Proteins in the Cell Wall and Membrane of Cryptococcus neoformans ...

INFECTION AND IMMUNITY, Nov. 1996, p. 4811–4819 0019-9567/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 64, No. 11

Proteins in the Cell Wall and Membrane of Cryptococcus neoformans Stimulate Lymphocytes from Both Adults and Fetal Cord Blood To Proliferate CHRISTOPHER H. MODY,1,2* KURTIS L. SIMS,2 CYNTHIA J. WOOD,2 RACHEL M. SYME,2 JASON C. L. SPURRELL,2 AND MARY M. SEXTON2 Department of Internal Medicine1 and Department of Microbiology and Infectious Diseases,2 University of Calgary, Calgary, Alberta, Canada Received 30 April 1996/Returned for modification 3 July 1996/Accepted 28 August 1996

Cryptococcus neoformans is an encapsulated yeast that infects patients who have defective cell-mediated immunity, including AIDS, but rarely infects individuals who have intact cell-mediated immunity. Studies of the immune response to C. neoformans have been hampered by a paucity of defined T-lymphocyte antigens, and hence, the understanding of the T-cell response is incomplete. The goal of this study was to separate C. neoformans into its component parts, determine whether those components stimulate lymphocyte proliferation, perform preliminary characterization of the proteins, and establish the potential mechanism of lymphocyte proliferation. The lymphocyte response to fungal culture medium, whole organisms, disrupted organisms, and the yeast intracellular fraction or cell wall and membrane was studied by determining thymidine incorporation and by determining the number of lymphocytes at various times after stimulation. The cell wall and membrane of C. neoformans stimulated lymphocyte proliferation, while the intracellular fraction and culture filtrate did not. The optimal response occurred on day 7 of incubation, with 4 3 105 peripheral blood mononuclear cells per well and with 13 mg of cryptococcal protein per ml. The number of lymphocytes increased with time in culture, indicating that thymidine incorporation was accompanied by proliferation. Proteinase K treatment of the cell wall and membrane abrogated lymphocyte proliferation, indicating that the molecule was a protein. [35S]methionine labeling, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography were performed to analyze the proteins contained in the cell wall and membrane, intracellular fraction, and culture filtrate. At least 18 discrete bands were resolved from the cell wall and membrane. Since a large percentage of healthy adults responded to the cryptococcal cell wall and membrane, a mitogenic effect was investigated by testing proliferation of fetal cord blood lymphocytes. The percentage of fetal samples that proliferated in response to the cell wall and membrane was similar to the percentage of fetal samples that proliferated in response to Staphylococcus enterotoxin B, a microbial mitogen. Thus, a protein in the cell wall and membrane of C. neoformans is a potent stimulant of lymphocyte proliferation and has mitogenic properties, which may have important implications for cell-mediated immunity to C. neoformans. purification of the cryptococcal antigens would permit experiments to separate these complex interactions. To determine the components of C. neoformans that stimulate lymphocyte proliferation, we stimulated normal human lymphocytes in vitro (29, 34). C. neoformans was disrupted and separated into the (i) cell wall and membrane (CCW/M) and (ii) intracellular fractions. The kinetics of the response to each fraction was determined. A preliminary identification of the proteins that are contained in CCW/M was performed, and the innate response of fetal cord blood lymphocytes was studied.

Failure of immunodeficient patients, such as those who have AIDS, to mount an immune response to Cryptococcus neoformans can result in a devastating infection (8, 12, 24, 55). C. neoformans is the most common invasive and fatal fungal infection in AIDS patients (9, 10), with the possible exception of Pneumocystis carinii. Despite the clinical importance of C. neoformans, the proteins that stimulate cell-mediated immune responses are incompletely defined. We have initiated investigations to determine the components of C. neoformans that activate cellular immune responses. Extensive investigations of the delayed-type hypersensitivity (DTH) to cryptococcal antigens have been performed with whole organisms or undefined antigens (1, 4, 13, 17, 23, 28, 30, 33, 36–39). These investigations have led to an incomplete understanding of the cell-mediated immune response to C. neoformans in which both CD4 and CD8 cells elicit DTH and are necessary for optimal host defense (13, 15, 17, 30–32). Both CD4 and CD8 cells also participate in a complex cascade of suppressor cells (38, 39). It is possible that different antigens, and perhaps even mitogens, are responsible for these different features of the immune response to C. neoformans and that

