CD4+ T cell-mediated protection against a lethal outcome of systemic infection with vesicular stomatitis virus requires CD40 ligand expression, but not IFN-
or IL-4
Camilla Andersen,
Teis Jensen,
Anneline Nansen,
Ole Marker and
Allan Randrup Thomsen
Institute of Medical Microbiology and Immunology, Panum Institute, University of Copenhagen, 3C Blegdamsvej, 2200 Copenhagen, Denmark
Correspondence to:
A. Randrup Thomsen
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Abstract
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To investigate the mechanism(s) whereby T cells protect against a lethal outcome of systemic infection with vesicular stomatitis virus, mice with targeted defects in genes central to T cell function were tested for resistance to i.v. infection with this virus. Our results show that mice lacking the capacity to secrete both IFN-
and perforin completely resisted disease. Similar results were obtained using IL-4 knockout mice, indicating that neither cell-mediated nor Th2-dependent effector systems were required. In contrast, mice deficient in expression of CD40 ligand were more susceptible than wild-type mice, and residual resistance in these mice was almost completely abrogated by depletion of CD8+ T cells. In keeping with this, mice lacking both MHC class I and class II expression succumbed to the infection, whereas most class II-deficient mice normally survive. Adoptive transfer experiments using B cell- and T cell-deficient recipients revealed that no protection could be obtained in the absence of B cells, whereas treatment with virus-specific immune (IgG) serum controlled viral spreading to the central nervous system (CNS), but did not necessarily accomplish virus elimination. Taken together, these results underscore that B cells are essential in preventing early infection of the CNS, but T cells are required for long-term survival. CD4+ T cells are most efficient in this context and the key function is to provide cognate help to B cells. However, if CD4+ cell function is compromised, CD8+ T cells become critical and may suffice for survival.
Keywords: antibodies, CD4+ T cells, T cell-mediated immunity, viral infection
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Introduction
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Both CD4+ and CD8+ T cells have the potential to contribute to the resolution of viral infections (1). Whereas CD8+ T cells primarily exert their function at close range either by killing virus-infected cells (24) and/or releasing pro-inflammatory and antiviral cytokines (5,6), CD4+ T cells may operate along two very different pathways. Thus virus-specific CD4+ T cells may either migrate to the site of infection and function in a manner very similar to that of CD8+ T cells (7) or they may stay in the secondary lymphoid organs, providing critical signals for the expansion and differentiation of virus-specific B cells (1,8). According to the terminology that has evolved in recent years, these different ways of functioning roughly correspond to the division into Th1 and Th2 cells, although one should be aware that also CD4+ T cells with the cytokine profile of Th1 cells may provide help to B cells (9,10). At present little is known for certain about which of the above CD4+ T cell effector mechanisms are most important in clearing a viral infection. From studies on respiratory viruses (8,1113) it appears that CD4+ T cells exert little antiviral activity in the absence of B cells indicating that the key function of CD4+ cells is to provide help to B cells. On the other hand, in mice infected with
-herpes virus, CD4+ T cells have been found to contribute significantly to the control of the infection even in B cell-deficient mice (J. Pravsgaard Christensen, pers. commun.). Therefore, to further investigate the mechanism(s) whereby CD4+ T cells contribute to antiviral protection, murine infection with vesicular stomatitis virus (VSV) was used as a model system. VSV is a cytopathic rhabdovirus closely related to rabies virus, and it is characterized by a high affinity for neuroepithelial cells (14). When injected i.v. into mice, little virus replication takes place in the peripheral tissues, but unless an immune response is induced, the virus spreads to the central nervous system (CNS) and a severe paralytic encephalitis develops with clinical symptoms similar to those of human rabies infection; death usually follows within a few days.
Besides inducing an early and potent IFN-
/ß response (15), systemic infection with VSV induces a transient IgM response peaking around day 4 post-infection (p.i.) (16), which is switched to a sustained T cell-dependent IgG response around day 68 p.i. (17). Also a virus-specific CTL response is induced, peaking around day 6 p.i. (18). CD8+ T cells are not, however, mandatory for protection against systemic infection with VSV in otherwise immunocompetent mice (1921), although studies in intranasally infected mice have revealed some increase in viral titers following depletion of this subset (22).
