Requirement for CD4+ T Cells in Vβ4+CD8+ T Cell Activation Associated with Latent Murine Gammaherpesvirus Infection

Emilio Flaño, David L. Woodland and Marcia A. Blackman
J Immunol September 15, 1999, 163 (6) 3403-3408;

Abstract

A CD8+ T cell lymphocytosis in the peripheral blood is associated with the establishment of latency following intranasal infection with murine gammaherpesvirus-68. Remarkably, a large percentage of the activated CD8+ T cells of mice expressing different MHC haplotypes express Vβ4+ TCR. Identification of the ligand driving the Vβ4+CD8+ T cell activation remains elusive, but there is a general correlation between Vβ4+CD8+ T cell stimulatory activity and establishment of latency in the spleen. In the current study, the role of CD4+ T cells in the Vβ4+CD8+ T cell expansion has been addressed. The results show that CD4+ T cells are essential for expansion of the Vβ4+CD8+ subset, but not other Vβ subsets, in the peripheral blood. CD4+ T cells are required relatively late in the antiviral response, between 7 and 11 days after infection, and mediate their effect independently of IFN-γ. Assessment of Vβ4+CD8+ T cell stimulatory activity using murine gammaherpesvirus-68-specific T cell hybridomas generated from latently infected mice supports the idea that CD4+ T cells control levels of the stimulatory ligand that drives the Vβ4+CD8+ T cells. As Vβ4+CD8+ T cell expansion also correlates with levels of activated B cells, these data raise the possibility that CD4+ T cell-mediated B cell activation is required for optimal expression of the stimulatory ligand. In addition, in cases of low ligand expression, there may also be a direct role for CD4+ T cell-mediated help for Vβ4+CD8+ T cells.

Intranasal infection of mice with the murine gammaherpesvirus, MHV-68,3 causes an acute respiratory infection that is rapidly resolved, followed by the establishment of lifelong viral latency in several cell types, including B cells (1, 2, 3), lung epithelial cells (4), and macrophages (5). Levels of latent virus in the spleen peak at about 14 days postinfection, but quickly drop to ∼1/106 spleen cells, and remain stable at this level (6). Late in the infection, after the clearance of lytic virus from the lung, a syndrome similar to the infectious mononucleosis associated with the human gammaherpesvirus EBV, develops. Hallmarks of this syndrome include splenomegaly, due to increased numbers of cycling CD4+ T cells, CD8+ T cells, and B cells, and lymphocytosis of the peripheral blood, due to increased numbers of activated CD8+ T cells, dominated by those bearing Vβ4+TCR (7, 8). The Vβ4+CD8+ T cell response does not appear to result from an outgrowth of cells responding to viral epitopes expressed during acute infection (9, 10). Rather, the response has several unusual features in that the Vβ4+ T cell expansion is not classically MHC restricted (8, 11) (C. L. Hardy, R. D. Cardin, E. Flaño, P. Nguyen, D. L. Woodland, R. W. Williams, and M. A. Blackman, manuscript in preparation), and appears to be independent of presentation by classical MHC class I or MHC class II molecules (11). Identification of the stimulatory ligand remains elusive, but its expression correlates with peak levels of splenic latency ∼2 wk after infection (11).

The requirement for CD4+ T cells in antiviral CD8+ T cell responses is not absolute, and may be controlled by variables such as the virulence or dose of the virus, and the activation state of the APC, including whether the APC are virally infected. It has been suggested that CD4+ T cells may be particularly important for the maintenance of cytolytic T cells and preventing reemergence of infectious virus in chronic infections (reviewed in Refs. 12, 13). Indeed, in the MHV-68 virus model, a progressive reactivation of lytic virus in the lung was seen in CD4-deficient, MHC class II−/− mice (6). Further analysis of CD8+ effector function for MHV-68 lytic epitopes in this model showed it to be intact, and it was concluded that the CD4+ T cells were either required for the Ab response, suggesting a critical role for Ab in controlling latent viral infection, or for the generation of CTL for epitopes other than those that dominated the acute response (14). The importance of Ab in controlling lytic virus after reactivation was directly shown in studies in MHV-68-infected, B cell-deficient μMT mice (4).

A role for CD4+ T cells in several aspects of the complex pathogenesis associated with MHV-68 infection has been previously reported. A requirement for CD4+ T cells in the splenomegaly induced by MHV-68 infection has been demonstrated (7, 15). In addition, CD4+ T cells were required for the nonspecific B cell activation and polyclonal Ab secretion associated with MHV-68 infection (16), as well as, not surprisingly, for the generation of the specific humoral response (17). Our previous experiments established that there was no Vβ4 expansion in MHC class II-deficient mice (8). These mice lack both MHC class II molecules and CD4+ T cells. Our early experiments with CD4 depletion suggested that the lack of Vβ4 expansion was due to the absence of MHC class II molecules, as CD4 depletion just before the time of Vβ4 expansion did not ablate the expansion (8). However, our subsequent analysis suggesting that MHC class II molecules are not necessary for stimulation of Vβ4+ hybridomas with latently MHV-68-infected spleen cells, reopened the possibility that CD4+ T cells were required early in the response (11). Thus, the experiments described in the present study analyze the requirement for CD4+ T cells in the Vβ4+CD8+ T cell expansion associated with early stages of latent MHV-68 infection.

