulfate at 60°C and exposed to a phosphorimaging plate for visualization on a Fuji BAS-2000 phosphorimaging system (Fuji Photo Film Co., Tokyo, Japan).

T-cell hybridoma stimulation assays for activity of SAG transfectants. The myeloma cell line FO, expressing the LTR with or without the Mtv-50 SAG gene, was incubated with 2 × 10⁵ cells of either KMls-8 (V6⁺) (16) or KJ25 (V3⁺) T-cell hybridomas (33) (kindly provided by J. W. Kappler and P. C. Marrack, National Jewish Center for Immunology and Respiratory Medicine, Denver, Colo.) per well in Costar flat-bottom 96-well plates (final volume, 200 µl) for 24 h at 37°C in a 5% CO₂-in-air atmosphere. After incubation, the amount of IL-2 in the supernatants was quantitated in a bioassay with the IL-2-dependent cell line CTLL-2 (6). CTLL-2 cells (2 × 10⁵ cells/well) were cultured in 150 µl of medium containing 50 µl of supernatants for 24 h at 37°C in a 5% CO₂-in-air atmosphere and pulsed with 1 µl (0.25 µCi) of [³H]thymidine ([³H]TdR) (Amersham, Buckinghamshire, United Kingdom) per well 24 h before being harvested. After the culture period, the cells were harvested onto glass fiber filter paper, and T-cell stimulatory activity was assessed by determining [³H]TdR incorporation with a liquid scintillation counter.

Tumor growth and survival in tumor-bearing mice. The various transfectants (2 × 10⁶ cells) were suspended in 100 µl of phosphate-buffered saline and inoculated subcutaneously into the back region of normal BALB/c and BALB/c nu/nu mice, and tumor growth was monitored. The size of tumors was determined by the formula (a² × b)/2, in which a defines the horizontal diameter and b defines the vertical diameter of the tumor mass, as determined by calipers. Data were representative of two experiments with five mice per group. For survival experiments, BALB/c mice were intraperitoneally inoculated with 2 × 10⁶ cells of transfectants or mock transfectants.

Flow cytometric analysis. Single-cell suspensions were prepared from popliteal LN cells of BALB/c mice after injection of FO cells (mock transfectant cells) or Mtv-50 SAG transfectant cells (2 × 10⁶ cells) into hind footpads. The prepared lymphocytes were preincubated with a culture supernatant from 2.4G2 (rat anti-FcrRII/III-specific IgG MAb) to prevent nonspecific staining. After being washed, cells were stained with various combinations of MAb (Pharmingen). Single-cell suspensions (10⁶ cells) were stained with fluorescein isothiocyanate (FITC)-conjugated MAb to CD3 (145-2C11), V6 (RR4-7), V8.1 or V8.2 (MR5-2), or V14 (14-2) and with phycoerythrin (PE)-conjugated MAb to CD45R/B220 (RA3-6B2) or CD4 (H129.19) and then analyzed with a FACSCalibur flow cytometer (Becton Dickinson, San Jose, Calif.). For analysis of activated V6⁺ CD4⁺ T cells, FITC-conjugated MAb to V6 or V14, PE-conjugated MAb to CD44 (IM7), CD69 (H1.2F3), or CD62L (MEL-14), and Cy-Chrome-conjugated CD4 were used. The live lymphocytes were carefully gated by forward and side scattering. The data were analyzed using CELLQuest software (Becton Dickinson).

Cytokine assay. Popliteal LN cells from mice inoculated with Mtv-50 SAG transfectants or mock transfectants 10 days previously were subjected to an in vitro stimulation assay for cytokine production. Nylon wool-passed LN cells were resuspended in RPMI 1640 medium and added to 96-well plates at a concentration of 5 × 10⁵ cells/well. Cells were cultured without any stimulation or with 50 µg of anti-CD3 MAb or with anti-TCR V6 MAb (44.22.1; kindly provided by H. Hengartner, University Hospital, Zurich, Switzerland) for 48 h at 37°C. IL-5 and IL-6 in the culture supernatants were measured with enzyme-linked immunosorbent assay kits provided by Cosmo Bio (Tokyo, Japan).

