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The Journal of Immunology, 2001, 167: 4566-4573.
Copyright © 2001 by The American Association of Immunologists

Distinct CD8 T Cell Functions Mediate Susceptibility to Histoplasmosis During Chronic Viral Infection1

Betty A. Wu-Hsieh2,*,{ddagger}, Jason K. Whitmire3,{dagger},{ddagger}, Rici de Fries4,{ddagger}, Jr-Shiuan Lin*, Mehrdad Matloubian5,{ddagger} and Rafi Ahmed2,{dagger},{ddagger}

* Graduate Institute of Immunology, National Taiwan University College of Medicine, Taipei, Taiwan; {dagger} Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322; and {ddagger} Department of Microbiology and Immunology, University of California, Los Angeles, School of Medicine, Los Angeles, CA 90064


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has long been recognized that some viral infections result in generalized immune suppression. In acute infections, this period of suppressed immunity is relatively short. However, chronic infections associated with a prolonged period of immune suppression present far greater risks. Here, we examined the role of CD8 T cell responses following viral infection in immunity to systemic histoplasmosis. Although wild-type mice with systemic histoplasmosis were able to control the infection, those simultaneously infected with lymphocytic choriomeningitis virus clone 13 showed reduced immunity with greater fungal burden and high mortality. The immune suppression was associated with loss of CD4 T cells and B cells, generalized splenic atrophy, and inability to mount a granulomatous response. Removing the anti-viral CD8 T cells in the coinfected mice enabled them to reduce the fungal burden and survive the infection. Their lymphoid organs were replenished with CD4 T and B cells. In contrast to wild-type mice, perforin-deficient mice infected with lymphocytic choriomeningitis virus clone 13 and Histoplasma showed an absence of immunopathology, but the animals still died. These results show that CD8 T cells can suppress immunity through different mechanisms; although immunopathology is perforin-dependent, lethality is perforin-independent.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
It has long been recognized that some viral infections result in generalized immune suppression. The serious complications of measles are often due to secondary bacterial and viral infections (1, 2). It is now well established that suppressed immunity is a common feature of many viral infections (3). In acute infections, this period of suppressed immunity is relatively short. However, a chronic infection associated with a prolonged period of immune suppression presents far greater risks.

Lymphocytic choriomeningitis virus (LCMV)6 infection in mice is a useful model for studying the pathogenesis of virus-induced immune suppression. Immune suppression is manifested as defects in both cell-mediated and Ab responses. Immunocompetent adult mice infected with LCMV clone 13 succumbed to opportunistic infection by Histoplasma capsulatum (4). The animals fail to mount an effective cellular response and do not form characteristic granulomas in tissue. Adult mice infected with LCMV WE strain generate a reduced or no T cell-independent IgM and T cell-dependent IgG Ab response to a subsequent vesicular stomatitis virus infection (5). However, CD8 T cell-depleted LCMV WE-infected and congenital LCMV carrier mice are able to make a normal Ab response indicating that CD8 T cells mediate immune suppression.

Attempts were made to explore the mechanisms by which CD8 T cells mediate immune suppression. Specific staining revealed a loss of marginal zone macrophages and follicle dendritic cells in mice infected with LCMV (6, 7). Based on these results, it was proposed that LCMV-specific CD8 T cells mediate immune suppression by lysis of LCMV-infected APCs. Certainly, the loss of APCs can have a profound impact on the immune response. However, the question of how antiviral (activated) CD8 T cells directly affect other immune cells has not been addressed.

In response to infection, CD8 T cells differentiate into effector cells. The CD8 T cells exert their effector functions through perforin-mediated lysis of pathogen-infected targets or secretion of cytokines (8). Recent studies have revealed that through the production of perforin and IFN-{gamma}, CD8 T cells can also play an important role in immune regulation. Perforin can induce immunopathology, control the expansion and exhaustion of virus-specific CD8 T cells, kill allogenic T cells, and destroy macrophages (9, 10, 11, 12, 13, 14). IFN-{gamma} not only is required to clear some viral infections, but it can also control hemopoiesis and maintain the hierarchy of immunodominance (9, 13, 15).

