Preactivation of B Lymphocytes Does Not Enhance Mouse Mammary Tumor Virus Infection

Abstract

We investigated whether mouse mammary tumor virus (MMTV) favors preactivated or naive B cells as targets for efficient infection. We have demonstrated previously that MMTV activates B cells upon infection. Here, we show that polyclonal activation of B cells leads instead to lower infection levels and attenuated superantigen-specific T-cell responses in vivo. This indicates that naive small resting B cells are the major targets of MMTV infection and that the activation induced by MMTV is sufficient to allow efficient infection.

Mouse mammary tumor virus (MMTV) is an oncogenic type B retrovirus which infects mainly B lymphocytes (4, 21). In the first hours after encounter with MMTV, a polyclonal T-cell-independent B-cell activation is observed (2). As much as 80% of B cells are activated by MMTV, but only a few of them become infected. The few infected B lymphocytes present a viral superantigen (Sag) bound to major histocompatibility complex (MHC) class II molecules to T cells expressing a specific T-cell receptor Vβ element. Subsequent Sag-specific T-helper-cell responses result in a strong preferential amplification and differentiation of infected B cells (for a review, see reference 21).

In all known retroviral infections, activated or cycling lymphocytes are required for infection to occur. For murine leukemia virus, Rous sarcoma virus, and spleen necrosis virus, the cell cycle of the target cells has been found to be necessary for viral integration and productive infection (3, 12, 14, 26). In addition, retroviral transcription requires an integrated proviral template (8). While nuclear breakdown during mitosis was shown to contribute to the integration of retroviral DNA into the nucleus, human immunodeficiency virus (HIV) has been shown to be independent of mitosis due to its karyophilic core protein (7). However, HIV requires T-lymphocyte activation for infection. In HIV infection, the inefficient reverse transcription in quiescent peripheral blood lymphocytes is caused by low levels of deoxynucleotides and contributes to a cytoplasmatic pool of mostly incomplete viral DNA which can be rescued after mitogenic stimulation (13).

For MMTV infection, there are two main interpretations for the early infection events. Either MMTV-induced activation facilitates infection of naive small resting B cells, or, alternatively, MMTV preferentially infects preactivated B cells. To address this question, we analyzed the effect of polyclonal B-cell stimulation on MMTV infection in mice.

To activate B lymphocytes, 7- to 8-week-old BALB/c mice were injected subcutaneously (s.c.) into the hind footpad with a single dose of either lipopolysaccharide (LPS) from Escherichia coli (Sigma, San Diego, Calif.) or monoclonal antibodies (MAbs) that induce a polyclonal B-cell stimulation via cross-linking of surface immunoglobulin (sIg) or CD40.

The following mitogenic MAbs were used in this study: b-7-6 (rat anti-mouse μ IgG1) (18), 1.19 (rat anti-mouse δ IgG2a) (5), or FGK 45 (anti-mouse CD40) (27). 2.4G2, a rat IgG2b that binds to mouse FcγRII, was injected s.c. prior to injection of the other MAbs in order to avoid binding of IgG to FcγRII (29). Blocking of FcγRII binding prevents induction of B-cell unresponsiveness (25) and by itself has no influence on MMTV infection and B-cell activation (Fig. 1 and data not shown).

FIG. 1.

Mitogen-induced B-cell activation and proliferation. A single dose of 2.4G2 (28 μg) or LPS (10 μg) was injected s.c. into the hind footpads of BALB/c mice, or mice were left untreated (c′). Anti-IgD MAb (50 μg), anti-CD40 (50 μg), anti-IgD plus anti-CD40 (50 μg each), or anti-IgM (10 μg) was injected s.c. into the hind footpad 30 min after injection of 2.4G2 (28 μg). The percentages of CD69⁺ B cells (A) and BrdU⁺ B cells (B) among B220⁺ B cells in the draining PO-LN were analyzed 24 (▧) and 48 ( ) h later. Each column represents the mean percentage ± standard deviation of B cells from four mice.

Induction of B-cell proliferation and differentiation by either LPS or cross-linking of sIg have been described previously (1, 6, 11, 24, 28). While the earliest biochemical events that parallel B-cell activation occur within seconds, events that reflect entry into the G₁ phase of the cell cycle occur within a few hours (9). Accordingly, we analyzed the percentage of B cells expressing the CD69 molecule, which is known as an early activation marker (Fig. 1A) and determined the percentage of cells having divided within 24 h or between 24 and 48 h after mitogen or antibody injection (Fig. 1B). Draining popliteal lymph node (PO-LN) cells (10⁶) were double stained with RA3-3A1 (fluorescein isothiocyanate [FITC]-conjugated anti-murine B220; Caltag) and H1-2F3 (biotin-conjugated anti-murine CD69 [31]). Flow cytometry was performed on a FACScan (Becton Dickinson & Co., Mountain View, Calif.) cell analyzer.

