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*
Evans Memorial Department of Clinical Research and Departments of Medicine and Microbiology, Boston University School of Medicine, Boston, MA 02118; and
Veterans Affairs Medical Center and Departments of Medicine and Microbiology, University of Iowa, Iowa City, IA 52242
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-elimination
inhibited MP-dependent stimulation of P1D6 and primary T cells by 89
and 90%, respectively. In addition, MP-dependent stimulation of P1D6
was abrogated after digestion with proteinase K, suggesting the protein
core of MP contributed the antigenic moiety presented by APC.
Stimulation of P1D6 by MP also was abolished using APC obtained from
invariant chain-deficient mice, demonstrating Ag presentation was MHC
class II restricted. Our data suggest that MP is a ligand for the MMR
and that T cell stimulation is functionally inhibited either by
competitive blockade of MR or by removal of carbohydrate residues
critical for recognition. The demonstration that efficient T cell
responses to MP require recognition of terminal mannose groups by MMR
provides both a molecular basis for the immunogenicity of cryptococcal
MP and support for vaccination strategies that target
MR. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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7% (4).
Considerably higher prevalence rates are seen in parts of the
developing world. Even with recent advances in therapy, morbidity and
mortality are high. In patients with AIDS, indefinite maintenance
therapy is required to prevent relapse (5). In light of
these daunting statistics, identification of immunoprotective
cryptococcal Ags that could serve as vaccine candidates has been
declared a public health priority (6).
Through observation of immunocompromised patients and the development
of infectious animal models, it has become clear that the cryptococcal
immune response relies heavily on an intact cell-mediated immune
response. Elimination of the CD4+ and/or
CD8+ T cell populations results in a diminished
capacity for clearance of C. neoformans
(7, 8, 9, 10). Other studies have also demonstrated a role for
Ab and B cells (11, 12). Initial attempts using whole
organism vaccination for protection were moderately successful
(13); however, the use of eukaryotic cells as vaccines
presents potential autoimmune and inflammatory complications.
Therefore, investigators have fractionated cryptococcal culture
supernatants to identify immunostimulatory components. Separation of
supernatants by Con A affinity chromatography yields a highly
mannosylated protein fraction termed mannoprotein
(MP)3
(14). MP can elicit delayed-type hypersensitivity
reactions and induces production of the cytokines TNF-
(15), IL-12, and IFN-
(16), shown to be
critical in decreasing fungal loads in murine models of cryptococcosis
(17, 18, 19, 20). Moreover, T cells from patients who have
recovered from cryptococcosis proliferate and secrete cytokines in
response to MP (21, 22). To further characterize the
immunoreactive moieties present in culture supernatants, we previously
generated CD4+ T cell hybridoma cell lines with
specificity for MP (23). One such hybridoma, designated
P1D6, has specificity for a serine/threonine-rich MP, MP98, with an
apparent molecular mass of 98 kDa. While other laboratories have
also identified immunoreactive cryptococcal Ags (24, 25),
it is likely that a successful vaccine will consist of multiple
components able to elicit both T and B cell-mediated immunity.
C-type lectins include a family of related proteins that have more than one putative lectin domain. These multilectin mannose receptors (MR) include the dendritic cell receptor DEC-205, phospholipase A2 receptor, and the macrophage mannose receptor (MMR), a well-characterized pattern-recognition receptor (reviewed in Ref. 26). Although these proteins are all on immune cells, their precise cellular distribution, ability to bind carbohydrates, and immune function appear to be quite different. Both the MMR and DEC-205 serve as links between innate and adaptive immunity by colocalizing acquired Ag with MHC class II-rich endosomes (27, 28, 29). The MMR can recognize patterns of exposed hexoses, including mannose and fucose, but not galactose (26). The MMR can interact with a range of mannose-decorated whole pathogens and Ags, including Borrelia burgdorferi (30) and lipoarabinomannans from pathogenic mycobacteria (31, 32, 33). We hypothesized that APCs, which express MMR and DEC-205, target cryptococcal MP for receptor-mediated uptake and subsequent efficient Ag presentation to T cells. To test this hypothesis, we assessed the capacity of MP to bind the MMR and be processed by APC to stimulate primary T cells from immunized mice as well as the MP-reactive T cell hybridoma P1D6. Moreover, we investigated the potential downstream consequences on T cell activation resulting from interference with MR-dependent MP uptake by APCs.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All materials were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise noted. RPMI 1640, FBS, and geneticin were purchased from Life Technologies (Rockville, MD). PBS and HBSS were purchased from BioWhittaker (Walkersville, MD). Tritiated thymidine was purchased from NEN Life Sciences (Boston, MA). Cell culture was performed in a humidified 37°C incubator supplemented with 5% CO2. RPMI-10 is defined as RPMI 1640 supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 10 mM HEPES buffer.