Materials and Methods

Mice

C57BL/6J (B6), C57BL/6-Ifngtm1Ts (IFN-γ−/−) (18), and C57BL/6-Cd4tm1Mak (CD4−/−) (19) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). H-2 IAb-deficient C2D mice (MHC class II−/−) (20) (licensed from GenPharm International, Mountain View, CA) were bred at St. Jude Children’s Research Hospital (Memphis, TN). Mice were housed under specific pathogen-free conditions before MHV-68 infection at 8–16 wk of age, and in BL 3 containment after infection.

Virus stocks and infection and sampling of mice

The stock of MHV-68 (clone G2.4) was obtained from Prof. A. A. Nash (Edinburgh, U.K.), propagated in OMK cells (ATCC 1566CRL), and titered on NIH-3T3 (ATCC CRL1568) monolayers, as previously described (6). Mice were anesthetized with Avertin (2,2,2-tribromoethanol) and infected intranasally with 600 PFU MHV-68 in a total volume of 40 μl of PBS. Splenocytes and/or peripheral blood were analyzed at various times after infection.

CD4+ T cell depletion

To deplete the CD4+ T cell subset, mice were i.p. injected with 500 μl of a CD4-specific mAb (GK1.5), in the form of either a 1/5 dilution of ascites or neat concentrated culture supernatant. Depletions were started at the time of infection or 1 wk later, and continued at 2-day intervals until the mice were sacrificed for analysis.

LacZ hybridoma assay

The characterization of Vβ4+CD8+ lacZ-inducible T cell hybridomas that specifically respond to MHV-68-infected spleen cells 14 days postinfection has been previously described (11). Spleen cells were T cell depleted using anti-Thy-1 mAb AT83 (21) and a mixture of rabbit and guinea pig complement (Cedarlane Laboratories, Hornby, Ontario, Canada), before being used as stimulator cells. A total of 106 stimulator cells were plated in 96-well flat-bottom plates, and titrated in 2-fold dilutions. LacZ-inducible T cell hybridomas 4BH-98 and 5BH-11 were added as 105 cells/well, and incubated overnight. β-galactosidase activity was assessed in individual cells, as previously described (10, 11), using 5-bromo-4-chloro-3-indolyl β-d-galactopyranoside (X-Gal) as the substrate, and blue cells were counted under the microscope. The background stimulation was determined by using naive APCs as stimulators.

Flow cytometry

After erythrocyte lysis in hemolytic Gey’s solution, spleen and blood cells were stained for FACS analysis using combinations of the following Abs and lectins: CD44 (clone IM7; PharMingen, San Diego, CA), CD62L (clone Mel-14; PharMingen), TCR Vβ4 (KT-4; PharMingen), CD8α (clone CT-CD8α; Caltag, Burlingame, CA), and peanut agglutinin (PNA; Sigma, St. Louis, MO). A total of 20,000 live cells were gated and acquired on a FACScan flow cytometer, and the data were analyzed using CellQuest software (Becton Dickinson Immunocytometry Systems, San Jose, CA).

Infective center assay

The frequency of latently infected cells was estimated by an infective center assay, dependent upon the ability of latently infected cells to reactivate upon in vitro culture with susceptible cells, as previously described (6). Serial dilutions (in triplicate) of T cell-depleted splenocytes were plated onto monolayers of NIH-3T3 cells, and the next day overlaid with carboxymethyl cellulose. After 6 days of culture, plaques were quantitated after methanol fixation and Giemsa staining. As this assay will also measure lytic virus, samples were simultaneously assayed after one cycle of freeze/thaw to assess the possible contribution of lytic virus to the titers. Control experiments, conducted to confirm that this procedure does not reduce the titer of cell-associated lytic virus, showed <20% reduction in lytic viral titers.

Results

CD4+ T cells are required for expansion of Vβ4+CD8+ T cells, but not for generalized CD8+ T cell activation

The requirement for CD4+ T cells in the activation of CD8+ T cells following the establishment of MHV-68 latency was analyzed in three experimental models of CD4-deficient mice: 1) B6 mice that had been depleted of CD4+ T cells by in vivo injection of anti-CD4 Abs, 2) CD4−/− (knockout) mice, and 3) CD4-deficient, MHC class II−/− (knockout) mice. Peripheral blood was analyzed on days 14 and 21 after infection, time points we have previously shown to precede and correlate, respectively, with increased levels of activated CD8+ T cells and expansion of the Vβ4+ subset (8). Analysis of all three models of CD4+ T cell deficiency showed that there was no increase of Vβ4+CD8+ T cells in the peripheral blood (Fig. 1⇓A) or spleen (data not shown). In contrast, there were variable and less marked effects on the increased percentage of CD8+ T cells in the peripheral blood (Fig. 1⇓B). It should be noted that, as expected in the absence of CD4+ T cells, there were higher levels of CD8+ T cells in the CD4−/− and MHC class II−/− mice before infection. Despite the variable increase in the proportion of CD8+ T cells in the peripheral blood in the absence of CD4+ T cells, the CD8+ T cells were still highly activated, as assessed by PNAhigh, CD62Llow, and CD44high expression (Fig. 2⇓).

