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The Journal of Immunology, 1998, 161: 5357-5365.
Copyright © 1998 by The American Association of Immunologists

Induction and Inhibition of CD40-CD40 Ligand Interactions: A New Strategy Underlying Host-Virus Relationships

Madhav D. Sharma*, Maria Leite de Moraes{dagger}, Flora Zavala{ddagger}, Christiane Pontoux* and Martine Papiernik1,*

* Institut National de la Santé et de la Recherche Médicale (INSERM) U345, Institut Necker, and {dagger} Centre National de la Recherche Scientifique, Unité de Recherche Associée 1461, and {ddagger} INSERM U25, Hôpital Necker, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Interaction between CD40 and the CD40 ligand (CD40L) is required for mouse mammary tumor virus (MMTV) propagation. We found that Fas was expressed on B cells and CD40L on a small subset of viral superantigen-cognate T cells 12 h after MMTV(SW) infection. CD40L and Fas were down-regulated after 24 h. All CD4 T cells then became resistant to anti-CD3-induced CD40L induction in vitro for 2 wk. Initiation of CD40L expression and its rapid shut-off was associated with IL-12 production and was controlled by IFN-{gamma} and shedding of soluble CD40. These results suggest that a rapid, transient CD40-CD40L interaction involving a small number of cells is sufficient for MMTV propagation. Modulation of CD40L expression may be a major mechanism regulating the balance between viral propagation and host defenses, allowing mutual survival.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Infectious mouse mammary tumor viruses (MMTV)2 use complex strategies to subvert the immune system and thereby spread and escape the host response. MMTV encode viral superantigens (vSAG), which are expressed on APCs and stimulate large families of vSAG-specific T cells (1, 2, 3). As B cells are the first to be infected and to express vSAG, B cell and T cell interaction is the first host response to infection detected when monitoring the vSAG-specific T cell response (activation followed by clonal deletion) (4, 5). This T cell-B cell interaction is a prerequisite for viral spread, as MMTV infection and its transmission to the mammary gland fail to occur in both T cell- and B cell-deficient mice (6, 7). CD40-CD40L interactions involved in T cell-B cell collaboration (8, 9, 10, 11) probably play a major role in viral spread, as MMTV cannot propagate in mice with defective CD40L expression (12).

We used the strong stimulatory capacity of the vSAG encoded by MMTV(SW) (13) to study early modifications of CD40L expression during infection and the functional consequence of these modifications. MMTV(SW) is an infectious MMTV encoding a vSAG nearly identical to that encoded by the endogenous Mtv-7 responsible for the Mls-1a phenotype (4, 5). Infection of adult mice by s.c. injection of MMTV(SW) induces strong activation and local trapping of vSAG-reactive Vß6+CD4+ T cells in the draining lymph nodes, followed by clonal deletion of the cells (5, 14). Despite the elimination of vSAG-cognate T cells, no functional immune abnormalities are detected in MMTV-infected mice. Infectious viral particles disseminate rapidly to all lymphoid organs (4), tolerance to vSAG occurs rapidly and is permanent, and virus is transmitted to offspring with no detriment to immune function. The mechanisms that lead to the nonpathologic relationship between this retrovirus and the host immune system are not fully understood. However, infection with MMTV is a good model for the study of strategies leading to permanent infection and mutual host/virus survival.

We show in the present report that T cell-B cell interaction is a very early event leading to CD40L expression on cognate T cells. T cell-B cell activation generates a cytokine cascade that leads to CD40L down-regulation and transient resistance to its induction. This phenomenon, which leads rapidly to the inhibition of CD40-CD40L molecular pairing, may limit immune reactivity against infected cells, but may also limit the host’s inflammatory responses, which in many types of infection are responsible for disease progression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

BALB/c (H-2d, Mls1b) mice were purchased from Charles River (Cleon, France) and maintained in our animal facilities.

Virus preparation

MMTV(SW) was purified from mammary tumors that developed in BALB/c mice infected by their mothers’ milk as newborns. Malignant tissue was cut into pieces and homogenized in 0.01 M Tris, pH 8.0, 0.1 M NaCl, and 1 mM EDTA-containing buffer and centrifuged at 600 x g for 10 min. The supernatant was again centrifuged at 12,400 x g for 10 min. The pellet was subjected to ultracentrifugation using Beckman SW41 swinging rotors (Beckman Instruments, Fullerton, CA) for 1 h at 105,000 x g. The virus-rich pellet was resuspended in 2–2.5 ml of 17% sucrose-PBS, pH 7.4. Aliquots of 50–100 µl were stored in liquid nitrogen until use. The viral preparation was tested for infectivity by injecting it into the hind footpad of BALB/c mice. Injection of the virus batch used in all of the following experiments led to an enhanced percentage of Vß6+ T cells within the CD4 subset (32–34% in the popliteal lymph nodes 5 days after infection).

Cell surface staining and flow cytometry

Popliteal or axillary lymph node cells were prepared as single-cell suspensions by using a Potter homogenizer (poly Labo Block, Paris, France) in RPMI medium, then centrifuging at 250 x g for 5 min. The pellet was resuspended in ice-cold PBS containing 5% FCS and 0.3 M sodium azide.

