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Originally published online as doi:10.1189/jlb.1002476 on June 16, 2003

Published online before print June 16, 2003
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(Journal of Leukocyte Biology. 2003;74:370-378.)
© 2003 by Society for Leukocyte Biology

The polysaccharide capsule of Cryptococcus neoformans interferes with human dendritic cell maturation and activation

Anna Vecchiarelli*,1, Donatella Pietrella*, Patrizia Lupo*, Francesco Bistoni*, Diane C. McFadden{dagger} and Arturo Casadevall{dagger}

* Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Italy; and
{dagger} Albert Einstein College of Medicine, Bronx, New York

1Correspondence: Microbiology Section, Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Via del Giochetto, 06122 Perugia, Italy. E-mail: vecchiar{at}unipg.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ability of encapsulated and acapsular strains of Cryptococcus neoformans to activate dendritic cells (DC) derived from monocytes stimulated with granulocyte macrophage-colony stimulating factor and interleukin-4 was evaluated. Profound differences in DC response to encapsulated and acapsular C. neoformans strains were observed. In particular, (i) the acapsular strain was easily phagocytosed by immature DC, and the process induced several molecular markers, such as major histocompatibility complex (MHC) class I and class II, CD40, and CD83, which are characteristic of mature DC; (ii) the encapsulated strain did not up-regulate MHC class I and class II and CD83 molecules; (iii) the soluble capsular polysaccharide glucuronoxylomannan (GXM) is unable to regulate MHC class I and class II molecules; (iv) the addition of monoclonal antibody to GXM (anti-GXM) to the encapsulated strain facilitated antigen-presenting cell maturation by promoting ingestion of C. neoformans via Fc receptor for immunoglobulin G (Fc{gamma}R)II (CD32) and Fc{gamma}RIII (CD16); (v) pertubation of FcR{gamma}II or Fc{gamma}RIII was insufficient to promote DC maturation; and (vi) optimal DC maturation permitted efficient T cell activation and differentiation, as documented by the enhancement of lymphoproliferation and interferon-{gamma} production. These results indicate that the C. neoformans capsule interferes with DC activation and maturation, indicating a new pathway by which the fungus may avoid an efficient T cell response.

Key Words: GM-CSF • interleukin-4 • major histocompatibility complex • glucuronoxylomannan


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dendritic cells (DC) are professional antigen-presenting cells, which can initiate primary and secondary T cell responses [1 ]. DC originate in the bone marrow and are transported by blood to the peripheral tissues, where they localize in the epithelial layer of the skin, gut, lung parenchyma, and the interstitial spaces of many solid organs [2 ]. Immature DC reside within nonlymphoid tissues, where they actively capture and process antigens, and their maturation can be induced by contact with proinflammatory cytokines and bacterial products. Mature DC produce chemokines that recruit macrophages, neutrophils, natural killer cells, and immature DC to the site of inflammation. They then migrate to lymphoid organs in search of antigen-specific T cells [3 ]. DC maturation is characterized by profound changes in major histocompatibility complex (MHC) class II distribution, antigen-processing capacity [4 ], expression of costimulatory molecules [5 ], and a marked rearrangement of adhesion molecules, which probably facilitate their migration to lymphoid organs [6 ]. When DC reach the lymph nodes, they lose their migratory capacity, are retained in T cell areas, and produce T and B cell stimulatory cytokines such as interleukin (IL)-12, IL-6, and interferon (IFN). In lymph nodes, DC present antigens that are derived mainly from phagocytosed microbes displayed on the cell surface as a stable peptide–MHC complex.

One well-known stimulus used to induce DC maturation/activation is lipopolysaccharide (LPS), a product of Gram-negative bacteria. LPS has major effects on the phenotype of immature DC, causing dramatic up-regulation of B7-1 (CD80), B7-2 (CD86), CD40, MHC class I and class II gene products, intercellular adhesion molecule-1, and very late antigen-4 in mice and humans [7 , 8 ]. Bacteria as well as some protozoa are also potent inducers of DC maturation in vitro. Whereas some of these stimuli promote maturation directly, others act indirectly through cytokines such as tumor necrosis factor and IL-1. Inflammatory cells or the DC themselves produce these cytokines. Mycobacterium tuberculosis bacilli are taken up by immature human DC in vitro, which results in their direct activation and maturation [9 ]. Live tachyzoites of Toxoplasma gondii up-regulate CD40, CD80, CD86, and MHC class II molecules, promoting differentiation of human DC [10 ].