Isolation of soluble C. neoformans MP fraction
MP was isolated from culture supernatants of C.
neoformans acapsular strain Cap67 (no. 52817; American Type
Culture Collection, Manassas, VA) as previously described
(34), with minor modifications. Briefly, fungi were grown
for 5 days at 30°C in an orbital shaker in medium consisting of 6.7
g/L yeast nitrogen base, 27 mM dextrose, 64 µM
L-histidine, 134 µM
L-methionine, and 97 µM
L-tryptophan. Cultures were allowed to settle
overnight at 4°C and the supernatant was collected, filter-sterilized
(0.22-µm pore size), and concentrated 60- to 80-fold using a
10-kDa-cutoff regenerated cellulose tangential filtration cassette
(Millipore, Bedford, MA). The concentrated crude cryptococcal
supernatant (CR) was exchanged for PBS and subjected to Con A Sepharose
chromatography using fast performance liquid chromatography (AKTA
model; Amersham Pharmacia Biotech, Piscataway, NJ). The nonbinding
flow-through (FT) fraction was collected followed by a MP fraction
eluted using 0.2 M methyl -D-mannopyranoside
(ManP). The fractions were dialyzed against dH2O
using 7-kDa-cutoff dialysis tubing (Pierce, Rockford, IL) and
subsequently lyophilized. After reconstitution with PBS, protein
concentration was assessed using the bicinchoninic acid assay (Pierce)
while total carbohydrate was measured using the phenol-sulfuric acid
assay (35). Aliquots were stored at -80°C. All
fractions were boiled for 5 min to inactivate any biologically active
Con A leached from the affinity column (36).
SDS-PAGE analysis
Culture supernatant fractions were run under denaturing conditions using 12% SDS-PAGE. Silver staining, periodic acid-Schiff (PAS), and Western blotting were performed using standard protocols. Briefly, for silver staining of proteins, gels were fixed by sequential incubations for 30 min in 50% methanol/10% acetic acid, overnight in 5% methanol/7% acetic acid, and for 30 min in 10% glutaraldehyde. Following extensive washing in dH20, the gel was stained for 15 min using 23.5 mM AgNO3/20 mM NaOH/2% NH4OH. After washing with dH20, the bands were visualized using a solution of 240 µM citric acid/0.02% formaldehyde. The development of the bands was stopped by incubating the gel for 20 min in 10% acetic acid. To assess carbohydrate association by PAS staining, gels were fixed overnight in PAS buffer (40% ethanol/25 mM NaCl/25 mM acetic acid). Carbohydrates were oxidized for 1 h using 1% sodium m-periodate in PAS buffer. After washing unreacted m-periodate, Schiff’s reagent was added in the dark for 1 h. The development of the bands was stopped with extensive washing. For Western blotting, following electrophoretic transfer (30 V, overnight, 4°C) polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA) were blocked using a 0.1 mg/ml polyvinyl alcohol solution for 1 min followed by a BSA block reagent (Kirkegaard & Perry Laboratories, Gaithersburg, MD) for 1 h. After washing in TBS (25 mM Tris/0.15 M NaCl/0.05% Tween 20, pH 7.5), membranes were incubated sequentially for 1 h each in 0.1 µg/ml biotinylated Con A probe (Pierce) and a 1/40,000 dilution of streptavidin-HRP conjugate (Zymed Laboratories, San Francisco, CA). The blot was visualized using the Supersignal West Pico chemiluminescent substrate (Pierce).
Fractionation, -elimination, and digestion of MP
After resolving the MP fraction using 12% SDS-PAGE, gels were sliced into three regions. Based on concurrently run, silver-stained molecular mass standards, region 1 contained bands with an apparent range of >60 kDa, region 2 contained bands with an apparent range of 60–30 kDa, and region 3 contained bands with an apparent range of <30 kDa. Gel slices were chopped into 2- to 3-mm2 cubes and placed in PBS overnight at 4°C to elute the glycoproteins. The gel slices were washed once with dH20 and the pooled buffer fractions were dialyzed against PBS using 3.5-kDa-cutoff dialyzer cassettes (Pierce).
For chemical O-linked deglycosylation, the MP fraction or OVA was incubated at a concentration of 0.65 mg/ml in PBS containing 0.1 M NaOH at 37°C. At the indicated time points, NaOH was neutralized using glacial acetic acid (37, 38).
For protein digestion, 0.4 mg/ml MP was incubated with 4% cross-linked agarose beads conjugated to proteinase K or protein G at a final concentration of 16 mg/ml. The mixtures were incubated rotating at 37°C for 4 h in RPMI 1640. The reaction was stopped by spinning the mixture through a 0.45-µm filter trap to remove the agarose beads.
