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The Journal of Immunology, 2005, 174: 7920-7928.
Copyright © 2005 by The American Association of Immunologists

Generation of Antifungal Effector CD8+ T Cells in the Absence of CD4+ T Cells during Cryptococcus neoformans Infection1

Dennis M. Lindell*,{dagger}, Thomas A. Moore*,{dagger}, Roderick A. McDonald*, Galen B. Toews* and Gary B. Huffnagle2,*,{dagger},{ddagger}

* Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine,{dagger} Immunology Graduate Program, and{ddagger} Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Immunity to the opportunistic fungus Cryptococcus neoformans is dependent on cell-mediated immunity. Individuals with defects in cellular immunity, CD4+ T cells in particular, are susceptible to infection with this pathogen. In host defense against a number of pathogens, CD8+ T cell responses are dependent upon CD4+ T cell help. The goal of these studies was to determine whether CD4+ T cells are required for the generation of antifungal CD8+ T cell effectors during pulmonary C. neoformans infection. Using a murine intratracheal infection model, our results demonstrated that CD4+ T cells were not required for the expansion and trafficking of CD8+ T cells to the site of infection. CD4+ T cells were also not required for the generation of IFN-{gamma}-producing CD8+ T cell effectors in the lungs. In CD4– mice, depletion of CD8+ T cells resulted in increased intracellular infection of pulmonary macrophages by C. neoformans, increasing the pulmonary burden of the infection. Neutralization of IFN-{gamma} in CD4–CD8+ mice similarly increased macrophage infection by C. neoformans, thereby blocking the protection provided by CD8+ T cells. Altogether, these data support the hypothesis that effector CD8+ T cell function is independent of CD4+ T cells and that IFN-{gamma} production from CD8+ T cells plays a role in controlling C. neoformans by limiting survival of C. neoformans within macrophages.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Cryptococcus neoformans is an encapsulated yeast that survives and replicates both intracellularly and extracellularly (1). In humans, cryptococcal pneumonia and disseminated cryptococcosis primarily affect immunocompromised hosts. Among these patient populations, CD4+ T cell deficiency results in markedly enhanced morbidity and mortality. Using murine models of pulmonary C. neoformans infection, our laboratory and others have demonstrated that both CD8+ and CD4+ T cells are essential for control of the fungus (2, 3, 4, 5, 6). However, it remains to be determined what role CD4+ T cells play in the development and regulation of CD8+ T cell immunity to C. neoformans.

The requirement for CD4+ T cell help for CD8+ T cell responses ranges from absolute to entirely dispensable, but the role of CD4+ T cells in the development of CD8+ T cell-mediated antifungal immunity has never been addressed. CD4+ deficiency can result in impaired CD8+ T cell activation and/or function during infection with some nonfungal pathogens (7, 8, 9). Originally, CD4+ T cell help was thought to occur via the production of cytokines. Based on studies of cross-presentation, it was proposed that CD4+ T cells were required for CD8+ T cell responses to exogenous Ags, but not endogenous ones. In this model, APCs become activated after engagement of CD40 by CD154 on activated CD4+ T cells (10, 11, 12). Similarly, a number of studies have found that ligation of CD40 can replace CD4+ T cell help for the development of CD8+ T cell responses (13, 14, 15, 16). Some studies have been consistent with this hypothesis, whereas others have not been. For instance, some viruses appear to bypass the requirement for CD4+ T cell help in the development of CD8+ T cell responses (17, 18). In murine Listeria monocytogenes infections (19), CD4+ T cell help via CD40L is dispensable for the CD8+ response to bacteria given i.v., but the mucosal CD8+ response is dependent on CD4+ T cells and CD40 (20). In many instances, microbes, through interactions with pattern recognition receptors such as TLR and mannose receptor, produce sufficient signals for CD8+ T cell priming by dendritic cells without CD4+ T cell help (21).

A number of intracellular pathogens including L. monocytogenes, Toxoplasma gondii, Mycobacterium tuberculosis, Trypanosoma cruzi, and Histoplasma capsulatum have evolved strategies to facilitate survival within macrophages. These mechanisms include escape from the phagosome, interfering with phagosome maturation and/or phagolysosomal fusion, and adaptations that allow growth within the phagolysosome (22, 23, 24). C. neoformans does not appear to use any of the strategies used by other microorganism for evasion of host immunity (1). C. neoformans is unusual among pathogenic fungi, however, in that it has a polysaccharide capsule, which accumulates within macrophages and disrupts function (25).

The objective of this study was to characterize the development of the CD8+ T cell response to C. neoformans in the lungs and secondary lymphoid tissues during CD4+ T cell deficiency to determine whether CD4+ T cells are required for the generation of a CD8+ T cell-mediated response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Female CBA/J mice (weight, 24 ± 4 g) were obtained from The Jackson Laboratory. Mice were housed under pathogen-free conditions in enclosed filter-topped cages. Clean food and water were given ad libitum. The mice were handled and maintained using microisolator techniques, with daily veterinarian monitoring. Bedding from the mice was transferred weekly to cages of uninfected sentinel mice that were subsequently bled at weekly intervals and found to be negative for Abs to mouse hepatitis virus, Sendai virus, and Mycoplasma pulmonis. All studies involving mice were approved by the University Committee on Use and Care of Animals at the University of Michigan.