The induction of a potent IFN-
/ß response (23,24) and an early IgM antibody response is critical in limiting viral replication in the periphery and in delaying the infection from reaching the CNS (21,25). However, T cells are also required for long-term protection, with CD4+ T cells being most important (21). VSV-specific CD4+ T cells are assumed to function primarily by providing help to B cells for isotype switching and thus ensuring a sustained antibody response (17,21,25). However, whether the capacity to help B cells is both necessary and sufficient for complete protection is not clear.
The main purpose of this study was therefore to examine the mechanism(s) whereby CD4+ T cells mediate antiviral protection in mice systemically infected with VSV. By following the outcome of VSV infection in either intact knockout mice or nude mice reconstituted with different cell populations, we show that most, but not all, antiviral activity of CD4+ T cells is dependent on CD40 ligand (CD40L) expression. Additionally, our results provide evidence that CD8+ T cells play a critical role as a back-up resistance mechanism when the function of CD4+ cells is compromised.
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Methods
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Mice
Female C57BL/6 and C57BL/6-nu/nu mice were purchased from Bomholtgaard (Ry, Denmark). Mice deficient in expression of both MHC class I and class II antigens (MHC class I/, II/) were obtained from Taconic Farms (Germantown, NY). Homozygous B cell-deficient (µMT/µMT) mice were the progeny of breeding pairs originally obtained from the National Institutes of Health (Bethesda, MD); these mice had been back-crossed 7 times to a C57BL/6 background followed by back-crossing 3 times to a C57BL/10 background. The local breeding of µMT/µMT mice and wild-type littermates (µMT/+) as well as the assessment of genotypes was performed as described in (26).
Female IL-4/ mice (C57BL/6J-IL4tm1Cgn), and breeding pairs of IFN-
/ (C57BL/6J- Ifgtm1Ts) and CD40L/ (B6,129-Cd40ltm1Imx) mice came from the Jackson Laboratory (Bar Harbor, ME). In addition, mice deficient in CD40L expression were generated by mating female B6,129-CD40ltm1Imx with male C57BL/6 mice. As the gene for CD40L is located on the X chromosome, all males in the F1 generation lacked CD40L expression while their female littermates were heterozygous for the Cd40l gene.
Mice deficient in both IFN-
and perforin (Pfp) production (IFN-
/, Pfp/) were produced in the following way: Pfp/ mice (B6,129-Ppf) purchased from Taconic Farms were mated with IFN-
/ mice to generate an F1 generation heterozygous for both targeted genes. These mice were then backcrossed to homozygous IFN-
/ mice, and offspring heterozygous at the perforin locus and homozygous for the disrupted Ifg gene were then selected and interbred. Assessment of genotypes was performed by PCR analysis and functional analysis was used to verify both defects (manuscript submitted). Mice at least 78 weeks old were used throughout and mice from outside sources were always allowed to acclimatize for at least 1 week. All animals were housed under specific pathogen-free conditions and sentinels were tested regularly according to FELASA guidelines.
Virus and virus quantitation
VSV of the Indiana serotype (originally provided by K. Berg of this institute) was used throughout this study. Stocks of virus were propagated in L929 cells (ATCC CCL 1) and stored at 70°C until use. Virus quantitation was performed by plaque assay on monolayers of L929 cells. In brief, serial 10-fold dilutions of virus were prepared in EMEM (F11) containing 1% L-glutamine, 1% penicillin/streptomycin, 5% NaHCO3 and 10% FCS. Then 1 ml of each dilution was added in duplicate to monolayers of L929 cells in Petri dishes plated 48 h earlier. After incubation for 90 min at 37°C in 5% CO2, medium containing the virus dilutions was aspirated, and the monolayers were overlaid with a mixture of 2.5 ml 1% agarose and 2.5 ml 2xF11. Monolayers were then incubated for 24 h at 37°C in 5% CO2 before staining with a mixture of 1 ml of 1% agarose and 1 ml of 2xF11 containing 1% neutralred. After a further 24 h of incubation, the number of p.f.u. were counted.
Virus infection
Mice were infected i.v with 0.3 ml virus dilution containing 1x106 p.f.u. of VSV. This dose of virus has previously been shown not to cause any disease in normal immunocompetent mice (21).
Survival study
Mortality was used as parameter for the severity of VSV infection, based on previous findings that virus titer in CNS correlate strongly with clinical symptoms (21). Mice were inspected daily for signs of VSV-induced paralysis, and sacrificed when severe paralysis was noted and the animals expected to succumb within the next 24 h (21).