Statistical analysis. The statistical significance of the data was determined by Student's t test. The statistical significance of the survival rate was determined by the generalized Wilcoxon test. A P value of less than 0.05 was considered significant.

	RESULTS

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Transfection of the Mtv-50 SAG gene in a mouse myeloma cell line. A BALB/c myeloma cell line, FO, was transfected with an expression plasmid containing the 3' LTR ORF of Mtv-50 using electroporation (Fig. 1A). After G418 selection, five clones, termed M14, M19, M110, M112, and M114, were cloned by limiting dilution. The expression of the Mtv LTR ORF in the isolated clones was examined by RT-PCR using Mtv-50 LTR ORF-specific or common Mtv LTR ORF-specific sense and antisense primers. As shown in Fig. 1B, all clones transfected with the 3' LTR ORF of Mtv-50 expressed the SAG gene. The in vitro growth properties of transfectants and mock transfectants, including cell morphology and growth rate, were similar to those of the parental FO cells and mock transfectants (data not shown).

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Transfection of the 3' LTR ORF of Mtv-50 into the FO cell line. (A) Schematic diagram of the construction of pCR3-Mtv-50. The 1.0-kb cDNA corresponding to the Mtv-50 3' LTR ORF was isolated from a low-melting-temperature agarose gel. The ORF construct was cloned in a mammalian expression vector, pCR3. (B) Detection of Mtv-50 SAG gene expression. Total RNA was prepared from Mtv-50 SAG gene transfectants and detected by RT-PCR with primers specific for the Mtv-50 LTR. The PCR products were run on a 1.0% agarose gel and analyzed by ethidium bromide staining. After hybridization, the membrane was exposed to X-ray film. Radioactivity was assessed using a Fuji BAS-2000 phosphorimaging system as described in Materials and Methods.

Mtv-50 SAG on transfectants is recognized by V6⁺ murine T-cell hybridomas. To examine if transfectants with the Mtv-50 LTR ORF gene expressed a functional SAG on their cell surfaces, IL-2 production from a V6⁺ T-cell hybridoma was measured after incubation with the transfectants. Mtv-50 ORF transfectants (10⁵ cells) were incubated with 10⁵ V6⁺ T-cell hybridomas (KMls-8) or V3⁺ T-cell hybridomas (KJ25) for 24 h, and serial dilutions of the culture supernatants were assayed for IL-2 activities by bioassays using IL-2-responsive CTLL-2 cells. A typical result, obtained with the M19 clone, is shown in Fig. 2A. M19 induced a strong IL-2 response in V6⁺ T-cell hybridomas but not in V3⁺ T-cell hybridomas. All clones produced very similar results, suggesting that all clones expressed functional Mtv-50 SAG on their surfaces (data not shown).

View larger version (48K): [in this window] [in a new window]   FIG. 2.   Transfectants stimulate V6 T-cell hybridomas in vitro. (A) IL-2 production by V6+ T-cell hybridomas (KMls-8) or V3+ T-cell hybridomas (KJ25) in response to FO cells transfected with the Mtv-50 LTR. (B) Inhibition of IL-2 production by anti-Mls-1a MAb (VS7-322-2; 50 µg/ml), anti-I-Ad and I-Ed MAb (2G9; 5 µg/ml), or control antibody (Ab) (rat IgG) (100 µg/ml). For both experiments, FO cells transfected with or without the Mtv-50 LTR (2 × 105 cells), twofold serially diluted, were incubated with the T-cell hybridoma KMls-8 or KJ25 (2 × 105 cells) in the presence or absence of MAb, and the amount of IL-2 in the supernatants was quantitated in a bioassay with 2 × 105 CTLL-2 cells. The T-cell stimulatory activity was assessed by measuring [3H]TdR incorporation. Data were obtained from at least three separate experiments and are expressed as the mean and standard deviation of triplicate cultures from a representative experiment. Statistical analysis was performed with Student's t test. *, significantly different from the value for mock transfectant (FO) cells (P < 0.05); **, significantly different from the value for Mtv-50 SAG transfectant (M19) cells treated with anti-Mls-1a or anti-I-Ad and I-Ed MAb (P < 0.01).