The following Abs were used: anti-CD4 (clone GK 1-5) (15), anti-Vß6 (clone 44.22.1) (16), anti-Vß8.2 (clone F23.2) (17) anti-B220 (clone 6B2) (18), anti-CD69 (H1.2F3) (19), anti-CD25 (clone pc61) (20), anti-CD40 (clone HM40–3 and clone 3-23) (21), anti-CD40L, gp39 (clone MR1) (22), anti-Fas (clone Jo2) (23), and anti-CD19 (24). Abs were directly coupled to FITC or phycoerythrin or were biotinylated. Cells were first incubated with biotinylated Ab, then with phycoerythrin-labeled Ab and FITC-conjugated Ab for triple surface staining. Biotinylated Abs were revealed with streptavidin tricolor (Caltag Laboratories, San Francisco, CA). Immunolabeled cells were analyzed by flow cytometry on a FACScan cytometer with LYSIS II software (Becton Dickinson, Mountain View, CA).

Anti-CD3 stimulation in vitro

CD4+ T cells were purified from popliteal or axillary lymph nodes from 6- to 8-wk-old BALB/c mice. Briefly, CD8+ T cells were eliminated by anti-CD8 Ab (clone Ly-2) binding, followed by depletion with magnetic beads coated with sheep anti-rat Ab. B cells were eliminated after binding to sheep anti-mouse IgG (Dynal, Oslo, Norway). CD4+ T cells were usually 95–97% pure. Purified CD4+ T cells were incubated at 37°C for 7 h in round-bottom 96-well culture plates (Nunc, Rosklide, Denmark). The wells were precoated with 10 µg/ml of purified anti-CD3 Ab for 2 h at 37°C (in RPMI culture medium supplemented with 100 IU/ml of penicillin and 1% sodium pyruvate) and saturated with the same medium plus 10% FCS. Anti-CD3-activated cells and control cells were labeled with anti-CD40L, anti-CD4, and anti-Vß6 or anti-Vß8.2 Abs for triple labeling, and flow cytometry was performed as described above. In another set of experiments, purified CD4+ T cells were labeled to measure the percentage of CD25+CD40L+ cells within the Vß6 and Vß8.2 CD4 T cell subsets.

In vitro production of IFN-{gamma}, IL-10, and IL-12

To test the capacity of activated T cells to produce IL-10 and IFN-{gamma}, CD4 T cells were purified from the lymph nodes of normal mice and from popliteal lymph nodes of MMTV(SW)-infected mice 1 to 5 days after infection. Purification was performed as described above. Purified CD4 T cells were cultured (106 cells/well in 100 µl of medium) for 48 h in the presence of immobilized anti-CD3 (see above) and 50 ng/ml of PMA.

To test the capacity of B cells to produce IL-12, popliteal lymph node B cells were purified from normal and MMTV(SW)-infected mice 24 h after infection. B cells were first enriched by depleting the cell suspension from CD4 and CD8 T cells using anti-CD4, anti-CD8 Abs, and magnetic beads as described above. To further purify B cells without dendritic cells and macrophages, the cell suspension was labeled with anti-CD19 FITC-conjugated Ab, which recognizes a specific pan-B cell Ag, and CD19+ cells were sorted on a FACSVantage (Becton Dickinson). Sorted CD19+ B cells were cultured (106 cells/well) for 48 h in the presence of 10 µg/ml of LPS from Escherichia coli (Sigma, Saint Fallavier, France). All of the supernatants were aliquoted and stored at -20°C until used for cytokine assay.

In vivo anti-IFN-{gamma} treatment

BALB/c mice were injected i.p. with anti-IFN-{gamma} Ab (clone R46A2) (25) (100 µg/mouse at each injection). Rat IgG was used as a control under the same conditions. Mice were injected with anti-IFN-{gamma} alone, rat IgG alone, or MMTV(SW) alone or with MMTV(SW) plus rat IgG or anti-IFN-{gamma}. Ab or rat IgG was administered twice, 24 h and 1 h before MMTV(SW) injection; mice were killed 24 h after MMTV(SW) injection, or were given a third injection 24 h after MMTV(SW) inoculation, and killed 48 h postinfection. Purified CD4+ popliteal lymph node T cells were then incubated in anti-CD3 Ab-coated wells for 7 h at 37°C with 5% CO2 in humidified air. After culture, cells were analyzed for CD40L expression on Vß6 and Vß8.2 CD4 cells.

In vitro anti-CD40 treatment

Serum was treated with rat anti-mouse CD40 Ab to deplete it of soluble CD40 (sCD40) molecules. MMTV(SW)-infected mice were bled 24 h after infection. Serum from normal and MMTV(SW)-infected mice was incubated with 50 µg/ml of rat anti-mouse CD40 Ab (clone 3/23; PharMingen, San Diego, CA) (21), then incubated for 30 min at 4°C on a roller with magnetic beads coated with sheep anti-rat Ig. Magnetic beads were removed with a magnet. Treated and untreated serum from normal and infected mice was used in the in vitro CD40L expression assay (25% mouse serum instead of 10% FCS).

Cytokine assays

Blood was collected from control and MMTV(SW)-infected BALB/c mice. Serum from five mice in each group was pooled and stored at -20°C until use. IL-12, IFN-{gamma}, and IL-10 were tested for by using commercial ELISA kits (Genzyme Diagnostics, Cambridge, MA, for IL-12, and R&D Systems, Minneapolis, MN, for IL-10 and IFN-{gamma}). Bioactive p70 heterodimer and total IL-12 (including p70, p402 homodimer, and p40 monomer) were also assayed. Cytokines were also tested in culture supernatants using the same procedure. Values were read from a standard curve for purifed cytokine (supplied by the manufacturer). The detection limit of the assay systems was ~5 pg/ml.