Adoptive transfer experiments
Two different protocols of adoptive transfer were applied in this study. In one model day 6-primed donor spleen cells were transferred into recipient mice infected with 106 p.f.u. of VSV i.v. 3 days earlier. In the other protocol nude recipient mice received naive donor splenocytes 78 days prior to i.v. infection with 106 p.f.u. of VSV i.v.
In vitro depletion of CD8+ cells prior to adoptive transfer
Donor spleen cells for adoptive transfer were depleted of Ly-2.2+ (CD8+) cells by complement-mediated lysis. In brief, single-cell suspensions of spleen cells were prepared and incubated for 1 h at 4°C with an appropriate dose of anti-Ly-2.2. (Cedarlane, Hornby, Ontario, Canada). Cells were then pelleted and resuspended in an optimal dilution of Low-Tox-M rabbit complement (Cedarlane). After 1 h at 37°C, cells were washed 3 times before transfer into recipients. Each recipient received 5x107 cells based on pretreatment numbers without compensation for cell losses. By the end of the experiments depletion of CD8+ cells was verified by flow cytometric analysis using a FACScan analyzer (Becton Dickinson, Mountain View, CA) (27).
In vivo depletion of T cell subsets
Ascitic fluid containing the mAb 2.43.1 or GK1.5 were used to deplete CD8+ and CD4+ cells respectively. Mice to be depleted were injected i.p. with mAb on days 1, 0 and +2 p.i., and once every week thereafter. The efficiency of the depletion was confirmed by flow cytometric analysis.
Serum neutralizations test
Serial 2-fold dilutions of serum in F11 were mixed with equal volumes of virus diluted to contain ~100 p.f.u./ml. After 1 h of incubation at room temperature, 1 ml of each serum/virus mixture was added in duplicate to monolayers of L929 cells in Petri dishes and assayed for the presence of residual virus by plaque assay (see virus and virus quantitation). The highest serum dilution that reduced the number of plaques by at least 50% was taken as the neutralizing titer. For determination of IgG titers, serum was preincubated with an equal volume of 0.1 M 2-mercaptoethanol for 1 h at room temperature. This treatment has been shown to inactivate IgM but not IgG (28). The neutralizing antibody was taken to be IgM only when the 2-mercaptoethanol-treated sample had at least a 4 times lower titer than the corresponding unreduced sample (17).
Treatment of mice with neutralizing antibodies
To produce neutralizing antibodies, groups of C57BL/6 or µMT/+ mice were infected with 106 p.f.u. of VSV i.v., and serum was harvested 5 and 14 days later. The antibodies present at these times have previously be shown to be predominantly IgM and IgG antibodies respectively, as evaluated by 2-mercaptoethanol resistance (17,21). Mice to be treated with IgM antibodies received i.p. injections of 0.5 ml serum on days 1 and 3 p.i., whereas mice given IgG antibodies were treated either on day 3 p.i. only or on days 3, 10 and 17 p.i.
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Results
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Impaired resistance to systemic VSV infection in CD40L-deficient mice, but not in mice deficient in Th2-dependent or cell-mediated immunity
T cells may operate to control of viral infections through cell-mediated and/or humoral effector mechanisms. With regard to the cell-mediated effector arm of the T cell response, perforin and IFN-
constitute two important effector mechanisms, of which either may suffice for virus clearance (20,2932). Therefore, to evaluate the importance of cell-mediated immunity in resistance to infection with VSV, mice with targeted defects of both the perforin gene and the IFN-
gene (Pfp/, IFN-
/) were tested for their capacity to resist infection with VSV. As seen in Fig. 1
such mice survived infection with 106 p.f.u. of VSV i.v., a virus dose that invariably kills nude mice. In contrast, five out of eight (62%) CD4+ T cell-depleted double knockout mice succumbed to the infection, indicating that an effective humoral immune response suffices for protection and that T cell help is essential in this context. Consequently, to determine the mechanism through which T cells influence the humoral response, IL-4/ mice, CD40L/ mice and, for comparison, B cell-deficient (µMT/µMT) mice were subjected to infection with the same dose of VSV. While all B cell-deficient mice died from the infection (the LD50 in these mice is <100 p.f.u.; data not shown), IL-4/ mice were completely resistant. In contrast, a substantial percentage (3050% in individual experiments) of infected CD40L/ mice developed severe paralysis and died; this mortality pattern closely resembles that previously observed for MHC class II-deficient mice (21) and therefore suggests that CD4+ T cell depends on CD40CD40L interaction for most of their protective effect.