Here, the roles of perforin and cytokine production in CD8 T cell function were examined in a coinfection model. We found that wild-type mice coinfected with LCMV clone 13 and H. capsulatum exhibited CD8 T cell-mediated immunopathology, which suppressed immunity to the fungal infection and resulted in death. In contrast, perforin-deficient mice with coinfection showed an absence of immunopathology but still died. These results show that CD8 T cells can suppress immunity through perforin-dependent and -independent mechanisms. Whereas immunopathology was perforin-mediated, lethality was perforin-independent.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Virus and mice

LCMV clone 13 was isolated from the spleen of a mouse infected at birth with the parent strain Armstrong CA 1371 (16). Viral titers were determined by plaque assay as previously described (16). BALB/cByJ, C57BL/6, and perforin-deficient mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Seven- to 9-wk-old adult mice were used in all experiments, except for carrier mice that were bred and infected with LCMV clone 13 at birth. Adult mice or neonates were injected with 1 x 106 PFU of LCMV clone 13 i.v. Carrier mice were made by injecting 1-day-old mice with LCMV clone 13; their susceptibility to Histoplasma was tested when they were 8 wk old. These neonatally infected carrier mice have high titers of virus in most of their organs (17).

Fungus

H. capsulatum strain 505 was used in coinfection experiments (4). Yeast phase of the fungus was maintained on blood-cysteine-glucose agar slant at 37°C for 72 h. Fresh cultures were used for each experiment. A sublethal dose of 2 x 105 yeast cells was given together with LCMV clone 13 at 1 x 106 PFU to mice i.v. as described above.

Antibody

Concentrated anti-CD8 Ab was prepared from the culture supernatant of hybridoma cell line 2.43 (American Type Culture Collection, Manassas, VA) by saturated ammonium sulfate precipitation. Before being used in experiments, all Ab preparations were tested in vivo for their ability to abrogate the development of a cytotoxic T lymphocyte response to LCMV Armstrong in adult mice. The Ab was administered by i.p. injection of 0.2 ml/inoculation/mouse on days 0 and 4 of infection.

Quantitation of fungal load in tissues

Homogenized suspensions of various tissues from infected mice were inoculated onto glucose-peptone agar plates. Mycelial colonies of H. capsulatum were counted after incubation at 30°C for 10 days.

Intracellular staining

To enumerate the number of IFN-{gamma}-producing cells, intracellular cytokine staining was performed as previously described (18). In brief, at day 8 of infection, 106 freshly explanted splenocytes and lymph node cells were cultured in flat-bottom 96-well plates. Cells were cultured in medium alone or stimulated with LCMV-specific NP118-126 peptide epitope (0.1 µg/ml) in RPMI 1640 medium containing recombinant human IL-2 (50 U/ml) for 6 h at 37°C in 6% CO2. Brefeldin A was added for the duration of the culture period to facilitate intracellular cytokine accumulation. After this period, cell surface staining with anti-CD8 Ab (clone 53-6.7) was performed, followed by intracellular IFN-{gamma} staining with anti-IFN-{gamma} Ab (clone XMG1.2) using the Cytofix/Cytoperm kit (BD PharMingen, San Diego, CA) in accordance with the manufacturer’s recommendations.

Flow cytometry and tetramer staining

MHC class I (H-2Ld) tetramers complexed with LCMV-NP118 peptide were produced as previously described (18). Freshly explanted splenocytes and lymph node cells (106) were stained in PBS containing 1% BSA (w/v) and 0.02% NaN3 using fluorochrome-conjugated Abs and allophycocyanin-conjugated LdNP118 tetramer. The Abs used were anti-CD8 (clone 53-6.7), anti-CD4 (clone RM4-5), anti-B220 (clone RA3-6B2), anti-CD44 (clone IM7), anti-CD62 ligand (clone MEL-14), anti-CD69 (clone H1.2F3), anti-CD11a (clone 2D7), and anti-CD25 (clone PC-61). All Abs were purchased from BD PharMingen. After staining, cells were fixed in PBS/1% paraformaldehyde, and acquired using a FACSCalibur flow cytometer (BD Biosciences, Mountain View, CA). Dead cells were excluded on the basis of forward and side light scatter. Data were analyzed using the computer program CellQuest (BD Biosciences).