FIGURE 1.
FIGURE 1.

CD4+ T cells are required for expansion of Vβ4+CD8+ T cells following MHV-68 infection. Kinetics of T cell expansion in peripheral blood lymphocytes after MHV-68 infection in B6 mice, B6 mice depleted of CD4+ T cells in vivo by injection with anti-CD4 Abs, CD4−/− (knockout) mice, and MHC class II−/− (knockout) mice that are also deficient in CD4+ T cells. A, Percentage of Vβ4+ T cells among CD8+ cells. B, Percentage of CD8+ cells among total blood lymphocytes. At least six individual mice from at least two different experiments were analyzed at 14 and 21 days after infection. Endogenous levels are shown for naive (−) animals. Error bars correspond to SD.

FIGURE 2.
FIGURE 2.

Activation profile of peripheral blood CD8+ T cells from MHV-68 infected, CD4-deficient mice. The percentage of CD8+ peripheral blood T cells that are PNAhigh (A), CD62Llow (B), and CD44high (C) are compared in naive (−) mice and mice at 21 days after MHV-68 infection. At least six individual mice from at least two different experiments were analyzed at each time point. Error bars correspond to SD.

Vβ4+CD8+ T cell expansion and generalized CD8+ T cell activation are relatively late events in the anti-MHV-68 response. We had previously shown that depletion of CD4+ T cells at day 11 of infection had little effect on the blood pathogenesis (8). These data suggest that there is an early requirement for CD4+ T cells, but the effect becomes CD4 independent later in the response. To more precisely determine at which stage of infection CD4+ T cells are required, CD4+ T cells were eliminated 7 days postinfection, a time point subsequent to the initiation of viral latency in the spleen. The data show that CD4 depletion at the initiation of infection and 7 days after infection was equally effective in inhibiting Vβ4+CD8+ T cell expansion (Fig. 3⇓).

FIGURE 3.
FIGURE 3.

CD4 Ab depletion of B6 mice beginning at the time of infection (day 0) or 7 days after infection (day 7) is comparable in preventing expansion of Vβ4+CD8+ T cells. B6 and B6 mice that had been depleted of CD4+ T cells with anti-CD4 Abs were analyzed for the percentage of Vβ4+CD8+ T cells among total CD8+ peripheral blood T cells at 21 days after infection. Data correspond to six individual mice from two different experiments. Error bars correspond to SD.

Expression of the latency-associated ligand that stimulates Vβ4+CD8+ T cell hybridomas is absent or reduced in CD4+ T cell-deficient mice

We have previously generated a panel of Vβ4+ T cell hybridomas from latently infected mice that can be used to monitor in vivo expression of the uncharacterized ligand that drives Vβ4+CD8+ T cell activation. Previous studies have shown that the hybridomas are specifically reactive to spleen cells isolated from MHV-68-infected mice, but not to spleen cells or cell lines that were lytically infected in vitro. In addition, the Vβ4+ T cell stimulatory activity is not detected in spleen cells during the early, lytic stages of the infection, despite the fact that low levels of latent virus are detectable as early as day 6. Rather, the Vβ4+ T cell stimulatory activity correlates precisely with the establishment of peak levels of splenic latency at ∼14 days after infection (11). Thus, to determine whether CD4+ T cells are required for generation of the stimulatory ligand, spleen cells were isolated from the three models of CD4-deficient mice 14 days after MHV-68 infection, and were used to stimulate Vβ4+ T cell hybridomas. The data in Fig. 4⇓ show that day 14 spleen cells from CD4−/− mice and CD4 Ab-depleted mice were unable to stimulate a representative hybridoma, and, consistent with our previous report (11), there was a dramatically reduced stimulation of the hybridoma by spleen cells from infected MHC class II−/− mice. Similar results were obtained with a second hybridoma (data not shown). The analysis also showed that Ab depletion initiated 7 days after infection was as efficient as depletion from the time of infection in preventing hybridoma stimulation (Fig. 4⇓A), consistent with the comparable effect on in vivo expansion of Vβ4+CD8+ T cells (Fig. 3⇑). As in vitro hybridoma stimulation is dependent on TCR ligation, but is independent of other factors such as costimulation and CD4+ T cell help, these data indicate that the ligand that drives Vβ4+CD8+ T cells is absent, or reduced below the threshold required for in vivo activation, in CD4-deficient mice, and suggest that the lack of Vβ4+CD8+ T cell expansion in the CD4-deficient mice is because CD4+ T cells are required for optimal expression of the stimulatory ligand.