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Fig. 1. Outcome of VSV infection in knockout mice with specified immune defects. Mice were infected with 106 p.f.u. of VSV i.v. The depicted data are pooled from several experiments.
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Impaired antibody response in CD40L/ mice
Whereas the antibody response in IL-4/ and pfp/,IFN-
/ mice did not differ significantly from that in wild-type mice (not shown), the antibody response in CD40L/ mice took a different course from that observed in similarly infected wild-type mice (Fig. 2
): 4 days after virus inoculation CD40L/ and wild-type C57BL/6 mice had neutralizing antibody titers within the same range (1:12,8001:25,600). However, while the antibody level in wild-type mice peaked on day 8 p.i. and stayed at about this level (1:12,8001:51,200) for the remainder of the observation period, the antibody response in CD40L/ mice was very transient and started to decline already from day 4 p.i. Thus, lack of CD40L expression resulted in a profoundly changed humoral immune response, consistent with a failure of CD40L-deficient Th cells to provide the appropriate signals required for normal isotype switching. Nevertheless, in most surviving CD40L/ mice low, but significant 2-mercaptoethanol-resistant neutralizing antibodies (titers: 1:2001:1600) were detected even at 40 days p.i., demonstrating that some virus-specific IgG could be produced despite the deficiency in CD40L expression. However, these low antibody titers did not seem to be mandatory for the protection observed as some mice survived the infection without having detectable neutralizing antibodies in serum at this time.

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Fig. 2. Time-course of the neutralizing antibody response against VSV in CD40L/ and C57BL/6 mice. Mice were infected with 106 p.f.u. of VSV i.v. and serum neutralizing antibody responses were determined on the indicated days. Sera collected day 14, 28 and 40 p.i. were tested for 2-mercaptoethanol resistance and found to be predominantly IgG antibodies. Data from individual mice are presented.
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Primed T cells restore resistance to VSV infection in nude mice, but not in B cell-deficient mice
To examine more closely the mechanism whereby T cells contribute to recovery from a systemic VSV infection, an adoptive transfer model was established. Groups of T cell-deficient nude mice and B cell-deficient µMT/µMT mice were infected with VSV. On day 3 p.i., nude recipients were adoptively transferred with day 6-primed spleen cells from either nu/nu or µMT/µMT donor mice (Fig. 3a
). In parallel, virus-infected µMT/µMT recipients received day 6-primed µMT/µMT splenocytes, and some of these mice were also given two injection of neutralizing IgM antibody on days 1 and 3 p.i. (Fig. 3b
). Control groups of nu/nu and µMT/µMT mice received virus only.

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Fig. 3. Reconstitution of nude mice with primed µMT/µMT cells restores resistance to 106 p.f.u. of VSV i.v. nu/nu (a) or µMT/µMT recipients (b) were on day 3 p.i. reconstituted with ~ 5x107 day 6-primed spleen cells from nu/nu or µMT/µMT donors. Some µMT/µMT recipients were also injected i.p. with serum containing neutralizing IgM antibodies (day 5 serum) on days 1 and 3 p.i. Non-transplanted recipients infected in parallel served as controls. The results depicted are the compiled data from four experiments.
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As can be seen in Fig. 3
(a), all nude mice receiving primed nude splenocytes developed paralytic disease and died of the infection as did non-transplanted controls. The same pattern was seen after transfer of primed µMT/µMT splenocytes into B cell-deficient recipients (Fig. 3b
). Even B cell-deficient mice treated with both IgM antibodies and effector T cells eventually succumbed to the infection, although mortality tended to be delayed in these mice. However, when nude recipients were reconstituted with primed µMT/µMT splenocytes, the majority (89%) survived without ever showing clinical signs of VSV infection (Fig. 3a
). Together these results demonstrate that primed T cells have little protective capacity in the absence of B cells and IgG antibodies.
