BALB/Mtv-null mice responding to strong mouse mammary tumor virus superantigens restrict mammary tumorigenesis

Abstract

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.

FIG. 1.

BALB/Mtv-null mice show T-cell proliferation specific to FM-MMTV Sag following footpad inoculation. Adult BALB/Mtv-null and BALB/c mice were inoculated in their left hind footpads with 50 μl of extracts (1:100 dilution) prepared from FM-MMTV-induced mammary tumors (kindly provided by Susan Ross). At 4 days postinfection, the draining popliteal lymph nodes were harvested from both infected and uninfected mice. Single cell suspensions were prepared from the lymph nodes and surface stained with phycoerythrin (PE)-labeled antibodies specific for mouse CD4 and fluorescein isothiocyanate (FITC)-labeled antibodies specific for mouse TCR Vβ8. The cells were analyzed on a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ) using CELLQuest software. The percentage of TCR Vβ8⁺ T cells in the gated CD4⁺ T-cell population was calculated. Three infected animals of each strain were used for the analysis. The mean percentage (± the standard deviation) of CD4⁺ TCR Vβ8⁺ T cells in each group is shown. The standard deviation is not shown on the control data because only one animal was used. However, the proportions of CD4⁺ TCR Vβ8⁺ T cells observed in the control BALB/c and Mtv-null lymph node populations (18 and 13%, respectively) are comparable to the proportions of these cells in the peripheral blood that has been analyzed from multiple animals of both strains (see Fig. 2A and B).

FIG. 2.

FM-MMTV infection induces peripheral deletion of FM Sag-reactive T cells but not detectable virus expression in spleens, salivary glands, or mammary glands. Adult BALB/c and BALB/Mtv-null mice were inoculated s.c. and i.p. with 250 μl (BALB/c) or 300 μl (Mtv-null) of extracts (1:100 dilution) prepared from FM MMTV-induced mammary tumors. Peripheral blood lymphocytes were prepared from infected and age-matched uninfected animals. Sag-specific T-cell deletion in BALB/c and Mtv-null mice was analyzed at 1.5 (A) and 6 (B) months postinfection. Three animals of each strain were used as uninfected controls, while five to six infected Mtv-null and two infected BALB/c mice were used for the analysis. The cells were dually labeled with PE-conjugated antibodies specific for mouse CD4 and FITC-conjugated antibodies specific for mouse TCR Vβ8 (FM-MMTV Sag-reactive) or TCR Vβ3 (FM-MMTV Sag nonreactive), followed by analysis on a FACSCalibur using CELLQuest software. The mean percentage (± the standard deviation) of TCR Vβ8⁺ or Vβ3⁺ T cells in the gated CD4⁺ T-cell population was calculated. The Sag-mediated deletion of the CD4⁺ TCR Vβ8⁺ T cells in the infected animals was statistically significant as determined by a two-tailed t test (P < 0.05). (C) RT-PCR analysis from tissues or tumors obtained from FM-MMTV-infected BALB/c and Mtv-null mice. Equal amounts of RNA (treated with DNase I) were used for reverse transcription, followed by 40 cycles of PCR using FM-MMTV-specific primers (upper panel). The same cDNA preparations also were used for PCR with primers specific for gapdh (lower panel). The results from multiple animals are shown. RNAs were derived from the following sources: mammary glands (MG), spleens (SP), mammary tumors (MT), or salivary glands (SG) of BALB/c (B) or BALB/Mtv-null (N) mice. Infection with FM MMTV is indicated by a plus sign. The results from RT-PCRs shown in lanes 5, 11, 17, 20, and 24 were obtained using RNA from tissues of mice injected with both FM MMTV and TBLV. Control reactions without added reverse transcriptase gave no products (data not shown). RT-PCRs were analyzed on 1.5% agarose gels and stained with ethidium bromide. The positions of molecular mass markers (M) are shown. The arrow indicates the position of the expected 402-bp product using the FM-MMTV-specific primers within the MMTV long terminal repeat.

FIG. 3.

Milk-borne transmission of FM MMTV does not occur in BALB/Mtv-null mice. Peripheral blood lymphocytes from three age-matched uninfected control mice and eight 3-month-old offspring of FM MMTV-infected Mtv-null mice were dually labeled with PE-conjugated antibodies specific for mouse CD4 and FITC-conjugated antibodies specific for mouse TCR Vβ8 (FM Sag-reactive) or TCR Vβ3 (FM Sag nonreactive), followed by analysis on a FACSCalibur using CELLQuest software. The mean percentage (± the standard deviation) of TCR Vβ8⁺ or Vβ3⁺ T cells in the gated CD4⁺ T-cell population was calculated.