C. neoformans

C. neoformans strain 52D was obtained from the American Type Culture Collection (ATCC 24067). For infection, yeasts were grown to stationary phase (48–72 h) at 34°C in Sabouraud dextrose broth (1% neopeptone and 2% dextrose; Difco) on an Adams Brand Nutator Mixer (BD Biosciences). The cultures were then washed three times in sterile nonpyrogenic saline (NPS),3 counted on a hemacytometer, and diluted to 3.3 x 105 CFU/ml in NPS. The precise number of organisms delivered was determined by CFU counts of inoculum plated on Sabaraud dextrose agar (Difco).

Intratracheal inoculation of C. neoformans

Mice were anesthetized by i.p. injection of ketamine (100 mg/kg; Fort Dodge Laboratories) and xylazine (6.8 mg/kg; Lloyd Laboratories) and were restrained on a small surgical board. A small incision was made through the skin over the trachea and the underlying tissue was separated. A 30-gauge needle was attached to a 1-ml tuberculin syringe filled with diluted C. neoformans culture. The needle was inserted into the trachea and 30 µl of inoculum (104 CFU) was dispensed into the lungs. The needle was removed and the skin was closed with cyanoacrylate adhesive. The mice recovered with minimal visible trauma.

T cell depletion using mAbs

Depletion of CD4+ and CD8+ T cell subsets was accomplished via i.p. administration of mAbs. Anti-CD4 (GK1.5, rat IgG2b) and anti-CD8{alpha} (YTS 169.04, rat IgG2b) (26) Abs were prepared from ascites by dilution in NPS and filtering through 0.45-µm syringe filter. Mice received 200 µg of GK1.5, both GK1.5 and YTS 169.04, or control rat IgG (Jackson ImmunoResearch Laboratories) in a volume of 200 µl of NPS at days –1 and 0 of infection, followed by 200 µg every 8 days. The efficiency of T cell depletion was assessed by flow cytometric analysis using Abs anti-CD4 (RM 4-4) and anti-CD8{beta} (53-5.8), which bind regions of CD4 and CD8 distinct from GK1.5 and YTS 169.04. Efficiency of T cell depletion in the lungs (CD4+ >98%, CD8+ >96%) and spleen (CD4+ >99%, CD8+ >99%) of mice was calculated by comparison of T cell numbers in treated mice with those in controls.

Lung and lymph node leukocyte isolation

Lungs from each mouse were excised, washed in PBS, minced, and digested enzymatically for 40 min in 15 ml/lung of digestion buffer (RPMI 1640, 5% FCS, 1 mg/ml collagenase (Boehringer Mannheim Biochemical) and 30 µg/ml DNase (Sigma-Aldrich)). After erythrocyte lysis using NH4Cl buffer, cells were washed, resuspended in complete medium, and centrifuged for 30 min at 2000 x g in presence of 20% Percoll (Sigma-Aldrich) to separate leukocytes from cell debris and epithelial cells. Total lung leukocyte numbers were assessed in the presence of trypan blue using a hemocytometer; viability was >85%. Subsets of isolated leukocytes (neutrophils, eosinophils, macrophages, and total lymphocytes) were determined by Wright-Giemsa staining of samples cytospun onto slides. Lung-associated lymph nodes (LALNs) were excised and cells were dispersed with the plunger of a 3-ml syringe. Erythrocytes were lysed using NH4Cl buffer, and cells were resuspended in complete medium.

Flow cytometry

For surface staining alone, leukocytes were washed and resuspended at a concentration of 107 cells/ml in FA Buffer, Dried (Difco) + 0.1% NaN3, and FcRs were blocked by the addition of anti-CD16/32 (Fc block; BD Pharmingen). After Fc receptor blocking, 106 cells were stained in a final volume of 120 µl in 12 x 75 polystyrene tubes (BD Pharmingen) for 20 min at 4°C. Leukocytes were stained with the following mAbs, per manufacturer’s instructions: CD4 (RM4-4 and H129.19), CD8{alpha} (5H10-1), CD8{beta} (53-5.8), CD11c (HL3), {alpha}{beta}TCR (H57-597), CD25 (7D4), CD44 (IM7), and CD69 (H1.2F3) (BD Pharmingen). Cells were washed twice with FA buffer, resuspended in 100 µl, and 200 µl of 4% formalin was added to fix the cells. A minimum of 20,000 events were acquired on a FACSCalibur flow cytometer (BD Pharmingen) using CellQuest software (BD Pharmingen). To minimize the possibility of contamination by other CD8{alpha}+ expressing cells (such as a subset of dendritic cells), lymphocytes were analyzed through a low forward light scatter, low side light scatter gate, which reliably excludes CD11c+ dendritic cells. In addition to CD8{alpha}, some experiments were conducted using anti-CD8{beta} Abs, with no difference in the number of CD8+ cells detected. Greater than 98% of CD8{alpha}+ cells from lung leukocyte preparations were CD8{beta}+ and TCR{alpha}{beta}+. For activation markers (i.e., CD25, CD44, CD69, CD62L, CD45RB), gates were set based on positive (splenocytes cultured with high-dose PMA (50 ng/ml) and ionomycin (500 ng/ml)) and negative (isotype) controls.

