Vol. 58, No. 6
INFECTION AND IMMUNITY, June 1990, p. 1914-1918 0019-9567/90/061914-05$02.00/0 Copyright © 1990, American Society for Microbiology
Opsonization of Cryptococcus neoformans by a Family of IsotypeSwitch Variant Antibodies Specific for the Capsular Polysaccharide ANNETTE M. SCHLAGETER AND THOMAS R. KOZEL* Department of Microbiology and Cell and Molecular Biology Program, School of Medicine, University of Nevada, Reno, Nevada 89557 Received 30 January 1990/Accepted 27 March 1990
A family of immunoglobulin isotype-switch variants was isolated by sib selection from a murine hybridoma which produced an immunoglobulin G subclass 1 (IgGl) antibody specific for the capsular polysaccharide of Cryptococcus neoformans. Antibodies of the IgGl, IgG2a, and IgG2b isotypes had similar serotype specificity patterns in double immunodiffusion assays which used polysaccharides of the four cryptococcal serotypes as antigens. A quantitative difference in the ability of the isotypes to form a precipitate with the polysaccharide was observed in a double immunodiffusion assay and confirmed in a quantitative precipitin assay. The relative precipitating activity of the antibodies was IgG2a > IgGl >> IgG2b. Analysis by enzyme-linked immunosorbent assay of the reactivity of the three isotypes with cryptococcal polysaccharide showed identical titers and slopes, suggesting that the variable region of the class-switch antibodies was unaltered. This system allowed us to examine the effect of the Fc portion of the antibody on opsonization of encapsulated cryptococci. Yeast cells were precoated with antibodies of each isotype and incubated with murine macrophages or cultured human monocytes. Antibodies of all three isotypes exhibited a dose-dependent opsonization for phagocytosis by both human and murine phagocytes. The relative opsonic activity of the antibodies was IgG2a > IgGl > IgG2b.
the affinity or epitope specificity of the antibody (7, 16, 21, 22). In a previous report, we described the production and characterization of MAbs reactive with cryptococcal polysaccharide (10). One of these antibodies, designated MAb 471, is reactive with an epitope shared by all four serotypes of C. neoformans. The broad specificity of this antibody makes it an ideal candidate for use in passive immunization. The objectives of this study were (i) to develop a family of isotype-switch variant antibodies from MAb 471 which differ only in their heavy-chain isotype, (ii) to verify that the different isotypes had identical reactivity with the capsular polysaccharide, and (iii) to compare the ability of the isotype-switch variants to opsonize encapsulated cryptococci for phagocytosis by murine macrophages and cultured human monocytes. Cryptococcosis frequently produces high levels of antigenemia, particularly in the patient with acquired immunodeficiency syndrome. Passive immunization has the possibility for producing in vivo immune complexes in patients with antigenemia, with accompanying pathology. As a consequence, our examination of the in vitro properties of the family of isotype-switch variants included an evaluation of their ability to produce precipitating antigen-antibody complexes. Our results indicate that IgG2b has a markedly weaker ability to precipitate cryptococcal polysaccharide than IgGl or IgG2a. Our results also indicate that the isotype-switch variants can opsonize cryptococci and that the opsonic activity can be ordered IgG2a > IgGl > IgG2b.
Cryptococcus neoformans is a pathogenic yeast surrounded by an antiphagocytic capsule. Nonencapsulated cryptococci are readily engulfed by phagocytes, but encapsulated yeast cells are resistant to phagocytosis. Previous studies have demonstrated that anticapsular immunoglobulin G (IgG) can opsonize the yeast for ingestion by phagocytes (14), suggesting a role for antibodies in host defense. The possible importance of antibody is supported by clinical evidence that circulating antibodies to the capsular polysaccharide are correlated with a positive prognosis (8). Direct evidence for the protective role of antibody was provided by Dromer et al., who demonstrated that passive immunization with a monoclonal antibody (MAb) specific for the capsular polysaccharide protected mice against the lethal acute pulmonary infection that occurs in complement component 5 (C5)-deficient mice (9). Since antifungal therapy against cryptococcosis is difficult in immunocompromised patients, particularly patients with acquired immunodeficiency syndrome, antibodies may have potential value in immunotherapy. Many biological properties of antibodies are related to the structure of the Fc portion of the molecule. The heavy-chain isotype of IgG determines biological activities such as complement fixation (20), serum half-life (28), antibody-directed cell-mediated cytotoxicity (13), and binding to monocyte receptors (2). Given the importance of the immunoglobulin isotype in determining the opsonic activity of an antibody, it is likely that the efficacy of an antibody for passive immunization is also influenced by the heavy-chain isotype. In a rare event, variant cells arise in antibody-producing hybridoma cell lines. These variant cells produce a different heavy-chain isotype than the parent cell line. The switch involves only the heavy-chain constant region, leaving the light chains and the heavy-chain variable region unaltered. Therefore, variation in the isotype occurs with no change in *