Tumor growth and survival in tumor-bearing mice. To analyze the tumorigenicity of FO cell lines expressing Mtv-50 SAG, BALB/c mice were injected subcutaneously with 2 × 10⁶ cells of Mtv-50 SAG transfectants, and tumor growth was compared with that obtained with mock transfectants (Fig. 3). All Mtv-50 clones grew faster than did mock transfectants, suggesting that FO myeloma cells became more tumorigenic in vivo with Mtv-50 SAG expression. To examine the possibility that the enhanced growth of the transfectants was caused by the presence of T cells reacting to SAG, we performed the same experiment using athymic nude mice. As shown in Fig. 4, the kinetics of the growth of M19 were much the same as those of the mock transfectants. These results indicate that T cells are essential for promoting tumor growth of clones expressing Mtv-50 SAG.

View larger version (23K): [in this window] [in a new window]   FIG. 3.   Accelerated tumor growth of FO cells expressing Mtv-50 SAG on BALB/c mice. BALB/c mice were injected subcutaneously (s.c.) with 2 × 106 cells of mock transfectants or Mtv-50 SAG transfectants. The size of the tumors was determined by the formula (a2 × b)/2, in which a defines the horizontal diameter and b defines the vertical diameter of the tumor mass, as determined by calipers. The average tumor for five mice per group is shown from two representative experiments.

View larger version (20K): [in this window] [in a new window]   FIG. 4.   Tumorigenicity of FO cells expressing Mtv-50 SAG on BALB/c or BALB/c nu/nu mice. Transfectants (2 × 106 cells) were inoculated subcutaneously (s.c.) into BALB/c mice or BALB/c nu/nu mice, and tumor growth was monitored. The size of the tumors was determined by the formula (a2 × b)/2. The average tumor for five mice per group is shown from two representative experiments. Statistical analysis was performed with Student's t test. *, significantly different from the value for mock transfectant cells (P < 0.01).

Kinetics of CD4⁺ T cells with V6 products after inoculation of Mtv-50 SAG transfectants. To determine whether SAG-reactive V6 T cells really respond in vivo after subcutaneous inoculation of Mtv-50 SAG transfectants, we examined the kinetics of SAG-reactive V6⁺ CD4⁺ T cells and non-SAG-reactive V14⁺ CD4⁺ T cells in the popliteal LN cell population at 0 (before), 3, 7, 10, and 14 days after injection with transfectants or mock transfectants by flow cytometry. As shown in Table 1, V6⁺ CD4⁺ T-cell populations were significantly increased on day 10 after inoculation of transfectants, whereas such increases were not evident after inoculation of mock transfectants. V14⁺ CD4⁺ T cells remained unchanged after inoculation with either transfectants or mock transfectants. In correlation with the increase in the number of V6⁺ CD4⁺ T cells, the number of IgM-positive B220⁺ B cells in the popliteal LN cell population was increased on day 10 after injection of Mtv-50 SAG transfectants (Table 1).

View this table: [in this window] [in a new window]   TABLE 1.   Kinetics of the numbers of B cells and V6+ or V14+ CD4+ T cells in the popliteal LN cell population after injection of mock transfectants or Mtv-50 SAG transfectantsa

View larger version (31K): [in this window] [in a new window]   FIG. 5.   Expression of CD44, CD69, and CD62L on V6+ CD4+ T cells from the popliteal LN cell population in BALB/c mice inoculated with mock transfectants or Mtv-50 SAG transfectants. BALB/c mice were inoculated with 2 × 106 transfectants or mock transfectants in the hind footpads, and popliteal LN cells were removed on day 10 after inoculation. Then cells were examined for the expression of V6 CD4 and CD44, CD69, or CD62L. The data shown were obtained after being gated on CD4+ T cells. The numbers in the quadrants indicate the proportions of cells falling in those quadrants. A typical result for six mice is shown.

Expression of IL-5 and IL-6 in the popliteal LN cells of mice inoculated with Mtv-50 SAG transfectants. To determine whether the T cells from mice inoculated with Mtv-50 SAG transfectants produced cytokines for tumor growth, we first examined the IL-5 and IL-6 mRNAs in the LN cells of the mice on day 10 after subcutaneous inoculation with transfectants. As shown in Fig. 6A, the popliteal LN cells from mice inoculated with Mtv-50 SAG transfectants expressed higher levels of IL-5 and IL-6 mRNAs, whereas those from mice inoculated with mock transfectants expressed only marginal levels of IL-5 and IL-6 mRNAs. We next examined IL-5 and IL-6 production by V6⁺ T cells in response to immobilized anti-TCR V6 MAb (Fig. 6B). The levels of IL-5 and IL-6 in the culture supernatants of the T cells from mice inoculated with Mtv-50 SAG transfectants 10 days previously were significantly higher than those from mice inoculated with mock transfectants.