Competition HRP uptake assay
The pVex expression vector containing human MMR cDNA (39) was used to generate a stably transfected MMR-expressing Chinese hamster ovary cell line (CHO-MMR) (40). Expression of MMR was confirmed by flow cytometry using a mAb specific for human MMR (BD PharMingen, San Diego, CA). CHO-MMR and untransfected wild-type CHO cells (CHO-K1) were cultured in 24-well plates in RPMI-10 supplemented with 0.5 mg/ml active geneticin (for selection of transfected cells only). When a confluent monolayer was achieved, the cells were washed twice with HBSS containing 1 mg/ml BSA. Cryptococcal supernatant fractions or mannosylated inhibitors of the MMR were preincubated with the CHO cells in binding buffer (1% BSA in HBSS) at the indicated concentrations for 5 min at room temperature. Following the preincubation, 50 µg/ml HRP type II was added for 1 h at 37°C. HRP type II is a glycoprotein decorated with mannose, fucose, and N-acetylglucosamine residues, making it a natural ligand for the MMR (41). Following the incubation, the CHO cells were washed four times, lysed, and solubilized using 300 µl 0.1% Triton X-100. HRP uptake was measured as previously described (42), with minor modifications. Briefly, 25 µl of cell lysate was incubated with 225 µl of o-dianisidine substrate solution (0.008% o-dianisidine/0.003% H2O2 in 0.05 M sodium phosphate, pH 5). Color conversion was read at an absorbance wavelength of 405 nm. HRP activity was compared with a standard curve of known HRP concentrations. There was no conversion of color substrate in the presence of CHO cell lysate alone. Total protein from the cell lysate was assessed using the bicinchoninic acid assay. HRP uptake is expressed as a ratio of HRP activity per milligram of total protein.
Naive splenocytes as source of APC
Spleens from naive C57BL/6 mice were collected, gamma irradiated (3000 rad), and macerated through sterile wire mesh. Contaminating erythrocytes were lysed using 0.15 M ammonium chloride. The resulting cell suspension was purified over a Ficoll-Hypaque gradient and the buffy layer was collected and washed in RPMI-10. The spleen cell population contains B cells, dendritic cells, and macrophages as potential sources of Ag presentation.
Immunization of mice
Pathogen-free 6-wk-old male C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were cared and housed under specific pathogen-free conditions at Boston University’s Laboratory Animal Science Center (Boston, MA). Immunizations were performed using the Ribi Adjuvant system. A total of 50 µg of CR were injected i.p. in 3-wk intervals for up to four immunizations. Seven days following the final boost, inguinal, para-aortic, axillary, and mesenteric lymph nodes (LN) were collected using aseptic technique.
Proliferation analysis
Proliferation of T cells from LN of CR-immunized C57BL/6 mice or whole splenocytes from DO11.10-transgenic mice (a gift of A. Marshak-Rothstein, Boston University School of Medicine) was assessed in response to MP or OVA, respectively.
LNs were macerated to a single cell suspension using frosted glass slides. RBCs were lysed with 0.15 M ammonium chloride. For purified T cells, contaminating B cells, macrophages, and other APCs were depleted using standard complement-mediated lysis techniques (43), with minor modifications. Supernatants from two Ab-secreting B cell hybridomas specific for MHC class II (clone TIB 120, anti-I-Ab,d,q, I-Ed,k; and clone TIB 229, anti-I-Ab,d; a gift of Dr. L. Wetzler, Boston University School of Medicine) were incubated with LN single cell suspensions for 45 min on ice at a concentration of 107 cells per milliliter. After washing, cells were resuspended in rabbit serum (a gift of Dr. L. Wetzler) at a dilution of 1/20 in RPMI-10 for 35 min at 37°C. Flow cytometric analysis of the purified T cell fraction revealed 70–90% CD3+, <10% B220+, and <7% I-Ab+. T cells were plated in 96-well flat-bottom plates at 2 x 105 cells per well in the presence or absence of Ag and inhibitors. Gamma-irradiated C57BL/6 splenocytes, added at 5 x 105 cells per well, served as a source of APC. Nonirradiated splenocytes from DO11.10 mice were prepared as described above and plated at 7 x 105 cells per well.
Cells were incubated for 4 days and pulsed with tritiated thymidine during the final 18 h. Plates were subjected to a freeze-thaw cycle, then collected onto fiberglass filter strips using a cell harvester (Cambridge Technology, Cambridge, MA). Samples were counted by scintillation spectroscopy (Delta 300; Searle Analytic, Des Plaines, IL). Results are expressed as stimulation index, defined as stimulated cpm divided by unstimulated cpm.
T cell hybridoma
The CD4+ T cell hybridoma cell line used in these studies, P1D6, is specific for an epitope found in cryptococcal MP, MP98 (23). Thus, in the presence of an APC and MP98, P1D6 will secrete IL-2. Hybridoma cell lines were maintained in RPMI-10 supplemented with 1% hypoxanthine/thymidine, 0.25 µg/ml amphotericin B, 10 µg/ml ciprofloxacin, and 55 µM 2-ME. Hybridoma cells at 1 x 105 cells per well were added to gamma-irradiated naive splenocytes at 2 x 105 cells per well in the presence or absence of Ag and inhibitors to a final volume of 200 µl. Stocks of inhibitors were prepared in RPMI 1640 and sterile filtered. After 24 h, 150 µl of supernatant was transferred to a 96-well plate and freeze/thawed to lyse any contaminating spleen or hybridoma cells. The amount of IL-2 in the supernatants was determined using the IL-2/IL-4-dependent CTLL-2 cell line (TIB 214; American Type Culture Collection) as in our previous studies (23). Results were compared with a standard curve generated with known amounts of IL-2 and are expressed in IL-2 units per milliliter.