MATERIALS AND METHODS Isolation of peripheral blood mononuclear cells (PBMC) and fetal umbilical cord blood mononuclear cells (FBMC). Human peripheral blood was obtained by venipuncture from healthy adult donors who had never had cryptococcosis or worked with C. neoformans. Blood was anticoagulated by adding 10 U of heparin (Organon Teknika-Cappel, Scarborough, Ontario, Canada) per ml. Fetal umbilical cord blood was collected in 8-ml tubes containing heparin (Becton-Dickinson, Rutherford, N.J.). For each experiment, PBMC or FBMC were obtained from different donors on different days. PBMC or FBMC were isolated by centrifugation (800 3 g for 20 min) on a Ficoll-Hypaque density gradient (Lymphoprep; Labquip, Woodbridge, Ontario, Canada). PBMC or FBMC were washed three times in Hanks’ balanced salt solution (Gibco, Burlington, Ontario, Canada), counted, and suspended in medium containing RPMI 1640 medium (Gibco), 5% human AB serum (BioWhitaker, Walkersville, Md.), 2 mM Lglutamine (Gibco), 100 U of penicillin per ml, 100 mg of streptomycin per ml, 0.2 mg of amphotericin B per ml (Gibco), 1 mM sodium pyruvate (Gibco), and 0.1 mM nonessential amino acids (Gibco).

* Corresponding author. 4811

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Preparation of C. neoformans. C. neoformans cap67 (ATCC 52817, acapsular mutant) (19) was obtained from the American Type Culture Collection (Rockville, Md.). The organisms were maintained on Sabouraud glucose slants (Difco, Detroit, Mich.) and passaged to fresh slants monthly as previously described (35). Periodically, the organism was examined by mucicarmine staining to ensure that it remained unencapsulated. The organisms were cultured in Sabouraud dextrose broth (1% Bacto Neopeptone, 2% glucose [pH 5.6]; Difco). Six 1-liter flasks were inoculated from a stationary-phase culture of C. neoformans cap67 in Sabouraud dextrose broth. The cultures were incubated at 308C for 96 h and then killed by autoclaving as previously described (34). Fractionation of C. neoformans. Organisms were washed three times in phosphate-buffered saline (PBS; 8.25 mM Na2HPO4, 1.74 mM NaH2PO4, 150 mM NaCl [pH 7.4]) and then suspended at 5 3 108/ml in PBS. C. neoformans was disrupted by rotating the organisms with 0.5-mm glass beads in a bead mill for 30 cycles of 1 min and 1 min off in an ice bath (Bead Beater; Biospec Products, Bartlesville, Okla.). The slurry of disrupted organisms was centrifuged at 200 3 g for 5 min to remove large particles. The supernatant was removed and stored at 2708C until it was used. The CCW/M was separated from the intracellular fraction by differential centrifugation at 13,000 3 g at room temperature for 30 min. The insoluble CCW/M was resuspended in an equal volume of PBS. The supernatant constituted the intracellular fraction. Purified mannoprotein was a generous gift of R. Cherniak, Georgia State University, Atlanta. Protein concentration was determined by the bicinchoninic acid protein assay (Pierce, Rockford, Ill.). Bovine serum albumin was used to prepare the standard curve. Carbohydrate concentration was determined by the colorimetric technique of Dubois (11), and glucose was used for the standard curve. The DNA content of each fraction was determined by measuring UV absorption at 260 nm. A standard curve was generated by using pure DNA (1-kb DNA ladder; Gibco) at various concentrations with 750 mg of bovine serum albumin per ml to control for UV absorption due to the protein. With the Limulus amebocyte lysate assay system (Sigma), which detects glucan, as well as bacterial endotoxin (47), and generation of a standard curve with Escherichia coli 0113H10 endotoxin (Associates of Cap Cod, Woods Hole, Mass.), microtiter wells containing CCW/M had less than 0.25 ng of endotoxin per ml. Proteolytic digestion of CCW/M. Proteinase K (0.1 mg/ml; Gibco) was added to CCW/M, and the mixture was incubated at 508C for 24 h. The mixture was then boiled for 5 min to inactivate proteinase K. As a control, previously inactivated proteinase K (boiled for 5 min) was also incubated with CCW/M. To ensure that boiled CCW/M was still capable of stimulating lymphocytes, it was boiled prior to incubation with previously inactivated proteinase K. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis of CCW/M proteins. Cryptococcal proteins were separated on either SDS–12.5% PAGE gels or SDS–4 to 20% PAGE gradient gels as described by Laemmli (25). CCW/M (40 mg of protein per lane) was boiled in reducing sample buffer containing 2.5% b-mercaptoethanol for 20 min and briefly centrifuged to remove insoluble material prior to loading on gels and electrophoresis. The gels were stained for protein by overnight incubation in 0.2% Coomassie brilliant blue R-250 (Bio-Rad, Richmond, Calif.) in 25% methanol–7.5% acetic acid and destained with 25% methanol–7.5% acetic acid (53). Silver staining (42), which was modified to use 25% isopropanol–7.5% acetic acid as an additional fixation step, was also used for protein detection. For carbohydrate detection, periodic acid-Schiff (PAS) staining was used (27). Briefly, gels were fixed overnight in 25% isopropanol–10% acetic acid and then soaked for 30 min in 7.5% acetic acid. The gels were then incubated in 0.2% periodic acid at 48C for 60 min, followed by Schiff’s reagent for 60 min at 48C in the dark. Finally, gels were fixed with three changes of 0.5% metabisulfite over 30 min in the dark. To enhance visualization of cryptococcal proteins on SDS-PAGE gels, [35S]methionine labeling, separation by SDS-PAGE, and visualization by 2,5diphenyloxazole (Sigma, St. Louis, Mo.) scintillation were performed (27). Briefly, C. neoformans was cultured for 4 days in minimal medium containing Bacto Yeast Nitrogen Base without amino acids and ammonium sulfate (Difco) to which 0.6 mCi of Trans[35S]label metabolic labeling reagent (70% L-methionine, 15% L-cysteine; specific activity, 1,000 Ci/mmol; ICN, Montreal, Quebec, Canada) was added. The organisms were killed by incubation at 568C for 2 h and washed in PBS. The culture medium was collected, and organisms were disrupted and separated into the CCW/M and intracellular fractions as described above. These fractions were placed in reducing SDS sample buffer and electrophoresed in SDS–12.5% PAGE gels as described above. The gels were dehydrated by two 30-min incubations in dimethyl sulfoxide (BDH, Toronto, Ontario, Canada) and then soaked for 3 h in 20% 2,5-diphenyloxazole scintillant in dimethyl sulfoxide. The 2,5-diphenyloxazole in the gels was precipitated by washing with water for 1 h, and after drying, the gels were placed in a cassette at 2708C for up to 48 h. The films were removed and developed. [3H]thymidine incorporation into stimulated PBMC. To determine whether components of C. neoformans stimulated [3H]thymidine incorporation, various numbers of PBMC (5 3 104 to 1 3 106 cells per well) were cultured in roundbottom wells of 96-well tissue culture plates (Corning, Corning, N.Y.). Various concentrations (see results of individual experiments) of the components of C. neoformans (described above) and whole C. neoformans cells (2 3 105 per well) were used to stimulate PBMC. Cultures were incubated for 3, 5, 7, 9, or 14 days at 378C in 5% CO2. Sixteen hours before the end of incubation, 1 mCi of