View larger version (33K): [in this window] [in a new window]   FIG. 6.   Expression of IL-5 and IL-6 in the popliteal LN cells of mice inoculated with mock transfectants or Mtv-50 SAG transfectants. BALB/c mice were inoculated with 2 × 106 transfectants or mock transfectants in the hind footpads, and popliteal LN cells were removed on day 10 after inoculation. (A) Expression of IL-5 and IL-6 mRNAs from popliteal LN cells. Total RNA extracted from popliteal LN cells pooled from five mice in each group by the AGPC method was reverse transcribed, and the cDNA was amplified using primers specific for IL-5 and IL-6. After amplification, the PCR products were resolved by electrophoresis on 1.0% agarose gels and blotted using IL-5- and IL-6-specific internal oligonucleotide probes. After hybridization, the membrane was exposed to X-ray film. Radioactivity was assessed with a Fuji BAS-2000 system. (B) Production of IL-5 and IL-6 by popliteal LN cells in response to immobilized anti-TCR V6 MAb. The culture supernatants of popliteal LN cells were recovered 48 h after culturing on immobilized anti-TCR V6 MAb, and IL-5 and IL-6 activities were determined by an enzyme-linked immunosorbent assay. Data were obtained from at least three separate experiments and were expressed as the mean and standard deviation for five mice in each group from a representative experiment. Data representative of three separate experiments are shown. Statistical analysis was performed with Student's t test. *, significantly different from the value for mock transfectant cells (P < 0.01).

	DISCUSSION

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In the present study, we obtained evidence for a direct link between SAG expression and tumor development. The BALB/c myeloma cell line FO transfected with a viral SAG gene from the 3' Mtv-50 LTR ORF selectively stimulated SAG-specific V6⁺ CD4⁺ T cells and, in turn, grew more rapidly than did mock transfectants in BALB/c mice after subcutaneous inoculation. Such accelerated tumor growth was not evident in athymic nude mice. These results suggested that the expression of viral SAG is related to the tumorigenicity of the myeloma cell line and that assistance by SAG-reactive T cells is essential for tumorigenicity.

FO cells express MHC class II on their surface and require IL-5 and IL-6 for their growth, like B cells in the later developmental stages. These cells showed accelerated tumorigenicity after transfection with the Mtv-50 SAG gene, as assessed by in vivo tumor growth and survival rate. There was no difference in tumor growth between the transfectants and the mock transfectants in athymic nude mice, supporting the notion of involvement of the T-cell response in accelerated tumor growth. It can be speculated that the SAG-MHC class II complex on the transfectants stimulated a large number of T cells bearing relevant TCR V to release cytokines, which in turn stimulated the transfectants to proliferate. In fact, a significant number of T cells bearing V capable of recognizing Mtv-50 SAG proliferated, expressed activation markers such as CD44 and CD69, and produced large amounts of IL-5 and IL-6. The number of B cells increased in correlation with the expansion of SAG-reactive T cells, suggesting that a significant fraction of B cells might expand in a "bystander" manner in the presence of cytokines released by SAG-stimulated T cells. It is known that CD40L is also required for B-cell expansion after MMTV infection (9). However, since our data revealed that V6⁺ CD4⁺ T cells expressed no CD40L, the CD40-CD40L pathway may not be involved in the tumor growth of transfectants expressing Mtv-50 SAG.