Intracellular flow cytometry

Leukocytes were cultured for 12 h at 2 x 106 cells/ml in 12-well plates in the presence of 0.1 µg/ml soluble anti-CD3 with or without 0.1 µg/ml anti-CD28. Brefeldin A or monensin (in the form of Golgi-stop or Golgi-block) were added for the last 4 h of culture per manufacturer’s instructions (BD Pharmingen). Nonadherent cells were harvested, washed twice with FA buffer, and incubated briefly with Fc block. After 10-min incubation at 4°C, staining for cell surface molecules was done as described above. Cells were washed of excess surface stains, fixed, permeabilized using Cytofix/Cytoperm (BD Pharmingen), and stained using anti-IFN-{gamma} (XMG1.2) or anti-TNF-{alpha} (MP6-XT22) in permeabilization buffer (FA buffer + 0.1% saponin (Sigma-Aldrich)) buffer at 4°C for 30 min. Flow cytometry was performed as for surface staining above, except that >50,000 events per sample were collected. The specificity of cytokine staining was tested by comparing staining of experimental samples with a minimum of two of three negative controls: 1) isotype control, 2) excess unlabelled Ab, and/or 3) preincubation of Ab with recombinant cytokine.

Proliferation

Cells were assayed for proliferation using an in vitro fluorescence-based assay. Briefly, 2 x 106 cells from the various organs were stained with 5 µM CFSE (Molecular Probes) in PBS 5% FCS for 7 min at room temperature. Cells were washed several times to remove excess CFSE and were cultured for 3 days in the presence or absence of anti-CD3 Abs (0.1 µg/ml). A minimum of 20,000 events were acquired on a FACSCalibur flow cytometer (BD Pharmingen) using CellQuest software (BD Pharmingen).

Statistical analysis

All values are means ± SEM, unless otherwise indicated. Differences between two means were evaluated using the Student t test (assuming unequal variance where dictated by F test). Differences between three means were evaluated using a one-way ANOVA, followed by Dunnett’s post test. p values <0.05 were considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The role of CD4+ T cells in the expansion and trafficking of CD8+ T cells

To determine the role of CD4+ T cells in the development of the CD8+ T cell response to C. neoformans, mice were treated with CD4-depleting mAbs and the effect on the expansion and trafficking of CD8+ T cells was quantified. The efficiency of CD4+ T cell depletion was >98% in the lungs and >99% in the spleen. LALNs are typically undetectable in uninfected mice housed under specific pathogen-free conditions and develop in response to the pulmonary infection. In control (CD4+) mice, the increase in the number of CD8+ T cells in the LALN paralleled the increase in lymph node size, increasing from wk 0–2 postinfection (Fig. 1A). With the exception of a modest delay at wk 1 postinfection, the increase in LALN CD8+ T cell number was not diminished in CD4-deficient (CD4–) mice (Fig. 1A). In the lungs, CD8+ T cell numbers in CD4+ mice displayed similar kinetics for increased CD8+ T cell numbers as that seen in the LALN (Fig. 1B). Whereas similar CD8+ T cell numbers were present in CD4+ and CD4– mice at wk 1 and 2, an exaggerated CD8+ T cell response was found in the lungs at wk 4 postinfection compared with CD4+ controls (Fig. 1B). We compared the proliferative capacity of CD8+ T cells from the spleen, LALN, and lungs at wk 2 postinfection using an in vitro CFSE-based proliferation assay. After 3 days in culture, 55.6% of spleen CD8+ T cells had undergone at least one cell division and 76.3% of LALN CD8+ T cells had undergone at least one cell division, whereas only 1.1% of lung CD8+ T cells had divided. Therefore, expansion of CD8+ T cells in the lungs largely represents trafficking, rather than proliferation of CD8+ T cells within the lungs. Thus, CD4+ T cells were not required for the expansion and trafficking of CD8+ T cells during pulmonary C. neoformans infection.



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  		FIGURE 1. Role of CD4+ T cells in the expansion of CD8+ T cells during pulmonary C. neoformans infection. Absolute CD8+ T cell numbers in the LALN (A) and lungs (B) of infected control and CD4– mice at wk 1, 2, and 4 postinfection. The wk 0 time point represents T cell numbers before infection. Each time point represents the mean of 8–10 animals ± SEM. Data are from three independent experiments. *, p < 0.05; {dagger}, p < 0.0001.