INFECT. IMMUN. TABLE 1. Composition of C. neoformans fractions Material analyzed

Protein concn (mg/ml)

Carbohydrate concn (mM)

DNA concn (mg/mg of protein)

Disrupted organismsa Intracellular fraction CCW/M Culture filtrate

3,505 724 2,672 3,225

13.2 5.1 7.5 8.3

0.4 2.8 0.05 0.26

a

5 3 108 C. neoformans cells per ml were used to prepare each fraction.

[3H]thymidine (ICN) was added. Cells were harvested on glass filters, and counts per minute were determined in a liquid scintillation counter. [3H]thymidine incorporation into cultures containing only whole killed C. neoformans cells or C. neoformans fractions was routinely less than 300 cpm. As a control, PBMC or FBMC were stimulated with 1 mg of Staphylococcus enterotoxin B (Sigma) or 1:10 Lf tetanus toxoid (Connaught Laboratories, Missisauga, Ontario, Canada) per ml. Analysis of proliferating lymphocytes. To determine whether lymphocytes proliferate in response to CCW/M, PBMC were harvested from stimulated or unstimulated cultures after 2 h or 5, 7, or 9 days. Lymphocytes were counted with a hemacytometer. Statistics. Data are given as the means 6 the standard errors of the means. A positive proliferative response was considered to be a stimulation index (stimulated counts per minute/unstimulated counts per minute) of greater than 2.5. To analyze the data statistically, we performed one-factor analysis of variance when allowed by the F test (Statview 5121; Brainpower Inc., Calabasas, Calif.). All experiments were repeated at least twice with material from different donors on different days. For these tests, P , 0.05 was considered significant.

