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The Journal of Immunology, 2001, 167: 5845-5851.
Copyright © 2001 by The American Association of Immunologists

The Capsular Polysaccharides of Cryptococcus neoformans Activate Normal CD4+ T Cells in a Dominant Th2 Pattern1

Geisy M. Almeida*, Regis M. Andrade* and Cleonice A. M. Bento2,{dagger}

* Programa de Imunobiologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; and {dagger} Instituto de Microbiologia e Parasitologia, Universidade do Rio de Janeiro, Rio de Janeiro, Brazil


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Capsular components of Cryptococcus neoformans induce several deleterious effects on T cells. However, it is unknown how the capsular components act on these lymphocytes. The present study characterized cellular and molecular events involved in immunoregulation of splenic CD4+ T cells by C. neoformans capsular polysaccharides (CPSs). The results showed that CPSs induce proliferation of normal splenic CD4+ T cells, but not of normal CD8+ T or B lymphocytes. Such proliferation depended on physical contact between CPSs and viable splenic adherent cells (SAC) and CD40 ligand-induced intracellular signal transduction. The absence of lymphoproliferation after fixation of SAC with paraformaldehyde has discarded the hypothesis of a superantigen-like activation. The evaluation of a cytokine pattern produced by the responding CD4+ T lymphocytes revealed that CPSs induce a dominant Th2 pattern, with high levels of IL-4 and IL-10 production and undetectable inflammatory cytokines, such as TNF-{alpha} and IFN-{gamma}. Blockade of CD40 ligand by relevant mAb down-regulated the CPS-induced anti-inflammatory cytokine production and abolished the enhancement of fungus growth in cocultures of SAC and CD4+ T lymphocytes. Our findings suggest that CPSs induce proliferation and differentiation of normal CD4+ T cells into a Th2 phenotype, which could favor parasite growth and thus important deleterious effects to the host.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cryptococcus neoformans is an important encapsulated fungal pathogen that causes cryptococcosis in patients with impaired cell-mediated immunity such as those with AIDS (1). The fungus enters the host via inhalation and establishes itself in the lungs, where it can produce either a local subclinical infection or an invasion through various organs, which frequently results in lethal meningitis (2). Several reports have demonstrated the importance of cell-mediated immunity in protection against C. neoformans (3, 4, 5, 6). Clinical and murine model studies have indicated that the clearance of pulmonary infection requires the presence of both CD4+ and CD8+ T cells (7, 8, 9). However, splenic CD4+ T cells appear to be the main determinant of protection to disseminated cryptococcosis (10, 11). Both lymphocyte subsets may have direct effects against yeast cells or act indirectly by stimulating anticryptococcal activity of effector cells, including cytotoxic cells and phagocytes (NK cells, neutrophils, and macrophages) (12, 13, 17, 18).

The cloned murine helper CD4+ T (Th) lymphocytes can be divided into functional subsets on the basis of the immunoregulatory cytokines (14). Thus, Th1 clones are characterized by production of IL-2, IFN-{gamma}, and TNF-{beta} and mediate delayed-type hypersensitivity responses. Th2 cells produce IL-4, IL-5, IL-6, and IL-10 and are largely responsible for B cell maturation and Ig isotype switching (14). Th0 clones, which can produce both Th1- and Th2-type cytokines, have been also identified (15). Th1 T cells, which secrete proinflammatory cytokines, are crucial for effective anticryptococcal response. Several studies have associated the Th1 cytokines IFN-{gamma} and IL-2 with resistance to C. neoformans infection (5, 6, 16). Other essential cytokines for establishment of resistance to C. neoformans include TNF-{alpha}, IL-12, and IL-18 (10, 17). The latter two cytokines seem to work synergistically to enhance IFN-{gamma} production by NK cells and facilitate the development of Th1 lymphocytes, which in turn produce more IFN-{gamma} and IL-12 (6, 18).