Statistics

Results were expressed as mean values ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MMTV(SW) infection induces early and transient expression of CD40L on T cells specific for MMTV(SW)-encoded vSAG

CD40L expression and CD40-CD40L interaction are critical steps in MMTV propagation (12). We studied the expression of CD40L on vSAG-reactive and -nonreactive T cells early after footpad inoculation of MMTV(SW). Four to six percent of the Vß6+CD4+ T cells expressed CD40L in the draining lymph node within 24 h after infection, compared with 1–1.5% in control BALB/c mice (Fig. 1GoA). This phenomenon did not occur in vSAG-nonreactive Vß8.2+CD4+ lymph node T cells or in Vß6+CD8+/Vß8.2+CD8+ lymph node T cells (not shown); it was restricted to the draining lymph node. Expression of CD40L on vSAG-specific T cells was transient, as Vß6+CD4+ lymph node T cells had a normal CD40L level on day 2 after MMTV infection (Fig. 1GoA). We have recently described a small vSAG-specific CD4 T cell subset with constitutive CD25 expression, which produces IL-10 mRNA and is resistant to vSAG-induced clonal deletion. CD25+ cells are infected and can induce infection and subsequent clonal deletion when injected into normal hosts (26). To determine whether this CD25 subset was specifically involved in the process leading to CD40-CD40L interaction, CD40L expression was analyzed within CD25+ and CD25- vSAG-specific Vß6+CD4+ subsets in the draining lymph node. As shown in Fig. 1GoB, the percentage of CD40L was slightly and transiently enhanced in the CD25- subset 24 h after infection (3.5% of the cells in infected mice compared with 1.5% of the control cells). On the CD25+ cell subset, CD40L expression began to increase 12 h after virus injection, was maximal 24 h after infection (15–17% of the CD25+Vß6+ T cells), and returned to normal after day 2. As CD25 is not induced on CD4+ T cells by MMTV(SW) infection in vivo (14), CD40L was induced on preexisting Vß6+CD25+ T cells. Expression of CD40L was not enhanced on vSAG-nonspecific T cells expressing CD25 (Vß8.2 subset).



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  		FIGURE 1. Expression of CD40L on vSAG-specific T cells after MMTV(SW) infection. Mice were infected by injection of MMTV(SW) in the hind footpads and CD40L expression was monitored in the draining lymph nodes during the following 5 days. A, Percentage of CD40L-expressing cells in the vSAG-specific (Vß6+CD4+) and nonspecific (Vß8.2+CD4+) draining lymph node T cell pools during the first 5 days following MMTV(SW) infection. CD40L was transiently expressed on vSAG-specific T cells 24 h after infection (mean value for 10 experiments, 3 mice/experiment) B, Expression of CD40L on the CD25+ and CD25- subpopulations of vSAG-reactive (Vß6+) and vSAG nonreactive (Vß8.2) CD4 T cells. CD40L expression was analyzed in control lymph node CD4+ T cells (day 0) and in infected lymph node cells 8 and 12 h and 1, 2, 3, and 4 days after infection. CD40L was expressed mainly on the CD25+ subset of Vß6+CD4+ T cells 24 h after infection (one kinetic experiment of two performed; three mice/day/experiment).

 
We have previously shown that CD69 expression increases on draining lymph node vSAG-reactive T cells (14). The kinetics of CD69 expression was studied early after infection. As shown in Fig. 2Go, CD69 expression occurred at least 24 h after peak CD40L expression on vSAG-reactive popliteal lymph node T cells. No change in the level of CD40 expression was found on B cells (data not shown).



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  		FIGURE 2. CD69 expression on vSAG-specific T cells. The activation marker CD69 was analyzed following MMTV(SW) infection on Vß6+CD4+ vSAG-specific T cells, Vß8.2+CD4+ nonspecific cells, and B cells (B220+). Expression of CD69 on vSAG-specific T cells and B cells was secondary to CD40L expression, which was maximal in the draining lymph node 24 h after infection (see Fig. 1Go). Mean value for six mice tested per time point (three separate experiments, two mice/experiment/day).

 
The B cell-T cell interaction is regulated by both CD40-CD40L and Fas-FasL molecular pairing (27). We therefore explored the expression of Fas on B cells after local MMTV(SW) infection. A positive shift in mean fluorescence intensity of Fas expression on B cells (B220+ cells) was observed on day 1 after infection. Increased Fas expression was transient and returned to control levels on day 2 (Fig. 3Go). Despite this increase in Fas expression, the absolute number of B cells was not modified (Table IGo), suggesting that B cells were not submitted to apoptosis. The absolute number of T cells was not modified either. The increase in CD69 expression on B cells in MMTV(SW)-infected popliteal lymph nodes occurred later than Fas expression and rose from the second day onward (Fig. 2Go).



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  		FIGURE 3. Up-regulation of Fas expression on B cells. Fas expression was analyzed day 1 and day 2 (D-1, D-2) after local MMTV(SW) infection on B cells (gated on B220+ cells). Fas expression was transiently up-regulated on B cells 24 h after infection. I C, isotype control (hamster IgG). Cells were isolated from lymph nodes of individual mice. One representative mouse is shown of three examined in each group.