Histology

Spleens were removed from mice and fixed in 10% buffered formalin. Paraffin-embedded spleens were sectioned and stained with H&E.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Mice with chronic viral infection are susceptible to opportunistic histoplasmosis

We have previously reported that infection of adult mice with LCMV clone 13 results in chronic infection associated with susceptibility to opportunistic histoplasmosis (4, 19). Mice infected with this viral isolate are unable to mount an effective immune response against Histoplasma and succumb to a sublethal dose of the fungus. To determine whether high titer of virus is the sole cause of increased susceptibility, mice infected with LCMV clone 13 as adults were compared with neonatally infected LCMV carrier mice that have high titers of virus in most of their organs. The results in Fig. 1Go show that despite a heavy virus load in the tissues, 10 of 10 carrier mice were able to survive the fungus infection. In striking contrast, 9 of 10 mice infected with LCMV clone 13 as adults died of histoplasmosis. These results show that factors in addition to virus infection are the cause of immune suppression as manifested by susceptibility to opportunistic infection.



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  		FIGURE 1. Neonatally infected LCMV carrier mice do not succumb to opportunistic histoplasmosis. Mice infected with LCMV clone 13 either at birth ({square}) or as adults ({blacksquare}) were given i.v. injection of yeast cells of Histoplasma at 8 wk of age. The number of mice surviving was recorded at 30 days after Histoplasma infection.

 
CD8 T cells cause immune suppression

The major biological difference between an adult carrier mouse infected with LCMV clone 13 at birth and a mouse infected with the virus as an adult is that the former is immunologically tolerant to the virus whereas the latter mounts a virus-specific CD8 T cell response (16). Adult mice coinfected with LCMV clone 13 and Histoplasma were tested for their ability to generate virus-specific responses. Fig. 2Go shows that CD8 T cells in the spleen of mice coinfected with the virus and the fungus as adults were activated at day 8 of infection. There were increased numbers of CD8 T cells expressing CD44high, CD62 ligandlow, CD69high, CD25, and CD11ahigh. Moreover, high percentages of CD8 T cells were IFN-{gamma} producers and LdNP118-126 reactive (Fig. 3Go). IFN-{gamma}-producing cells constituted 12.4% (6.8 x 105 in cell number) and 25% (1.3 x 105) splenic and lymph node CD8 T cells, respectively.



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  		FIGURE 2. CD8 T cells are activated in coinfected mice. Spleen cells from uninfected mice or from mice coinfected 8 days earlier with LCMV clone 13 and Histoplasma were stained with the indicated Abs and analyzed by flow cytometry. Numbers indicate the percentage of CD8 T cells expressing the activation phenotype.

 


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  		FIGURE 3. CD8 T cells in coinfected mice are reactive to LCMV peptide LdNP118-126. At 8 days postinfection, virus-specific CD8 T cells were measured in the spleen (A) and lymph nodes (B) of coinfected mice by tetramer staining. LdNP118-reactive cells were identified by surface staining with FITC-conjugated anti-CD8 Ab and allophycocyanin-conjugated LdNP118 tetramer. The ability of these cells to make IFN-{gamma} was determined by intracellular staining. Spleen cells (A) and lymph node cells (B) were cultured in the presence or absence of NP118-126 peptide and then stained for surface CD8 and intracellular IFN-{gamma} as described in Materials and Methods. Numbers shown indicate the percentage of CD8 T cells that were tetramer-positive or IFN-{gamma}-positive. Some of the NP 118-126-specific cells that have reduced tetramer levels are capable of making IFN-{gamma}.