Statistical significance
Student’s t test with a value of p < 0.05 was used as a measure of statistical significance. Representative data are expressed as means ± SE.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mean protein concentrations in the CR and MP fraction were 5.5
and 3.3 mg/L, respectively. As calculated by mass, the MP and FT
fractions had carbohydrate:protein ratios of 5.4:1 and 6.5:1,
respectively. SDS-PAGE analysis of the MP fraction resolved at least 10
distinct bands ranging from <20 to >100 kDa (Fig. 1
A, MP). Several bands
exhibited negative staining on silver stain, suggesting the presence of
heavy carbohydrate conjugation. The presence of polysaccharide was
verified by PAS staining (Fig. 1B, MP) as well as an
ultraviolet light-visible glycostain (Pro-Q Emerald 300 Glycoprotein
Gel Stain kit; Molecular Probes, Eugene, OR) (data not shown). Western
blotting with a biotinylated Con A probe showed five bands measuring at
apparent molecular masses of 111, 96, 71, 36, and 29 kDa (Fig. 1C, MP). Staining with Con A suggests terminal mannose
conjugation. As expected, the FT fraction did not stain (Fig. 1C, FT).
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Several members of the MR family have been described that exist on
innate immune cells, including MMR and DEC-205 (26). We
used a CHO cell line engineered to heterologously express functional
MMR (CHO-MMR) (40) to determine whether MP binds
specifically to the MMR. To detect binding of MP to the CHO-MMR cells,
a known ligand of the MMR, HRP, was used in a competition uptake assay.
To confirm the specificity of the HRP/CHO-MMR uptake assay, competitive
inhibitors were used. A total of 50 mM ManP and mannans from
Saccharomyces cerevisiae inhibited HRP uptake by 90 and
94%, respectively, as compared with 8% by methyl
-D-galactopyranoside (GalP) (Fig. 2). MP incubated at various
concentrations demonstrated dose-dependent inhibition of HRP uptake,
reaching a maximum of 82% at 50 µg/ml. FT, at 50 µg/ml, inhibited
HRP uptake by 37%, suggesting that this fraction contains ligands that
recognize the MMR but not Con A. CR, which contains significant amounts
of MP, inhibited HRP uptake by 74%. The parent cell line, CHO-K1,
demonstrated only a minor capacity for HRP acquisition. To test the
possibility that individual components of the HRP uptake assay could
have interfered with HRP enzymatic activity, 400 µg/ml MP, FT, and
CR, as well as 100 mM ManP, 100 mM GalP, and 15 mg/ml mannan were
coincubated with 50 pg/ml HRP at 37°C for 3 h. There was no
inhibition of HRP-mediated substrate conversion with any of the
coincubations (data not shown).
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We next assessed the contribution of MR to the stimulation of the
T cell hybridoma P1D6 by MP. Using gamma-irradiated naive splenocytes
as sources of APC, MP with or without sugar analogs were incubated
overnight in the presence of T cell hybridoma cells as described in
Materials and Methods. Addition of the MMR competitors, ManP
and mannans, inhibited IL-2 production by 99% at 40 µg/ml MP (Fig. 3). The osmotic control, GalP,
demonstrated nonsignificant decreases of IL-2 as compared with control
(PBS). MP, mannan, or the sugar analogs did not nonspecifically affect
individual components of the hybridoma/CTLL-2 bioassay, as
MP-stimulated IL-2 production was seen only if both hybridoma cells and
splenocytes were present in the wells (data not shown). In addition,
neither mannan nor the sugar analogs had direct nonspecific effects on
the proliferation of CTLL-2 cells (data not shown). Thus, these data
suggest dependence on MR for hybridoma stimulation.
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Glycoprotein and proteoglycan carbohydrate residues may be
conjugated to protein cores by O-, N-, or
GPI-links (44). The amino acids serine and threonine serve
as donors for O-links, whereas asparagine provides the means
for N-links. Investigators have established the presence of
abundant serines and threonines in MP fractions from C.
neoformans (45). The MP98 sequence contains 103
serine/threonine residues to serve as potential sites for
O-links, as well as 12 possible N-link sites
(23). Removal of carbohydrates manifests as a shift in
apparent molecular mass when glycoproteins are resolved on SDS-PAGE.
Digestion of the MP fraction with enzymes specific for
N-linked carbohydrates such as peptide N-glyco
idase F or EndoHf (New England Biolabs,
Beverly, MA) resulted in only slight decreases in the apparent
molecular mass (data not shown). To assess the presence of
O-links, chemical deglycosylation was used in the form of
-elimination. -Elimination uses mild sodium hydroxide treatment
to specifically cleave O-links via nucleophilic attack
(37). Following -elimination for 3 or 24 h, high
molecular mass bands found in the MP fraction shifted to lower
apparent molecular mass, as demonstrated by silver staining (Fig. 4A). Moreover, -elimination
resulted in loss of carbohydrate as demonstrated by PAS staining (Fig. 4B). This suggests O-links as the dominant form
of carbohydrate conjugation used by C. neoformans strain
CAP 67.
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-eliminated MP was incubated with primary T cells isolated from
CR-immunized mice as well as with P1D6 (Fig. 4
C).