 

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  		Table I. Absolute number of cells in the draining lymph nodes during the 48 h following MMTV(SW) injection1

 
The first cellular modification detected after MMTV(SW) infection in vivo was thus a transient rise in CD40L expression on Vß6+CD4+ vSAG-reactive T cells, specifically on those constitutively expressing CD25. Fas was up-regulated on B cells within 24 h after infection.

Anti-CD3-induced expression of CD40L on CD4+ T cells in vitro is impaired in MMTV(SW)-infected mice

In vitro, anti-CD3 Ab stimulation leads to high-level CD40L expression on CD4+ T cells (28). The injection of bacterial superantigen SEB (staphylococcal enterotoxin B) into mice results in impaired anti-CD3-induced CD40L expression on T cells, inhibiting their B cell-stimulating activity (29). To see whether anti-CD3-induced CD40L expression was also impaired after infection by MMTV(SW), purified popliteal CD4+ lymph node T cells from normal and MMTV(SW)-infected BALB/c mice were incubated for 7 h with anti-CD3 Ab. Most (62.3 ± 5.1%) CD4+ T cells from noninfected mice expressed CD40L under these conditions; after MMTV(SW) infection, CD40L expression induced by anti-CD3 in vitro was inhibited by 50% after 24 h and by 80% after 4 days, returning to normal on day 16 (Fig. 4GoA). Surprisingly, inhibition of CD40L expression was not restricted to vSAG-reactive Vß6+CD4+ T cells but was a generalized phenomenon also involving Vß8.2+CD4+ vSAG-nonspecific T cells, as well as CD4+ T cells in the draining and nondraining lymph nodes (Fig. 4GoB). However, this inhibition was specific for CD40L expression, as CD3-induced expression of CD69 and CD25 in vitro was not inhibited (data not shown).



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  		FIGURE 4. In vitro anti-CD3-induced expression of CD40L. Draining popliteal and nondraining axillary lymph nodes were recovered from normal and MMTV(SW)-infected mice 1 to 16 days after infection. CD4 T cells were purified and incubated for 7 h in vitro with plastic-bound anti-CD3 Ab. Anti-CD3-induced expression of CD40L on CD4+ T cells was analyzed, and results were expressed as the percentage of inhibition of CD40L expression on cells of infected mice compared with cells of normal mice (62.3 ± 5.1% of normal CD4 T cells expressed CD40L in these experimental conditions). A, Inhibition of anti-CD3-induced CD40L expression on vSAG-specific Vß6+CD4+ and vSAG-nonspecific Vß8.2+ T cells in the draining lymph node. B, Inhibition of anti-CD3-induced CD40L expression on total CD4+ T cells from draining and nondraining lymph nodes. In vitro CD3-induced expression of CD40L was rapidly impaired on all CD4+ T cells from infected mice. At least three experiments were done for each time point. For each experiment, cells from two mice were pooled. Results are expressed as mean value for three experiments ± SD.

 
In summary, anti-CD3-induced CD40L expression on popliteal and axillary CD4+ lymph node T cells in vitro was considerably inhibited following MMTV(SW) infection. This inhibition of CD40L expression occurred within 24 h, was maximal 4 days after infection, and was reversible.

Serum of infected mice induces resistance to CD3-induced CD40L expression in vitro: role of sCD40

sCD40 may be involved in the observed down-regulation of CD40L expression. Indeed, sCD40 down-modulates CD40L expression and reduces CD40L mRNA levels in T cells (30, 31, 32). Normal CD4 T cells were incubated with anti-CD3 in vitro in the presence of normal or infected mouse serum preincubated or not with anti-CD40 Ab. Serum of infected mice sampled 24 h after infection (and not later) inhibited anti-CD3-induced CD40L expression on CD4+ T cells (Fig. 5A). sCD40 was specifically involved in this effect, as preincubation of infected serum with anti-CD40 Ab (followed by depletion of possible sCD40-anti-CD40 Ab complexes on anti-rat Ab-coated magnetic beads) abrogated the inhibitory effect of infected serum on anti-CD3-induced CD40L expression (Fig. 5GoB). It was important to demonstrate that sCD40 present in the serum of infected mice has a direct effect on CD40L expression and was not just masking the expression of CD40L on activated CD4 T cells. Purified CD4 T cells were cultured for 7 h with plastic-bound anti-CD3 with normal or day 1-infected mice serum. CD40L and CD40 were detected by immunofluorescence (Fig. 5Go). CD40L was up-regulated in the presence of normal serum, and CD40 could not be detected on these cells (Fig. 5GoC, top). When CD4 T cells were activated in vitro for 7 h at 37°C and then incubated with infected mice serum for 30 min at 4°C, sCD40 present in the infected serum bound to CD40L and could be detected by rat anti-CD40 Ab (clone 3–23). Under these experimental conditions (incubation at 4°C), the sCD40-CD40L complex could not be internalized and CD40L was masked by sCD40 on part of the CD40L-expressing cells (Fig. 5GoC, middle). When CD4 T cells were activated for 7 h at 37°C with infected mice serum, the percentage of CD40L-expressing cells was reduced, but sCD40 could no longer be detected on activated cells (Fig. 5GoC, bottom). These experiments demonstrate that sCD40 could bind to CD40L expressing cells and had a direct effect on CD40L expression.



