The Capsular Polysaccharides of
Cryptococcus neoformans Activate Normal
CD4 + T Cells in a Dominant Th2 Pattern
This information is current as
of June 17, 2017.
Geisy M. Almeida, Regis M. Andrade and Cleonice A. M.
Bento
J Immunol 2001; 167:5845-5851; ;
doi: 10.4049/jimmunol.167.10.5845
http://www.jimmunol.org/content/167/10/5845
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References
The Capsular Polysaccharides of Cryptococcus neoformans
Activate Normal CD4ⴙ T Cells in a Dominant Th2 Pattern1
Geisy M. Almeida,* Regis M. Andrade,* and Cleonice A. M. Bento2†
C
ryptococcus 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– 6). Clinical and murine model studies have indicated that the clearance of pulmonary infection requires the presence of both CD4⫹ and CD8⫹ T cells (7–9). 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-␥, and TNF-␤ 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*Programa de Imunobiologia, Instituto de Biofı́sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; and †Instituto de Microbiologia e Parasitologia, Universidade do Rio de Janeiro, Rio de Janeiro, Brazil
Received for publication March 6, 2001. Accepted for publication September
17, 2001.
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 grants from Programa de Apoio ao Desenvolvimento
Cientı́fico 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.
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.
Copyright © 2001 by The American Association of Immunologists
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-␥ and IL-2 with resistance to C. neoformans infection (5, 6, 16). Other essential cytokines for establishment of resistance to C. neoformans include TNF-␣, IL-12, and
IL-18 (10, 17). The latter two cytokines seem to work synergistically to enhance IFN-␥ production by NK cells and facilitate the
development of Th1 lymphocytes, which in turn produce more
IFN-␥ and IL-12 (6, 18).
The fungal capsule is clearly the major virulence factor of C.
neoformans (19 –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-␣
secretion from LPS-stimulated human monocytes (21) while inducing IL-10 production by these cells in vitro (21, 25). This antiinflammatory 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.
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.
0022-1767/01/$02.00
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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 CD4ⴙ T cells by C. neoformans capsular polysaccharides (CPSs). The results showed that CPSs induce
proliferation of normal splenic CD4ⴙ T cells, but not of normal CD8ⴙ 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 superantigenlike activation. The evaluation of a cytokine pattern produced by the responding CD4ⴙ 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-␣
and IFN-␥. 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 CD4ⴙ T lymphocytes. Our findings suggest that CPSs
induce proliferation and differentiation of normal CD4ⴙ T cells into a Th2 phenotype, which could favor parasite growth and thus
important deleterious effects to the host. The Journal of Immunology, 2001, 167: 5845–5851.
5846
Materials and Methods
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
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 ⫻ 106/well in 48-well vessels (Corning Glass
Works, Corning, NY) in 500 ␮l complete medium (DMEM supplemented
with 10% FCS, 2 mM glutamine, 5 ⫻ 10⫺5 M 2-ME, 10 ␮g/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 ⫻ 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 (antiThy-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 ␬-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 ⫻ 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 ⫻
106/ml) obtained from columns were treated with a saturating dose of antiCD8 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 ␮l) 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䡠H2O (1.36 g/L), urea (1.29 g/L), sodium glutamate
(1 g/L), MgSO4䡠7H2O (0.3 g/L), thiamine-HCl (2 mg/L), and biotin (10
␮g/ml). Results are presented as mean ⫾ SEM of Cap 67 cell number in
triplicate cultures.
Cell proliferation assay
Primarily, whole splenocytes (1.5 ⫻ 106/ml) were cultured in the presence
or absence of crescent doses of the CPSs (0.3 to 60 ␮g/ml) in 0.2 ml
complete medium. In other experiments, CD4⫹ T cell (1.5 ⫻ 106/ml), in
the presence or absence of B lymphocytes (1.5 ⫻ 106/ml), were set up with
or without 30 ␮g/ml CPSs. All cultures were established in 96-well flatbottom 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 ⫻
106/ml) with SAC (5.0 ⫻ 105/ml) were kept under the same conditions in
the presence of 30 ␮g/ml CPSs, biochemically treated or not, in 48-well
vessels (500 ␮l). 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 ␮g/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 ␮g/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 ␮g/ml) of the anti-B7.1 and/or anti-B7.2, antiCD40 ligand (CD40L), rat (IgG2a) or hamster (IgG) control isotypes were
used. The cultures were pulsed with 0.5 ␮Ci/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 CPStreated 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 ␬-chain mAb MAR 18.5 were
gifts from Dr. Ethan Shevach (National Institutes of Health, Bethesda,
MD). Anti-TCR␤ 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 ␮l/well) were collected for cytokine determination
from cultures containing SAC (5.0 ⫻ 105/ml) with or without CD4⫹ T
splenocytes (1.5 ⫻ 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-␣ and rat anti-mouse IFN-␥,
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-␥,
TNF-␣, 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.