RESULTS 3

Stimulation of [ H]thymidine incorporation into PBMC by the CCW/M of C. neoformans. C. neoformans was disrupted with rotating glass beads. With this technique, microscopic inspection of the resultant slurry revealed no intact organisms. The CCW/M and intracellular fractions were separated by differential centrifugation, and the protein and carbohydrate contents were determined (Table 1). To ensure that the CCW/M had been separated from the intracellular fraction, the DNA content of each fraction was determined (Table 1). The CCW/M contained minimal amounts of DNA, indicating that adequate separation had been achieved. When the CCW/M and intracellular fractions derived from disrupted organisms were used to stimulate PBMC, the CCW/M stimulated lymphocytes while the intracellular fraction did not (Fig. 1). Both disrupted and whole organisms stimulated lymphocytes, while the Sabouraud medium that had supported cryptococcal growth did not. The average maximum stimulation index for CCW/M among the responders was 17.4 6 1.5 (n 5 50). Kinetics of [3H]thymidine incorporation. To determine the optimal conditions for [3H]thymidine incorporation in response to CCW/M, various numbers of PBMC were stimulated with different concentrations of CCW/M for various lengths of time. A positive response occurred with as little as 0.13 mg/ml (Fig. 2A), and the maximum response occurred in cultures containing 13 mg of CCW/M per ml. The average stimulation index among responders to 13 mg of CCW/M per ml was 15.7 6 1.4 (n 5 46). A positive response occurred as early as day 3 (Fig. 2B), and the maximum response occurred on day 7. The average stimulation index among responders on day 7 was 16.0 6 1.6 (n 5 48). A positive response occurred with 2 3 105 PBMC per well (Fig. 2C), and the maximum response occurred with 4 3 105 PBMC per well. The average stimulation index among responders with 4 3 105 PBMC per well was 17.4 6 2.0 (n 5 25), and with 2 3 105 PBMC per well it was 15.1 6 1.5 (n 5 49). Lymphocyte proliferation in response to CCW/M. [3H]thymidine uptake indicates that cells have entered the S phase of

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FIG. 1. The CCW/M of C. neoformans stimulates [3H]thymidine incorporation into PBMC. PBMC were incubated for 7 days with whole C. neoformans cells, disrupted organisms, the intracellular fraction, CCW/M, or the Sabouraud medium that had supported cryptococcal growth (culture filtrate). Values for individual experiments (E) and the means 6 the standard errors of the means (SEM) (h) are shown. ✮, P , 0.05; †, P , 0.01 (compared with unstimulated PBMC), NS, not statistically significantly different from unstimulated PBMC.

the cell cycle and synthesized new DNA, which is commonly used as a surrogate of lymphocyte proliferation. To determine whether lymphocyte proliferation had occurred, PBMC were stimulated with CCW/M and the viable lymphocytes (as determined by trypan exclusion) were counted after various times. There was an increase in the lymphocyte number at 5 days (Fig. 3) and a further increased on day 9. There was no increase in the number of unstimulated lymphocytes. Thus, the [3H]thymidine uptake was accompanied by an increase in lymphocyte numbers indicating that CCW/M induces lymphocyte proliferation. Proteinase K treatment of CCW/M abrogates lymphocyte proliferation. To determine whether a cryptococcal protein was responsible for lymphocyte stimulation, CCW/M was digested with a protease. Treatment of CCW/M with proteinase K abrogated lymphocyte proliferation (Fig. 4). Heat-inactivated proteinase K had no effect on lymphocyte proliferation, indicating that proteinase K itself was not responsible for the decrease in proliferation. Heating of the CCW/M had no effect on proliferation, indicating that the heat used to inactivate proteinase K did not inactivate CCW/M. This indicates that a CCW/M protein that is susceptible to proteinase K digestion is responsible for lymphocyte proliferation. SDS-PAGE analysis of CCW/M. To determine the molecular weights of SDS-extractable proteins of CCW/M, SDSPAGE analysis was performed. CCW/M (40 mg per lane) was boiled in 1% SDS for 5 min and then centrifuged at 13,000 3 g for 5 min to remove insoluble material. The supernatant was run in an SDS–4 to 20% PAGE gradient gel under reducing conditions. The gel was stained with Coomassie brilliant blue (Fig. 5A). This revealed a discrete band at an Mr of 17,500; minor bands at Mrs of 18,500 and 22,500; diffuse, strong staining from 24,000 to 33,000 and 39,000 to 46,000; and heavy staining with poor resolution at higher molecular weights. PAS staining was performed to visualize carbohydrates. PAS