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  		FIGURE 5. Role of sCD40 in the inhibition of in vitro CD3-induced CD40L expression on T cells from MMTV(SW)-infected mice. Lymph node CD4+ T cells from normal mice were purified and incubated for 7 h with plastic-bound anti-CD3. A, Incubation was done either with normal mouse serum (D-0) or with infected mouse serum recovered 24 h (D-1) or 48 h (D-2) after infection. B, Incubation was done either with normal serum (histogram A) or with infected-mouse serum recovered 24 h after infection (histogram B), showing the reduction of the percentage of CD40L-expressing cells by infected serum. Normal mouse serum (histogram C) and infected serum (histogram D) were preincubated with anti-CD40 Ab before culture. Percentage of CD40L expression on CD4+ T cells in each experimental group: A = 61.1, B = 32.6, C = 63.1, D = 56.3. Preincubation with anti-CD40 Ab overcame the inhibitory effect of infected serum (histogram D compared with B). C, Incubation of CD4 T cells with plastic bound anti-CD3 was done with normal serum 7 h at 37°C (top); with normal mouse serum for 7 h at 37°C, then with D 1-infected serum at 4°C for 30 min (middle); with D 1 infected serum at 37°C for 7 h (bottom). Cells were then washed and labeled with either rat anti-CD40L Ab or rat anti-CD40 Ab (clone 3–23). Experiments shown in this figure were done with pooled serum from six mice. One experiment of two performed under each experimental condition is shown.

 
In summary, the serum of infected mice sampled 24 h after infection contained sCD40 molecules able to block CD40L expression on anti-CD3-stimulated CD4 T cells.

Modification of CD40L expression is partly dependent on IFN-{gamma} production in vivo

IFN-{gamma} down-regulates anti-CD3-induced CD40L expression in vitro (33). IFN-{gamma} production in vivo during MMTV(SW) infection may be responsible for the secondary down-regulation of CD40L in vivo and for impaired anti-CD3-induced CD40L expression in vitro. IFN-{gamma} was not detected in the serum of either control or infected mice (data not shown), but may have been below the detection limit of the assay. To test the ability of CD4 T cells of infected mice to produce IFN-{gamma}, lymph node CD4 T cells of normal and popliteal lymph node CD4 T cells of infected mice were purified and stimulated by anti-CD3 and PMA. The level of IFN-{gamma} was tested in the culture supernatant. The level of IFN-{gamma} production increased from day 1 up to day 5 after infection (Table IIGo).


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  		Table II. In vitro production of cytokine by B cells and CD4 T cells from normal and in vivo MMTV(SW)-infected mice1

 
To further investigate the role of IFN-{gamma}, anti-IFN-{gamma} Ab was injected in mice before and after infection with MMTV(SW). Control mice were injected with rat IgG under the same conditions. The kinetics of in vivo CD40L expression on vSAG-specific T cells following MMTV(SW) infection was not modified by in vivo injection of anti-IFN-{gamma} (data not shown). However, when CD4+ T cells from anti-IFN-{gamma}-treated mice were purified and submitted to anti-CD3 cross-linking for 7 h in vitro, anti-CD3-induced expression of CD40L was partially restored (Fig. 6Go). Results were identical when mice were treated twice with Ab and tested on day 1 or treated three times and tested on day 2 after infection. This suggests that production of IFN-{gamma} in vivo is involved in resistance to CD40L induction on CD4 T cells from MMTV(SW)-infected mice in vitro.



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  		FIGURE 6. Role of IFN-{gamma} production in vivo on the inhibition of anti-CD3-induced CD40L expression in vitro. Mice were injected with rat IgG alone, anti-IFN-{gamma} alone and MMTV(SW) alone or combined with rat IgG or anti-IFN-{gamma} Ab. Popliteal lymph nodes were recovered 2 days after MMTV injection. CD4+ T cells were incubated for 7 h in anti-CD3 Ab-coated plates. Results are expressed as percentage of inhibition of CD40L expression compared with control nonmanipulated mice. Injection of anti-IFN-{gamma} partly restored anti-CD3-induced CD40L expression in MMTV(SW)-infected mice. One experiment of four separate experiments performed is shown. Each experiment was done with the pooled lymph node cells of two mice in each category.

 
In summary, anti-IFN-{gamma} Ab treatment in vivo corrected the impaired expression of CD40L induced by anti-CD3 in vitro, suggesting that in vivo IFN-{gamma} secretion is involved in this inhibition.

Infection by MMTV(SW) induces a transient rise in IL-12 production within 24 h, followed by an increased capacity of CD4 T cells to produce IL-10