The fungal capsule is clearly the major virulence factor of C. neoformans (19, 20, 21). It is well established that the circulating levels of cryptococcal capsular Ags directly correlate with disease progression in patients and in experimental animals with disseminated cryptococcosis (1). Gadesbusch (22) described an "immunological paralysis"-like phenomenon after injection of a large dose of C. neoformans capsular polysaccharides (CPSs)3 in mice. Murphy and Cozad (23) found that low doses of cryptococcal CPSs injected i.v. induce a cascade of suppressor splenic T cells that secrete factors that down-regulate humoral and cellular anticryptococcal response. C. neoformans-derived CPSs reduce the production of proinflammatory cytokines and elicit the production of other cytokines that have been associated with inhibitory effects on the anticryptococcal response. These glycans inhibit TNF-{alpha} secretion from LPS-stimulated human monocytes (21) while inducing IL-10 production by these cells in vitro (21, 25). This anti-inflammatory cytokine, when added to a mixture of infected human monocytes and normal T lymphocytes, inhibits lymphoproliferation. Added to macrophage cultures, it affects IL-12 secretion and expression of class II MHC molecules (6, 21, 25, 26). In this study, we characterized the cellular and molecular events involved in the activation of the murine splenocytes induced by C. neoformans-derived CPSs.


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

Male BALB/c mice, 6 to 8 wk of age, were obtained from Oswaldo Cruz Institute (Instituto Oswaldo Cruz-Fundaçao Oswaldo Cruz, Rio de Janeiro, Brazil) animal facility and used as a source of normal splenocytes.

C. neoformans strains

The encapsulated form of C. neoformans (strain 444) of serotype A, obtained from an AIDS patient with cryptococcal meningitis, and the nonencapsulated mutant Cap 67 (54) of serotype D were kindly provided by Dr. L. Mendonça-Previato (Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil).

CPS purification and biochemical treatments

C. neoformans wild-type strain was maintained on Sabouraud dextrose agar at 4°C, and yeast cells were grown for use for 5 days in a chemically defined liquid medium (27) at 37°C in a shaker at 100 rpm. The cells were then collected by centrifugation (7000 x g, 14 min, 4°C) and washed three times with cold 150 mM NaCl (pH 7.0). To purify the CPSs, washed yeasts (~250 g wet weight) were suspended in 250 ml 40 mM citrate buffer (pH 7.0), and the suspension was autoclaved for 90 min at 121°C. After cooling, the supernatant was recovered by centrifugation at 6000 x g for 15 min at 4°C. The resulting pellet was suspended in 400 ml 20 mM citrate buffer, and the extraction procedure was performed again. The CPSs were isolated from vacuum-concentrated combined supernatants by precipitation with 3 volumes of ethanol at 4°C. The pellet was dissolved in water, some insoluble material was removed by centrifugation (6000 x g for 15 min at 4°C), and the supernatant (CPSs) was lyophilized. The CPSs were dissolved in HBSS, irradiated (10,000 rad) and stored at -20°C.

To evaluate the possible influence of nonpolysaccharide contaminants of CPSs on the activation of CD4+ T lymphocyte, our CPS samples were solubilized in 20 mM Tris-HCl buffer (pH 7.4) and digested exhaustively by Pronase E from Streptomyces griseus (Merck, Darmstadt, Germany) for 3 h at 37°C; by DNase 1 (Sigma, St. Louis, MO) in 20 mM Tris-HCl (pH 7.5), containing 1 mM MgCl2 and 1 mM MnCl2 for 3 h; or by RNase A from Sigma (previously heated at 80°C for 30 min to inactivate DNase) in 150 mM NaCl (pH 7.0) at 37°C for 3 h.

Finally, the contribution of the polysaccharide part in our system was evaluated by successive sodium periodate (IO4-) oxidation and sodium borohydride reduction of CPSs, as follows. The CPSs were treated with an excess of aqueous sodium periodate overnight at 4°C, in dark and the reaction was stopped by the addition of ethylene glycol. The product was reduced with sodium borohydride, and after 1 h the solution dialyzed.

