The role of neutralizing antibodies for mouse mammary tumor virus transmission and mammary cancer development

Abstract

Mouse mammary tumor virus (MMTV) infection establishes chronic germinal centers and a lifelong neutralizing Ab response. We show that removal of the draining lymph node after establishment of the germinal center reaction led to complete loss of neutralizing Abs despite comparable infection levels in peripheral lymphocytes. Importantly, in the absence of neutralization, only the exocrine organs mammary gland, salivary gland, pancreas, and skin showed strikingly increased infection, resulting in accelerated mammary tumor development. Induction of stronger neutralization did not influence chronic infection levels of peripheral lymphoid organs but strongly inhibited mammary gland infection and virus transmission to the next generation. Taken together, we provide evidence that a tight equilibrium in virus neutralization allows limited infection of exocrine organs and controls cancer development in susceptible mouse strains. These experiments show that a strong neutralizing Ab response induced after infection is not able to control lymphoid MMTV infection. Strong neutralization, however, is capable of blocking amplification of mammary gland infection, tumor development, and virus transmission to the next generation. The results also indicate a role of neutralization in natural resistance to MMTV infection.

Figure 1

Loss of neutralization after LN removal. (A) Titration of a serum of MMTV(SW)-infected BALB/c mice obtained 3 months after MMTV injection. Conc., concentrated milk. (B) Titration of MMTV. Different doses of MMTV were injected into the footpad of BALB/c mice, and the SAg response was determined 4.5 days later in the draining popliteal LN. (C) Mice were infected s.c. with MMTV, and 4, 9, or 23 days later the draining popliteal LN was either left in place or removed. Sera were taken 90 days after operation. The amount of neutralizing Abs in the sera was tested by neutralization assay. Two to three individual mice were used for the experiments and the mean (A and B) and individual values (C) are shown.

Figure 2

Infection levels of peripheral organs after LN removal. After determination of the linear range of the PCR, PCR and liquid hybridization of 500 ng of DNA were repeated three times with DNA from a total of six mice with similar results. The results shown are from operated mice (dr LN-removed) and mock-operated control mice (no) 6 months after operation. (A) Peripheral lymphoid organs of day 23-operated mice. (B) Peripheral nonlymphoid organs of day 23-operated mice. (C) Mammary gland from day 4-, 9-, or 23-operated or mock-operated mice. (D) Exocrine epithelial tissues from day 23-operated and mock-operated mice. (E) Dilution of 50, 5, 0.5, 0.05, and 0.005 ng of DNA isolated from BALB.D2 (Mtv-7⁺) diluted in a total amount of 500 ng of BALB/c DNA.

Figure 3

Inverse correlation among virus dose, mammary gland infection, and virus transmission. (A) Mice were injected with serial dilutions of MMTV. Sera of these mice were tested in a neutralization assay 20 days later. Individual mice and mean values (curve) are shown. The experiment was repeated twice. (B) Serial dilutions of MMTV(SW) were injected into adult mice, and the amount of reverse-transcribed retroviral DNA isolated from mammary gland was tested 5 weeks later from 500 ng. As control 50, 5, 0.5, 0.05, and 0.005 ng of DNA from BALB/D2 mice mixed with 500 ng of BALB/c DNA was used. (C) Different virus doses were injected into mice, and 3 months later infection in mothers and their first litters was monitored by deletion of SAg-reactive T cells.

Figure 4

Reduced neutralization results in accelerated cancer development. (A) The number of tumor-free mice after s.c. infection of either control or LN-operated mice (day 18 after virus injection). The highly tumorigenic MMTV(C3H) was used. (B) The number of tumor-free mice after s.c. infection of either control or LN-operated mice (day 18 after virus injection). The low tumorigenic MMTV(SW) was used.

Figure 5

Mice lacking endogenous Env copies induce stronger neutralization titers after MMTV infection. (A) Thirteen Mtv-free or Mtv-expressing littermates were injected with MMTV(SW), and the neutralizing Abs were measured in a neutralization assay 4 weeks later. Immune and preimmune sera were used as controls from both mouse strains. A Student's t test showed P < 0.01 for Mtv-free compared with Mtv-expressing littermates. (B) Mtv-free and littermate (LM) mice were injected with MMTV and the anti-Env IgG Abs were measured 3 months later by ELISA. Dilutions of sera from three animals are shown. Experiments were repeated three times with similar results. Background values of 0.06–0.08 OD were subtracted.