Partial resistance in nude mice treated with IgG antibodies
To explore the antiviral potential of neutralizing IgG antibodies, groups of VSV challenged nu/nu mice were either left untreated or treated with i.p. injections of 0.5 ml immune serum (IgG) either once (day 3 p.i.) or 3 times (days 3, 10 and 17 p.i.) and mortality was recorded. For comparison, a group of VSV-infected CD40L/ mice, which like nude mice are deficient in the ability to produce neutralizing IgG antibodies, were also treated with antibody on day 3 p.i. As seen in Table 1
, all nude mice receiving a single injection of antibody succumbed to the infection, while the mortality in mice repeatedly treated with immune serum/IgG antibodies was reduced to 40%. Generally, it should be noted that antibody-treated mice that died from VSV infection did so after an extended asymptomatic period. In comparison, five of six antibody-treated CD40L/ mice survived the infection. Thus, these results show that although IgG antibodies are able to prevent the virus from spreading, complete virus clearance is difficult to accomplish with antibodies alone. Additionally, our findings indicate that T cells are functional and act in synergy with IgG antibodies even in the absence of CD40L expression.
Primed CD8-depleted cells from CD40L-deficient mice significantly protect nude recipients
Since we have previously found that CD4+ T cells suffice for protection (21), we next sought to define the central function of these cells using the adoptive transfer model. Nude recipients infected 3 days before cell transfer were tranfused with CD8+ cell-depleted splenocytes from either IFN-
/ or CD40L/ mice primed 6 days earlier.
Whereas non-transplanted controls invariably succumbed to the infection, all nude recipients except one given donor cells deficient in IFN-
production survived the infection (Fig. 4
). In addition, when the neutralizing antibody response was analyzed on day 28 p.i., we found high neutralizing antibody titers comparable to those found in normal immunocompetent mice at this time. Thus, even in the absence of CD8+ cells, lack of secretion of IFN-
from CD4+ and 
T cells did not seem to affect either the total amount of virus-specific neutralizing antibodies produced or the predisposition for VSV induced paralysis.
Surprisingly, the majority (83%) of nude mice reconstituted with primed CD8-depleted CD40L/ spleen cells also survived the infection (Fig. 4
). Evaluation of the level of neutralizing antibodies in these mice 40 days after infection revealed low but significant antibody titers (1:2001:800) similar to what was found in intact CD40L/ mice. Apparently, the transplanted cells were able to mediate some protection despite the lack of both CD40L expression and CD8+ cells. This finding therefore indicates that under certain conditions CD8 T cells are able to exert an antiviral effect which does not require collaboration with B cells through the CD40CD40L interaction.
Reconstitution of nude mice with naive CD8-depleted, CD40L spleen cells significantly increases resistance to VSV infection
In the experiments described above, the donor cells comprised splenocytes from mice primed 6 days prior to the adoptive transfer which was carried out 3 days after infection of the recipients. Therefore, it could argued that part of the resistance seen in the nude recipients was due to the presence of donor B and T cells in a more activated state than usually found 3 days after virus challenge. To test whether this was the underlying explanation, a survival experiment was carried out in which naive CD8-depleted spleen cells from CD40L/ donors were transferred into nude recipients 7 or 8 days before i.v. infection with VSV.
Further, to reduce the risk of a graft versus host reaction under these conditions, the transferred splenocytes in this case came from CD40L-deficient males created by mating female CD40L knockout mice (/) with male C57BL/6 mice (+/0). As the gene for CD40L is located on the X chromosome, male F1 progeny were deficient in CD40L (/0), while their female littermates had a wild-type phenotype (/+). This pattern of inheritance was further confirmed by challenging F1 mice of both sexes with 106 p.f.u. of VSV i.v. Female mice all produced high levels of neutralizing antibodies and survived, while both the capacity to produce antibodies and the susceptibility of their male littermates roughly corresponded to that of ordinary CD40L/ mice (Fig. 5b
).

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Fig. 5. Naive CD8CD40L cells increase resistance to VSV infection in nude mice. (a) At 78 days prior to i.v. infection with 106 p.f.u. of VSV, a group of nude mice was reconstituted with naive CD8-depleted CD40L donor cells. A control group of VSV-infected nude mice did not receive any cells. (b) Resistance to 106 p.f.u. of VSV i.v. in male and female mice of the donor strain (C57BL/6xCD40L/); neutralizing antibody titers day 40 p.i.: females, 1:128001:51200; males, <1:200. At the termination of the experiment splenocytes were analyzed for presence of CD8+ T cells, median of group: 1.5%.