Cryptococcus neoformans is an opportunistic fungus that causes serious infections in the immunocompromised host. Its principal virulence factor is capsular polysaccharide, composed of 80% glucuronoxylomannan (GXM), which has been shown to be antiphagocytic and to mediate a variety of deleterious effects on host immune function [11 ]. Cryptococcal infection normally occurs via inhalation of infectious particles, and in the immunocompetent host, it is limited to the lung. The effective immune response to infectious agents is thought to represent a polarization toward the T helper cell type 1 (Th1) or Th2 subset, depending on the type of pathogen. There is consensus in the field that a Th1 response is important for control of C. neoformans infection in the lung. However, the complex interplay between humoral and cellular immunity is also widely recognized as being critical for the control of infection [12 ]. In fact, there is convincing evidence that certain antibodies can make a decisive contribution to host defense [13 , 14 ]. Thus, it appears that C. neoformans infection could be efficiently controlled by the concomitant presence of the Th1 response and specific immunoglobulin G (IgG)1 to GXM. It has been reported that a murine IgG1 monoclonal antibody (mAb) to GXM (mAb 18B7) prolongs the survival of mice with lethal C. neoformans infection [15 ]. mAb 18B7 was shown to bind to all four serotypes of C. neoformans, enhance human and mouse effector-cell antifungal activity, and activate the complement pathway leading to deposition of complement component 3 on the cryptococcal capsule [13 ]. This mAb is currently in phase I clinical evaluation for treatment of cryptococcal meningitis.

Despite the importance of the functional status of DC in influencing the generation of the T cell immune response, very little is known about interaction of DC with this fungus, and the available information is limited to murine cells. DC have been shown to be important for the generation of a protective cell-mediated response against C. neoformans in mice [16 ]. Furthermore, Syme et al. [17 ] demonstrated that mannose receptors and Fc receptor for IgG (Fc{gamma}R)II are required for uptake and presentation of C. neoformans. In this study, we explored the maturation of human DC cells in vitro following their interaction with C. neoformans in the presence and absence of capsular material and specific antibody. The results indicate new mechanisms by which the capsular polysaccharide can interfere with the development of effective immune responses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and media
RPMI 1640 with glutamine and fetal calf serum (FCS) were obtained from Gibco-BRL (Grand Island, NY). Human serum type AB was purchased from Sigma (Milan, Italy). Mouse monoclonal anti-human MHC class I fluorescein isothiocyanate (FITC) conjugate (IgG2a), mouse monoclonal anti-human MHC class II FITC (IgG1), mouse monoclonal anti-human CD14 (IgG2a), mouse monoclonal anti-human CD40 FITC (IgG1), mouse monoclonal anti-human CD83 (IgG1), mouse monoclonal anti-human CD86 FITC (IgG1), mouse monoclonal anti-human Fc{gamma}RI (CD64, IgG1), mouse monoclonal anti-human Fc{gamma}RII (CD32, IgG1), mouse monoclonal anti-human CD32 FITC (IgG1), and mouse monoclonal anti-human Fc{gamma}RIII (CD16, IgG1) were obtained from Ancell (Alexis Italia, Florence, Italy). Mouse monoclonal anti-human CD80 FITC was purchased from Calbiochem (San Diego, CA). Mouse monoclonal anti-human CD16 FITC (IgG2a) was purchased from Euroclone (Milan, Italy). Rabbit anti-mouse IgG FITC, isotype-control mouse IgG1 FITC, and mouse IgG2a FITC were obtained from Sigma.

GXM was isolated from culture supernatant fluid of serotype D, strain B3501, by differential precipitation with ethanol and cetyl-trimethyl-ammonium bromide [18 ]. IgG1 mAb to GXM mAb 18B7 (referred to as anti-GXM mAb) was purified by protein G affinity chromatography (Pierce, Rockford, IL), and concentration was determined by enzyme-linked immunosorbent assay (ELISA) relative to isotype-matched standards of known concentrations. The characteristics of anti-GXM mAb have been described previously [13 ].