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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 ⫻ 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 ⫻ 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 ⫻ 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.
ACTIVATION OF CD4⫹ T CELLS BY CRYPTOCOCCAL CPSs
The Journal of Immunology
Results
C. neoformans CPSs induce proliferative response of normal
murine CD4⫹ T cells by a mechanism dependent on splenic
adherent cells
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. 2A). Additionally,
the SAC monolayers were washed exhaustedly and cocultured
with CD4⫹ T lymphocytes (Fig. 2B). It was observed that supernatants collected from the adherent cells were unable to induce the
in vitro proliferative response of CD4⫹ T cells (Fig. 2A). However,
the direct contact between splenocytes provided mitogenic activity
of CPSs on CD4⫹ T cells (Fig. 2B). 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.
2C, 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 antiCD40L mAbs to the cultures. Effective blockade of the CPS-induced lymphoproliferation was achieved only by anti-CD40 mAbs
(Fig. 3). Neither anti-B7.1 nor anti-B7.2 modified the level of
[3H]thymidine uptake on these in the presence of CPSs.
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 I). Additionally, we also assayed cytokine
production by cocultures of SAC with CD4⫹ T cells in the presence of CPSs (Fig. 4). Clearly, CPSs induced IL-10 secretion by
both culture types (Table I and Fig. 4). IL-4 production was detected only on wells containing CD4⫹ T lymphocytes and was
augmented by CPSs (Fig. 4). 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-␣ and
IFN-␥) and anti-inflammatory (IL-4 and IL-10) cytokines assayed
(Fig. 4).
FIGURE 1. C. neoformans-derived CPSs induce proliferation of CD4⫹ T cells in a SAC-dependent manner. A, Normal whole splenocytes (1.5 ⫻ 106/ml;
200 ␮l/well) were cultured with crescent doses of CPSs of C. neoformans (0.3– 60 ␮g/ml); B, Normal whole splenocytes (1.5 ⫻ 106/ml; 200 ␮l/well) were
cultured with or without saturating doses of blocking anti-CD4 (␣-CD4; 5 ␮g/ml) and/or anti-CD8 (␣-CD8; 30% v/v supernatants of clone 53.6.7) mAb,
in the presence of the CPSs (30 ␮g/ml); C, CD4⫹ T lymphocytes (1.5 ⫻ 106/ml) were cocultured in the presence or absence of purified B lymphocytes
(1.5 ⫻ 106/ml; 200 ␮l/well) or SAC (5.0 ⫻ 105/ml; 500 ␮l/well), all obtained from normal spleen; D, CD4⫹ T lymphocytes (1.5 ⫻ 106/ml) with SAC (5.0 ⫻
105/ml; 500 ␮l/well) were cocultured in the presence of CPSs (30 ␮g/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 ␮g/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 ␮Ci per well) added during the final 16 h of culture. Results
indicate the means ⫾ SEM of triplicate cultures.
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C. neoformans CPSs induced maximum proliferative response of
normal murine splenocytes at 30 ␮g/ml (Fig. 1A). 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. 1B). 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. 1C).
Nevertheless, the ability to induce proliferative response of CD4⫹
T cells was acquired when these lymphocytes were cocultured
with SAC monolayers (Fig. 1C). 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. 1D, only
the treatment with periodate ablated CPSs induced in vitro lymphoproliferative responses of murine CD4⫹ T cells.
5847
ACTIVATION OF CD4⫹ T CELLS BY CRYPTOCOCCAL CPSs
5848
⫹
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 ⫻ 105/ml; 500 ␮l/well) were cultured overnight with
CPSs (30 ␮g/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 ⫻
106/ml; 200 ␮l/well). B, These adherent cells were additionally washed and
cocultured with CD4⫹ T lymphocytes (1.5 ⫻ 106/ml; 500 ␮l/well). C,
Normal SAC (5.0 ⫻ 105/ml) were fixed with paraformaldehyde solution
(1% v/v) for 15 min, washed, and cocultured with CD4⫹ T cells (1.5 ⫻
106/ml; 500 ␮l/well) in the presence or absence of CPSs. As positive control, SEB was used (2 ␮g/ml). All cultures were kept for 3 days, and
proliferation was assessed by [3H]TdR uptake (0.5 ␮Ci per well) added
during the final 16 h of culture. Results are indicated as 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-␥ and TNF-␣ (Fig. 4). 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. 5, 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 II). Production of inflammatory cytokines was undetectable
either before or after CPS addition (Table II).