staining of CCW/M showed major bands at .200 and ,14 kDa with diffuse staining in the region of 60 to 120 kDa (Fig. 5B). Highly glycosylated yeast proteins may not be visualized by Coomassie or silver staining (48). To improve the visualization of cryptococcal proteins, [35S]methionine labeling and 2,5-diphenyloxazole fluorography were performed (27). To ensure efficient uptake of [35S]methionine, a minimal medium was used to culture C. neoformans. To ensure that growth in minimal medium did not alter the ability of the proteins to stimulate lymphocyte proliferation, CCW/M from C. neoformans grown in minimal medium was compared with CCW/M from C. neoformans grown in Sabouraud medium. Lymphocytes proliferated in response to both preparations of CCW/M (data not shown). C. neoformans fractions were visualized by fluorography following metabolic labeling of cultures (Fig. 5C). Distinct banding patterns were visible in the culture supernatant and the intracellular and CCW/M fractions. In the CCW/M fraction, bands were resolved over the complete molecular mass range of the gel (.200 to 14 kDa). At least 18 bands were seen in CCW/M. Many of these bands were not visible on Coomassie brilliant blue staining (Fig. 5A) or on silver staining (data not shown). Response to mannoprotein. Although CCW/M is a complex array of proteins, the major protein of the cryptococcal cell wall is mannoprotein (45). To determine whether mannoprotein causes lymphocyte proliferation, PBMC were stimulated with purified mannoprotein. Purified mannoprotein did not stimulate PBMC proliferation, while CCW/M caused significant proliferation (Fig. 6). This suggests that a protein(s) other than the mannoprotein is responsible for lymphocyte proliferation. Fetal lymphocyte proliferation in response to C. neoformans. Our studies indicate that a high percentage of healthy adults respond to CCW/M, suggesting that it contains an immuno-

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FIG. 2. Optimal conditions for [3H]thymidine incorporation into PBMC. (A) Various concentrations of CCW/M were used to stimulate 4 3 105 PBMC for 7 days. (B) PBMC (4 3 105 per well) were cultured with (■) or without (3) CCW/M (13 mg/ml) for various lengths of time. (C) Various numbers of PBMC were stimulated with (■) or without (3) CCW/M (13 mg/ml) for 7 days. Each experiment was repeated at least three times with similar results. ✮, P , 0.001 compared with unstimulated PBMC. SEM, standard errors of the means.

dominant antigen and/or a mitogen. To determine whether CCW/M has mitogenic properties, FBMC were stimulated with CCW/M. FBMC responded to CCW/M with kinetics similar to that of PBMC (Fig. 7). The best response occurred on day 7 with 13 mg of protein per ml. A large percentage of healthy adults had a positive PBMC response (85% had a stimulation index of greater than 2.5) (Fig. 8). This was comparable to the percentage of healthy adults who responded to Staphylococcus enterotoxin B and more than the percentage who responded to tetanus toxoid. A smaller percentage of fetal samples responded to CCW/M and Staphylococcus enterotoxin B, but none responded to tetanus toxoid (Fig. 8). The fetal samples that did not respond to

Staphylococcus enterotoxin B were the same samples that did not respond to CCW/M. This suggests that CCW/M has mitogenic properties. DISCUSSION We have made four observations. (i) PBMC from healthy adults proliferated in response to CCW/M but did not proliferate in response to the intracellular fraction or the Sabouraud medium used to support cryptococcal growth. (ii) The optimal response ([3H]thymidine incorporation) to CCW/M occurred on day 7 at 13 mg of CCW/M per ml and with 4 3 105 PBMC per well. (iii) A protein in CCW/M is responsible for lympho-

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FIG. 3. Lymphocyte proliferation in response to CCW/M. PBMC (2 3 105) were placed in each well of a 96-well culture plate. These PBMC were cultured either with medium alone (h) or with 13 mg of CCW/M protein per ml (u). After the incubation times indicated, nonadherent cells were washed out of the wells and lymphocytes were counted on a hemacytometer. Four separate groups of cells were counted at each time point. The experiment was repeated with similar results. ✮, P , 0.05 compared with unstimulated cell counts; †, P , 0.05 compared with the number of stimulated cells on day 5.

cyte proliferation, and proteins in CCW/M migrated on SDSPAGE with at least 18 discrete bands with molecular masses between 200,000 and 14,000 kDa. (iv) The percentages of PBMC (85%) and FBMC (50%) that proliferated in response to CCW/M were similar to the percentages that proliferated in response to Staphylococcus enterotoxin B, suggesting that CCW/M has mitogenic properties. The current studies were performed to identify the components of C. neoformans that could stimulate T-lymphocyte pro-