In vitro generation and culture of human DC
The generation of DC from human peripheral blood monocytes was performed as described previously with minor modifications [19 ]: Heparinized venous blood was obtained from healthy volunteers and diluted with RPMI 1640 (Gibco-BRL). Peripheral blood mononuclear cells (PBMC) were separated by density-gradient centrifugation over Ficoll-Hypaque PLUS (Pharmacia Biotech, Uppsala, Sweden) [20 ], recovered, washed twice and suspended in RPMI 1640 supplemented with 5% FCS, penicillin (100 U/ml), and streptomycin (100 µg/ml), plated onto cell-culture Petri dishes (Nunc Inter Med, Roskilde, Denmark), and incubated for 1 h at a density of 2 x 106-3 x 106/ml. Adherent peripheral blood monocytes were recovered using a cell scraper (Falcon, Oxnard, CA), washed twice, and purified by E-rosetting to remove contaminating T cells. The cells recovered were >98% CD14+ monocytes, as evaluated by flow cytometry analysis. Isolated monocytes (2x106/ml) were incubated in RPMI 1640 plus 10% FCS, which contained 50 ng/ml human recombinant granulocyte macrophage-colony stimulating factor (hrGM-CSF; Sigma) and 30 ng/ml hrIL-4 (Sigma). After 6 days of culture, DC were harvested, washed, and suspended in RPMI 1640 plus 10% human serum type AB (Sigma), penicillin (100 U/ml), and streptomycin (100 µg/ml), referred to as cRPMI and used for subsequent experiments.

Microorganisms
An encapsulated strain of C. neoformans var neoformans serotype D ATCC B3501 (Cap-Cn) and an acapsular mutant derived from this strain, CAP67 (Acap-Cn) were obtained from the American Type Culture Collection (Manassas, VA). The CAP67 acapsular phenotype is a result of a mutation in a single gene, which when complemented, restores the capsule and the virulence of the strain [21 ]. The cultures were maintained by serial passage on Sabouraud agar (BioMérieux, Lyon, France). Log-phase yeasts were harvested by suspending a single colony in RPMI 1640, washed twice with saline, counted on a hemocytometer, and adjusted to the desired density in cRPMI [20 ]. All yeasts were inactivated at 60°C for 30 min. Yeast-killing is necessary, as live cells would grow rapidly in the cell media and overwhelm the DC culture.

Flow cytometry analysis of surface molecules
Surface-molecule expression was quantified by flow cytometry after various culture incubation times [22 ]. Suspensions of immature DC (1x106) in cRPMI were stimulated with different strains of C. neoformans at effector:target ratios of 1:2 or LPS (1 µg/ml) as a positive control and were incubated for different times. After incubation at 37°C in the presence of 5% CO2, cells were collected by centrifugation, fixed in 3% paraformaldehyde in phosphate-buffered saline (PBS), washed twice in PBS containing 0.5% bovine serum albumin and 0.1% sodium azide, and mixed with mouse IgG2a anti-human MHC class I FITC-conjugate, mouse IgG1 anti-human MHC class II FITC-conjugate, mouse IgG1 anti-human CD40 FITC-conjugate, mouse IgG1 anti-human CD14, mouse IgG1 anti-human CD80 (B7-1) FITC-conjugate, mouse IgG1 anti-human CD86 (B7-2), mouse IgG1 anti-human CD83, mouse IgG1 anti-human CD32, or mouse IgG2a anti-human CD16 FITC. After 30 min of incubation on ice, cells were washed and analyzed using a flow cytometer (FACScan, Becton Dickinson, San Jose, CA). An irrelevant FITC-conjugate isotype-matched Ab was used as a negative control for each experiment.

Determination of C. neoformans binding and phagocytosis by DC
Uptake of C. neoformans by flow cytometry was performed as described previously [23 ]. Briefly, heat-killed C. neoformans cells were suspended in PBS at a density of 108 yeasts/ml. Yeast cells were labeled with FITC (Sigma) at 1 µg/ml in PBS at 22°C for 10 min. Labeled C. neoformans (107) were incubated with DC (106) at 37°C for varying time intervals.

Binding was evaluated after 30 min, and phagocytosis was determined after 2, 4, and 18 h by adding 1 ml ice-cold PBS to the suspension. Trypan blue (200 µg/ml; Sigma) was added to each test suspension and incubated for 10 min to quench fluorescence of noninternalized fungi. Unbound trypan blue was then removed by centrifugation, and the percentage of binding cells or phagocytic DC was determined by flow cytometry.

To block the phagocytosis of C. neoformans, DC were incubated for 30 min at 37°C with 100 µg/ml ß-glucan (Sigma) or 5 mg/ml mannan (Sigma) before yeast cells were added.

Lymphoproliferation assay
DC (2x104) were incubated with Cap-Cn or Acap-Cn in the presence or absence of anti-GXM mAb (5 µg/ml) for 2 days. Irrelevant isotype-matched mouse IgG1 was used as a negative control. After incubation, autologous lymphocytes (2x105) were added to the culture. After 7 days, cultures were pulsed overnight with 0.5 µCi (methyl-3H) thymidine (Amersham Life Science, Buckinghamshire, UK); thereafter, cells were collected onto filter paper using a cell harvester (PBI International, Milan, Italy). The dried filters were counted directly in a ß counter (Packard Instruments, Downers Grove, IL). Proliferation values were expressed as mean ± SE of indicated replicates [20 ].