Gamma-irradiated splenocytes from naive mice served as APCs in both
reactions. Compared with the 0-h -elimination time point, which was
immediately neutralized, progressively longer treatment resulted in
maximal inhibitions of 90 and 89% as measured by incorporation of
tritiated thymidine and IL-2 production, respectively. As a control for
potential mitogens in the cryptococcal fractions, 50 µg/ml MP, FT,
CR, and -eliminated MP were coincubated with naive C57BL/6 single
splenocyte suspensions for 4 days and proliferation was measured by
tritiated thymidine incorporation. The results demonstrate that there
was no stimulatory activity attributable to the fractions alone
(stimulation indices for MP, FT, CR, -eliminated MP, and Con A (5
µg/ml as a positive control) were 1.07 ± 0.14, 2.68 ±
0.36, 0.56 ± 0.02, 0.81 ± 0.07, and 29.99 ± 1.21,
respectively).
Extreme pH conditions can degrade disulfide bonds and hydrolyze the
peptide backbone, altering primary and secondary protein structure
(46). To control for possible cellular toxicities or
protein degradation resulting from the process of -elimination, we
took advantage of the transgenic DO11.10 mouse strain, which has been
genetically engineered to express an TCR specific for a peptide
sequence found in OVA (47). OVA was subjected to the
-elimination procedure by incubation with sodium hydroxide for 3 and
24 h. Isolated DO11.10 splenocytes were then incubated with
various concentrations of OVA pre- and post--elimination, and
proliferation was measured by tritiated thymidine
incorporation. In three independent experiments,
proliferation of the DO11.10 splenocytes was undiminished by
progressively longer -elimination treatments (stimulation indices
using 100 µg/ml OVA -eliminated for 0, 3, and 24 h were
57 ± 3, 83 ± 3.26, and 81 ± 2.73, respectively).
These results suggest that the conditions used for O-linked
deglycosylation leave the protein core intact.
Determination of the immunoreactive Ags
The next set of experiments was designed to determine which of the
multiple bands in the MP fraction were immunodominant Ags. Separation
of the cryptococcal MP fraction into three molecular mass ranges (>60,
60–30, and <30 kDa) was performed by slicing 12% SDS-PAGE gels and
eluting them into PBS. Approximately 90 µg of protein were collected
per fraction per gel. The apparent molecular masses of the fractions
and the efficiency of the elution procedure were confirmed by silver
and PAS stains (data not shown). To determine where the immunodominant
Ag(s) existed, T cells from LN of CR-immunized C57BL/6 mice were
purified and coincubated with gamma-irradiated naive splenocytes. ManP
and GalP were included to assess the contribution of MR to the
stimulation by competitive blockade. Only the Ags found within the high
molecular mass range (>60 kDa) elicited T cell proliferation as
measured by thymidine incorporation (Fig. 5). Blockade of mannose receptors using
ManP resulted in a 72% inhibition as compared with the osmotic
control, GalP (Fig. 5).
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0.05 comparing the highest molecular mass fraction with either the
middle or the low molecular mass fraction). When the hybridoma cell
line was coincubated with the >60-kDa fraction and a competitive
inhibitor of the MMR (3 mg/ml mannan), IL-2 concentrations were similar
to levels seen in the absence of stimulation (0.052 ± 0.001 and
0.050 ± 0.037 U/ml IL-2, respectively). MP core protein is required for hybridoma stimulation
The above experiments established the requirement for the
carbohydrate portion of MP for optimal responses to adaptive immune
cells. This final set of experiments was designed to determine 1)
whether the protein core of MP contributes to T cell stimulation and 2)
whether stimulation occurs via MHC. Neither can be assumed, as other
MHC-like molecules, particularly CD1, have the capacity to stimulate T
cells by presenting nonprotein Ags, such as lipids (48).
To determine whether the T cell response to MP is dependent on the core
protein, MP was digested by incubation with agarose beads coupled to
proteinase K (PK beads). As a control for nonspecific adsorption of MP
by the agarose beads themselves, agarose beads coupled to protein G (PG
beads) were used. IL-2 production by P1D6 hybridoma cells was used to
assess MP immunogenicity following protease treatment. A complete loss
of reactivity to MP after treatment with PK beads, but not PG beads,
was observed (Fig. 6), suggesting that
the protein core of MP is necessary for its immunoreactivity.
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To determine whether the response to MP was MHC restricted,
invariant chain (Ii)-deficient mice were used. APCs from
Ii-/- mice cannot traffic functional or
sufficient MHC class II molecules to the surface (49).
Thus, Ii-/- APCs lack the ability to present Ag
via the endocytic pathway. In contrast, presentation via CD1 molecules
should remain intact, as CD1 trafficking to endocytic vesicles is
independent of Ii (50). MP was incubated with P1D6
hybridoma cells in the presence of gamma-irradiated splenocytes
isolated from naive wild-type C57BL/6 and Ii-/-
mice. Deficiency of the MHC class II pathway resulted in an 88%
decrease in IL-2 production in response to 40 µg/ml MP (Fig. 7). These data suggest MP-dependent
stimulation is MHC class II restricted.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although their function is largely unknown, cryptococcal MPs account for a large component of the secreted material from C. neoformans. Therefore, it is likely that MPs are among the initial Ags that professional APCs engage at a cryptococcal focus of infection. In preliminary experiments using an i.v. model of cryptococcosis, compared with groups treated with adjuvant only, MP-vaccinated C57BL/6 mice demonstrated significantly increased survival and decreased organ fungal loads in kidneys and brain (M. K. Mansour and S. M. Levitz, unpublished data). MP can also be isolated from other fungi such as Candida albicans and Penicillium marneffei (51, 52). Consistent with cryptococcal MP, candidal MP has been shown to elicit anticandidal T cell responses (52). Thus, the development of MPs as antifungal T cell vaccines may have extended efficacy in the prevention of a variety of pathogenic fungi.