 
To assess the role of CD8 T cells in mediating immune suppression, we used anti-CD8 Ab to deplete CD8 T cells from adult coinfected mice. This treatment depleted over 98% of CD8 T cells in the lymph nodes and 93% of CD8 T cells in the spleen. CD8 T cell depletion enabled coinfected mice to mount an anti-fungal immune response. The fungal burdens were reduced from log10 7.67 ± 0.29 to 6.87 ± 0.05 CFU/g (6.3-fold reduction) in the spleen and from log10 7.70 ± 0.21 to 7.04 ± 0.42 CFU/g (4.5-fold reduction) in the liver (Fig. 4Go). Most strikingly, anti-CD8 Ab treatment resulted in 100% survival of coinfected mice (Fig. 5Go). Although coinfected mice without Ab treatment succumbed as early as day 9 after infection, 100% of those with Ab treatment still survived at day 30. These data demonstrate that CD8 T cells in coinfected mice are the cause of immune suppression and susceptibility to histoplasmosis.



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  		FIGURE 4. Depletion of CD8 T cells lowers the number of fungi in spleen (A) and liver (B) of mice coinfected with LCMV clone 13 and Histoplasma. Six mice were included in each group.

 


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  		FIGURE 5. CD8 T cell-depleted mice survive coinfection. Coinfected mice were treated ({blacksquare}, n = 10) or not treated ({square}, n = 11) with monoclonal anti-CD8 Ab at days 0 and 4 of infection and then followed over time for survival.

 
Maintenance of lymphoid tissue cellularity in the absence of CD8 T cells

We then examined the changes in lymphoid organs to understand how CD8 T cells in coinfected mice affected the immune system. As shown in Fig. 6Go, coinfection with LCMV clone 13 and Histoplasma dramatically changed the gross splenic morphology. The spleen was small, hollow, and pale (Fig. 6GoB). However, when given anti-CD8 Ab, spleens of the coinfected mice became large in size and deep red in color (Fig. 6GoC), similar to the spleen of normal mice responding to Histoplasma infection (4, 20). In addition to splenomegaly, lymphadenopathy was also observed in these mice (data not shown). Histological pictures (Fig. 7GoB) show massive tissue destruction in spleens of coinfected mice. The spleens were necrotic and devoid of cells, and the follicular structure were unidentifiable. Most remarkably, depleting CD8 T cells in these mice restored the integrity of splenic structure (Fig. 7GoC). The spleen was replenished with cells, and the follicular structure could clearly be identified. Lymphoid cell replenishment is further documented in Table IGo. Coinfected mice had only one-half the number of cells in the spleen and one-fourth that in the lymph nodes as compared with a normal uninfected mouse. CD8 T cell depletion dramatically increased the total cell numbers in the lymphoid organs, which consisted of mostly CD4 T cells and B cells.



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  		FIGURE 6. Coinfected mice with CD8 T cell depletion exhibit splenomegaly. Spleens from uninfected (A), coinfected (B), and anti-CD8-treated coinfected mice (C) are shown. The scale is shown in centimeters.

 


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  		FIGURE 7. CD8 T cell depletion restores the structural integrity of splenic follicles. Spleen sections are from normal (A), coinfected (B), and coinfected with anti-CD8 Ab treatment (C). Spleen sections were stained with H&E. Magnification, x100.

 

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  		Table I. CD8 T cell depletion restores cellularity in lymphoid tissues of coinfected mice1

 
Analyzing CD4 T cells and B cells in coinfected mice after CD8 T cell depletion, we found that activated cells in both populations were greatly enhanced (Table IIGo). B cell blasts in the spleen of CD8 T cell-depleted coinfected mice were 4.3-fold higher than in undepleted coinfected mice. Activated CD4 T cells as assessed by expression of CD44high and CD11a phenotypes were 3.9- to 5.4-fold higher in the spleens and 18.4- to 16.2-fold higher in the lymph nodes when CD8 T cells were removed.