Determination of IFN-{gamma} production
DC (2x105) were incubated with Cap-Cn or Acap-Cn in the presence or absence of anti-GXM mAb (5 µg/ml) for 2 days. Irrelevant isotype-mouse IgG1 was used as a negative control. After incubation, autologous lymphocytes (2x106) were added to the culture. After 7 days, supernatant fluids were recovered and stored at -80°C. Cytokine levels in culture supernatant fluids were measured with an ELISA kit for human IFN-{gamma} (EuroClone Ltd., Devon, UK). The IFN-{gamma} ELISA kit detected a dose of <3 pg/ml.

Statistical analysis
Statistical significance was determined using Student’s paired t-test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of DC and stimulation with C. neoformans
We derived immature DC from human peripheral blood monocytes by incubating the latter in medium supplemented with GM-CSF and IL-4 according to established protocols [19 ]. Phase-contrast light microscopy revealed that they exhibited morphological features of DC, such as prominent cytoplasmic processes and/or ruffled membranes. These cells lost their antigen-capturing capacity and acquired T cell stimulatory capacity with LPS.

Given that mature DC express high levels of MHC class I and class II molecules, as well as the costimulatory molecules CD40, CD80, CD83, and CD86, we tested whether C. neoformans, in encapsulated and acapsular forms, could induce DC maturation. To this end, immature DC were stimulated for 48 h with one of two isogenic strains of C. neoformans (Cap-Cn or Acap-Cn) or LPS as a positive control. After incubation, surface-marker expression was analyzed, and the results demonstrate that both strains of C. neoformans induced several antigens characteristic of mature DC (Fig. 1 ). However, neither strain was able to promote significant enhancement of CD86 expression. It is interesting that the Cap-Cn and Acap-Cn forms differed in their ability to induce expression of MHC class I and class II molecules. In particular, the Acap-Cn was a positive regulator of MHC class I [migration inhibitory factor (MIF)=484±36 vs. MIF=206±19 of unstimulated cells] and class II (MIF=1660±102 vs. MIF=760±68 of unstimulated cells) molecules, whereas the Cap-Cn was unable to modulate MHC class I and induced only a slight increase in MHC class II expression.



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  		Figure 1. Cell-surface expression of MHC and costimulatory molecules on DC upon addition of C. neoformans or LPS. Immature DC were not treated or treated with Cap-Cn or Acap-Cn C. neoformans or with LPS (1 µg/ml) for 48 h, stained with FITC-conjugated mAb to CD40, CD86, and MHC I and II, and then examined by flow cytometry. The data shown are a representative fluorescein-activated cell sorter (FACS) profile of one of three independent experiments using DC from three different donors. Black lines indicate the FACS profile of untreated cells, and gray lines show treated cells. The mean fluorescence intensity (MFI) of the isotype-matched control antibody was less than 6 ± 1 in all determinations performed.

 
Acap-Cn cells but not Cap-Cn cells are phagocytosed through mannose or glucan receptors. For encapsulated cells, the presence of the capsule prevents the interaction of these receptors with structures in the fungal cell wall. To verify whether it is the phagocytic process that is responsible for DC activation or whether it is the perturbation of mannose or glucan receptors by the Acap-Cn, DC were stimulated with glucan (100 µg/ml) or mannan (5 mg/ml). As shown in Figure 2 , mannan inhibited the internalization of Acap-Cn by 35–40% and was unable to inhibit phagocytosis of unopsonized Cap-Cn. Glucan was less effective than mannan in inhibiting phagocytosis (reduction of 10–15%) and had no significant effect on ingestion of encapsulated yeast. Furthermore, mannan treatment increased CD40 and MHC class I expression, whereas glucan treatment did not (data not shown).



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  		Figure 2. Inhibition of phagocytosis of C. neoformans by mannan or glucan. Immature DC were incubated for 30 min at 37°C with 100 µg/ml ß-glucan or 5 mg/ml mannan. After incubation, FITC-labeled Cap-Cn or FITC-labeled Acap-Cn was added for 4 h. Trypan blue was added to quench fluorescence of noninternalized fungi, and the percentage of phagocytic cells was determined by flow cytometry. Results are expressed as a percentage of phagocytic cells. Results represent the mean ± SE of three separate experiments with cells from three different donors. *, P< 0.05 (treated cells vs. untreated cells).