 
The role of CD4+ T cells in the activation status of CD8+ T cells

To determine whether CD4+ T cells played a role in the activation of CD8+ T cells during C. neoformans infection, CD8+ T cells from the LALN and lungs of CD4– and CD4+ mice were assayed by flow cytometry for the expression of various activation markers. LALNs are undetectable in uninfected, specific pathogen-free mice. In response to the pulmonary infection, LALNs increase dramatically in size during the first week of infection. In CD4+ mice, low frequencies of CD8+ T cells expressing CD25+, CD69+, CD62Llow, or CD45RBlow were found in the LALNs throughout the course of the infection, although a modest up-regulation of the activation marker CD69 was observed between wk 1 and 2 (Fig. 2). Similarly, in CD4– mice, low frequencies of CD8+ T cells expressing activations markers were found in the LALNs throughout the course of the infection (Fig. 2). The activation status of CD8+ T cells in the LALNs of CD4+-deficient mice was not impaired relative to that in CD4+ mice. At wk 2 postinfection, LALN CD8+ T cells from CD4– mice had a trend toward higher frequencies of CD44, CD69, and CD62Llow than in CD4+ mice (Fig. 2). Thus, little activation of CD4+ or CD8+ T cells occurred in the LALN, and the activation of CD8+ T cells in the LALN was unchanged in the absence of CD4+ T cells.



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  		FIGURE 2. Role of CD4+ T cells in the activation of CD8+ T cells. Activation marker expression on CD8+ T cells from the lungs and LALN of CD4– and control CD4+ mice at 1, 2, and 4 wk postinfection. The expression of CD44, CD25, CD69, CD62L, and CD45RB was determined by flow cytometry of lymph nodes and whole lung dispersions, as described in Materials and Methods. Data points represent the mean of three to five mice per time point, with error bars representing the SEM. Data are from three independent experiments. The wk 0 time point for LALN is not shown because the number of cells present in LALN of uninfected animals was insufficient for analysis. *, p < 0.05.

 
In contrast with the relatively low levels of activation markers detected in the LALN, a much larger proportion of the CD8+ T cells in the lungs of both groups of mice expressed an activated phenotype (Fig. 2). In the lungs of CD4+ mice, the frequency of CD44high CD8+ T cells increased during the course of infection. In CD4– mice, as early as wk 2 postinfection, a significantly higher proportion of CD8+ T cells were CD44high (Fig. 2). CD8+ T cells from the lungs of CD4+ mice up-regulated CD25 during the first 2 wk of the infection, rising to a peak of ~20% (Fig. 2). At this one time point, a small decrease in the frequency of CD25+CD8+ T cells was found in the lungs of CD4– mice. In contrast, however, no decrease in the frequencies of CD8+ T cells expressing CD69, CD62Llow, or CD45RBlow was found in the lungs of CD4– mice at any time point (Fig. 2). In fact, a trend toward higher frequencies of activated phenotype CD8+ T cells in CD4– mice predominated. Therefore, during C. neoformans infection, the activation of T cells occurred in the lungs, and CD4+ T cells were not required for the activation of CD8+ T cells.

The role of CD4+ T cells in the development of IFN-{gamma}-producing CD8+ effector T cells in the lungs

To assess the effector function of CD8+ T cells in the lungs of CD4+ and CD4– mice, we determined the frequency of IFN-{gamma}-secreting CD8+ T cells by intracellular flow cytometry. Lymphocytes were recovered from the lungs after enzymatic digest, as described in Materials and Methods. The frequency of IFN-{gamma}+CD8+ T cells in the lungs of both groups of mice tended to increase with time (Fig. 3B). The frequency of IFN-{gamma}+CD8+ T cells in the lungs ranged from 5 to 10% for both groups at wk 1 and 2, with the percentage reaching 15% in the CD4– group at wk 4 (Fig. 3B).



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  		FIGURE 3. Role of CD4+ T cells in the generation of IFN-{gamma}-producing (A–C) and TNF-{alpha}-producing (D–F) CD8+ T cell effectors. Lung leukocytes from whole lungs were isolated from CD4+ and CD4– mice, as described in Materials and Methods, and were cultured in the presence of low-dose anti- CD3 and anti- CD28 Abs. The production of IFN-{gamma} and TNF-{alpha} by CD8+ T cells was assayed using intracellular flow cytometry. A, Representative dot plots from wk 4 postinfection. The percentage of IFN-{gamma}+CD8+ T cells (B) was multiplied by the number of CD8+ T cells in the lungs to determine the absolute number of IFN-{gamma}+CD8+ T cells per lung (C). D, Representative dot plots from wk 2 postinfection. The percentage of TNF-{alpha}+CD8+ T cells (E) was multiplied by the number of CD8+ T cells in the lungs to determine the absolute number of TNF-{alpha}+CD8+ T cells per lung (F). Time points were generated using pooled samples of three to five mice per time point. Data are representative of three independent experiments.

 
When these data are analyzed for the absolute number of IFN-{gamma}+CD8+ cells, there is clearly a marked increase in IFN-{gamma}+CD8+ cells in CD4– mice compared with CD4+ mice at wk 4 postinfection (Fig. 3C). Thus, CD4+ T cells were not required for the development of IFN-{gamma}-producing CD8+ T cells in the lungs of C. neoformans-infected mice. We also investigated the requirement for CD4+ T cells in the generation of TNF-{alpha}-producing CD8+ T cell effectors. Similarly, the ability of lung CD8+ T cells to produce TNF-{alpha} was unimpaired in the absence of CD4+ T cells (Fig. 3, D–F). In summary, these studies demonstrated that, during pulmonary C. neoformans infection, CD8+ T cells trafficked to the lungs, were activated, and acquired effector function independently of CD4+ T cells. Therefore, the remainder of our studies focused on the role and function of CD8+ T cells during CD4+ T cell deficiency.