FIGURE 4.
FIGURE 4.

CD4+ T cells are required for generation of the ligand that stimulates Vβ4+CD8+ T cell hybridomas. Stimulation of a representative MHV-68-specific Vβ4+CD8+ T cell hybridoma (4BH-98) by T cell-depleted spleen cells 14 days after MHV-68 infection. A, B6 mice that were depleted of CD4+ T cells at the time of infection (day 0) or 7 days after infection (day 7); B, CD4−/− mice; C, MHC class II−/− mice. Comparable results were obtained with a second hybridoma (5BH-11, data not shown). Positive controls are specific hybridoma reactivity to B6 T cell-depleted spleen cells 14 days after infection (B6 MHV-68). Naive T cell-depleted splenocytes were used as negative controls (B6 naive).

Because expression of the ligand that drives Vβ4+CD8+ T cell activation correlates with latency (11), the effect of CD4 depletion on titers of latent virus, assessed by the infective center assay, was also analyzed. In agreement with previous data (6, 11), MHC class II−/− mice had ∼10-fold lower levels of latently infected spleen cells at day 14, the peak of latency (Fig. 5⇓). Analysis of the CD4−/− and Ab-depleted mice showed that there was an even greater (>100-fold) reduction in latency levels at day 14. As expected, latency levels were declining in the control B6 spleen cells by day 21 (6). However, the levels of latency in the MHC class II−/− and Ab-depleted animals remained unchanged, and latency levels increased ∼10-fold in the CD4−/− animals between days 14 and 21. Consistent with a previous report (16), the absence of CD4+ T cells resulted in reduced percentages of activated splenic B cells in all three models of CD4 depletion (Fig. 6⇓).

FIGURE 5.
FIGURE 5.

MHV-68 latency in CD4-deficient mice. Latent viral titers in T cell-depleted spleen cells at 14 and 21 days after infection were estimated by an infective center assay. The infective center numbers are the mean values of triplicate wells from at least two different experiments. Levels of lytic virus assessed in parallel samples that had been freeze thawed before assay were always less than 1 viral particle/106 cells.

FIGURE 6.
FIGURE 6.

Reduced levels of B cell activation in CD4-deficient mice. Splenic B cells from naive mice (−) and MHV-68-infected mice (14 and 21 days after infection) were analyzed for levels of PNA binding. Data are expressed as a percentage of PNA+ cells among CD19+ spleen cells. Error bars represent SD.

Despite the finding that there was no Vβ4 expansion at day 21 in either the CD4−/− or MHC class II−/− animals (Fig. 1⇑), differences in these models became apparent with extended kinetic analysis. Unexpectedly, there was a late emergence of Vβ4+CD8+ T cells in the CD4−/−, but not MHC class II−/− animals (Fig. 7⇓), that correlated with the late appearance of activated B cells in the CD4−/− mice (Fig. 8⇓). These results suggest that there may be additional factors controlling the effect of CD4+ T cells on Vβ4+ T cell expansion that differ between the two experimental models, and/or that CD4−/− animals develop compensatory mechanisms that are not found in the MHC class II−/− animals.

FIGURE 7.
FIGURE 7.

Late expansion of Vβ4+CD8+ T cells in CD4−/− mice. The percentage of Vβ4+ cells among peripheral blood CD8+ T cells was assessed for MHC class II−/− mice (A) and CD4−/− mice (B) at various time points after MHV-68 infection. At least six mice from at least two different experiments were analyzed. Error bars represent SD.

FIGURE 8.
FIGURE 8.

Time course analysis of B cell activation in MHV-68-infected mice. The percentage of PNAhigh cells among CD19+ spleen cells was assessed for B6, MHC class II−/−, and CD4−/− mice at various time points after infection. Between 3 and 12 mice from one to four separate experiments were analyzed at each time point. Error bars represent SD.

Dependence of Vβ4+CD8+ T cell expansion on CD4+ T cells does not reflect a requirement for IFN-γ

IFN-γ is produced at high levels in the spleen and lymph nodes during MHV-68 infection (22). Numbers of IFN-γ-secreting CD4+ T cells, including virus-specific as well as bystander-activated CD4+ T cells, increase gradually in the spleen, attaining peak levels at ∼14 days after infection (23). It has been shown that these cells mediate virus-specific effector function through IFN-γ (24), raising the possibility that the requirement for CD4+ T cells for Vβ4+CD8+ T cell activation is also mediated through IFN-γ. To test this possibility, levels of Vβ4+CD8+ T cells were assessed in MHV-68-infected IFN-γ−/− mice. The data show normal levels of Vβ4+CD8+ T cells (Fig. 9⇓) and CD8+ T cell activation (data not shown) in IFN-γ−/− mice. The ability of infected spleen cells from IFN-γ−/− mice to stimulate a representative MHV-68-specific Vβ4 hybridoma was also assessed (Fig. 10⇓). Interestingly, there was an ∼8-fold increase in hybridoma stimulation by day 14 spleen cells from MHV-68-infected IFN-γ−/− mice relative to B6 controls, which correlates with an increase in latency levels at day 14 (data not shown). Both the hybridoma reactivity and latency had returned to normal levels by day 17 (data not shown). These data reinforce the correlation between latency and expression of the ligand for Vβ4+CD8+ T cell stimulation (11). The increased hybridoma stimulation and the high levels of Vβ4+CD8+ T cells in MHV-68-infected IFN-γ−/− mice rule out the possibility that the requirement for CD4+ T cells is mediated through IFN-γ.