 
These data prompted us to evaluate the expression of other markers known to be up-regulated during DC maturation. As described previously, LPS stimulation led to increased expression of CD80 and CD83 [9 , 24 ]. Figure 3 shows that these markers were optimally expressed 2 days after stimuli addition (day 8). Both strains failed to produce significant enhancement of CD80 molecules; indeed, Acap-Cn increased expression of CD83, whereas Cap-Cn only induced a slight increase. The enhancement of spontaneous CD83 after 8 days of culture could be a result of a spontaneous maturation of unstimulated cells.



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  		Figure 3. Expression of maturation markers on DC exposed to C. neoformans. Monocytes were cultured for 6 days with GM-CSF and IL-4 and analyzed for expression of CD80, CD83, MHC class I, MHC class II, Fc{gamma}RII, and Fc{gamma}RIII molecules. On day 6, Cap-Cn, Acap-Cn, or LPS was added to DC, and surface expression of CD80, CD83, MHC class I, MHC class II, Fc{gamma}RII, and Fc{gamma}RIII was determined after 24 h (day 7) and 48 h (day 8) of incubation. Results are expressed as MFI. Results represent the mean ± SE of three separate experiments with cells from three different donors. *, P< 0.05 (treated vs. respective untreated cells).

 
The differentiation of monocytes to DC was also evaluated by loss of CD14 from monocytes. We observed a rapid and strong decrease of CD14 expression during maturation of DC-derived monocytes, and C. neoformans addition did not significantly alter the progressive disappearance of this marker (data not shown).

The kinetic study of DC MHC class I and class II molecule expression after stimulation with C. neoformans revealed that Cap-Cn did not significantly modify MHC class I and class II expression after 24 h (day 7) or 48 h (day 8) of incubation. In contrast, the Acap-Cn produced a slight up-regulation of MHC class II expression by 24 h (day 7), which reached a significant level after 48 h (day 8) of incubation (Fig. 3) . LPS was used as a positive control [25 ].

DC express several receptors for the Fc{gamma}R, which mediates internalization of antigen–IgG complexes. However, in mature DC, there is a down-regulation of Fc{gamma}R consistent with the reduced ability of these cells to capture and present antigen [26 , 27 ]. To evaluate the ability of C. neoformans to induce DC maturation, cells were incubated for 24 h in the presence of Cap-Cn, Acap-Cn, or LPS and were then analyzed for surface expression of Fc{gamma}RII and Fc{gamma}RIII. No effect on Fc{gamma}RIII expression was observed, but stimulation of DC with both strains of C. neoformans rapidly down-regulated expression of Fc{gamma}RII molecules, as did LPS (Fig. 3) .

Internalization of C. neoformans by human DC
Immature DC derived from PBMC by culture with cytokines have been reported to phagocytose bacteria [9 , 28 ] and Candida albicans [29 ]. We tested the ability of cultured human DC to internalize both strains of heat-inactivated C. neoformans. Given that antibodies to the capsular polysaccharide are opsonic and that enhanced internalization may promote maturation of DC [14 , 30 ], we studied the effects on DC expression of an IgG1 mAb to GXM–anti-GXM mAb complexed with Cap-Cn. Phagocytosis was allowed to proceed for 2, 4, or 18 h and was then analyzed by cytofluorimetry. Acap-Cn was readily internalized, reaching high levels after 2 h of incubation with DC. In contrast, phagocytic activity of Cap-Cn was significantly lower than that of Acap-Cn (Fig. 4 ). Anti-GXM mAb was opsonic for Cap-Cn but not for Acap-Cn.



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  		Figure 4. Phagocytosis of Cap-Cn or Acap-Cn by immature DC in the presence or absence of anti-GXM mAb. Immature DC were incubated with FITC-labeled C. neoformans yeasts for 2, 4, or 18 h at 37°C in the presence or absence of anti-GXM mAb (5 µg/ml). Trypan blue was added to quench fluorescence of noninternalized fungi, and the percentage of phagocytic cells was determined by flow cytometry. Results are expressed as a percentage of phagocytic cells. Results represent the mean ± SE of three separate experiments with cells from three different donors. *, P< 0.05 (anti-GXM mAb-treated cells vs. anti-GXM mAb-untreated cells).

 
Given that phagocytic activity of DC for Cap-Cn was highly inefficient within 2–4 h, the incubation was prolonged until 18 h in the presence of 10% human serum. For Acap-Cn, the percentage of phagocytic cells was 52 ± 4 and was not altered by the presence of anti-GXM mAb. For Cap-Cn, the percentage of phagocytic cells after this longer interval in the absence and presence of anti-GXM mAb was 31 ± 4 and 58 ± 5, respectively.