Preparation of murine cells

Splenocytes obtained from normal mice were depleted of RBC by treatment with Tris-buffered ammonium chloride. This cellular preparation was washed three times in HBSS (Life Technologies, Gaithersburg, MD), counted, and suspended in medium containing DMEM (Life Technologies), 5% heat-inactivated FCS. To obtain splenic adherent cells (SAC), whole splenocytes, 2.5 x 106/well in 48-well vessels (Corning Glass Works, Corning, NY) in 500 µl complete medium (DMEM supplemented with 10% FCS, 2 mM glutamine, 5 x 10-5 M 2-ME, 10 µg/ml gentamicin, sodium pyruvate, MEM nonessential amino acids, and 10 mM HEPES), were cultured at 37°C and 7% CO2 in humid atmosphere. After 4 h incubation, nonadherent cells were discarded, and adherent cells were washed, leaving ~2.5 x 105 SAC/well. To obtain B lymphocytes, suspensions of single spleen cells were washed three times with DMEM plus 10% FCS and treated with a mixture of culture supernatants of anti-T cell Abs (anti-Thy-1, anti-CD4, and anti-CD8) (28) for 30 min on ice. This was followed by treatment with Low Tox-M rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada) in the presence of tissue culture fluid containing the anti-rat {kappa}-chain mAb MAR 18.5 (1% ascites) at 37°C for 45 min. The B cell fractionation was performed as previously described (29). Briefly, discontinuous Percoll gradient (Pharmacia, Uppsala, Sweden; at 70%, with density of 1.086 g/ml) was used to separate B cells. A suspension of splenic B cells obtained as described above was layered on the cold Percoll and spun at 1900 x g for 15 min. Flow cytometry analysis of the B cell preparations showed 85–90% of B220+ cells and a contamination <3% with residual (CD3+) T cells. The CD4+ T cells were obtained by two sequential passages through nylon wool columns, followed by complement-mediated cytotoxicity, as described (30). Briefly, enriched T cells (20–50 x 106/ml) obtained from columns were treated with a saturating dose of anti-CD8 mAb 53.6.7 for 30 min at 4°C, washed, and treated with anti-rat Ig mAb MAR 18.5 (1% ascites) plus 10% Low Tox-M rabbit complement for 45 min at 37°C. Viable cells were recovered after centrifugation and counted by trypan blue exclusion for each individual well in hemocytometer. Mean viable cell recovery in control unstimulated cultures was taken as reference. By flow cytometry, 92–97% of the resulting T cells were CD4+, and 1–3% were CD8+.

Determination of fungus burden

At the indicated days of culture infection with C. neoformans (Cap 67 strain), the whole content of each individual well (500 µl) was gently pipeted up and down and transferred to a synthetic fungus-specific medium (CDC-2500). After growth for 10 days at 37°C, viable fungi were identified in the counting chamber. This CDC-2500 medium is composed by D-glucose (40 g/L), KH2PO4·H2O (1.36 g/L), urea (1.29 g/L), sodium glutamate (1 g/L), MgSO4·7H2O (0.3 g/L), thiamine-HCl (2 mg/L), and biotin (10 µg/ml). Results are presented as mean ± SEM of Cap 67 cell number in triplicate cultures.

Cell proliferation assay

Primarily, whole splenocytes (1.5 x 106/ml) were cultured in the presence or absence of crescent doses of the CPSs (0.3 to 60 µg/ml) in 0.2 ml complete medium. In other experiments, CD4+ T cell (1.5 x 106/ml), in the presence or absence of B lymphocytes (1.5 x 106/ml), were set up with or without 30 µg/ml CPSs. All cultures were established in 96-well flat-bottom microtiter (Corning Glass), incubated for 3 days at 37°C and 7% CO2 in a humid incubator. The cocultures containing CD4+ T cells (1.5 x 106/ml) with SAC (5.0 x 105/ml) were kept under the same conditions in the presence of 30 µg/ml CPSs, biochemically treated or not, in 48-well vessels (500 µl). To evaluate whether CPSs work as a superantigen (SAg), SAC monolayers were fixed with 1% (v/v) paraformaldehyde solution, and, as positive control, we used an optimal dose (2 µg/ml) of Staphylococcus aureus enterotoxin B (SEB), a classical SAg (Sigma). For blocking experiments, murine cells were cultured in the presence of saturating doses of various mAbs. Anti-CD8 (30% v/v of supernatants of 53.6.7 clone) and/or anti-CD4 (4 µg/ml GK1.5 clone) were added to each well. In some wells, to evaluate the effect of costimulator molecules in proliferation assay, saturating doses (10 µg/ml) of the anti-B7.1 and/or anti-B7.2, anti-CD40 ligand (CD40L), rat (IgG2a) or hamster (IgG) control isotypes were used. The cultures were pulsed with 0.5 µCi/well [3H]TdR (Sigma); 16 h later, the cells were harvested using a cell-harvesting device. The amount of [3H]TdR incorporated into DNA was assessed by liquid scintillation spectroscopy. All cultures were done in triplicate. The SEM were 10% of the mean cpm values. Results are presented as the difference between CPS-treated and control (medium alone) mean cpm values.