The contribution of CD8+ T cells that develop in the absence of CD4+ T cells to controlling growth of C. neoformans in the lungs

The next objective was to determine whether CD8+ T cells that develop in the absence of CD4+ T cells during pulmonary C. neoformans infection play a role in controlling the growth of the pathogen. Previous studies demonstrated that CD4+ T cells are required for optimal clearance of C. neoformans, but the absence of CD8+ T cells in CD4– mice results in progressive fungal growth (2, 3, 6). Consistent with previous studies, CD4–CD8+ mice did not clear the infection, and the pulmonary burden remained greater than 105 CFU at wk 4 postinfection (Fig. 4A). However, the combination of CD4+ and CD8+ T cell deficiencies resulted in progressive growth of C. neoformans in the lungs (Fig. 4A). As early as wk 2 postinfection, a significant difference in lung CFU was found between CD4–CD8+ and CD4–CD8– mice (Fig. 4A). By wk 4 postinfection, greater than 30-fold more organisms were present in the lungs of CD4–CD8– mice than in CD4–CD8+ mice (Fig. 4A). Thus, during CD4+ T cell deficiency, CD8+ T cells contribute significantly to the clearance of C. neoformans from the lungs.



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  		FIGURE 4. Role of CD8+ T cells in pulmonary fungal clearance and recruitment of lung leukocytes in CD4– mice. Pulmonary fungal burden (A) and whole lung leukocyte numbers (B) in the lungs of CD4–CD8+, CD4–CD8–, and CD4+CD8+ mice at wk 1–4 postinfection were assessed as described in Materials and Methods. Each data point represents the mean ± SEM of five to six mice. Data are from two independent experiments. *, p < 0.05 vs both CD4–CD8+ and CD4+CD8+; {dagger}, p < 0.05 vs CD4+CD8+.

 
The contribution of CD8+ T cells that develop in the absence of CD4+ T cells to lung leukocyte recruitment

The next objective was to determine what role CD8+ T cells that develop in the absence of CD4+ T cells play in leukocyte recruitment during pulmonary C. neoformans infection. At wk 2 postinfection, CD4–CD8+ mice were impaired in their ability to recruit leukocytes to the lungs compared with CD4+CD8+ mice (Fig. 4B). CD4–CD8– mice, however, had a near-complete abrogation of the lung leukocyte recruitment observed in CD4–CD8+ mice (Fig. 4B). This defect was consistent across several cell types, including macrophages, small monocytes/lymphocytes, and neutrophils (Fig. 5A). By wk 4 postinfection, CD4–CD8– mice had similar total numbers of leukocytes in their lungs, but the leukocyte infiltrate contained a significantly larger proportion of neutrophils, compared with the leukocyte infiltrate in CD4–CD8+ mice (Fig. 5B). The importance of early leukocyte recruitment for containing the pathogen was apparent from histological analysis. In the lungs of CD4–CD8+ mice at wk 2 postinfection, cryptococci were typically surrounded by mononuclear cells (Fig. 6A). In contrast, in the lungs of CD4–CD8– mice, far fewer mononuclear cells were present and many clusters of cryptococci continued to grow beyond the edges of inflammatory foci, suggesting a lack of containment of the infection (Fig. 6B). Thus, in CD4–CD8+ mice, CD8+ T cells mediate the early pulmonary leukocyte recruitment in response to C. neoformans infection.



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  		FIGURE 5. Role of CD8+ T cells in recruitment of various lung leukocyte subsets in CD4– mice. Lung leukocyte differentials from the lungs of CD4– and CD4–CD8– mice at wk 2 (A) and 4 (B) postinfection. The absolute numbers of the various leukocyte subsets were determined by differential counts of enzymatically digested whole lungs, as described in Materials and Methods. Each data point represents the mean ± SEM of six mice. Data are from two independent experiments. *, p < 0.05.

 


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  		FIGURE 6. Histological analysis of C. neoformans-infected lungs from CD4–CD8+ (A and C) and CD4–CD8– (B and D) mice. Photomicrographs are of H&E-stained lung sections from mice 2 wk postinfection. Note that in CD4–CD8+ mice (A), clusters of cryptococci are surrounded by a primarily mononuclear infiltrate. In contrast, clusters of cryptococci are present outside the perimeter of inflammatory foci, suggesting a lack of containment of the infection. Macrophages from the lungs of CD4–CD8+ mice are more vacuolar and foamy (C), whereas macrophages in the lungs of CD4–CD8– mice are less vacuolar, with many more intracellular C. neoformans. A and B, x100 magnification; C and D, x400 magnification.

 
Intracellular infection of macrophages during CD4/CD8 T cell deficiency

C. neoformans is a pathogen that survives and replicates both extracellularly and intracellularly. To determine whether CD8+ T cells contributed to control of the intracellular infection, we examined histologic lung sections from CD4–CD8+ and CD4–CD8– mice. A much larger proportion of macrophages in the lungs of CD4–CD8– mice had intracellular cryptococci (Fig. 6D), which were not present in the lungs of CD4–CD8+ mice (Fig. 6C).