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  		Table II. CD4 T cells and B cells are activated in coinfected mice after T cell depletion1

 
Perforin deficiency ablates CD8 T cell-mediated tissue destruction

To delineate the role of CD8 T cell cytotoxic functions in destruction of lymphoid tissues, we compared coinfection of perforin-deficient mice with wild-type mice. Histological pictures in Fig. 8Go show that coinfection in perforin-deficient mice did not cause the splenic destruction or cellular depletion that was seen in the C57BL/6 control mice. These data indicate that the cytotoxic functions of CD8 T cells mediated tissue destruction and cellular depletion.



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  		FIGURE 8. Perforin-deficient coinfected mice exhibit intact splenic follicular structure. Spleen sections are from wild-type C57BL/6 (A) and perforin-deficient (B) mice coinfected with LCMV clone 13 and Histoplasma. Spleen sections were stained with H&E. Magnification, x100.

 
Perforin-deficient mice still succumb to coinfection

Interestingly, although perforin-deficient mice escaped tissue destruction, they still succumbed to coinfection like their wild-type counterparts (Table IIIGo). The kinetics of death was also similar with both groups dying between days 11 and 13 after infection. Depleting CD8 T cells rescued both the wild-type and perforin-deficient mice. These data demonstrate that in this coinfection model CD8 T cells mediate lethality and tissue destruction/cellular depletion by two different mechanisms. Although tissue destruction/cellular depletion was mediated by perforin-dependent cytotoxic functions, lethality was perforin-independent.


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  		Table III. Perforin-deficient mice succumb to coinfection

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we showed that LCMV clone 13-infected mice succumbed to concurrent Histoplasma infection and exhibited massive tissue destruction and cellular depletion. Although the coinfected mice had functionally active CD8 T cells, eliminating these cells restored lymphoid tissue cellularity and allowed the animals to control the fungal infection and survive. By use of perforin-deficient mice, we delineated the mechanisms of CD8-mediated immune suppression. Our data show that CD8 T cell-mediated tissue destruction and cellular depletion are perforin-dependent, whereas CD8 T cell-mediated lethality is perforin-independent.

Immune activation as a cause for immunopathology and exacerbation of disease has been shown in different models. In a peripheral tolerance model, adoptive transfer of CD8 T cells from LCMV TCR transgenic mice into LCMV Ag transgenic mice expressing glycoprotein epitope 33-41 led to induction of peripheral tolerance (21). When recipient mice were challenged with a secondary infection or a proinflammatory stimulus, tolerance was prevented and CD8 T cells become activated and expanded. The vigorous T cell response led to reduction of CD4 T and B cell counts, immunopathology, weight loss, and death (21). In SIV-infected rhesus monkeys, Mycobacterium bovis bacillus Calmette-Guérin (BCG) coinfection enhanced viral pathogenicity and accelerated SIV-induced disease (22). BCG coinfection not only enhanced a decline of CD4 T cell counts in the peripheral blood but also increased viral replication, both of which correlated with T cell activation. The animals developed T cell activation-related toxic shock syndrome. Activation-induced sensitivity to secondary challenge was also shown in a mouse model of acute LCMV-Armstrong infection (23). During acute LCMV infection, mice were much more sensitive to the lethal effects of LPS. Dysregulated cytokine production by NK cells, CD4 T cells, and CD8 T cells was responsible for the disease. The results reported here extend these observations to a situation where live viral infection makes a sublethal fungal infection lethal.

LCMV clone 13, a macrophage and endothelial variant of LCMV, causes persistent infection with depressed but detectable CTL activity (16, 24). Infected animals are not able to clear the infection, and virus titer is high in all tissues (19). Although T cell-mediated immunopathology is observed in these mice, mortality is rarely seen (25). Mortality is also rarely seen in mice given Histoplasma at the doses used here. A sublethal infection in an immune competent mouse can be cleared within 4–5 wk (26). In response to Histoplasma infection, CD4 and CD8 T cells and NK cells are activated (26, 27, 28). However, animals depleted of CD4 T cells but not of CD8 T cells succumb to sublethal dose of infection (27, 28, 29). Although single infection of either LCMV clone 13 or Histoplasma alone does not cause overwhelming disease, mice coinfected with LCMV clone 13 and Histoplasma manifested immune activation-related immune suppression. Animals experienced drastic weight loss of up to 30% of body weight before they died (data not shown).