FIGURE 9.
FIGURE 9.

Vβ4+CD8+ T cell expansion is not dependent on IFN-γ. B6 and IFN-γ−/− mice were assessed for the percentage of Vβ4+ T cells among CD8+ peripheral blood T cells. Mice were analyzed at 14 and 21 days after MHV-68 infection. Naive mice (−) were used as controls.

FIGURE 10.
FIGURE 10.

Stimulation of a representative MHV-68-specific Vβ4+CD8+ hybridoma, 4BH-98, by T cell-depleted IFN-γ−/− spleen cells from mice 14 days after infection (IFN-γ−/− MHV-68). Similar results were obtained with a second hybridoma (data not shown). Reactivity to B6 T cell-depleted spleen cells at the same time postinfection is shown as a positive control (B6 MHV-68). Reactivity to T cell-depleted splenocytes from naive IFN-γ−/− and B6 mice is shown as a negative control.

Discussion

Analysis of MHV-68 infection in three independent models of CD4-deficient mice has shown a requirement for CD4+ T cells in the development of Vβ4+CD8+ lymphocytosis in the peripheral blood. The reduced ability of splenocytes from infected CD4-deficient mice to stimulate Vβ4+ T cell hybridomas suggests that the CD4+ T cells play a role in generation of the ligand driving the Vβ4+CD8+ T cells. This appears not to be their only role, however, because MHC class II−/− mice, which are also CD4 deficient and fail to exhibit expanded levels of Vβ4+CD8+ T cells, do stimulate Vβ4+ T cell hybridomas in vitro, albeit less efficiently. Analysis of more than one model of CD4 depletion in this study points out that the role for CD4+ T cells in Vβ4+CD8+ T cell expansion may be complex.

A requirement for CD4+ T cells for generation of the ligand is consistent with the idea that B cell activation is associated with the activation of Vβ4+CD8+ T cells. The demonstration that late expansion of Vβ4+CD8+ T cells observed in CD4−/−, but not MHC class II−/− mice, is accompanied by the late emergence of activated (PNAhigh) B cells supports this possibility. Previous analysis of CD4−/− mice described the development of a population of CD4−CD8− (double-negative) TCRαβ+ T cells that compensate functionally for CD4+ T cell help for B cells and facilitate isotype switching (25). This population of cells has not been described for MHC class II−/− mice, and these mice are incapable of undergoing isotype switching (20, 26). An association between Vβ4+CD8+ T cell expansion and B cell activation is also consistent with the finding that there is no Vβ4+CD8+ T cell expansion in B cell-deficient μMT mice or CD40L−/− mice 27 .

It is possible that the stimulatory ligand is expressed exclusively by latently infected, activated B cells. Alternatively, Vβ4 stimulation may be dependent on a threshold level of expression of the ligand, controlled by the level of latent infection, which in turn may be dependent on CD4+ T:B interactions. The latter possibility is consistent with the recent report that B cells may control levels of latency despite the fact that they are not required for the establishment of MHV-68 latency (28). In support of a threshold effect is the finding that spleen cells from MHV-68-infected MHC class II−/− mice are capable of weak hybridoma stimulation, although these mice do not show Vβ4 expansion after MHV-68 infection. Of relevance to this point is our finding that there were activated B cells in the MHC class II−/− mice 14 days after infection, although reduced compared with the B6 mice, but there was no evidence for B cell activation at this time point in the CD4−/− or CD4 Ab-depleted mice.

The finding that spleen cells from infected MHC class II−/− mice can stimulate T cell hybridomas in vitro, but not drive expansion of naive Vβ4+CD8+ T cells in vivo, raises the possibility that CD4+ T cells are also required as helper cells for Vβ4+CD8+ T cell expansion, in addition to their role in generation of the ligand. CD4+ T cell help might only be required in vivo in situations of reduced levels of latency, and correspondingly low levels of stimulatory ligand.

It should be emphasized that, in contrast to the essential role for B cell activation in the establishment of EBV latency (29), B cells do not appear to be required for MHV-68 latency. Latency can be established in B cell-deficient mice, and cells other than B cells can be latently infected (4, 5, 30). In addition, there is no conservation of latency-associated genes between the two viruses (31). The current studies do not address requirements for the establishment of latency, but do suggest that activated B cells may be a requirement for Vβ4+CD8+ T cell expansion. As Vβ4+CD8+ T cell expansion has also been linked to early stages in the establishment of latency, further characterization of latent gene expression in activated B cells is clearly important.