Effect of capsule-specific anti-GXM mAb on fungal-induced DC maturation
Both strains of C. neoformans up-regulated CD40 expression on the DC surface after 48 h of incubation (Fig. 1) . No modulation of CD40 expression was observed after 24 h (day 7), but after 48 h (day 8), the expression of CD40 was comparable with that observed when LPS stimulation was used as a positive control (see Fig. 5A ).



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  		Figure 5. Expression of CD40 and CD86 molecules on DC exposed to Cap-Cn or Acap-Cn in the presence or absence of anti-GXM mAb. (A) Monocytes were cultured for 6 days with GM-CSF plus IL-4 and analyzed for expression of CD40 or CD86 molecules. On day 6, Cap-Cn, Acap-Cn, or LPS was added, and surface expression of CD40 or CD86 was determined after 24 h (day 7) and 48 h (day 8) of incubation. (B) CD40 and CD86 expression on DC (monocytes cultured with GM-CSF plus IL-4 for 6 days) treated for 48 h with Cap-Cn or Acap-Cn in the presence or absence of anti-GXM mAb or with LPS. Results are expressed as MFI. Results represent the mean ± SE of three separate experiments with cells from three different donors. *, P< 0.05 (treated Cap-Cn, Acap-Cn, or LPS cells vs. respective untreated cells).

 
Addition of anti-GXM mAb to the suspension of DC and C. neoformans cells significantly increased CD40 expression for the Cap-Cn strain but not for the Acap-Cn (Fig. 5B) . As anti-GXM mAb does not bind to the Acap-Cn, this result proves the specificity of the mAb effect and provides an important negative control that excludes nonspecific effects.

When CD86 expression was analyzed, both strains of C. neoformans induced a weak up-regulation after 48 h (day 8) of incubation. No difference was evident between the isogenic strains (Fig. 5) . Addition of anti-GXM mAb significantly increased CD86 expression on DC when incubated with cells of the encapsulated strain, which was avidly ingested in the presence of specific antibody. In contrast, addition of anti-GXM mAb to suspensions of DC and Acap-Cn had no effect on CD86 expression.

The analysis of other maturation markers revealed that a combination of Cap-Cn with anti-GXM was able to up-regulate CD83 on DC with respect to Cap-Cn alone (MIF=50±4.1 vs. MIF=40±2.5, respectively); on the contrary, no modulation of CD80 expression was observed. Moreover, the opsonization of Cap-Cn with anti-GXM mAb did not modulate the expression of MHC class I or MHC class II (data not shown).

Given that the capsular material of C. neoformans plays an important role in DC maturation, we tested whether soluble GXM, the major component of capsular material, influenced this process. Expression of CD40, CD86, and MHC class II and class I was examined after incubation with GXM (250 µg/ml) or GXM plus anti-GXM mAb (5 µg/ml). The former concentration of GXM is comparable with that found in lung tissue during infection [31 ]. The results in Figure 6 show that GXM (250 µg/ml) was unable to modulate the expression of MHC class I and class II, CD40, and CD86. The addition of specific anti-GXM mAb (5 µg/ml) produced significant changes in the pattern of cellular surface-molecule expression only for MHC class II, probably as a consequence of ingestion of Ab–GXM complexes through Fc receptors.



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  		Figure 6. Expression of CD40, CD86, and MHC I and II molecules on DC after stimulation with GXM in the presence or absence of anti-GXM mAb. Immature DC (monocytes cultured with GM-CSF plus IL-4 for 6 days) were treated with GXM (250 µg/ml) in the presence or absence of anti-GXM mAb (5 µg/ml) for 48 h. Results are expressed as MFI. Results represent the mean ± SE of three separate experiments with cells from three different donors. *, P< 0.05 (treated vs. untreated cells).

 
DC capability to induce proliferation
The efficiency of DC antigen presentation is strictly correlated to its maturation state [19 ]. To verify whether efficient maturation corresponds to efficient capability of DC to induce T cell activation, DC were treated with Cap-Cn and Acap-Cn in the presence or absence of anti-GXM mAb and were mixed with autologous T cells. The magnitude of T cell activation was measured by lymphoproliferation assay. The Acap-Cn stimulated a stronger T cell proliferative response than the Cap-Cn (Fig. 7A ). However, the proliferative response to the encapsulated strain used in combination with anti-GXM mAb was similar to that of the acapsular mutant. Moreover, addition of anti-GXM mAb resulted in an augmented production of IFN-{gamma} (Fig. 7B) .