Abs and other reagents

Anti-CD8 mAb 53.6.7 was purchased from BD PharMingen (La Jolla, CA). Anti-CD4 mAb GK 1.5 and anti-rat Ig {kappa}-chain mAb MAR 18.5 were gifts from Dr. Ethan Shevach (National Institutes of Health, Bethesda, MD). Anti-TCR{beta} mAb H57.597 (33) was a gift from Dr. M. Bélio (Instituto de Microbiologia Paulo de Góes, Universidad Federal do Rio de Janeiro, Rio de Janeiro, Brazil). All mAbs used in culture were obtained from BD PharMingen, as follows: anti-B7.1 (CD80) mAb 1G10, anti-B7.2 (CD86) mAb GL1, anti-CD40L (CD154) mAb MR1, hamster IgG (4B3), or rat IgG1 (R3-34) mAbs as control isotypes. Primary and biotinylated secondary mAb from BD PharMingen were also used in cytokine assay.

Cytokine determination

Supernatant fluids (500 µl/well) were collected for cytokine determination from cultures containing SAC (5.0 x 105/ml) with or without CD4+ T splenocytes (1.5 x 106/ml) that were submitted to different treatments. The supernatants were collected 72 h after the treatments and submitted to evaluations of cytokine contents by sandwich ELISA, according to the protocol provided by the manufacturer, using two cytokine-specific mAbs for each cytokine (rabbit anti-mouse TNF-{alpha} and rat anti-mouse IFN-{gamma}, IL-4, and IL-10), one of which was biotinylated (BD PharMingen). The reaction was revealed with alkaline phosphatase-conjugated streptavidin (Southern Biotechnology Associates, Birmingham, AL), using p-nitrophenyl phosphate (Sigma) as substrate. Recombinant murine IL-4, IFN-{gamma}, TNF-{alpha}, and IL-10 (BD PharMingen), ranging from 1 to 50 ng/ml were used to construct standard curves.

Statistical analysis

The Student t test for paired means was applied to all data reported in this article. The experimental means were compared with control means for each experiment.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
C. neoformans CPSs induce proliferative response of normal murine CD4+ T cells by a mechanism dependent on splenic adherent cells

C. neoformans CPSs induced maximum proliferative response of normal murine splenocytes at 30 µg/ml (Fig. 1GoA). To analyze the cellular mechanism involved in this immunostimulatory effect, splenic T subsets were blocked with neutralizing mAb against CD4+ and/or CD8+ T cells. Functional blockade of the CD4+ T subset reduced, in ~60%, the uptake of tritiated thymidine, when compared with the control (medium alone) (Fig. 1GoB). These results suggest immunoregulatory effects by CPSs on CD4+ T cells. However, the addition of CPSs to CD4+ T cell-enriched cultures, with or without splenic B cells, did not induce proliferation (Fig. 1GoC). Nevertheless, the ability to induce proliferative response of CD4+ T cells was acquired when these lymphocytes were cocultured with SAC monolayers (Fig. 1GoC). In all subsequent experiments, cocultures containing CD4+ T cells were standardized with SAC to evaluate the events triggered by CPSs under the conditions used here. To verify whether the proliferative effect induced by C. neoformans CPSs on the murine CD4+ T cells was indeed mediated by glycan components, the CPS preparation was submitted to different biochemical pretreatments. As indicated in the Fig. 1GoD, only the treatment with periodate ablated CPSs induced in vitro lymphoproliferative responses of murine CD4+ T cells.



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  		FIGURE 1. C. neoformans-derived CPSs induce proliferation of CD4+ T cells in a SAC-dependent manner. A, Normal whole splenocytes (1.5 x 106/ml; 200 µl/well) were cultured with crescent doses of CPSs of C. neoformans (0.3–60 µg/ml); B, Normal whole splenocytes (1.5 x 106/ml; 200 µl/well) were cultured with or without saturating doses of blocking anti-CD4 ({alpha}-CD4; 5 µg/ml) and/or anti-CD8 ({alpha}-CD8; 30% v/v supernatants of clone 53.6.7) mAb, in the presence of the CPSs (30 µg/ml); C, CD4+ T lymphocytes (1.5 x 106/ml) were cocultured in the presence or absence of purified B lymphocytes (1.5 x 106/ml; 200 µl/well) or SAC (5.0 x 105/ml; 500 µl/well), all obtained from normal spleen; D, CD4+ T lymphocytes (1.5 x 106/ml) with SAC (5.0 x 105/ml; 500 µl/well) were cocultured in the presence of CPSs (30 µg/ml), which had been submitted to different treatments (T) as indicated: In T1, the CPSs preparation was exhaustively dialyzed to release the glucose from culture medium; in T2, the CPSs preparation was treated with Pronase (500 µg/ml); in T3 and T4, the CPSs preparation was treated with DNase or RNase, respectively; and in T5, the CPSs preparation was oxidized with periodate. All the cultures were kept for 3 days, and cell proliferation was assessed by [3H]TdR uptake (0.5 µCi per well) added during the final 16 h of culture. Results indicate the means ± SEM of triplicate cultures.