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VSV challenge of nu/nu mice reconstituted with naive CD8-depleted CD40L/xC57BL/6 cells of male genotype resulted in a high survival rate (78%) (Fig. 5a
), even though most intact male donors succumbed to the same dose of virus (Fig. 5b
). Thus, these results underscore that in the nude environment, an antiviral effect may be mediated by CD8 T cells which do not require the expression of CD40L. Again, when sera collected 4050 days after infection were tested in a serum neutralization test, low neutralizing antibody titers (1:2001:1600) were detected in reconstituted nude mice. Thus, like CD40L/ mice, reconstituted nude mice were capable of mounting an impaired yet sustained antibody response.
Severely reduced resistance to systemic VSV infection in CD40L/ mice depleted of CD8+ T cells
Having demonstrated a CD40L independent antiviral effect mediated by CD8-depleted cells in nude mice, we next wished to investigate the importance of this antiviral activity in intact CD40L/ mice. For this purpose CD40L/ and C57BL/6 mice were infected with 1x106 p.f.u. of VSV, and simultaneously in vivo depleted of CD8+ cells by i.p. injections of the mAb 2.43.1 at days 1, 0, +2 p.i. and once every week thereafter. For comparison, a group of MHC class I/, II/ mice lacking both CD4 and CD8 T cells, but not 
T cells (33), was infected i.v. with the same dose of virus. When survival was monitored (Fig. 6
), all MHC class I/, II/ mice developed paralytic disease and succumbed to the infection within 21 days after virus challenge. Given that ~60% of VSV-infected MHC class II-deficient mice resist the infection (21), this finding points to an important back-up function of class I-restricted T cells when the CD4+ T cell subset is compromised. In keeping with this interpretation, nine of 10 CD8-depleted CD40L/ mice (90%) developed clinical signs of disease and subsequently died, whereas all CD8-depleted wild-type mice resisted the infection. Thus, in contrast to the situation in nude mice, CD8-depleted T cells had little or no protective capacity in the absence of CD40L expression. Consequently, although class I-restricted CD8+ T cells are not required for virus resolution in otherwise immunocompetent animals, these cells serve a critical function in mice lacking either MHC class II or CD40L expression.

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Fig. 6. CD8-depleted T cells have little or no protective capacity in the absence of CD40L. CD40L/ and C57BL/6 mice were infected i.v. with 106 p.f.u. of VSV and concurrently in vivo depleted of CD8+ cells by i.p. injections with the mAb 2.43.1; flowcytometric analysis of splenocytes on day 6 p.i. revealed <1% CD8+ cells. MHC class I/, II/ mice infected in parallel served as controls. For comparison, the cumulative mortality of all VSV-infected intact CD40L/ mice included in this study are shown.
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Discussion
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The present study confirms and extends our understanding of the role of CD4+ T cells in resistance to systemic infection with VSV. In addition, our results for the first time directly demonstrate a role for CD8+ T cells as a critical back-up effector system in this infection model. Thus the effector mechanisms involved in controlling systemic infection with VSV appear to be more complex than previously realized.
With regard to the CD4+ T cell subset, our results clearly show that expression of CD40L is central to the activity of this cell subset, whereas IFN-
is irrelevant even when CD8+ cells are absent. This latter point is pertinent, based on our finding that CD8+ T cell may participate in preventing a lethal outcome. Thus, previous studies (21,24) showing the redundancy of this cytokine could have yielded false-negative results based on the presence of cytotoxic CD8+ T cells. The fact that mice lacking both major effector mechanisms involved in cell-mediated immune responses resist a lethal outcome further support the hypothesis that B cells help to constitute the primary T cell function required for long-term survival in this model. In this context it is important to note that although passive transfer of IgG antibodies to nude mice effectively prevents the virus from spreading to the CNS [in a previous study we have clearly demonstrated a direct correlation between virus titer in CNS and clinical symptoms (21)], complete virus clearance is not invariably obtained as evidenced by the finding that antibody-treated nude mice may develop paralytic disease following clearance of the transfused antibody. Therefore, it is possible that CD4+ cells perform some additional function that may contribute to virus elimination. However, although the CD4+-dependent protection against systemic VSV infection superficially resembles the result of a Th2 response, IL-4 is redundant in otherwise immunocompetent mice.