Discussion
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 mu-
rine 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– 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. 1B). 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. 1C). 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
Table I. C. neoformans-derived CPSs induce IL-10 secretion by SAC
monolayersa
Treatment
Control
CPS
IL-10 (ng/ml)
TNF-␣ (ng/ml)
8.10 ⫾ 1.60
36.70 ⫾ 4.70
⬍1
⬍1
a
Normal SAC monolayers (5.0 ⫻ 105/ml) were kept in the presence or absence
of CPSs (3.0 ␮g/ml). Supernatants were collected (500 ␮gl/well) 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 triplicates.
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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 ⫻ 105/ml) were cocultured with autologous CD4⫹ T cells (1.5 ⫻
106/ml) for 3 days in the presence of CPSs (30 ␮g/ml). The role of different
signaling pathways in the mitogenic activity was evaluated by addition of
saturating doses (10 ␮g/ml) of anti-B7.1 (␣-B7.1; 1G10), anti-B7.2 (␣B7.2; GL1) or anti-CD40L (␣-CD40L; MR1) mAbs. As control isotype,
we used a hamster IgG (hamsterIgG) or rat IgG2a (ratIgG2a), both at 10
␮g/ml. Proliferation was assessed by [3H]TdR uptake (0.5 ␮Ci per well)
added during the final 16 h of culture. Results are indicated as means ⫾
SEM of triplicate cultures.
The Journal of Immunology
(Fig. 1B). 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 pu-
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 ⫻ 105/ml) with CD4⫹ T cell (1.5 ⫻ 106/ml; 500 ␮l)
were kept with the nonencapsulated C. neoformans strain Cap 67 (103/ml)
in the presence or absence of CPSs (30 ␮g/ml). To evaluate the role of the
signaling through CD40L in the course of the in vitro infection, saturating
doses (10 ␮g/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 ␮l) was transferred to 1.5 ml synthetic medium (CDC2500) 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.
rified 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 ␣136- and ␣134-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. 2C).
SAgs, characteristically, are able to activate T lymphocytes by
simultaneously associating, without processing, the outside of the
MHC class II molecules with some V␤ 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. 1B). However,
oxidation of CPSs by periodate abolished the mitogenic activity on
CD4⫹ T cells induced by intact CPSs (Fig. 1B). 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 I). 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
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
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 ⫻ 106/ml) and
SAC monolayers (5.0 ⫻ 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 ␮g/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.
5849
ACTIVATION OF CD4⫹ T CELLS BY CRYPTOCOCCAL CPSs
5850
Table II. Enhancement of anti-inflammatory cytokine production induced by C. neoformans-derived CPSs depends on signaling triggered by CD40La
Control
Cap 67
Cap 67 ⫹
Cap 67 ⫹
Cap 67 ⫹
Cap 67 ⫹
Cap 67 ⫹
Treatment
IL-10 (ng/ml)
IL-4 (ng/ml)
IFN-␥ (ng/ml)
TNF-␣ (ng/ml)
hamster IgG
anti-CD40L
CPS
CPS ⫹ hamster IgG
CPS ⫹ anti-CD40L
4.10 ⫾ 1.61
10.70 ⫾ 2.33
12.50 ⫾ 1.15
12.10 ⫾ 1.60
36.20 ⫾ 5.60
29.40 ⫾ 4.72
12.60 ⫾ 1.35
2.20 ⫾ 1.25
9.70 ⫾ 2.47
9.60 ⫾ 1.60
7.80 ⫾ 1.30
17.40 ⫾ 2.60
17.60 ⫾ 1.13
4.60 ⫾ 0.78
⬍1
⬍1
⬍1
⬍1
⬍1
⬍1
⬍1
⬍1
6.5 ⫾ 2.12
3.1 ⫾ 0.67
3.8 ⫾ 1.03
⬍1
⬍1
⬍1
a
CD4⫹ T lymphocytes (1.5 ⫻ 106/ml) were cocultured with SAC monolayers (5.0 ⫻ 105/ml; 500 ␮l/well) in the presence of 103 Cap 67/ml and with or without CPSs (30
␮g/ml). In some wells, we added saturating doses (10 ␮g/ml) of neutralizing anti-CD40L mAb, or a control hamster isotype (hamster IgG). As control, the cocultures were
maintained in the medium alone. After 3 days, the supernatants of cocultures were collected (500 ␮l/well) 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 triplicates.
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.
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