As shown above, both IFN-{gamma} production and sCD40 release were involved in the resistance to CD40L expression after MMTV(SW) infection. However, other cytokines may also be involved in a regulatory cytokine cascade. IL-10 and IL-12 are candidates, as they can be produced in the course of immune responses, which require engagement of the CD40-CD40L pathway, as shown in many infectious diseases. IL-12 up-regulates the production of IFN-{gamma} (34), while IL-10 has an inhibitory effect on IFN-{gamma} production by inhibiting IL-12 production (35, 36). Total IL-12 and dimeric biologically active p70 were tested for in the serum of infected mice. There was an early increase in total IL-12 (within 6 h), followed by a second increase 18 h later. Biologically active p70 levels showed a sharp and transient increase 24 h after infection, with a second peak 3 days later (Fig. 7). As B cell-T cell interaction and CD40-CD40L molecular pairing play a major role in MMTV(SW) infection (6, 7, 12), it was of interest to know whether B cells were directly involved in IL-12 production. B cells were sorted on the basis of CD19 expression, a pan-B cell-specific Ag, and stimulated in vitro by LPS for 48 h. The biologically active form of IL-12, p70, was tested in the culture supernatant by ELISA. As shown in Table IIGo, a low level of IL-12 p70 was produced in vitro by normal, sorted B cells stimulated by LPS, and this production was enhanced when B cells were sorted from the popliteal lymph node cells of mice infected by MMTV(SW) 24 h earlier. This result showed that B cells were directly involved in the production of IL-12 p70. IL-10 was not detected in the serum during the 5 days following MMTV(SW) infection (data not shown). The level of IL-10 could be too low to be detected in the serum or rapidly used locally. To test the capacity of CD4 T cells activated by MMTV(SW) in vivo to produce IL-10, CD4 T cells were purified and stimulated by immobilized anti-CD3 and PMA for 48 h. IL-10 was tested in the supernatant of control and in vivo MMTV(SW)-activated CD4 T cells recovered 1 to 4 days after infection. As shown on Table IIGo, CD4 T cells of infected mice had an enhanced capacity to produce IL-10, and this capacity increased from day 2 to a plateau level at day 3.

In conclusion, CD40L expression on vSAG-specific Vß6CD4+ T cells was associated with a rapid and transient increase in IL-12 production, 6 to 24 h after infection, followed by an increased capacity of CD4 T cells to produce IL-10.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Infectious MMTV cohabits with its host for life, provided that mammary tumors do not develop. B cell-T cell interactions are needed for MMTV dissemination and are dependent on CD40-CD40L pairing (4, 12). In this study, we found that CD40L expression on vSAG-specific T cells was an early, transient phenomenon, followed 24 h after infection by resistance to CD40L induction on all CD4 T cells for 2 wk. This sequence of events may favor infection, limit the host’s immune responses (which involve T cell-B cell cooperation), and be part of the strategy leading to mutual host and virus survival.

Activation of T cells induced by infectious MMTV(SW) injected into the footpads of adult mice has been extensively studied. MMTV(SW) increases the number of vSAG-reactive T cells in the draining lymph node within 3–5 days (4, 5). This cell accumulation is linked mainly to the trapping of vSAG-cognate T cells (14). A high percentage of these cells expressed CD69 activation molecules, down-regulated CD62L, and gradually disappeared from all lymphoid areas. However, CD40L expression on vSAG-specific T cells preceded the above modifications by at least 24 h. Indeed, CD40L was expressed within 24 h after infection, suggesting that infected cells very rapidly expressed the vSAG, which can be recognized by a small number of specific T cells. CD40-CD40L pairing, which is a highly potent signaling system, was functional a few hours after infection. CD40 expression on the surface of B cells was not modified. However, Fas (another molecule belonging to the TNF receptor family (37)), which is induced by CD40-mediated signaling (38, 39), was up-regulated on B cells within 24 h, showing that B cells were also activated very early. Both CD40-CD40L and Fas-FasL pairing are involved in B and T cell responses to Ag (27). However, engagement of the Fas-FasL system is not a prerequisite for virus propagation (40), contrary to CD40-CD40L molecular pairing (12). Interestingly, although Fas was induced early on B cells, there was no modification in B cell (or T cell) number, suggesting that, indeed, apoptosis was not induced early after infection by MMTV(SW). An explanation for this result may be the control of apoptosis by the expression of antiapoptotic molecules: indeed, Yang et al. (41) have shown that T cells activated by SEB in vivo are resistant to early Fas-mediated apoptosis by the expression of apoptosis-preventing genes at the same time as apoptosis-inducing genes. However, we have to consider that a small proportion of CD4+Vß6+CD40L+-activated T cells might have disappeared from the lymph node either by migrating away or by dying locally, even if the absolute number of CD4 T cells was not modified 24 and 48 h after infection. The percentage of activated vSAG-reactive T cells expressing CD69 continued to increase significantly from day 2 onward (Ref. 14 and Fig. 2Go), whereas CD40L expression did not increase. This suggests that even if some activated T cells may disappear early from the lymph node, most of those that accumulate in the lymph node after day 2 are unable to express CD40L.

Expression of CD40L on vSAG-specific T cells and Fas expression on B cells was an early, transient phenomenon. Both parameters returned to baseline after 24 h. This was followed by long-lasting resistance of T cells to CD40L expression after anti-CD3 stimulation in vitro. Surprisingly, this impaired response involved not only vSAG-reactive T cells in the draining lymph node but all CD4+ T cells, including those in nondraining areas. Anti-CD3-induced expression of CD40L in vitro was inhibited by 80% 4 days after infection and returned to normal within 2 wk. Interestingly, this occurrence was specific for CD40L expression, as anti-CD3-induced expression of CD69 and CD25 in vitro was not impaired. These results show that the cells were still susceptible to activation, despite their inability to use one of the major signaling pathways for T cell-B cell interactions. Such inhibition of CD3-induced CD40L expression in vitro has already been described in anergic CD4+ T cells, which are induced in vivo by bacterial SAG and lose their B cell stimulatory capacity (29, 42). However, we found in this study that unresponsiveness involved all CD4 T cells, irrespective of their vSAG specificity, including T cells in nondraining areas.