Immunopathology in coinfected mice was evident by the complete absence of splenic follicles and dramatic cell loss (Fig. 7Go and Table IGo). A role for perforin in CD8 T cell-mediated immunopathogenesis has been implicated in different models. In viral infections, perforin is of critical importance in CTL-mediated viral clearance (10, 13, 30, 31). Besides its antiviral function, perforin is also known to regulate the exhaustion of activated antiviral T cells and T cell-mediated immunopathology (9, 13, 32). In animals immunized with staphylococcal enterotoxin B-epitope peptide and given a rechallenge, perforin was also responsible for the regulation of the expansion and persistence of CD8 effector T cells (11). Perforin as an effector molecule was involved in CTL-mediated killing of macrophages in a mouse pancreatitis model (14). In our coinfection model, mice with perforin deficiency were able to maintain lymphoid tissue cellularity and structural integrity in the face of coinfection. These results indicate that coinfection enhances CD8 T cell cytotoxic functions and causes immunopathology via a perforin-dependent mechanism.

CD4 down-regulation as a result of infection has been observed in several viral infections. Human herpesvirus-7 (HHV-7) infection of SupT1 lymphoblastoid CD4 T cell line induces drastic loss of total CD4 protein. Both intracellular and surface CD4 molecules are lost, but the loss of surface CD4 is more dramatic (33). The suppressive effect of HHV-7 infection was found to be on CD4 transcription (33). The down-modulation of cell surface CD4 is by a process involving a variety of viral gene products (34, 35). HIV-1 infection down-regulates CD4 at different levels, including the internalization of surface CD4, reduction of CD4 transcript levels, impaired translation of CD4 mRNA, formation of CD4-gp160 intracellular complexes and HIV-1 Vpu gene-mediated degradation of CD4 (35). In SIV-infected cells, the Nef gene product mediates the degradation of a newly synthesized CD4 protein in an acidic cellular compartment (34). A common feature of these three viruses is their selective tropism for CD4 T cells, and the CD4 molecule has a central importance to their life cycles. LCMV clone 13 is macrophage-tropic and does not infect lymphocytes efficiently (32). Our data suggest that the mechanism of coinfection-induced CD4 T cell down-regulation is different from that observed for HHV-7, HIV-1, and SIV. Instead of being an event directly related to virus tropism for CD4 T cells or to the expression of viral gene products, perhaps cells producing factors important for CD4 T cell homeostasis were destroyed by perforin-secreting CD8 T cells. Alternatively, it could be that Fas ligand expression by CD8 T cells following LCMV infection induced these cells to undergo apoptosis (36).

Recently, Dalton’s laboratory showed that IFN-{gamma} plays a critical role in suppressing CD4 T cell proliferation and inducing ex vivo apoptosis. They showed that CD4 T cells are the disease-mediating cells in progressive and fatal experimental autoimmune encephalomyelitis in IFN-{gamma}- or IFN-{gamma} receptor-deficient mice. Activated CD4 T cells in the spleen and CNS of IFN-{gamma}-deficient mice proliferate more and undergo decreased apoptosis compared with those in the wild-type mice (37). Furthermore, they showed that during BCG infection of IFN-{gamma}-deficient mice, the CD4 T cells expanded markedly to comprise 30–50% of total lymphocytes in the spleen and liver. There was also evidence that IFN-{gamma}-deficient mice fail to induce apoptosis of activated CD4 T cells during BCG infection resulting in accumulation of activated CD4 T cells (38). Here we show active IFN-{gamma} production by CD8 T cells and a specific loss of CD4 T cells that depends on CD8 T cells. It is possible that IFN-{gamma} regulates the proliferation and apoptosis of activated CD4 T cells in our coinfection model as it does in the experimental autoimmune encephalomyelitis and BCG infection models.