Expansion of Vβ4+CD8+ T cells is prevented whether CD4+ T cells are depleted at the time of infection or 7 days after infection. Taking the current results together with our previously published report showing that CD4+ T cell depletion at day 11 did not affect Vβ4 expansion (8), the data suggest that Vβ4 expansion becomes independent of CD4+ T cells sometime between 7 and 11 days of infection, just before the major increase in levels of latency that peaks at 14 days. Again, this is consistent with the hypothesis that Vβ4+CD8+ T cells are responding to a ligand expressed during latent infection, and that CD4+ T cells are required for ligand expression.

It should be noted that, despite the lack of Vβ4+CD8+ T cell expansion, there was still substantial CD8+ T cell activation in the CD4-deficient animals. This is consistent with data showing that Vβ4+ T cells are only one component of the CD8+ T cell lymphocytosis in the peripheral blood. Although there is not a relative increase in the percentage of T cells bearing TCR Vβ elements other than Vβ4, there is generalized CD8+ T cell activation indicated by the characteristic CD44high, CD62Llow, PNAhigh phenotype. This may represent both nonspecific activation driven by the cytokine-rich milieu of the spleen during the infection (22), and specific activation, driven by latent Ags (32) (E. Usherwood and D. L. Woodland, manuscript in preparation) or late lytic Ags (9, 10). The presence of CD8+ T cells in the peripheral blood reactive to latent or lytic viral epitopes would be consistent with recent studies in EBV (33, 34).

MHV-68 infection elicits high levels of IFN-γ-secreting CD4+ T cells (22, 23, 24). However, the present studies showed no reduction of Vβ4+CD8+ T cell expansion in IFN-γ−/− mice, indicating that IFN-γ is not functioning in the CD4 requirement for Vβ4+CD8+ T cell expansion. Although high levels of IFN-γ are induced during MHV-68 infection, analysis of MHV-68-infected IFN-γ−/− mice showed that IFN-γ was not essential for recovery from acute intranasal infection, and there appeared to be little effect on splenomegaly and the establishment of viral latency (35). In contrast, disruption of the characteristic splenomegaly was observed in IFN-γR-deficient mice, characterized by infiltration of the spleen with granulocytes, reduced numbers of CD4+ and CD8+ T cells and B cells, and a 10–100-fold increase in levels of latent virus (36). The reasons for differences in the two models are unclear, but it was suggested previously that the IFN-γR might act as the receptor for another cellular or viral cytokine (36).

Previous studies have shown that expression of the Vβ4+CD8+ T cell stimulatory ligand correlates with the peak of splenic latency as measured by the infective center assay, consistent with the hypothesis that a viral or cellular gene product expressed during latency is driving the unusual T cell activation (11). In the current study, we have shown that the striking expansion of Vβ4+CD8+ T cells in the peripheral blood of MHV-68-infected mice is dependent on CD4+ T cells. It is our hypothesis that CD4+ T cells are required for optimal expression of the stimulatory ligand, perhaps due to a requirement for CD4+ T cell help for B cell activation leading to establishment of a particular pattern of latency. In addition, CD4+ T cells may also be important for conventional T cell help for Vβ4+CD8+ T cells, particularly in cases in which there is suboptimal expression of the ligand. Whether Vβ4+CD8+ T cells are stimulated by a ligand expressed exclusively on B cells, whether quantitative and/or qualitative differences in latency control their stimulation, and the identification of the stimulatory ligand are currently under investigation.

Acknowledgments

We thank Drs. Edward Usherwood and Charles Hardy for helpful discussions and critical evaluation of the manuscript, Dr. Jan Christensen for anti-CD4 ascites, Phuong Nguyen for technical assistance, and Mahnaz Paktinat for help with the flow cytometry.

Footnotes

  • ↵1 This work was supported by National Institutes of Health Grants AI42927 (M.A.B.) and P30 CA21765 (CORE Grant), and the American Lebanese Syrian Associated Charities.

  • ↵2 Address correspondence and reprint requests to Dr. Marcia A. Blackman, Department of Immunology, St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105. E-mail address: marcia.blackman{at}stjude.org

  • ↵3 Abbreviations used in this paper: MHV-68, murine gammaherpesvirus-68; PNA, peanut agglutinin.

  • Received April 30, 1999.
  • Accepted July 7, 1999.