The mechanisms leading to this marked and rapid inhibition of the ability to express CD40L may be under the control of cytokines, especially IFN-{gamma} and sCD40.

IFN-{gamma} down-regulates CD3-induced CD40L expression by T cells in vitro (33) and could also be an in vivo mechanism involved in the acquired resistance to CD40L expression after MMTV(SW) infection. IFN-{gamma} was not detected in the serum of infected mice. However, purified popliteal lymph node CD4 T cells from these mice have an increased capacity to produce IFN-{gamma} in vitro, starting from day 1 after infection. IFN-{gamma} production was indeed involved in the resistance to anti-CD3-induced expression of CD40L, as in vivo treatment of infected mice by anti-IFN-{gamma} partly overcame this resistance. IL-12 is a potent inducer of IFN-{gamma} production (34), and production of IL-12 during MMTV(SW) infection may be the first induction signal for IFN-{gamma} production. Indeed, induction of resistance to CD40L expression was preceded by a peak of IL-12 in the serum of infected mice. The role of the CD40-CD40L interaction in the production of IL-12 and IFN-{gamma} is clear in CD40-/- and CD40L-/- mice, which both produce much lower levels of IL-12 and IFN-{gamma} upon infection than their wild-type controls (reviewed in 11 . One may wonder whether B cells were directly responsible for IL-12 production. We showed that, indeed, B cells sorted from popliteal lymph node cells 24 h after infection have an enhanced capacity to produce the biologically active form of IL-12. Production of IL-12 by infected B cells has been demonstrated, whereas IL-12 production by normal B cells is controversial. Maruo et al. (43) could not detect production of IL-12 by stimulated B cells, but our own results and those of Fujimoto et al. (44) show low levels of IL-12 production by in vitro-activated B cells. Experimental conditions may explain this discrepancy. Interestingly, Fujimoto et al. (44) show that macrophages produce much higher levels of IL-12 than B cells. However, although B cells of MMTV(SW)-infected mice produced enhanced level of IL-12, it does not rule out the possibility that dendritic cells and/or macrophages were also involved. IL-12 may be involved not only in the induction of IFN-{gamma} production, but may also be directly involved in the up-regulation of CD40L expression on vSAG-reactive T cells, as it was recently shown that IL-12 is able to up-regulate CD40L expression on human T cells (45). The peak of IL-12 p70 in the serum also correlates with the peak of CD40L expression 24 h after infection. Production of IL-12 is sharply down-regulated after 24 h, which may induce down-regulation of IFN-{gamma} production and prepare recovery from resistance to CD40L induction 2 wk later. Down-regulation of IL-12 production may be linked to IL-10 production, which is known to suppress IL-12 production and thereby inhibit IFN-{gamma} production. IL-10 was not detected in the serum of infected mice. However, CD4 T cells recovered from MMTV(SW)-infected mice produced an increased level of IL-10 upon stimulation in vitro, starting from day 2 after infection, following the peak of IL-12 production in the serum. Interestingly, vSAG-reactive T cells, which constitutively express CD25 and produce IL-10 (26), were the first ones to express CD40L, starting 12 h after infection. Additionally, five times more CD25+ cells than CD25- cells were induced to express CD40L. This small CD4 CD25+ cell population may thus be a major regulatory T cell subset in MMTV(SW) infection, by interacting with vSAG-presenting cells during the initial phase of infection; it may be responsible for IL-12 down-regulation through its ability to produce IL-10 (26).

The second possible mechanism of in vivo down-regulation of CD40L and resistance to CD40L induction in vitro was the shedding of sCD40 after B cell activation by CD40-CD40L interaction. This seems to be an important mechanism of regulation for T helper cell function by B cells, leading to down-regulation of CD40L mRNA production in T cells (30, 31, 32). This hypothesis was also confirmed, as serum recovered from infected mice 24 h after infection inhibited anti-CD3-induced expression of CD40L on CD4 T cells, and treatment of the serum by anti-CD40 Ab nearly abrogated this effect. The mechanisms leading to the release of CD40 in the blood of infected mice are unknown. Correlations should be sought between sCD40 release and levels of other cytokines produced during infection. sCD40 and IFN-{gamma} may act sequentially in this process. sCD40 was rapidly and transiently produced, being detected in the serum during the first 24 h after infection only. sCD40 may be responsible for the early down-modulation of CD40L on vSAG-specific T cells in vivo. IFN-{gamma}, which was produced for a longer period of time by MMTV(SW)-infected mice CD4 T cells, may have a persistent, long-lasting effect. Indeed, anti-IFN-{gamma} Ab treatment in vivo did not correct the rapid down-regulation of CD40L on vSAG-responsive T cells, but corrected the long-lasting acquired resistance of CD4 T cells to anti-CD3-induced expression of CD40L. This finding is consistent with the immunosuppressive effects of IFN-{gamma} in vivo, which has been demonstrated in other experimental systems including those using IFN-{gamma}-/- mice (46).