FIG. 5. SDS-PAGE analysis of C. neoformans fractions. (A) 4 to 20% gradient gel stained with Coomassie brilliant blue. Lanes: 1, molecular weight markers; 2, CCW/M at 40 mg per lane. (B) 12.5% gel stained with PAS. Lane 1, CCW/M at 150 mg per lane. (C) Autoradiograph of SDS–12.5% PAGE gel in which 35S-labeled C. neoformans fractions were resolved. Lanes: 1, culture filtrate (50,000 cpm per lane); 2, CCW/M (100,000 cpm per lane); 3, intracellular fraction (100,000 cpm per lane).

liferation. Although other mechanisms participate in the host defense against C. neoformans, T-cell-mediated immunity is the most important. A great deal of work has been done on the antigens of C. neoformans that elicit an antibody response (2,

FIG. 4. Abrogation of lymphocyte proliferation by proteinase K (Prot K) treatment of CCW/M. Proteinase K was added to CCW/M, and the mixture was incubated at 508C for 24 h. The mixture was then boiled for 5 min to inactivate any further activity and added to 2 3 105 PBMC (p). As a control, proteinase K was inactivated by boiling for 5 min before incubation with CCW/M (u). To show that CCW/M itself is not inactivated by boiling, it was boiled prior to incubation (u). After the 24-h incubation, the mixtures were added to 2 3 105 PBMC and cultured for 7 days. In all cases, the CCW/M concentration used was 13 mg/ml. This experiment was repeated with an incubation temperature of 558C, and similar results were obtained. ✮, P , 0.001 compared with unstimulated values; §, P , 0.01 compared with untreated CCW/M.

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FIG. 6. Failure of purified mannoprotein to stimulate lymphocyte proliferation. PBMC (2 3 105) were placed in each well of a 96-well culture plate. These PBMC were cultured either with medium alone (h), with various concentrations of purified mannoprotein (■), with 2 3 105 whole C. neoformans cells (u), or with 13 mg of CCW/M protein per ml (u). ✮, P , 0.001 compared with PBMC alone. This experiment was repeated with similar results.

6, 7, 16, 22); however, much less is known about the cryptococcal antigens that elicit a protective T-cell response. Because T cells, which are central to cell-mediated immunity, respond primarily to protein antigens, we were interested in studying the T-cell response to cryptococcal proteins. We found that a

protein(s) in the CCW/M of C. neoformans caused vigorous lymphocyte proliferation, while proteins in the intracellular fraction and proteins released into the yeast culture medium did not stimulate human lymphocyte proliferation. Our studies attempted to minimize the influence of B-cell

FIG. 7. Fetal lymphocyte proliferation in response to CCW/M. FBMC (2 3 105 per well) were cultured either with medium alone (h), with 1.3 mg of CCW/M per ml (u), or with 13 mg of CCW/M per ml (3) for the times indicated. ✮, P , 0.05 compared with unstimulated counts per minute. This experiment was repeated twice with similar results.

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FIG. 8. Similarity of percentages of donors responding to stimulation by CCW/M or staphylococcal (Staph) enterotoxin B. Lymphocytes were considered to be proliferating when the stimulation index (SI) (unstimulated counts per minute/stimulated counts per minute) was greater than 2.5. A total of 2 3 105 PBMC (A; n 5 20) or FBMC (B; n 5 5) were stimulated with 13 mg of CCW/M protein per ml.

antigens. Glucuronoxylomannan is the major component of the capsular polysaccharide and is a T-independent B-cell antigen. Glucuronoxylomannan also induces T-cell-mediated suppression of the primary response (49) but does not stimulate T cells to produce a cell-mediated immune response (40). To avoid the effect of glucuronoxylomannan, we selected C. neoformans cap67, which is an acapsular mutant that does not contain glucuronoxylomannan (52). CCW/M was separated from the intracellular components by disrupting the organism in a glass bead mill and using centrifugation to separate the components. We found that the intracellular fraction contained large amounts of DNA, while CCW/M did not, suggesting that very little intracellular contamination of CCW/M occurred. By using SDS-PAGE analysis, we found that CCW/M contained concanavalin A-binding proteins while the intracellular fraction did not (data not shown) and that CCW/M stimulated lymphocytes to proliferate while the intracellular fraction did not, suggesting that CCW/M did not contaminate the intracellular fraction. Thus, separation of the CCW/M and intracellular components was achieved and the protein(s) that stimulates lymphocyte proliferation is in CCW/M. The cell envelope of C. neoformans consists of a glucuronoxylomannan capsule (absent in strain cap67), a fibrillar-wall– membrane matrix of glucan-chitin, and a readily soluble galactoxylomannan complex. The mannoprotein is the major protein of the cell wall of C. neoformans; however, a detailed analysis of the proteins in CCW/M has not been performed (45). The proteins of CCW/M could not be adequately resolved by SDS-PAGE and Coomassie or PAS staining. By [35S]methionine labeling, we found that multiple bands were present, suggesting that CCW/M is a complex mixture of proteins. In particular, we found at least nine bands in CCW/M that were not present in the intracellular fraction or culture supernatant. Many of these bands were not visible on Coomassie brilliant blue staining (Fig. 5A) or on silver staining (data not shown); this may be due to the increased sensitivity of [35S]methionine labeling. It is unlikely that growth in minimal medium (which was used to facilitate uptake of [35S]methionine) significantly altered the production of the protein(s) that stimulates lymphocytes, since growing C. neoformans in minimal medium causes the cell wall to be less compact than does growth in complex medium without structural changes in the cell wall (46), and lymphocytes proliferated in response to CCW/M grown in minimal medium. [35S]methionine labeling