With regard to the role of CD40L, our results confirm and extend a recent study by Borrow et al. (34) also revealing an increased susceptibility to VSV infection in CD40L/ mice. However, in that study no analysis of the underlying mechanism nor of the resistance mechanism leading to the survival of the majority of infected mice was carried out. Here we clearly demonstrate that depletion of CD8+ cells results in almost 100% mortality in CD40L/ mice, demonstrating that under physiological conditions CD4+ cells have little or no capacity to control VSV infection if expression of CD40L is prevented. Precisely how this molecule is involved in the CD4+ response against VSV is not known with certainty. Clearly CD40CD40L interaction could be involved both in the afferent and efferent phases of the antiviral immune response. Recently, however, Oxenius et al. (35) have provided evidence indicating that CD40CD40L interaction is critical only in TB cooperation but not for other anti-viral CD4+ T cell functions. Particularly it should be noted that virus-specific CD4+ T cells could be efficiently primed in the absence of this interaction. Consequently, it seems most likely that the basis for the compromised resistance of CD40L/ mice to VSV infection is to be found in the efferent phase and that impaired capacity to provide the help required for B cells to produce neutralizing IgG antibodies is the essential factor. Confirming this, a single transfusion of IgG antibodies suffices for protection of CD40L/ mice, whereas nude mice treated in the same way all succumb. Moreover, we find that preactivated T cells even in conjunction with IgM antibodies do not protect B cell-deficient mice (present report), whereas neutralizing IgG antibodies do (21), thus demonstrating that T cell-mediated protection is critically dependent on collaboration with B cells.
Contrary to Oxenius et al. (35), we detect low but significant titers of neutralizing IgG in most of the VSV-infected CD40L/ mice. However, other studies addressing the importance of CD40L in isotype switching also reveal that some IgG may be produced independently of CD40CD40L interaction (34,36,37). The mechanism underlying this phenomenon has been suggested to be virus-induced T cell-independent B cell activation based on the finding that CD4+ cell depletion did not reduce the magnitude of the response (34). Alternatively, 
T cells might support isotype switching in a non-cognate fashion (38). On the other hand, two independent studies have failed to reveal production of neutralizing IgG antibodies in MHC class II-deficient mice (21,39). Consequently, our findings point to the possibility that Th may provide some help to B cells even in the absence of CD40L expression. Notably, all MHC class I/, II/ mice succumbed to the infection. Since these mice have been reported to have normal numbers of 
T cells (33), this finding indicates that this T cell subset cannot provide sufficient help for the mice to produce protective levels of antibody.
The ability of CD8-depleted cells from CD40L/ mice to protect nude mice is remarkable given the almost complete failure of these cells to protect in situ. The explanation for this phenomenon could be an increased natural resistance of the nude environment which may delay the virus spread sufficiently for these cells to cope with the infection in most of the nude recipients. Many studies have documented that natural resistance mechanisms are preactivated in nude mice. The cellular mechanism underlying protection apparently does not involve help to B cells as antibody levels in transplanted mice do not differ from those in intact CD40L/ mice. Whatever the mechanism, CD8-depleted T cells may exert limited antiviral activity independent of CD40L.
In conclusion, our studies demonstrate that B cells are mandatory to prevent the early spreading of VSV to the CNS. However, T cell-mediated immunity is clearly required for long-term survival. CD4+ T cells are of primary importance and expression of CD40L is central to their antiviral function, while capacity to secrete IFN-
and IL-4 is redundant. Neutralizing IgG antibodies can at least in part substitute for Th cells, but may not suffice for complete virus elimination. CD8+ T cells and a cellular immune response are not required if these primary defences are operating efficiently. If, however, CD4+ T cell function is compromised, CD8+ T cells become critical and may suffice for survival. In contrast, 
T cells appear to play little, if any, role.
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Acknowledgments
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This work was supported in part by the Danish Medical Research Council, the Biotechnology Center for Cellular Communication, and the Novo Nordisk Foundation. T. J. and A. N. are the recipients of PhD scholarships from the Faculty of Health Sciences, University of Copenhagen.
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Abbreviations
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CD40L CD40 ligand |
CNS central nervous system |
µMT/µMT B cell-deficient mice |
Pfp perforin |
p.i. post-infection |
VSV vesicular stomatitis virus |
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Notes
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Transmitting editor: A. McMichael
Received 14 June 1999,
accepted 10 September 1999.
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