APCs can use a variety of pathways to acquire Ag, including macro- and micropinocytosis, as well as receptor-mediated endocytosis. In the case of the MMR, this receptor recognizes whole pathogens and microbial products decorated with terminal mannose groups, such as mycobacterial lipoarabinomannans (31, 32, 33). Unlike lower eukaryotes, such as yeast, extracellular mammalian glycoproteins rarely exhibit terminal mannosylation, but instead integrate mannose within the internal branching structure of complex oligosaccharides (44). In the case of DEC-205, the structural determinants of Ags for binding to the receptor are less clearly defined. It is uncertain whether DEC-205 binds carbohydrates (26). Based on competition HRP uptake studies, cryptococcal MP is a ligand for the MMR. Moreover, acquisition of MP through MR is essential for proper Ag presentation to T cells. Experimental evidence suggests that Ag acquired through multilectin receptors, such as MMR and DEC-205, is targeted for efficient processing and presentation (28, 29). Consistent with these observations, blocking MR on APC with mannose analogs and mannans resulted in a significant reduction in stimulation of primary T cells and CD4+ T cell hybridomas. Our data do not rule out that other multilectins on the surface of APCs may also participate with the MMR in the recognition of MP.
Terminal mannosylation on MP would be required to allow recognition by
the MR. The lectin Con A, which recognizes terminal mannoses and
glucoses (53), was used to purify MP from CR. Not
surprisingly, MP, but not the FT fraction, resolved by SDS-PAGE stained
with a biotinylated Con A probe. In addition, two components of the MP
fraction, MP98 and MP88, have been sequenced and contain C-terminal
serine/threonine-rich regions that presumably serve to form
O-linked glycosylation domains (Ref. 23 and
S. M. Levitz, unpublished data). Our deglycosylation experiments
lend support to the importance of O-links for MP terminal
mannosylation. Cleavage of these links by -elimination
dramatically decreased the apparent molecular mass of silver-stained
bands and eliminated reactivity with the carbohydrate-specific PAS
stain. In contrast, digestion of MP with glycosidases specific for
N-links had little effect on the apparent molecular mass of
MP. These O-linked carbohydrates appear to be critical for
MP stimulation, as deglycosylation of MP by -elimination profoundly
decreased the capacity of APC to stimulate primary T cells and the
MP-reactive T cell hybridoma P1D6. These results did not appear to be
secondary to nonspecific effects of -elimination on protein
structure or cellular toxicity, as no decrease in Ag-specific responses
to a control -eliminated protein, OVA, was observed.
Ags acquired and processed through the endocytic pathway are normally presented on MHC class II (54). This appears to be the method used for MP presentation as well. Digesting MP with proteinase K, a nonspecific protease, dramatically diminished P1D6 stimulation, indicating a requirement for the protein core. However, the possibility that the protein was required to maintain proper carbohydrate conformation while being presented cannot be entirely ruled out, especially as crystallographic evidence supports the ability of MHC to accommodate monosaccharides (55). A requirement for an MHC class II-restricted pathway was suggested by our data demonstrating splenocytes from Ii-/- fail to stimulate the MP-dependent CD4+ T cell hybridoma cell line.
To date, a notable problem with vaccine technology has been the difficulty designing formulations that specifically elicit protective cell-mediated immune responses. The potential for carbohydrate patterning to increase antigenic potency has led to novel approaches to vaccine development. Examples include chemically conjugating mannans from S. cerevisiae to weak Ags (56), expressing recombinant proteins in S. cerevisiae (57), and targeting of DEC-205 with mAbs coupled to peptides (58). Thus, our data demonstrating that efficient T cell responses to MP require recognition of terminal mannose groups by mannose receptors provide both a molecular basis for the immunogenicity of cryptococcal MP and support for vaccination strategies that target MR members.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stuart M. Levitz, Evans Memorial Department of Clinical Research and Department of Medicine, Boston University School of Medicine, Room X626, Boston Medical Center, 650 Albany Street, Boston, MA 02118. E-mail address: slevitz{at}bu.edu
3 Abbreviations used in this paper: MP, mannoprotein; FT, flow-through; CR, crude cryptococcal supernatant; MR, multilectin mannose receptor; MMR, macrophage mannose receptor; PAS, periodic acid-Schiff; ManP, methyl -D-mannopyranoside; GalP, methyl -D-galactopyranoside; PK bead, proteinase K-conjugated agarose bead; PG bead, protein G-conjugated agarose bead; LN, lymph node; CHO, Chinese hamster ovary; Ii, invariant chain.
Received for publication November 1, 2001. Accepted for publication January 4, 2002.