 
The role of SAC monolayers in the events induced by CPSs on normal CD4+ T cells

To characterize the molecular and cellular mechanisms involved in the immunoregulation by SAC, these adherent cells were pretreated with CPSs for 18 h, and supernatant was harvested and added in various dilutions to CD4+ T cells (Fig. 2GoA). Additionally, the SAC monolayers were washed exhaustedly and cocultured with CD4+ T lymphocytes (Fig. 2GoB). It was observed that supernatants collected from the adherent cells were unable to induce the in vitro proliferative response of CD4+ T cells (Fig. 2GoA). However, the direct contact between splenocytes provided mitogenic activity of CPSs on CD4+ T cells (Fig. 2GoB). Our results showed that mitogenic activity of CPSs is dependent on contact with APC. Next, we tried to determine whether CD4+ T cell activation depended on TCR cross-link by a SAg-like mechanism. As demonstrated in Fig. 2GoC, paraformaldehyde-fixed SAC did not stimulate CD4+ T cells to proliferate in the presence of CPSs. These results suggest the need of induced events on viable APC to activate CD4+ T cell in response to CPSs. The next step was to analyze the contribution of costimulatory molecules expressed by the APC and CD4+ T cells that could participate in CPS-induced lymphoproliferation. For this purpose, we added neutralizing anti-B7.1, anti-B7.2, or anti-CD40L mAbs to the cultures. Effective blockade of the CPS-induced lymphoproliferation was achieved only by anti-CD40 mAbs (Fig. 3Go). Neither anti-B7.1 nor anti-B7.2 modified the level of [3H]thymidine uptake on these in the presence of CPSs.



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  		FIGURE 2. Mitogenic effect of C. neoformans-derived CPSs on CD4+ T cells is dependent on physical contact with viable SAC. A, Murine SAC monolayers (5.0 x 105/ml; 500 µl/well) were cultured overnight with CPSs (30 µg/ml). The next day, the supernatant was collected and added, in different dilutions (1:2, 1:10, or 1:50), to CD4+ T lymphocytes (1.5 x 106/ml; 200 µl/well). B, These adherent cells were additionally washed and cocultured with CD4+ T lymphocytes (1.5 x 106/ml; 500 µl/well). C, Normal SAC (5.0 x 105/ml) were fixed with paraformaldehyde solution (1% v/v) for 15 min, washed, and cocultured with CD4+ T cells (1.5 x 106/ml; 500 µl/well) in the presence or absence of CPSs. As positive control, SEB was used (2 µg/ml). All cultures were kept for 3 days, and proliferation was assessed by [3H]TdR uptake (0.5 µCi per well) added during the final 16 h of culture. Results are indicated as means ± SEM of triplicate cultures.

 


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  		FIGURE 3. Blockade of signalizing triggered by the CD40L ablates CD4+ T cells proliferation induced by CPSs of C. neoformans. Normal SAC (5.0 x 105/ml) were cocultured with autologous CD4+ T cells (1.5 x 106/ml) for 3 days in the presence of CPSs (30 µg/ml). The role of different signaling pathways in the mitogenic activity was evaluated by addition of saturating doses (10 µg/ml) of anti-B7.1 ({alpha}-B7.1; 1G10), anti-B7.2 ({alpha}-B7.2; GL1) or anti-CD40L ({alpha}-CD40L; MR1) mAbs. As control isotype, we used a hamster IgG (hamsterIgG) or rat IgG2a (ratIgG2a), both at 10 µg/ml. Proliferation was assessed by [3H]TdR uptake (0.5 µCi per well) added during the final 16 h of culture. Results are indicated as means ± SEM of triplicate cultures.