The number of CD69⁺ B220⁺ B cells increased from 5% in untreated control mice to as much as 95% in mitogen-treated BALB/c mice 24 or 48 h after antibody or mitogen injection (Fig. 1A). Due to the high amount of endogenous IgM in the sera of normal mice, the level of activation of B cells following anti-IgM MAb treatment was relatively low. We confirmed the effect of each antibody treatment by surface staining of several lymphocyte activation markers. In Fig. 1B, we show the percentage of B cells having incorporated 5′-bromo-2′-deoxyuridine (BrdU; Sigma) incorporation. After staining with RA3-3A1 (phycoerythrin-conjugated anti-murine B220; Caltag), fixation in formaldehyde, and DNase treatment, flow cytometry was performed with anti-BrdU–FITC (Becton Dickinson) (for the method used, see reference 28). One group of mice received 3 mg of BrdU intraperitoneally and thereafter 1 mg/ml continuously in the drinking water at the same time as mitogen or antibody, and the mice were analyzed after 24 h. The other group of mice received BrdU 24 h after mitogen or antibody injection during a 24-h pulse. Among the different treatment protocols, anti-CD40 induced the lowest level of cell division, followed by LPS, and the strongest induction of the cell cycle was found with anti-IgD or anti-IgD plus anti-CD40. Anti-FcγRII treatment did not lead to significant activation and proliferation.

BALB/c mice received a single dose of mitogen s.c. (10 to 50 μg) either before, concomitantly with, or after s.c. injection of MMTV(SW), a retrovirus which expresses a Vβ6-specific Sag (10⁸ virus particles [16]). We analyzed the percentage of Sag-reactive Vβ6⁺ T cells in the CD4⁺ T-cell population and the amount of viral DNA in the draining PO-LN at days 3.5 to 4 after infection. This time point allows measuring both reduced or enhanced responses, since maximal stimulation was observed on days 5 to 6. In order to quantify infection levels, 500 ng of DNA of cells extracted from the draining PO-LN was analyzed by PCR. Trace amounts of [α-³²P]dATP were added, and 30 cycles (1 cycle consisting of 5 min at 95°C; 30 cycles with 1 cycle consisting of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C; and, finally, an extension step for 10 min at 72°C) were used. The Sag sequences of the endogenous proviruses Mtv-6, Mtv-8, and Mtv-9 were amplified as internal standards relative to MMTV(SW) in the same tube with previously described primers (17). This PCR method is linear for MMTV(SW) in the range of 3 to 95% of Mtv-6 signals. For weaker signals, the PCR slightly overestimates the signals. DNA extracted from lymphocytes derived from uninfected BALB/c mice or from PO-LN of mice 3.5 days after injection of MMTV (3 × 10⁶ to 3 × 10⁸ viral particles) was used as the control. PCR products were separated on a 6% denaturing polyacrylamide gel, which was dried and then exposed to Kodak X-Omat film (Eastman Kodak Company, Rochester, N.Y.). The linear range of the PCR was determined by PhosphorImager analysis. In Fig. 2, the PCR was linear after injection of more than 10⁶ viral particles.

FIG. 2.

(A) Impaired MMTV infection of mice treated with LPS. Viral DNA was amplified from 100,000 cells isolated from the draining PO-LN of mice 3.5 days after s.c. footpad injection of MMTV-infected milk. LPS injection was performed at days −2, −1, 0, or +1 relative to hind footpad injection with MMTV. LN cells from uninfected BALB/c mice and LN cells from mice injected with virus doses ranging from 3 × 10⁵ to 3 × 10⁷ viral particles were used as controls. Each lane represents the result obtained from one mouse. The experiment was repeated three times with similar results. (B) Impaired Sag stimulation in LPS-treated mice. In parallel to the PCR analysis of PO-LN, LN cells were analyzed by flow cytometry 3.5 days after MMTV infection. Vβ6⁺ cells among CD4⁺ T cells were analyzed in LPS-treated mice (•), in MMTV-infected mice (upper hatched horizontal bar), and in uninfected BALB/c mice (lower hatched horizontal bar). Results were obtained with four mice.

As shown in Fig. 2A, treatment of mice with LPS before or during MMTV challenge strongly reduced the amount of detectable viral DNA in the draining PO-LN cells. In agreement with this, the expansion of MMTV(SW) Sag-specific Vβ6⁺ CD4⁺ T cells was impaired in LPS-treated mice (Fig. 2B). The T-cell stimulation by MMTV was intermediate when LPS was injected 48 h before infection, indicating that the effect of LPS was transient and partially reversible after 48 h. One day after MMTV infection, LPS had only a marginal inhibitory effect on the stimulation of Vβ6⁺ CD4⁺ T cells, as well as on infection levels. It is likely that the events influencing the efficiency of MMTV infection were completed within the first 24 h, before the mitogen had been given (2).