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  		Figure 7. Proliferative T cell response to DC treated with the indicated stimuli. DC were treated with Cap-Cn or Acap-Cn in the presence or absence of anti-GXM mAb (5 µg/ml) for 2 days. Autologous lymphocytes were added, and proliferative response (A) or IFN-{gamma} production (B) in supernatant fluids after 7 days was evaluated. The results are expressed as counts per minute (CPM) for proliferative response and as pg/ml for IFN-{gamma} production. Results represent the mean of three separate experiments with cells from three different donors. *, P< 0.05 (anti-GXM mAb+C. neoformans-treated cells vs. respective C. neoformans-treated cells).

 
Anti-GXM mAb binding on human DC
Given that anti-GXM mAb affects DC function, which may be related to enhancement of phagocytosis and perturbation of Fc{gamma}RII and/or Fc{gamma}RIII, we identified the receptor, which was preferentially used by DC to bind immunocomplexes. To this end, DC were pretreated for 1 h with mAb to CD64 (Fc{gamma}RI), CD32 (Fc{gamma}RII), or CD16 (Fc{gamma}RIII), and the binding of immune complexes Cap-Cn opsonized with anti-GXM mAb was quantified. The results reported in Figure 8 show that the binding and phagocytic processes are not affected by the presence of mAb to CD64 (Fc{gamma}RI). This is consistent with the fact that CD64 has low expression or indeed is not expressed at all [26 ]. In contrast, both processes are greatly inhibited by the presence of mAb to CD16 (Fc{gamma}RIII) and CD32 (Fc{gamma}RII).



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  		Figure 8. Effect of mAb to Fc{gamma}RIII, Fc{gamma}RII, or Fc{gamma}RI on phagocytosis of Cap-Cn opsonized with anti-GXM mAb. DC were incubated for 1 h with mAb to CD16, CD32, or CD64 (5 µg/ml), washed, and then treated for 4 h with FITC-labeled Cap-Cn and anti-GXM mAb (5 µg/ml) or isotype-matched control antibody (5 µg/ml). The histograms show the fluorescence and percentage, determined by flow cytometry, of binding cells (upper panels) or phagocytic cells (lower panels). Black lines indicate binding or phagocytic DC in the presence of anti-GXM mAb; gray lines show binding or phagocytic DC in the presence of the isotype-matched control antibody. The data are representative of one of three independent experiments with cells from three different donors.

 
We also investigated the possibility that immunocomplexes could influence the activation of DC via CD16 and/or CD32. By blocking these receptors separately, we observed that CD80 expression on DC stimulated with anti-GXM–Cap-Cn was significantly down-regulated (~35%) relative to DC stimulated with Cap-Cn alone (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our results show that two isogenic strains of C. neoformans, Cap-Cn and Acap-Cn, induce two distinct functional responses in blood-derived human DC. The Acap-Cn was a strong inducer of cell-surface markers related to DC maturation, such as MHC class I and class II, CD83, and CD40. In contrast, the Cap-Cn was a slow and relatively poor inducer of DC maturation. In particular, incubation of the encapsulated strain with DC up-regulated CD40 expression but not MHC class I and class II and CD83 molecules. Both strains were unable to regulate CD80 and CD86 markers. Indeed, the encapsulated strain was only able to up-regulate the CD40 marker. Moreover, the possibility exists that the DC population may already be somewhat activated. If that is the case, then the differences measured for the interaction with Cap-Cn and Acap-Cn could appear reduced relative to those that would be expected for nonactivated cells.

We consistently noted a major difference in the ability of DC to phagocytose Cap-Cn or Acap-Cn. However, addition of anti-GXM mAb was opsonic for Cap-Cn and enhanced the expression of several receptors. Notably, anti-GXM added to suspensions of DC, and Cap-Cn increased the expression of CD40, CD86, and CD83 but failed to regulate MHC class I and CD80. Different receptor-mediated phagocytosis (Fc{gamma}R vs. mannose receptor) could lead to different stages of DC maturation. In addition, the fact that only in the presence of opsonized cells is there an increase in the expression of CD86 could suggest that DC activated with mAb-opsonized cells are different from those activated with the acapsular strain. Moreover, the CD86 modulation that we observed suggests that DC maturation, induced by C. neoformans, is not mediated by Toll-like receptors (TLRs).