References

  1. ↵
    Sunil-Chandra, N. P., S. Efstathiou, A. A. Nash. 1993. Interactions of murine gammaherpesvirus 68 with B and T cell lines. Virology 193: 825
    OpenUrlCrossRefPubMed
  2. ↵
    Sunil-Chandra, N. P., S. Efstathiou, A. A. Nash. 1992. Murine gammaherpesvirus 68 establishes a latent infection in mouse B lymphocytes in vivo. J. Gen. Virol. 73: 3275
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Usherwood, E. J., J. P. Stewart, K. Robertson, D. J. Allen, A. A. Nash. 1996. Absence of splenic latency in murine gammaherpesvirus 68-infected B cell-deficient mice. J. Gen. Virol. 77: 2819
    OpenUrlAbstract/FREE Full Text
  4. ↵
    Stewart, J. P., E. J. Usherwood, A. Ross, H. Dyson, T. Nash. 1998. Lung epithelial cells are a major site of murine gammaherpesvirus persistence. J. Exp. Med. 187: 1941
    OpenUrlAbstract/FREE Full Text
  5. ↵
    Weck, K. E., S. S. Kim, H. W. Virgin, S. H. Speck. 1999. Macrophages are the major reservoir of latent murine gammaherpesvirus 68 in peritoneal cells. J. Virol. 73: 3273
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Cardin, R. D., J. W. Brooks, S. R. Sarawar, P. C. Doherty. 1996. Progressive loss of CD8+ T cell-mediated control of a gamma-herpesvirus in the absence of CD4+ T cells. J. Exp. Med. 184: 863
    OpenUrlAbstract/FREE Full Text
  7. ↵
    Usherwood, E. J., A. J. Ross, D. J. Allen, A. A. Nash. 1996. Murine gammaherpesvirus-induced splenomegaly: a critical role for CD4 T cells. J. Gen. Virol. 77: 627
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Tripp, R. A., A. M. Hamilton-Easton, R. D. Cardin, P. Nguyen, F. G. Behm, D. L. Woodland, P. C. Doherty, M. A. Blackman. 1997. Pathogenesis of an infectious mononucleosis-like disease induced by a murine gamma-herpesvirus: role for a viral superantigen?. J. Exp. Med. 185: 1641
    OpenUrlAbstract/FREE Full Text
  9. ↵
    Stevenson, P. G., G. T. Belz, J. D. Altman, P. C. Doherty. 1999. Changing patterns of dominance in the CD8+ T cell response during acute and persistent murine gamma-herpesvirus infection. Eur. J. Immunol. 29: 1
    OpenUrlCrossRefPubMed
  10. ↵
    Liu, L., E. Flaño, E. J. Usherwood, S. Surman, M. A. Blackman, D. L. Woodland. 1999. Lytic cycle T cell epitopes are expressed in two distinct phases during MHV-68 infection. J. Immunol. 163: 868
    OpenUrlAbstract/FREE Full Text
  11. ↵
    Coppola, M. A., E. Flaño, P. Nguyen, C. L. Hardy, R. D. Cardin, N. Shastri, D. L. Woodland, M. A. Blackman. 1999. Apparent MHC-independent stimulation of CD8+ T cells in vivo during latent murine gammaherpesvirus infection. J. Immunol. 163: 1481
    OpenUrlAbstract/FREE Full Text
  12. ↵
    Kalams, S. A., B. D. Walker. 1998. The critical need for CD4 help in maintaining effective cytotoxic T lymphocyte responses. J. Exp. Med. 188: 2199
    OpenUrlFREE Full Text
  13. ↵
    Lanzavecchia, A.. 1998. Immunology: license to kill. Nature 393: 413
    OpenUrlCrossRefPubMed
  14. ↵
    Stevenson, P. G., G. T. Belz, J. D. Altman, P. C. Doherty. 1998. Virus-specific CD8+ T cell numbers are maintained during γ-herpesvirus reactivation in CD4-deficient mice. Proc. Natl. Acad. Sci. USA 95: 15565
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Ehtisham, S., N. P. Sunil-Chandra, A. A. Nash. 1993. Pathogenesis of murine gammaherpesvirus infection in mice deficient in CD4 and CD8 T cells. J. Virol. 67: 5247
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Stevenson, P. G., P. C. Doherty. 1999. Non-antigen-specific B-cell activation following murine gammaherpesvirus infection is CD4 independent in vitro but CD4 dependent in vivo. J. Virol. 73: 1075
    OpenUrlAbstract/FREE Full Text
  17. ↵
    Stevenson, P. G., R. D. Cardin, J. P. Christensen, P. C. Doherty. 1999. Immunological control of murine gammaherpesvirus independent of CD8+ T cells. J. Gen. Virol. 80: 477
    OpenUrlAbstract
  18. ↵
    Dalton, D. K., S. Pitts-Meek, S. Keshav, I. S. Figari, A. Bradley, T. A. Stewart. 1993. Multiple defects of immune cell function in mice with disrupted interferon-γ genes. Science 259: 1739
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Rahemtulla, A., W. P. Fung-Leung, M. W. Schilham, T. M. Kundig, S. R. Sambhara, A. Narendran, A. Arabian, A. Wakeham, C. J. Paige, R. M. Zinkernagel. 1991. Normal development and function of CD8+ cells but markedly decreased helper cell activity in mice lacking CD4. Nature 353: 180
    OpenUrlCrossRefPubMed