In order to compare the effects of LPS- and other mitogen-induced B-cell activations, we treated BALB/c mice with a single dose of either anti-μ MAb, anti-δ MAb, anti-CD40 MAb plus anti-δ MAb, or anti-CD40 MAb alone. The cross-linking of CD40 molecules on B cells has been shown to mimic a T-helper-cell response that activates resting B lymphocytes in vivo and to enhance the survival of B cells after cross-linking of membrane IgD (10). Treatment of mice with a single dose of either anti-μ MAb (Fig. 3A) or anti-δ MAb (Fig. 3B) before, during, or after MMTV challenge reduced the amount of viral DNA recovered from the draining PO-LN in all but two samples, in which anti-μ MAb-treated mice showed infection levels comparable to those for controls (shown in Fig. 2A). Simultaneous injection of anti-CD40 MAb and anti-δ MAb similarly reduced the PCR signals (Fig. 3C). The effect of CD40 cross-linking on resting B cells subsequent to MMTV infection is represented in Fig. 3D. The ratio of PCR amplification products of MMTV(SW) versus Mtv-6 was quantitated by gel electrophoresis and subsequent PhosphorImager analysis. Calculations of the ratios between the MMTV(SW) and Mtv-6 PCR bands showed that in none of the samples obtained from antibody- or LPS-treated mice were the MMTV(SW) PCR products stronger than in mice injected with 10⁷ viral particles. The different treatments did not influence the absolute numbers of B cells (data not shown). Taken together, the in vivo activation of B cells by LPS treatment or by cross-linking of surface Ig or CD40 predominantly reduced the efficiency of MMTV infection. There was an inverse correlation between B-cell proliferation by mitogens on the one hand and levels of infection and Sag response on the other hand. Therefore, we conclude that MMTV preferentially infects naive B cells which are activated upon infection. One of the explanations for the reduced infection levels after preactivation might be the reduction of accessible surface receptor structures.

FIG. 3.

Impaired MMTV infection of Ig- or anti-CD40-treated mice. Anti-IgM (A), anti-IgD (B), anti-IgD plus anti-CD40 (C), or anti-CD40 alone (D) was injected s.c. into the hind footpad, and Mtv-6, Mtv-8, Mtv-9 and MMTV(SW) was amplified by PCR from 500 ng (10⁵ cells) of DNA of the draining PO-LN 3.5 days after MMTV(SW) injection. The virus titration is shown in Fig. 2A. Each lane represents the result obtained from one mouse. The experiment was repeated three times with similar results.

In vitro studies clearly demonstrated that the B-cell activators used in this study (LPS, anti-CD40, or anti-Ig) upregulated B7-2 and MHC class II expression on B cells (15, 22, 23). MHC class II expression is a prerequisite for Sag presentation of B cells, Sag-derived T-cell–B-cell interaction, and subsequent amplification of MMTV-infected B cells (20). Therefore, even smaller numbers of infected cells should result in sufficient T-cell stimulation. Analysis of the T-cell Sag response of Vβ6⁺ CD4⁺ T cells in LPS-, anti-IgD-, and anti-IgD-plus-anti-CD40-treated mice, however, resulted in a strong reduction of the Sag response (Table 1). For anti-IgM-treated mice, only a weak inhibition was observed, most likely due to high levels of IgM in serum. In vivo cross-linking of CD40 did not change the percentages of Vβ6⁺ CD4⁺ T cells compared with infected control mice.

TABLE 1.

Kinetics of Vβ6⁺ CD4⁺ T-cell increase in mitogenor antibody-treated, MMTV-infected mice

Day of MAb treatmentb	% (± SD) of Vβ6+ among CD4+ T cells treated with the following MAbsa:
	LPS	Anti-IgM	Anti-IgD	Anti-IgD + anti-CD40	Anti-CD40
−2	23.3 (2.9)	32.5 (0.3)	20.4 (1)	29.4 (1.5)	32.2 (0.75)
−1	20.3 (4.2)	32.7 (0.7)	25.4 (1)	28.7 (3.5)	32.4 (0.3)
0	18.5 (2.3)	24.8 (1.9)	24.8 (0.1)	26.5 (0.2)	32.5 (0.9)
1	27.7 (1.8)	32.5 (0.1)	27.9 (1.7)	33.2 (1.6)	32.9 (0.6)

^aPercentages of Vβ6⁺ T cells among CD4⁺ T cells in the draining PO-LN were determined by flow cytometry 3.5 days after MMTV infection. Values are means ± standard deviations for three to six lymph nodes. Uninfected mice had 11.0 ± 0.2% and infected mice had 33.2 ± 0.9% Vβ6⁺ T cells among CD4⁺ T cells. 

^bDay of MAb treatment relative to MMTV infection. 

Our studies clearly show that MMTV infection of B cells occurs efficiently without preactivation of the target cells, as has been shown for HIV infection of nonproliferating monocytes and HeLa cells (19, 30). The capability of MMTV to activate its target cell in the early phase of infection might be an efficient strategy of retroviruses to facilitate infection of naive target cells.