The recognition and internalization by DC of mAb-opsonized C. neoformans suggest that specific mAb may play the dual role of facilitating yeast uptake and promoting DC surface markers. These mAb-induced processes appear to be sufficient to allow a more efficient antigen delivery by enhancing antigen-presenting function, which in turn, results in a significant enhancement of T cell activation and IFN-{gamma} production.

The above results indicate that DC interaction with the two strains of C. neoformans produces different effects. Following stimulation with Acap-Cn, DC underwent maturation, whereas after stimulation with Cap-Cn, DC failed to mature fully. Given that many pathogens have a polysaccharide capsule, the concept that such structures interfere with the efficiency of DC could potentially be extended to other microorganisms. In addition, the fungal form responsible for human infection has not been conclusively established. Pulmonary infection is believed to require inhalation of small, poorly encapsulated yeast capable of alveolar penetration. Conceivably, the rapidity of immature DC in capturing and ingesting C. neoformans soon after infection could in part avoid the emergence of large capsule fungal forms, which are observed in vivo. Interference with the emergence of large, encapsulated forms would be expected to foster an efficient T cell response. This may be related to DC capacity to internalize acapsular or encapsulated strains. Consistent with this hypothesis, the monoiodoacetic acid-induced inhibition of C. neoformans phagocytosis resulted in significant inhibition of up-regulation of CD40 and CD86 induced by the acapsular strain (A. Vecchiarelli, unpublished). To further support this concept, anti-GXM mAb added to suspensions of DC, and the Cap-Cn greatly increased CD40, CD83, and CD86 expression.

The facilitation phagocytosis by anti-GXM mAb did not influence MHC class I and CD80 expression, indicating that regulation of these cell markers may be dependent on signal-transduction differences as a result of engagement of different receptors. In fact, up-regulation of MHC class I may be a result of DC activation through mannose [32 , 33 ] or glucan [34 ] receptors, which are likely to be used by acapsular C. neoformans to bind myeloid cells [35 ]. However, it seems likely that the perturbation of mannan receptors, rather than glucan, is involved in the DC maturation process, as demonstrated by the fact that mannan, but not glucan, is able to inhibit phagocytosis and to increase CD40 and MHC class II expression. This suggests that the acapsular strain may use the phagocytic process as well as mannose receptor engagement to promote DC maturation.

The inability of free GXM or GXM plus anti-GXM mAb to up-regulate MHC class I, CD40, CD83, and CD86 expression by DC indicates that engagement of Fc{gamma}R alone is insufficient to stimulate DC maturation, but we have to consider that cross-linking Fc{gamma}R after binding GXM–anti-GXM mAb complexes may be different from that resulting with opsonized Cap-Cn.

However, the evidence that blockage of CD16 and CD32 results in inhibition of CD80 expression suggests that Fc cross-linking and phagocytosis of intact yeast are required for this process. In addition, engagement of several receptors, for example, the mannose receptor, may determine or contribute to DC maturation. The inability of free GXM to modulate some surface DC markers could indicate the difficulty of DC in binding and internalizing GXM. This may be consistent with the fact that GXM binds to CD14 and TLR-4 and that DC lack CD14. However, studies are in progress to clarify this point.

It is well established that maturation of DC results in a loss of phagocytic activity [36 ]. This observation is consistent with down-regulation of Fc{gamma}R in mature DC. Both strains of C. neoformans inhibited Fc{gamma}RII, thereby possibly contributing to the transformation of immature DC into mature. Given that Fc{gamma}R represents a privileged antigen-internalization route for MHC class II-restricted antigen presentation on DC [19 ] and that the immune complexes are internalized by Fc{gamma}R (Fig. 8) , optimal stimulation of CD4 T cells can be expected to occur. This is consistent with T cell proliferation and enhancement of IFN-{gamma} production (Fig. 7) , suggesting that anti-GXM mAb, by stimulating DC, favors a Th1-type response. The identification of substances that regulate the function and maturation of DC may help to elucidate or predict the efficiency of antigen presentation under pathological conditions.

Taken together, these results suggest the existence of additional connections between humoral immunity and DC sentinel function. This phenomenon has important implications in initiating, driving, and maintaining T cell response and suggests that the immune response may be different when specific antibody is present.


   ACKNOWLEDGEMENTS
 
This study was supported by a grant from the National Research Program on AIDS, "Opportunistic Infections and Tuberculosis", contract No. 50D.31, Italy, and by National Institutes of Health Grant 1R03TW01201-01. The authors thank Jo-Anne Rowe for secretarial and editorial support.

Received October 2, 2002; revised April 15, 2003; accepted April 16, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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