  20. ↵
    Grusby, M. J., R. S. Johnson, V. E. Papaioannou, L. H. Glimcher. 1991. Depletion of CD4+ T cells in major histocompatibility complex class II-deficient mice. Science 253: 1417
    OpenUrlAbstract/FREE Full Text
  21. ↵
    Sarmiento, M., A. L. Glasebrook, F. W. Fitch. 1980. IgG or IgM monoclonal antibodies reactive with different determinants on the molecular complex bearing Lyt 2 antigen block T cell-mediated cytolysis in the absence of complement. J. Immunol. 125: 2665
    OpenUrlAbstract
  22. ↵
    Sarawar, S. R., R. D. Cardin, J. W. Brooks, M. Mehrpooya, R. A. Tripp, P. C. Doherty. 1996. Cytokine production in the immune response to murine gammaherpesvirus 68. J. Virol. 70: 3264
    OpenUrlAbstract/FREE Full Text
  23. ↵
    Christensen, J. P., P. C. Doherty. 1999. Quantitative analysis of the acute and long-term CD4+ T-cell response to a persistent gammaherpesvirus. J. Virol. 73: 4279
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Christensen, J. P., R. D. Cardin, K. C. Branum, P. C. Doherty. 1999. CD4+ T cell mediated control of a γ-herpesvirus in B cell-deficient mice is mediated by IFN-γ. Proc. Natl. Acad. Sci. USA 96: 5135
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Rahemtulla, A., T. M. Kundig, A. Narendran, M. F. Bachmann, M. Julius, C. J. Paige, P. S. Ohashi, R. M. Zinkernagel, T. W. Mak. 1994. Class II major histocompatibility complex-restricted T cell function in CD4-deficient mice. Eur. J. Immunol. 24: 2213
    OpenUrlCrossRefPubMed
  26. ↵
    Grusby, M. J., L. H. Glimcher. 1995. Immune responses in MHC class II-deficient mice. Annu. Rev. Immunol. 13: 417
    OpenUrlCrossRefPubMed
  27. ↵
    Brooks, J. W., A. M. Hamilton-Easton, J. P. Christensen, R. D. Cardin, C. L. Hardy, and P. C. Doherty. 1999. Requirement for CD40 ligand, CD4+ T cells and B cells in an infectious mononucleosis-like syndrome. J Virol. In press.
  28. ↵
    Weck, K. E., S. S. Kim, H. W. Virgin, IV, S. H. Speck. 1999. B cells regulate murine gammaherpesvirus 68 latency. J. Virol. 73: 4651
    OpenUrlAbstract/FREE Full Text
  29. ↵
    Hurley, E. A., D. Thorley-Lawson. 1988. B cell activation and the establishment of Epstein-Barr virus latency. J. Exp. Med. 168: 2059
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Weck, K. E., M. L. Barkon, L. I. Yoo, S. H. Speck, H. W. Virgin, IV. 1996. Mature B cells are required for acute splenic infection, but not for establishment of latency, by murine gammaherpesvirus 68. J. Virol. 70: 6775
    OpenUrlAbstract/FREE Full Text
  31. ↵
    Virgin, H. W., P. Latreille, P. Wamsley, K. Hallsworth, K. E. Weck, A. J. Dal Canto, S. H. Speck. 1997. Complete sequence and genomic analysis of murine gammaherpesvirus 68. J. Virol. 71: 5894
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Husain, S. M., E. J. Usherwood, H. Dyson, C. Coleclough, M. A. Coppola, D. L. Woodland, M. A. Blackman, J. P. Stewart, J. T. Sample. 1999. Murine gammaherpesvirus M2 gene is latency-associated and its protein a target for CD8+ T lymphocytes. Proc. Natl. Acad. Sci. USA 96: 7508
    OpenUrlAbstract/FREE Full Text
  33. ↵
    Callan, M. F., N. Steven, P. Krausa, J. D. Wilson, P. A. Moss, G. M. Gillespie, J. I. Bell, A. B. Rickinson, A. J. McMichael. 1996. Large clonal expansions of CD8+ T cells in acute infectious mononucleosis. Nat. Med. 2: 906
    OpenUrlCrossRefPubMed
  34. ↵
    Callan, M. F., L. Tan, N. Annels, G. S. Ogg, J. D. Wilson, C. A. O’Callaghan, N. Steven, A. J. McMichael, A. B. Rickinson. 1998. Direct visualization of antigen-specific CD8+ T cells during the primary immune response to Epstein-Barr virus in vivo. J. Exp. Med. 187: 1395
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Sarawar, S. R., R. D. Cardin, J. W. Brooks, M. Mehrpooya, A. M. Hamilton-Easton, X. Y. Mo, P. C. Doherty. 1997. γ Interferon is not essential for recovery from acute infection with murine gammaherpesvirus 68. J. Virol. 71: 3916
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Dutia, B. M., C. J. Clarke, D. J. Allen, A. A. Nash. 1997. Pathological changes in the spleens of γ interferon receptor-deficient mice infected with murine gammaherpesvirus: a role for CD8 T cells. J. Virol. 71: 4278
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

The Journal of Immunology: 163 (6)
The Journal of Immunology
Vol. 163, Issue 6
15 Sep 1999
Print
Download PDF
Article Alerts
Email Article
Citation Tools

Jump to section