Histologic analysis demonstrated that growth of cryptococci in the lungs of CD4–CD8– was poorly contained, and macrophages in the lungs of CD4–CD8– mice had many intracellular C. neoformans. From histologic sections, however, it is difficult to determine the boundaries of individual macrophages, as well as determine whether yeast lie inside or outside of these boundaries. Additionally, many of the macrophages in the lungs of CD4–CD8– mice are multinucleated giant cells and, thus, quantifying the number of macrophages is subjective. For these reasons, we chose to use cytospins of cell preparations from enzymatically digested lungs for quantitative analysis of intracellular fungal burden. We counted the number of intracellular C. neoformans per 100 macrophages in cytospins of lung leukocytes from CD4–CD8+ and CD4–CD8– mice. Isolated macrophages from CD4–CD8– mice had higher numbers of intracellular cryptococci than macrophages from CD4–CD8+ mice (Fig. 7). The histology and analysis of isolated macrophages both demonstrate that CD8+ T cells play a role in limiting the intracellular persistence of C. neoformans in macrophages.



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  		FIGURE 7. Role of CD8+ T cells in limiting intracellular infection of macrophages in CD4– mice. Macrophages were recovered from the lungs of CD4– and CD4–CD8– mice at wk 2 post-C. neoformans infection. The numbers of intracellular C. neoformans were determined from cytospins of leukocyte aliquots from enzymatically digested lungs, as described in Materials and Methods. Data are from two independent experiments, with a total of six mice per group. *, p < 0.01.

 
Role of efferent/effector phase IFN-{gamma} in the clearance of C. neoformans from the lungs of CD4-deficient mice

The previous results indicated that CD8+ T cells that arose in the absence of CD4+ T cells could produce IFN-{gamma} and played a significant role in control of C. neoformans growth in the lungs. The next objective was to determine the role of efferent IFN-{gamma} production in the clearance of C. neoformans from the lungs of CD4–CD8+ mice. CD4–CD8+ mice were given an intratracheal challenge of C. neoformans, and the infection was allowed to progress for 2 wk. Beginning at wk 2 postinfection, mice were given either control rat Ig (IFN-{gamma}+) or IFN-{gamma} neutralizing Ab (IFN-{gamma}–), as described in Materials and Methods. This protocol was chosen to focus on the effects of IFN-{gamma} in the effector phase of the response, while minimizing effects on the afferent phase. At wk 3 postinfection (wk 1 post-IFN-{gamma} neutralization), similar numbers of C. neoformans were present in the lungs of CD4–CD8+IFN-{gamma}+ mice and CD4–CD8+IFN-{gamma}– mice (Fig. 8). By wk 4 postinfection, however, CD4–CD8+IFN-{gamma}+ mice had begun to clear C. neoformans from the lungs, whereas lung CFU in CD4–CD8+IFN-{gamma}– mice remained high (Fig. 8). The observed difference in lung CFU was not due to a defect in the recruitment of lung leukocytes in CD4–IFN-{gamma}– mice (data not shown). Thus, IFN-{gamma} plays a significant role in the clearance of C. neoformans during the effector phase of pulmonary immunity in CD4–CD8+ mice.



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  		FIGURE 8. Role of IFN-{gamma} in controlling the growth of C. neoformans in the lungs of CD4– mice. Pulmonary fungal burden in CD4–CD8+IFN-{gamma}+ and CD4–CD8+IFN-{gamma}– mice at wk 3 and 4 was assessed as described in Materials and Methods. Each data point represents the mean ± SEM of eight mice. Data are from two independent experiments. *, p < 0.05.

 
Intracellular infection of macrophages during IFN-{gamma} deficiency

The previous results demonstrated that IFN-{gamma} plays a role in the efferent/effector phase of the pulmonary immune response to C. neoformans, independent of lung leukocyte recruitment. The next objective was to determine whether, similar to CD8+ T cell deficiency, IFN-{gamma} deficiency would result in increased intracellular survival of C. neoformans in lung macrophages of CD4–CD8+ mice. To determine the intracellular fungal burden in macrophages from the lungs of CD4–CD8+IFN-{gamma}+ and CD4–CD8+IFN-{gamma}– mice, we counted the number of intracellular cryptococci in macrophages from cytospins of lung leukocytes at wk 3 postinfection (wk 1 post-IFN-{gamma} neutralization). Compared with control CD4–CD8+IFN-{gamma}+ mice, macrophages from CD4–CD8+IFN-{gamma}– mice had higher numbers of intracellular cryptococci (Fig. 9). These macrophages came from lungs at wk 3 postinfection, where no significant difference in lung CFU was present. It may be argued that the increased frequency of intracellular cryptococci in macrophages in CD4–CD8– mice may have been due to a difference in fungal burden in the lungs (Fig. 4). The increased intracellular survival of C. neoformans during IFN-{gamma} deficiency demonstrates, however, that differences in macrophage parasitism exist in lungs with similar fungal burden. Thus, during CD4+ T cell deficiency, IFN-{gamma} plays a role in limiting intracellular survival of C. neoformans in lung macrophages.