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in human peripheral blood mononuclear cells by the mannoprotein of Cryptococcus neoformans involves human mannose binding protein. J. Immunol. 159:2979.[Abstract]
are required for initiating the protective Th1 response to pulmonary cryptococcosis in resistant C.B-17 mice. Am. J. Respir. Cell Mol. Biol. 17:733. (TNF-) in host defence mechanism against Cryptococcus neoformans. Clin. Exp. Immunol. 106:468.[Medline]
in protecting mice during pulmonary and disseminated infection with Cryptococcus neoformans. FEMS Immunol. Med. Microbiol. 13:123.[Medline]
in human peripheral blood mononuclear cells. Infect. Immun. 65:272.[Abstract]
-elimination from Candida albicans cell walls. FEMS Microbiol. Lett. 123:131.[Medline]
-1,3-mannosyltransferases responsible for adding the terminal mannose residues of O-linked oligosaccharides. Glycobiology 9:1045.This article has been cited by other articles:
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  N. Marina-Garcia, L. Franchi, Y.-G. Kim, Y. Hu, D. E. Smith, G.-J. Boons, and G. Nunez Clathrin- and Dynamin-Dependent Endocytic Pathway Regulates Muramyl Dipeptide Internalization and NOD2 Activation J. Immunol., April 1, 2009; 182(7): 4321 - 4327. [Abstract] [Full Text] [PDF]
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 D. Pietrella, P. Lupo, A. Rachini, S. Sandini, A. Ciervo, S. Perito, F. Bistoni, and A. Vecchiarelli A Candida albicans Mannoprotein Deprived of Its Mannan Moiety Is Efficiently Taken Up and Processed by Human Dendritic Cells and Induces T-Cell Activation without Stimulating Proinflammatory Cytokine Production Infect. Immun., September 1, 2008; 76(9): 4359 - 4367. [Abstract] [Full Text] [PDF]
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J. M. Dan, R. M. Kelly, C. K. Lee, and S. M. Levitz Role of the Mannose Receptor in a Murine Model of Cryptococcus neoformans Infection Infect. Immun., June 1, 2008; 76(6): 2362 - 2367. [Abstract] [Full Text] [PDF]
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 S. Yamagami, S. Yokoo, S. Amano, and N. Ebihara Characterization of Bone Marrow Derived Cells in the Substantia Propria of the Human Conjunctiva Invest. Ophthalmol. Vis. Sci., October 1, 2007; 48(10): 4476 - 4481. [Abstract] [Full Text] [PDF]
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J. C. Panepinto, K. W. Komperda, M. Hacham, S. Shin, X. Liu, and P. R. Williamson Binding of Serum Mannan Binding Lectin to a Cell Integrity-Defective Cryptococcus neoformans ccr4{Delta} Mutant Infect. Immun., October 1, 2007; 75(10): 4769 - 4779. [Abstract] [Full Text] [PDF]
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J. M. Vyas, Y.-M. Kim, K. Artavanis-Tsakonas, J. C. Love, A. G. Van der Veen, and H. L. Ploegh Tubulation of Class II MHC Compartments Is Microtubule Dependent and Involves Multiple Endolysosomal Membrane Proteins in Primary Dendritic Cells J. Immunol., June 1, 2007; 178(11): 7199 - 7210. [Abstract] [Full Text] [PDF]
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L.-Z. He, A. Crocker, J. Lee, J. Mendoza-Ramirez, X.-T. Wang, L. A. Vitale, T. O'Neill, C. Petromilli, H.-F. Zhang, J. Lopez, et al. Antigenic Targeting of the Human Mannose Receptor Induces Tumor Immunity J. Immunol., May 15, 2007; 178(10): 6259 - 6267. [Abstract] [Full Text] [PDF]
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 G. M. Olson, D. S. Fox, P. Wang, J. A. Alspaugh, and K. L. Buchanan Role of Protein O-Mannosyltransferase Pmt4 in the Morphogenesis and Virulence of Cryptococcus neoformans Eukaryot. Cell, February 1, 2007; 6(2): 222 - 234. [Abstract] [Full Text] [PDF]
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D. M. Lindell, M. N. Ballinger, R. A. McDonald, G. B. Toews, and G. B. Huffnagle Diversity of the T-Cell Response to Pulmonary Cryptococcus neoformans Infection. Infect. Immun., August 1, 2006; 74(8): 4538 - 4548. [Abstract] [Full Text] [PDF]
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S. O. Dionne, A. B. Podany, Y. W. Ruiz, N. M. Ampel, J. N. Galgiani, and D. F. Lake Spherules Derived from Coccidioides posadasii Promote Human Dendritic Cell Maturation and Activation Infect. Immun., April 1, 2006; 74(4): 2415 - 2422. [Abstract] [Full Text] [PDF]
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M. K. Mansour, E. Latz, and S. M. Levitz Cryptococcus neoformans Glycoantigens Are Captured by Multiple Lectin Receptors and Presented by Dendritic Cells. J. Immunol., March 1, 2006; 176(5): 3053 - 3061. [Abstract] [Full Text] [PDF]
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 C. M. Galdeano and G. Perdigon The Probiotic Bacterium Lactobacillus casei Induces Activation of the Gut Mucosal Immune System through Innate Immunity Clin. Vaccine Immunol., February 1, 2006; 13(2): 219 - 226. [Abstract] [Full Text] [PDF]