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demonstrated that there are a large number of protein candidates for the mitogenic activity seen in the CCW/M. Very little is known about the cryptococcal antigens that elicit a protective T-cell response. DTH can be elicited in mice by the proteins in the culture filtrate (30, 33, 37, 41). The major proteoglycan of cryptococcal culture filtrates is a mannoprotein. The mannoprotein elicits a DTH response in mice immunized either with the cryptococcal culture filtrate in complete Freund’s adjuvant or whole heat-killed C. neoformans yeast cells (40). It is likely that the strong, diffuse staining at .200 and 60 to 120 kDa we observed with PAS staining is the mannoprotein. However, human lymphocytes proliferate poorly in response to culture filtrate antigens which contain the mannoprotein (29, 34). Further, we observed that lymphocytes did not proliferate in response to purified mannoprotein, suggesting that it is not the protein responsible. Other proteins have been extracted from whole C. neoformans cells by using 11.6 M urea in 0.1 borate buffer (pH 9), and these proteins elicited DTH in guinea pigs (3). The extract migrated at low molecular weights on SDS-PAGE. By using similar extraction procedures, we were unable to extract a protein that stimulated human lymphocytes (data not shown). In contrast to patients with defective cell-mediated immunity, normal adults are unlikely to become infected with C. neoformans. This suggests that normal adults have a protective immune response to C. neoformans. We reasoned that peripheral T cells would reflect this protective immunity and that they would proliferate in response to C. neoformans in an antigenspecific fashion. To test whether the CCW/M of C. neoformans causes antigen-specific proliferation or whether it contained a mitogen, we studied the ability of the CCW/M to cause proliferation of FBMC, which are unlikely to have been exposed to C. neoformans. When FBMC respond, it indicates that an antigen-nonspecific or mitogenic response occurs. Since the optimal response occurred with 4 3 105 PBMC per well on day 7 and with 13 mg of protein per ml, which are characteristics of a recall antigen, we were surprised to find that FBMC proliferated in response to CCW/M. Some researchers have suggested that C. neoformans stimulates lymphocytes as a mitogen (14), while others have suggested that adult-lymphocyte proliferation represents in vitro priming (29). Our data do not exclude an antigen-specific response, since the CCW/M is a complex mixture of proteins, of which some may be mitogenic and others may be antigenic. Additionally, mitogens can function as recall antigens since Staphylococcus exotoxin B can elicit an antigen-specific response (44, 50). However, our data do suggest that the CCW/M of C. neoformans possesses a mitogen in addition to possible antigens. Previous studies have demonstrated that proteins released into the culture filtrate elicit a DTH reaction (40). This suggests that proteins easily released from the organism are responsible for a cognate cell-mediated immune response. Our studies indicate that a protein(s) that is not easily released from the CCW/M of C. neoformans is responsible for an innate lymphocyte response. If this were the case, then this component might only be produced following degradation of the yeast during the immunoinflammatory response. Thus, we speculate that an active phagocytic cell is required to release this component in vivo to activate lymphocytes. There are a number of mechanisms by which the mitogen in CCW/M could contribute to the pathogenesis of cryptococcosis. Microbial mitogens could have either positive or negative consequences for host defense. Mitogens, superantigens in particular, stimulate lymphocytes to produce antigen-nonspecific cytokines (5, 21) that could either activate mechanisms of host defense or mediate a destructive tissue response. Mito-