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  		FIGURE 9. Role of IFN-{gamma} in limiting intracellular infection of macrophages in CD4– mice. Macrophages were recovered from the lungs of CD4–IFN-{gamma}+ and CD4–IFN-{gamma}– mice at wk 3 post-C. neoformans infection. The numbers of intracellular C. neoformans were determined from cytospins of leukocyte aliquots from enzymatically digested lungs, as described in Materials and Methods. Data are from two independent experiments, with a total of eight mice per group. *, p < 0.01.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In the current study, we report that CD4+ T cells were not required for the CD8+ T cell-mediated immune response to the encapsulated fungus C. neoformans. CD8+ T cells expanded and trafficked to the lungs in the absence of CD4+ T cells. CD8+ T cells acquired activation markers with similar magnitude and kinetics in the absence of CD4+ T cell help. Differentiation of CD8+ T cells to IFN-{gamma}-producing effectors in the lungs occurred without CD4+ T cell help. The CD8+ T cells generated in CD4+-deficient mice conferred a significant degree of protection against C. neoformans infection. Depletion of CD8+ T cells from CD4– mice resulted in enhanced parasitism of macrophages in the lungs. Similar to depletion of CD8+ T cells, Ab-mediated neutralization of IFN-{gamma} also impaired clearance of C. neoformans from the lungs of CD4– mice. Neutralization of IFN-{gamma} also led to increased macrophage parasitism in the lungs of CD4– mice. These data demonstrate that protective CD8+ T cell-mediated immunity to C. neoformans is generated without the requirement for CD4+ T cell help and support the hypothesis that protection by CD8+ T cells in the absence of CD4+ T cells is mediated via the production of IFN-{gamma}.

Our results demonstrated that CD8+ T cell activation and expansion occurred during fungal infection in the absence of CD4+ T cells. In a variety of experimental systems, CD4+ T cell deficiency results in impaired CD8+ T cell function (9, 27, 28, 29, 30, 31, 32, 33), though the nature of help provided by CD4+ T cells remains controversial. The requirement for CD4+ T cell help in CD8+ T cell responses can depend upon whether Ag is presented via the endogenous (classical) or exogenous (cross-presentation) pathways (34). The exogenous pathway is dependent upon CD4+ T cells via CD40-CD154 interactions with APCs (10, 11, 12, 35). Other signals, such as type I IFNs (36), CD40 signaling (13, 14), or direct microbial stimulation via pattern recognition receptors (19), can bypass the requirement for CD4+ T cells in the response to exogenous Ags. Because C. neoformans is a facultative intracellular pathogen that survives in various intracellular compartments (1), our data indicating CD4+ T cell-independent CD8+ T cell activation suggest that both endogenous and exogenous Ag presentation pathways likely take place during C. neoformans infection.

Another paradigm of CD4+ T cell help for CD8+ T cells suggests that CD8+ T cell responses to acute infections are often independent of CD4+ T cell help, but maintenance of long-term responses depends on CD4+ T cell help (30, 37, 38, 39). Our studies found that, at 4 wk postinfection, CD8+ T cell numbers in the lungs (Fig. 1B) and the number of IFN-{gamma}-producing effectors (Fig. 3C) are enhanced in the absence of CD4+ T cells. These results suggest that the CD8+ T cell response to C. neoformans is maintained in the absence of CD4+ T cell help.

In the absence of CD4+ T cells, CD8+ T cells were not deficient in trafficking to the lungs, but instead reached the lungs in exaggerated numbers. During HIV infection, up to 60% of patients develop CD8+ T cell lymphocytic alveolitis (40, 41, 42). In animal models, CD4– mice infected with Pneumocystis carinii also develop pulmonary CD8+ lymphocytosis (43). A common theme from each of these situations is CD4+ T cell deficiency combined with concurrent infection. Other evidence suggests that effector molecules play a role in modulation of the CD8+ T cell response. After infection with L. monocytogenes, IFN-{gamma}–/– mice exhibit increased expansion and delayed contraction of the Ag-specific CD8+ T cell response (44). This is even more pronounced in the context of additional perforin deficiency (44). Similarly, pulmonary C. neoformans infection in IFN-{gamma}–/– BALB/c mice leads to hyperexpansion of CD8+ T cells in the lungs (G.B.H., unpublished observations). Thus, during infection with C. neoformans or other pathogens, CD4+ T cells and effector molecules can limit the expansion of CD8+ T cells.

Our results demonstrated that IFN-{gamma}-producing effector CD8+ T cells in the lungs were generated in the absence of CD4+ T cells. The role of CD4+ T cells in the generation of IFN-{gamma}-producing CD8+ T cell effectors during pulmonary M. tuberculosis infection has been investigated. Similar to the results reported here, CD8+ T cells in M. tuberculosis-infected CD4–/– mice acquired an activated phenotype with similar kinetics as CD8+ T cells in wild-type mice (45). Additionally, the ability of CD8+ T cells from the lungs to make IFN-{gamma} was similarly unimpaired (45). The consequences of depleting CD8+ T cells from these mice were not addressed. Pulmonary C. neoformans infection shares some similarities with M. tuberculosis infection. Both are facultative intracellular pathogens that can establish long-term latent infection (46, 47). In CD4+ mice, the majority of IFN-{gamma}+ T cells in the lungs are CD4+ T cells, and not CD8+ T cells.4 In CD4– mice, the absolute number of IFN-{gamma}-producing T cells in the lungs approximates that in CD4+ T cell-sufficient mice; however, IFN-{gamma} production comes from the CD8+ T cell compartment, which has a higher number of IFN-{gamma}+ cells than in CD4+ mice. Thus, our results demonstrate that IFN-{gamma}-producing CD8+ T cell effectors were generated during pulmonary fungal infection in the absence of CD4+ T cells.