On the basis of these results, the following sequence of events can be postulated (Fig. 8). 1) Infection of adult mice by MMTV(SW) leads to rapid infection of B cells and expression of vSAG. Recognition of vSAG by specific CD4+ T cells induces B cell-T cell interaction, activation, and expression of CD40L on T cells, as well as activation and expression of Fas on B cells. vSAG expression, B cell-T cell interaction and thus CD40L expression occurred 12 h after infection. 2) CD40-CD40L interaction is switched off after 24 h by down-regulation of CD40L on vSAG-reactive T cells. 3) At the same time, CD4 T cells become resistant to further CD40L induction in a non-TCR-restricted way, an effect that is reversible within 2 wk.

The mechanisms of these regulatory phenomena may be: 1) production of sCD40, which is known to bind to CD40L and down-modulate its expression; and 2) production of IL-12, within 24 h, which is responsible for production of IFN-{gamma} which, in turn, is known to induce CD40L down-regulation in vitro.

MMTV infection is one of the best examples in which the virus and host immune system reach a steady state leading to mutual survival. The rapid switching on and off of CD40L expression and inhibition of one of the main T-B signaling pathways may be part of the strategy used. T-B interaction through CD40-CD40L signaling is known to be required for MMTV propagation (4, 12), but our results showed that a rapid, transient interaction involving a small number of cells was sufficient for MMTV(SW) dissemination in adults. Indeed, a small number of specific T cells were initially induced to express CD40L by interaction with B cells. Obviously, there is no need for an extensive activation process for viral amplification: MMTV (C3H), known to give very poor activation of vSAG-reactive T cells in vivo (13), leads to massive infection of the mammary gland and viral transmission to offspring. While infection is set up, down-regulation of T-B interaction by inhibition of CD40-CD40L interaction may limit host inflammatory responses detrimental to both host and virus. Indeed, long-lasting and extensive T-B interaction may have a detrimental effect on the host, as overactivation of the immune system and overproduction of inflammatory cytokines often facilitate disease progression (47, 48). Down-regulation of CD40L expression on vSAG-specific T cells and inhibition of CD40L induction of nonspecific T cells may limit both specific and nonspecific inflammatory responses. By the time CD40L inducibility is back to normal (within 2 wk), most vSAG-reactive clones have been deleted, and specific T cell-B cell interactions are limited. Interestingly, CD40 cross-linking by T cells expressing CD40L also induces Fas on B cells. During CD40 ligation, a balance is established between rescue and induction of B cell apoptosis (39, 49). Early switching off of Fas expression may limit apoptosis of infected vSAG-expressing B cells and protect the initial reservoir of virus. However, one may wonder whether the high initial response to the infection through SAG recognition may also limit virus spreading. Indeed, while neonatal infection by MMTV(SW) leads to low or undetectable activation, progressive clonal deletion, massive and rapid mammary gland infection, and susceptibility to mammary tumor development, infection in adult mice induces rapid local activation, rapid clonal deletion, progressive mammary gland infection, and no mammary tumor development (50, 51). It is likely, therefore, that the rapid induction and control of inflammatory responses following primary infection in adult mice first favor viral infection and then limit viral spread.

It remains to be established whether inhibition of the CD40-CD40L interaction described here, following activation by vSAG, is a general mechanism of immune regulation or perhaps even the basic mechanism involved in antigenic competition.



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  		FIGURE 7. Detection of IL-12 in the serum of MMTV(SW)-infected mice. Serum of normal mice (day 0) and of mice infected by MMTV(SW) and collected 24, 48, 72, and 96 h after infection were tested in an ELISA for the presence of IL-12. Both total IL-12 and biologically active IL-12 p70 were tested for. An early transient peak of biologically active IL-12 p70 was found in the serum of infected mice. Pooled serum from at least five mice for each time point was used in this experiment.

 


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  		FIGURE 8. Postulated model for the MMTV(SW)-host relationship. Induction and inhibition of the CD40-CD40L interaction. A, Infection and CD40L modulation. Step 1, Infection leads to vSAG presentation and recognition by vSAG-specific T cells. CD40L on T cells and Fas on B cells are up-regulated 24 h after infection. Step 2, Expression of CD40L and Fas is transient and down-regulated after 24 h. Step 3, CD4+ T cells are resistant to anti-CD3-induced expression of CD40L in a non-TCR-restricted way. B, Putative mechanism underlying down-regulation and resistance to induction of CD40L expression. CD40-CD40L interaction led to the production of IL-12 (step 1), which is the most potent inducer of IFN-{gamma} production; step 2, CD40L down-regulation and resistance to CD40L induction are under the control of both sCD40 and IFN-{gamma}; step 3, reduction in IL-12 production may be under the control of IL-10 (step 4), which may down-regulate IFN-{gamma} production, leading to CD40L induction recovery. Early and transient vSAG-specific T-B interaction through CD40-CD40L pairing permits initial viral infection. Down-regulation of CD40L expression may limit T-B interaction and signaling and the inflammatory response, leading to a balance between the virus and the immune system, permitting mutual survival.

 

   Acknowledgments
 
We thank Drs. Benedita Rocha, Claude Penit, and Michel Dy for discussions and for critically reviewing the manuscript, and Sonia Hamon for preparing the manuscript. We are indebted to Corinne Garcia, who performed the B cell sorting.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Martine Papiernik, INSERM U345, Institut Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15. Back

2 Abbreviations used in this paper: MMTV, mouse mammary tumor virus; CD40L, CD40 ligand; FasL, Fas ligand; vSAG, viral superantigen; sCD40, soluble CD40; SEB, staphylococcal enterotoxin B. Back

Received for publication December 5, 1997. Accepted for publication July 15, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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