 
CPSs induce anti-inflammatory cytokines production

To extend our observations about stimulating effects of CPSs on splenocytes, cytokine secretion by SAC was measured in the presence of CPSs (Table IGo). Additionally, we also assayed cytokine production by cocultures of SAC with CD4+ T cells in the presence of CPSs (Fig. 4Go). Clearly, CPSs induced IL-10 secretion by both culture types (Table IGo and Fig. 4Go). IL-4 production was detected only on wells containing CD4+ T lymphocytes and was augmented by CPSs (Fig. 4Go). These results indicate that CPSs mainly induce a Th2-type cytokine pattern. Addition of saturating doses of anti-CD4 mAb reduces by 80% the ability of CPSs to induce IL-4 and IL-10 secretion. Addition of a polyclonal activator (anti-TCR), however, induced both inflammatory (TNF-{alpha} and IFN-{gamma}) and anti-inflammatory (IL-4 and IL-10) cytokines assayed (Fig. 4Go).


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  		Table I. C. neoformans-derived CPSs induce IL-10 secretion by SAC monolayers1

 


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  		FIGURE 4. CPSs of C. neoformans induce a Th2-type cytokine pattern in cocultures of CD4+ T cells with SAC. CD4+ T cell (1.5 x 106/ml) and SAC monolayers (5.0 x 105/ml), both purified from normal mice spleen, were cocultured in the presence of anti-TCR (10% v/v supernatant of H57 clone), CPSs (30 µg/ml), or medium alone (control). Supernatants were collected after 72 h culture and assayed for cytokine contents by sandwich ELISA. The concentrations were obtained by comparison with standard curves of respective recombinant cytokines, ranging from 1 to 50 ng/ml. The results are means ± SEM of triplicate cultures.

 
CD40L-dependent CD4+ T cell activation enhances in vitro infection

As demonstrated above, the ability of CPSs to activate normal CD4+ T cells cocultured with SAC was CD40L dependent. These activated cocultures secrete IL-10 and IL-4, but not inflammatory cytokines, such as IFN-{gamma} and TNF-{alpha} (Fig. 4Go). Therefore, we analyzed the contribution of the CD40L signaling pathway on nonencapsulated C. neoformans growth in normal coculture, in the presence or absence of the CPSs. As indicated in Fig. 5Go, the blockade of signaling through CD40L, by anti-CD40L mAb, reverted the fungus growth to that observed in CPS-free cocultures. The matching isotype did not change fungus survival. The evaluation of cytokine contents in these cocultures subjected to CD40L blockade demonstrated a marked reduction of IL-10 and IL-4 secretion (Table IIGo). Production of inflammatory cytokines was undetectable either before or after CPS addition (Table IIGo).



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  		FIGURE 5. Induction of CD4+ T cell with suppressor activity by CPSs was dependent on signaling through CD40L. Normal cocultures containing SAC monolayers (5.0 x 105/ml) with CD4+ T cell (1.5 x 106/ml; 500 µl) were kept with the nonencapsulated C. neoformans strain Cap 67 (103/ml) in the presence or absence of CPSs (30 µg/ml). To evaluate the role of the signaling through CD40L in the course of the in vitro infection, saturating doses (10 µg/ml) of neutralizing anti-CD40L mAb or control hamster isotype (hamsterIgGH), were added. The cultures were maintained in humid chamber at 37°C and 7% CO2 atmosphere. After 3 days, the whole content in each well (500 µl) was transferred to 1.5 ml synthetic medium (CDC-2500) for fungus growth, and the viable yeast cells were counted after 10 days at 37°C. The results are indicated as means ± SEM of triplicate cultures.

 

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  		Table II. Enhancement of anti-inflammatory cytokine production induced by C. neoformans-derived CPSs depends on signaling triggered by CD40L1