Several lines of evidence suggest that T cells play a crucial role in MMTV infection and transport to the mammary gland. It has been reported that infection with MMTV occurs vertically by transmission from mother to offspring by milk and that the amplification of MMTV infection may be due mainly to the expansion of MMTV-infected B cells in response to the cognate interaction with SAG-reactive T cells (3, 5, 41). MMTV infection of the mammary gland in athymic nude mice occurred only after transfer of T cells from infected mice (40), suggesting that T cells play important roles not only in the spread of MMTV via expansion of infected B cells but also in mediating mammary gland infection. Although MMTV SAGs play only an indirect role in the development of mammary carcinoma, allowing stable MMTV infection, a clear example of SAG-dependent tumor development is found in SJL mice (23, 37-39). SJL mice develop follicular B-cell lymphomas spontaneously after 1 year of age. It was recently shown that these tumors arise because they overexpress endogenous MMTV SAGs and that they depend on cytokines produced by SAG-reactive T cells expressing TCR V16 or V17 (23, 37-39). The results of the present study support this finding in that newly expressed SAG in B-cell tumors may enhance tumorigenicity via activation of T cells.

Earlier studies on V-specific responses of CD4⁺ T cells to SAG in vivo showed that the majority of the responding T cells were rapidly eliminated after a strong proliferative response (4, 20, 21). Injection with exogenous MMTV leads to the proliferation of CD4⁺ T cells bearing a TCR V chain specific for SAG and the subsequent depletion of SAG-reactive T cells during the course of infection. Held et al. showed that as early as 5 days after infection, systemic injection of BALB/c mice with MMTV (SW) triggered a large increase in the amount SAG-responding V6⁺ CD4⁺ T cells from 12 to 35% and consequently led to the deletion of SAG-reactive T cells (21). Similarly, injection of cells expressing Mtv-7 SAG from BALB.D2 mice into adult BALB/c mice induced strong local immune responses, including an initially large expansion of V6⁺ CD4⁺ T cells and a subsequent deletion or anergy of reactive T cells (4, 20). In our study, the number of V6⁺ CD4⁺ T cells was increased but not eliminated completely at late stages in BALB/c mice inoculated with Mtv-50 SAG transfectants (Table 1). We found that only a portion of the V6⁺ CD4⁺ T cells expressed activation markers, including CD44^high, CD69^high, and CD62L^low, after injection of transfectant cells (Fig. 5).

The specific role of MMTV SAG in the initiation of the T-cell response is still controversial. In transgenic mice expressing the MMTV SAG gene alone or in combination with the viral envelope genes, only antigen-presenting cells from transgenic mice expressing both env and the SAG gene ORF were capable of stimulating a proliferative response of primary T cells, indicating that the MMTV envelope protein participates in the presentation of SAG to T cells (17). In contrast, in another system of transgenic mice, Mtv-6 (which lacks env) stimulated T-cell proliferation perfectly well (42). Our current data seem to support the former findings. Since we transferred the Mtv SAG gene ORF alone to the myeloma cell line, it is possible that transfectants expressing SAG alone may not have efficiently induced expansion and subsequent deletion of T cells expressing relevant V chains. Hayden et al. reported that the surviving cells either failed to make contact with SAG or were unresponsive to SAG (18).

Immobilization with anti-TCR V6 MAb significantly induced cytokine production in CD4⁺ cells from mice inoculated with the transfectants, suggesting that the V6⁺ T cells remaining after the inoculation may not be subject to clonal anergy. Since transfectants with Mtv-50 SAG grew slowly in vivo and stimulated only a minor population of V6⁺ T cells, clonal deletion or anergy of SAG-reactive T cells may not be obvious in tumor-bearing mice. We previously reported that the amount bacterial SAG-reactive T cells increased after several treatments with bacterial SAG (7, 8). Kuroda et al. reported that clonal deletion of SAG-reactive T cells after in vivo injection of SAG was not detected in mice given IL-2 continuously, and they suggested that starvation of growth factors may cause clonal deletion of SAG-reactive T cells in vivo after SAG injection (25). Therefore, it is also possible that continuous SAG stimulation from growing transfectants may stimulate SAG-reactive T cells to produce IL-2 continuously and, in turn, prevent apoptosis of SAG-activated T cells in mice inoculated with Mtv-50 SAG transfectants. However, further experiments are needed to confirm these hypotheses.

In conclusion, we have shown a direct link between SAG expression and tumorigenicity using a B-cell tumor cell line transfected with the Mtv-50 LTR ORF. Assistance by SAG-reactive T cells is essential for the accelerated tumor growth of the transfectants.