Although it is clear that perforin was critically important in immunopathology and cellular depletion in the LCMV clone 13 and Histoplasma coinfection model, it is also apparent that perforin was only partially responsible for CD8 T cell-mediated immune suppression. The perforin-deficient mice succumbed to coinfection even in the absence of immunopathology (Table IIIGo and Fig. 8Go). This suggests that cytokine or some other molecule produced by CD8 T cells is responsible for mortality. It has been documented that LCMV infection induces cytokines, most prominently IFN-{gamma} and TNF-{alpha} (18, 24, 39). The same set of cytokines is also produced in mice infected with Histoplasma. The ability of the host to clear the fungus is dependent on its competence in producing these cytokines (40, 41, 42, 43, 44, 45). Our ongoing work has shown that in Histoplasma-infected mice CD8 T cells, in addition to CD4 T cells, are vigorous producers of IFN-{gamma} (J.-S. Lin and B. A. Wu-Hsieh, unpublished observations, and Ref. 26). There are a number of reports showing that hyperstimulation of the immune system by concurrent infection and biological insults leads to exaggerated expression of cytokines. One of these showed that mice given a challenge of LPS during LCMV-Armstrong infection died of cytokine-mediated disease and IFN-{gamma} was important for lethality (23). Our unpublished data show that spleen cells of coinfected mice produce at least 2-fold higher levels of IFN-{gamma} than cells from the mice with Histoplasma infection alone. Although it is possible that IFN-{gamma} causes the immune suppression, it is difficult to test this directly. Infection of IFN-{gamma} knockout mice or mice depleted of IFN-{gamma} by Ab with Histoplasma results in death (45). Our data indicate that IFN-{gamma} likely serves a protective role at physiological concentrations induced by Histoplasma infection alone but causes immune suppression at higher levels induced by coinfection. These results support the conclusion drawn from studies of concurrent LPS challenge with LCMV infection that the normally protective cytokines in single infections work synergistically in causing cytokine-mediated disease in coinfection (23).

In summary, we demonstrated in LCMV clone 13 and Histoplasma coinfection model that CD8 T cell-mediated immune suppression was the sum of disparate functions of CD8 T cells. Perforin was responsible for tissue destruction and cellular depletion, whereas animal lethality was perforin-independent, and possibly the result of cytokine-mediated disease.


   Acknowledgments
 
We acknowledge Sylvia Odesa for her excellent technical assistance. The contribution of Mary Brandt to this study is also gratefully acknowledged.


   Footnotes
 
1 This work was supported by funds from National Science Council, Republic of China Grants 88-2320-B-002 and 89-2320-B-002 (to B.A.W.-H.) and U.S. Public Health Service Grants AI 30048 and NS 21496 (to R.A.). Back

2 Address correspondence and reprint requests to Dr. Betty A. Wu-Hsieh, Institute of Graduate Immunology, National Taiwan University College of Medicine, No. 1 Jen-Ai Road, Section 1, Taipei, Taiwan, Republic of China; E-mail address: wuhsiehb{at}ha.mc.ntu.edu.tw; or Dr. Rafi Ahmed, Emory Vaccine Center and Department of Microbiology and Immunology, Emory University School of Medicine, G211 Rollins Research Center, 1510 Clifton Road, Atlanta, GA 30322. E-mail address: ra{at}microbio.emory.edu Back

3 Current address: Department of Molecular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121. E-mail address: jwhitmire{at}liai.org Back

4 Current address: Department of Immunology, University of Washington; 1959 NE Pacific Street, Seattle, WA 98195-7650. Back

5 Current address: Department of Microbiology and Immunology, University of California, Box 111R, Veteran Administration Medical Center, San Francisco, CA, 94143. Back

6 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; HHV-7, human herpesvirus-7; BCG, bacillus Calmette-Guérin. Back

Received for publication May 9, 2001. Accepted for publication August 23, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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