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The C. neoformans has many virulence factors such as mannitol, melanin, and extracellular proteinases (33). However, the ability to synthesize CPSs is the most relevant evasion mechanism. The murine model is very useful for immunopathological studies on C. neoformans infection, because uncontrolled growth of Cryptococcus in the lungs of these animals results in disseminated infection with intense shedding of CPSs (34). Injection of CPSs in experimental animals results in immunosuppression (19, 22, 26, 31, 32, 35, 36, 37, 38, 39, 40). These soluble molecules induce a set of immunoregulatory T cells that inhibits the inflammatory response induced by specific and unspecific Ags (31, 32, 40). Similar mechanisms have been suggested for the phenomenon of Ab unresponsiveness to some bacterial polysaccharides (41). Results shown here suggest that C. neoformans-derived CPSs, in the presence of viable SAC, induce in vitro the proliferation of normal CD4+ T lymphocytes and the production of anti-inflammatory cytokines. The ability to induce CD4+ T cells activation, in our system, was maintained by SAC pretreated for 4 or 18 h with CPSs (Fig. 1GoB). We do not believe that dendritic cells are involved because they attach only transitorily to plate surfaces (42). CPSs did not augment the level of [3H]TdR uptake when they were added to cultures containing highly purified splenic B cells (Fig. 1GoC). This result eliminates the possibility of LPS contamination in the capsular preparation. The involvement of CD8+ T cells was also discarded by the failure of the relevant neutralizing mAb to alter CPS-induced proliferative response. The failure of total blockade of proliferation in cultures of whole splenocytes treated with anti-CD4 may be explained by the presence of other T cell subsets that may be responsive to CPSs (Fig. 1GoB). NK cells, for example, are able to recognize complex polysaccharides (43), and these lymphocytes are not eliminated by the procedure we used to purify CD4+ T cells.

When C. neoformans CPSs were fractionated by anion exchange chromatography on a Mono Q column, only the bound fraction, containing mannose, xylose, and glucuronic acid residues, was responsible for the mitogenic activity on whole splenocytes and purified CD4+ T cells (data not shown), indicating that the ability of CPSs to activate CD4+ T cells, in our system, is due to the presence of glucuronoxylomannan (GXM), the major CPS of C. neoformans (55). This observation is in agreement with those of others who noted that many deleterious effects induced by the C. neoformans capsule are mediated by GXM (24, 25). Other published results also demonstrated structural diversity on GXM molecule from strains of the same serotype (27). Therefore, a detailed molecular analysis, which is being conducted by our group, will permit a closer definition between the function and structure of CPSs.

The assays to characterize molecular events involved in the CD4+ T cells activation by CPSs of C. neoformans suggest that the proliferation of these lymphocytes is dependent on physical contact with SAC and involves signaling through CD40L. The CD40 expression on CD4+ T cells is an early event following activation through the TCR:CD3 complex (47). Furthermore, the ability of activated NK cells to express CD40L strengthens the possibility of these lymphocytes to participate in up-regulating the response to CPSs (48). The capacity of microbial polysaccharides to activate classical T lymphocytes has been described by other authors. Muzuno et al. (44) reported that {alpha}1->6- and {alpha}1->4-glucans from Agaricus blazei induce murine CD4+ and CD8+ T cells to proliferate. The work done by Mattern et al. (45) established a direct correlation between activation of human T lymphocytes and LPS. These authors demonstrated that mitogenic activity on T cells was dependent on physical contact with LPS-treated monocytes and involved signaling through the B7 molecule but was not MHC restricted. More recently, a work by Brubaker et al. (46) reported that CPS A, purified from Bacteroides fragilis, induces intra-abdominal abscess in rodents by activating CD4+ T cells via a mechanism dependent on physical contact with viable macrophages. Probably, these nonproteic molecules mentioned above can induce by standard activation of T lymphocytes by stimulating expression of costimulatory molecules, such as B7 family (45) and CD40L (46).

In our model, results from cocultures containing paraformaldehyde-fixed SAC monolayers with freshly purified autologous CD4+ T cells suggested that 1) intracellular events induced by C. neoformans CPSs on SAC are pivotal to assure activation of CD4+ T cells and 2) the pathway used by CPSs to activate these lymphocytes do not behave like a SAg-induced activation (Fig. 2GoC). SAgs, characteristically, are able to activate T lymphocytes by simultaneously associating, without processing, the outside of the MHC class II molecules with some V{beta} family of TCR (49). We considered the possibility of class II MHC involvement in the lymphoproliferation. When C. neoformans-derived CPSs were submitted to Lowry’s method, a 1% protein contamination was detected (data not shown). The next step was to treat the CPSs with Pronase and submit them to SDS-PAGE, followed by silver staining. Nevertheless, extensive digestion of CPSs with Pronase did not change the ability of CPSs to activate CD4+ T cells (Fig. 1GoB). However, oxidation of CPSs by periodate abolished the mitogenic activity on CD4+ T cells induced by intact CPSs (Fig. 1GoB). It is possible that CPS-treated APC may achieve the ability to activate autoreactive CD4+ T cells by presenting endogenous peptides through molecules of class II MHC. The use of anti-I-E or anti-I-A mAb will reveal the exact contribution of this pathway in CD4+ T cell activation in our model.