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J. S. Lam, M. K. Mansour, C. A. Specht, and S. M. Levitz A Model Vaccine Exploiting Fungal Mannosylation to Increase Antigen Immunogenicity J. Immunol., December 1, 2005; 175(11): 7496 - 7503. [Abstract] [Full Text] [PDF]
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C. Biondo, L. Messina, M. Bombaci, G. Mancuso, A. Midiri, C. Beninati, V. Cusumano, E. Gerace, S. Papasergi, and G. Teti Characterization of Two Novel Cryptococcal Mannoproteins Recognized by Immune Sera Infect. Immun., November 1, 2005; 73(11): 7348 - 7355. [Abstract] [Full Text] [PDF]
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N. M. Ampel, D. K. Nelson, L. Li, S. O. Dionne, D. F. Lake, K. A. Simmons, and D. Pappagianis The Mannose Receptor Mediates the Cellular Immune Response in Human Coccidioidomycosis Infect. Immun., April 1, 2005; 73(4): 2554 - 2555. [Abstract] [Full Text] [PDF]
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D. Pietrella, C. Corbucci, S. Perito, G. Bistoni, and A. Vecchiarelli Mannoproteins from Cryptococcus neoformans Promote Dendritic Cell Maturation and Activation Infect. Immun., February 1, 2005; 73(2): 820 - 827. [Abstract] [Full Text] [PDF]
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L. E. Yauch, M. K. Mansour, S. Shoham, J. B. Rottman, and S. M. Levitz Involvement of CD14, Toll-Like Receptors 2 and 4, and MyD88 in the Host Response to the Fungal Pathogen Cryptococcus neoformans In Vivo Infect. Immun., September 1, 2004; 72(9): 5373 - 5382. [Abstract] [Full Text] [PDF]
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 J. Masuoka Surface Glycans of Candida albicans and Other Pathogenic Fungi: Physiological Roles, Clinical Uses, and Experimental Challenges Clin. Microbiol. Rev., April 1, 2004; 17(2): 281 - 310. [Abstract] [Full Text] [PDF]
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M. K. Mansour, L. E. Yauch, J. B. Rottman, and S. M. Levitz Protective Efficacy of Antigenic Fractions in Mouse Models of Cryptococcosis Infect. Immun., March 1, 2004; 72(3): 1746 - 1754. [Abstract] [Full Text] [PDF]
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S. Bellocchio, C. Montagnoli, S. Bozza, R. Gaziano, G. Rossi, S. S. Mambula, A. Vecchi, A. Mantovani, S. M. Levitz, and L. Romani The Contribution of the Toll-Like/IL-1 Receptor Superfamily to Innate and Adaptive Immunity to Fungal Pathogens In Vivo J. Immunol., March 1, 2004; 172(5): 3059 - 3069. [Abstract] [Full Text] [PDF]
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 L. Romani, C. Montagnoli, S. Bozza, K. Perruccio, A. Spreca, P. Allavena, S. Verbeek, R. A. Calderone, F. Bistoni, and P. Puccetti The exploitation of distinct recognition receptors in dendritic cells determines the full range of host immune relationships with Candida albicans Int. Immunol., January 1, 2004; 16(1): 149 - 161. [Abstract] [Full Text] [PDF]
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M. Chieppa, G. Bianchi, A. Doni, A. Del Prete, M. Sironi, G. Laskarin, P. Monti, L. Piemonti, A. Biondi, A. Mantovani, et al. Cross-Linking of the Mannose Receptor on Monocyte-Derived Dendritic Cells Activates an Anti-Inflammatory Immunosuppressive Program J. Immunol., November 1, 2003; 171(9): 4552 - 4560. [Abstract] [Full Text] [PDF]
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J. N. Steenbergen, J. D. Nosanchuk, S. D. Malliaris, and A. Casadevall Cryptococcus neoformans Virulence Is Enhanced after Growth in the Genetically Malleable Host Dictyostelium discoideum Infect. Immun., September 1, 2003; 71(9): 4862 - 4872. [Abstract] [Full Text] [PDF]
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I. Bose, A. J. Reese, J. J. Ory, G. Janbon, and T. L. Doering A Yeast under Cover: the Capsule of Cryptococcus neoformans Eukaryot. Cell, August 1, 2003; 2(4): 655 - 663. [Full Text] [PDF]
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D. Pietrella, R. Mazzolla, P. Lupo, L. Pitzurra, M. J. Gomez, R. Cherniak, and A. Vecchiarelli Mannoprotein from Cryptococcus neoformans Promotes T-Helper Type 1 Anticandidal Responses in Mice Infect. Immun., December 1, 2002; 70(12): 6621 - 6627. [Abstract] [Full Text] [PDF]
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C. Huang, S.-h. Nong, M. K. Mansour, C. A. Specht, and S. M. Levitz Purification and Characterization of a Second Immunoreactive Mannoprotein from Cryptococcus neoformans That Stimulates T-Cell Responses Infect. Immun., October 1, 2002; 70(10): 5485 - 5493. [Abstract] [Full Text] [PDF]
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