In this study, we report that CD8+ T cells provide substantial protection against pulmonary C. neoformans infection in a CD4– host. Previous studies in our laboratory, as well as others, have demonstrated that CD8+ T cells can play a protective role in pulmonary C. neoformans infection (2, 3, 6, 48). CD8+ T cells play a protective role in immunity to H. capsulatum infection in both perforin-dependent and perforin-independent manners (49, 50). CD8+ T cells play a protective role against pulmonary paracoccidioidomycosis (51). In the absence of CD4+ T cells, CD8+ T cells have been shown to play a protective role in host defense against other pathogenic fungi. In murine P. carinii infection of CD4+-deficient mice, depletion of CD8+ T cells dramatically increases susceptibility (43). CD4+ T cell-independent CD8-mediated vaccine immunity to Blastomyces dermatitidis and H. capsulatum has also been reported (52). Although CD4– mice had impaired clearance relative to controls (Fig. 4), CD4–CD8– mice were dramatically more susceptible to pulmonary C. neoformans infection. Thus, during C. neoformans infection, CD8+ T cells, which develop in the absence of CD4+ T cells, play a critical role in controlling fungal growth.

Our studies support the concept that, during CD4+ T cell deficiency, CD8+ T cells recognize C. neoformans-infected macrophages and produce IFN-{gamma} in response to intracellular infection. This CD8+ T cell-derived IFN-{gamma}, in turn, acts on the macrophage to decrease intracellular survival of C. neoformans. The production of IFN-{gamma} is associated with protection against a variety of fungal pathogens including Penicillium marneffei (53), H. capsulatum (54, 55), B. dermatitidis (56), Paracoccidioides brasiliensis (57), and Candida albicans (58, 59). IFN-{gamma} depletion during C. neoformans infection in CD4+CD8+ mice leads to enhanced susceptibility and mortality (60), and exogenous IFN-{gamma} enhances protection (61, 62).

Mice genetically deficient in IFN-{gamma} (G. B. Huffnagle, unpublished observations) or IFN-{gamma}R (63) are dramatically more susceptible to pulmonary C. neoformans infection. In the lungs of CD4+CD8+ mice, the majority of T cell-derived IFN-{gamma} is produced by CD4+ T cells.4 The results presented here demonstrate that even when the most prominent source of IFN-{gamma} is absent (in CD4– mice), IFN-{gamma} still plays a critical role in the efferent response to pulmonary C. neoformans infection.

IFN-{gamma}-mediated activation of macrophages is a common mechanism of antifungal immunity. Preincubation of macrophages with IFN-{gamma} results in NO-dependent antifungal activity against C. albicans, C. neoformans, and P. brasiliensis (57, 64, 65, 66). In vivo, inducible NO synthase mRNA and systemic NO release are up-regulated at the onset of cryptococcal clearance in resistant mice (67). Inflammatory cells recovered from the lungs inhibit C. neoformans growth in vitro in an NO-dependent manner (67). Treatment of mice with either anti-IFN-{gamma} neutralizing Abs or the NO synthesis inhibitor NG-L-monomethyl arginine blocks clearance of C. neoformans (67). Our data demonstrate increased parasitism of macrophages by C. neoformans 1) during neutralization of IFN-{gamma} or 2) in the absence of IFN-{gamma}-producing effector CD8+ T cells. Thus, these results demonstrate in vivo the importance of CD8+ T cells and IFN-{gamma}-mediated macrophage activation to control C. neoformans infection and show that anti-C. neoformans effector CD8+ T cells can be generated in the absence of CD4+ T cells.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants R01-HL065912 (to G.B.H.), R01-AI059201 (to G.B.H.), R01-HL051082 (to G.B.T.), R01-AI049448 (to T.A.M.), and T32-AI07413 (to D.M.L.) and by a Department of Veterans Affairs Merit Grant (to G.B.T.). Back

2 Address correspondence and reprint requests to Dr. Gary B. Huffnagle, Pulmonary and Critical Care Medicine, 6301 MSRB III, University of Michigan Medical Center, Ann Arbor, MI 48109-0642. E-mail address: ghuff{at}umich.edu Back

3 Abbreviations used in this paper: NPS, nonpyrogenic saline; LALN, lung-associated lymph node. Back

4 D. M. Lindell, T. A. Moore, R. A. McDonald, G. B. Toews, and G. B. Huffnagle. Distinct compartmentalization of CD4+ T cell effector function versus proliferative capacity during pulmonary cryptococcosis. Submitted for publication. Back

Received for publication November 23, 2004. Accepted for publication March 30, 2005.


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