The results obtained from cytokine dosage showed that CPSs induce IL-10 secretion in SAC monolayers (Table IGo). This result is in agreement with other data demonstrating in vitro IL-10 production by CPSs such as GXM on murine and human adherent cells (25, 26). The cytokine dosage from cocultures containing SAC with purified CD4+ T cells suggested that CPSs induce a Th2 cytokine pattern, with high levels of IL-4 and IL-10 production (Fig. 4Go). The contribution of this T lymphocyte subset for the resulting cytokine pattern was confirmed by treatment with anti-CD4 mAb, which ablated IL-4 secretion and significantly reduced IL-10 secretion (Fig. 4Go). We believe that the remaining IL-10 production is originated from the SAC monolayers (Table IGo). As described in the literature, these anti-inflammatory cytokines, besides making macrophages refractory to TNF-{alpha} and IFN-{gamma} (50), are involved in induction of humoral immunity (51). The ability of polyclonal activator to induce inflammatory cytokines in our system negated the possibility of a previously established Th2 phenotype. In our system, the functional blockade of signaling through CD40L by mAb significantly diminished the anti-inflammatory cytokines production induced in vitro by CPSs (Table IIGo) and reduced the fungus growth to control levels (Fig. 5Go). Additionally, the mouse strains most sensitive to cryptococcosis develop exacerbated humoral response (56). Probably, the ability of CPSs to induce IL-4 and IL-10 secretion is detrimental for the host by favoring the humoral response. The possible contribution of other nonclassical lymphocytes, such as CD4+NK1+ T cells, may be through recognition of CD1 coupled to polysaccharides and secretion of high levels of IL-4 (52, 53). Preliminary observations by our group on the murine cryptococcosis model revealed that CD1 knockout (CD1-/-) mice are more resistant to cryptococcosis than wild-type strains. It is well established that CD1-/- animals lack CD4+NK1+ T cells, indicating that these nonclassical lymphocytes play an important role in the pathogenesis of cryptococcosis. The procedures used for CD4+ T cells purification in the present work did not eliminate this T cell subset; thus, the involvement of CD4+ NK1+ T lymphocytes on the immunological events induced by CPSs cannot be discarded.

In summary, our results suggest that the ability to induce in vitro CD4+ T cells to proliferate and secrete Th2 cytokines, such as IL-10 and IL-4, makes CPS an important evasion mechanism of C. neoformans via inhibition of cellular immune response and thus exacerbation of in vitro infection. We are now investigating the precise mechanism by which CPS-induced CD4+ T cells down-regulate protective cellular immune response to fungal infection and their impact on the course of murine cryptococcosis.


   Acknowledgments
 
We thank Dr. Marise P. Nunes (Instituto Oswaldo Cruz-Fundaçao Oswaldo Cruz, Rio de Janeiro, Brazil) for providing the animals used in this work, Dr. Cerli Gattas for SEB, and Drs. Miercio E. A. Pereira (Department of Pathology, Tufts University School of Medicine, Boston, MA) and Arnaldo F. B. Andrade (Departamento de Microbiologia e Imunologia, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil) for reviewing the manuscript.


   Footnotes
 
1 This work was supported by grants from Programa de Apoio ao Desenvolvimento Científico e Tecnológico/World Bank, Financing Agency of Studies and Projects, National Research Council, Rio de Janeiro State Foundation for the Advancement of Science, and Programa de Núcleos de Excelência of the Brazilian Ministry of Science and Technology. Back

2 Address correspondence and reprint requests to Dr. Cleonice A. M. Bento, Rua Barão do Flamengo, 28/701, Flamengo, Rio de Janeiro, RJ 22220-080, Brazil. Back

3 Abbreviations used in this paper: CPS, capsular polysaccharide; SAC, splenic adherent cell; GXM, glucuronoxylomannan; SEB, Staphylococcus aureus enterotoxin B; SAg, superantigen; CD40L, CD40 ligand. Back

Received for publication March 6, 2001. Accepted for publication September 17, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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