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gen-induced lymphocyte activation can be followed by unresponsiveness (18, 26, 43) or programmed cell death (apoptosis) (20, 51, 54) leading to immunosuppression. Thus, a cryptococcal mitogen could stimulate cell-mediated immunity for enhanced host defense, or it could mediate an immunosuppressive effect that enhances the survival of the organism within the host. To facilitate the study of how these mechanisms of lymphocyte activation affect the pathogenesis of cryptococcosis, it is necessary to purify the antigenic and/or mitogenic components of C. neoformans. In summary, we have determined that a protein in the CCW/M of C. neoformans stimulates lymphocyte proliferation. The proteins in the CCW/M produce a complex banding pattern on gel electrophoresis. The mechanism of lymphocyte activation is likely to be that of a mitogen, but we cannot exclude the presence of a recall antigen(s). Separation of the cryptococcal CCW/M proteins may provide the reagents necessary to dissect the complex cell-mediated immune response to C. neoformans. ACKNOWLEDGMENTS This work was supported by grants from the National Health Research and Development Program/Medical Research Council of Canada initiative for AIDS and the Canadian Foundation for AIDS Research. C.H.M. is a clinical investigator of the Alberta Heritage Foundation for Medical Research. R.M.S. is supported by a studentship from the National Health Research and Development Program. We thank M. Retzer for reviewing the manuscript, the staff at the Foothills Hospital maternity unit, and Lindsay Davidson for secretarial assistance. REFERENCES 1. Abrahams, J., and T. G. Gilleran. 1960. Studies on actively acquired resistance to experimental cryptococcosis in mice. J. Immunol. 85:629–635. 2. Belay, T., and R. Cherniak. 1995. Determination of antigen binding specificities of Cryptococcus neoformans factor sera by enzyme-linked immunosorbent assay. Infect. Immun. 63:1810–1819. 3. Bennett, J. E. 1981. Cryptococcal skin test antigen: preparation variables and characterization. Infect. Immun. 32:373–380. 4. Buchanan, K. L., P. L. Fidel, and J. W. Murphy. 1991. Effects of Cryptococcus neoformans-specific suppressor T cells on the amplified anticryptococcal delayed-type hypersensitivity response. Infect. Immun. 59:29–35. 5. Carlsson, R., and H. O. Sjogren. 1985. Kinetics of IL-2 and interferon-g production, expression of IL-2 receptors and cell proliferation in human mononuclear cells exposed to staphylococcal enterotoxin A. Cell. Immunol. 96:175–183. 6. Casadevall, A., J. Mukherjee, S. J. Devi, R. Schneerson, J. B. Robbins, and M. D. Scharff. 1992. Antibodies elicited by a Cryptococcus neoformanstetanus toxoid conjugate vaccine have the same specificity as those elicited in infection. J. Infect. Dis. 165:1086–1093. 7. Cherniak, R., and J. B. Sundstrom. 1994. Polysaccharide antigens of the capsule of Cryptococcus neoformans. Infect. Immun. 62:1507–1512. 8. Chuck, S. L., and M. A. Sande. 1989. Infections with Cryptococcus neoformans in the acquired immunodeficiency syndrome. N. Engl. J. Med. 321: 794–799. 9. Coker, R. J. 1992. Cryptococcal infection in AIDS. Int. J. Sex. Transm. Dis. AIDS 3:168–172. 10. Dismukes, W. E. 1988. Cryptococcal meningitis in patients with AIDS. J. Infect. Dis. 157:624–628. 11. Dubois, M., J. K. Gilles, P. A. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for the determination of sugars and related substances. Anal. Chem. 28:350–356. 12. Eng, R. H., E. Bishburg, and S. M. Smith. 1986. Cryptococcal infection in patients with acquired immune deficiency syndrome. Am. J. Med. 81:19–23. 13. Fidel, P. L., and J. W. Murphy. 1990. Characterization of a cell population which amplifies the anticryptococcal delayed-type hypersensitivity response. Infect. Immun. 58:393–398. 14. Graybill, J. R., and R. H. Alford. 1974. Cell mediated immunity in cryptococcosis. Cell. Immunol. 14:12–21. 15. Hill, J. O., and A. G. Harmsen. 1991. Intrapulmonary growth and dissemination of an avirulent strain of Cryptococcus neoformans in mice depleted of CD41 or CD81 T cells. J. Exp. Med. 173:755–758. 16. Houpt, D. C., G. S. Pfrommer, B. J. Young, T. A. Larson, and T. R. Kozel. 1994. Occurrences, immunoglobulin classes, and biological activities of antibodies in normal human serum that are reactive with Cryptococcus neofor-

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