MATERIALS AND METHODS Cell lines and selection of class-switch variants. The parent murine hybridoma cell line 471 produces an IgGl antibody reactive with an epitope shared by polysaccharides of C. neoformans serotypes A, B, C, and D. Procedures for production and characterization of this MAb have been described elsewhere (10). We used sib selection to sequentially isolate spontaneously produced isotype-switch vari-
Corresponding author. 1914
VOL. 58, 1990
ants secreting IgG2b and IgG2a (26). Cells of hybridoma line 471 were cultured in a 96-well flat-bottomed plate at a density of 1,000 cells per well and grown to confluency. All wells were screened by enzyme-linked immunosorbent assay (ELISA), as described below, for the presence of immunoglobulins of the IgG2b subclass. Cells from wells which contained class-switch antibodies were placed in 1-ml cultures and grown to confluency. The supernatant fluid was screened to ensure that class-switch antibodies were present, and positive wells were subcultured at a density of 20 cells per well. After 10 days, the supernatant fluid from the wells was screened for the presence of the class-switch variant. Wells containing IgG2b-secreting cells were further cloned at least twice by limiting dilution. A cell line producing antibody of the IgG2a isotype was isolated from the IgG2b-secreting line in an identical manner. The lineage of the complete family of isotype-switch variants was IgGl
IgG2b -+ IgG2a. ELISA for detection of class-switch antibodies. A sandwich ELISA assay was used to screen supernatant fluids for the presence of IgG2b or IgG2a antibodies. Microtiter plates (Immunol 1; Dynatech Laboratories, Inc., Chantilly, Va.) were coated with affinity-purified goat anti-mouse IgG2b or IgG2a (cat. no. OB1174-21 or OB1165-21; Fisher Scientific Co., Orangeburg, N.Y.) at 0.5 ,ug per well in 50 mM phosphate buffer, pH 7.0, for 1.5 h at 37°C. The plates were washed with phosphate-buffered saline (PBS; pH 7.0) containing 0.05% Tween 20 and 0.5% gelatin (PBS-Tweengelatin). Supernatant fluid from cell cultures was diluted 1:2 in PBS-Tween-gelatin, and 200 ,ul of diluted sample was added to the wells and incubated for 1.5 h at 37°C. After washing, 200 pI of peroxidase-labeled goat anti-mouse IgG (heavy and light chains) (cat. no. 1030-05; Southern Biotechnology Associates, Birmingham, Ala.) diluted 1:5,000 in PBS-Tween-gelatin was added to the wells. The plates were incubated for 1.5 h at 37°C and washed, and the substrate solution (40 mg of o-phenylenediamine and 40 ,ul of 30% H202 in 100 ml of 0.1 M phosphate-citrate, pH 5.0) was added. The reaction was stopped after 30 min with 4 N H2SO4, and the optical density at 490 nm was read on an enzyme immunoassay plate reader (Bio-Tek Instruments, Inc., Burlington, Vt.). Purification of antibodies. BALB/c mice were primed with pristane and injected intraperitoneally with 3 x 10' hybridoma cells secreting antibody of the IgGl, IgG2a, or IgG2b isotype. Ascites fluid was collected, and the antibody was affinity purified on a column in which cryptococcal polysaccharide serotype A was coupled to AH-Sepharose (15). A stepwise pH gradient was used with a protein ASepharose CL-4B (Pharmacia, Uppsala, Sweden) column to further purify the antibodies and to ensure the absence of antibody of a contaminating subclass. Antibodies were equilibrated in binding buffer (3.0 M NaCl, 1.5 M glycine, pH 8.9), bound to protein A-Sepharose, and eluted sequentially with citric acid buffers (0.1 M citric acid) at pH values of 6.0, 5.0, and 4.0. Antibodies were assessed for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The purified antibodies were dialyzed against PBS, filter sterilized, and frozen at -700C. Immunochemical analysis of class-switch antibodies. The isotypes of the class-switch antibodies was confirmed by double immunodiffusion against antiserum specific for the various murine IgG isotypes (IgGl, cat. no. 1070-01; IgG2b, cat. no. 1090-01; IgG2a, cat. no. 1080-01; Fisher Scientific). The serotype specificity of the isotype-variant antibodies (2
ANTICRYPTOCOCCAL ISOTYPE-SWITCH VARIANTS
1915
mg/ml) was determined by double immunodiffusion against polysaccharides (2 mg/ml) of the four cryptococcal serotypes. Cryptococcal polysaccharides of serotypes A, B, C, and D were isolated (respectively) from culture supernatant fluids of cryptococcal strains ATCC 24064, ATCC 24065, ATCC 24066, and ATCC 24067 (American Type Culture Collection, Rockville, Md.). Polysaccharides were purified by sequential precipitation with ethanol and cetyltrimethylammonium bromide (4). Reactivity of the antibodies with cryptococcal polysaccharide was further demonstrated by a variation of the ELISA assay described by Eckert and Kozel (10). Microtiter plates were coated with serotype A polysaccharide and incubated with dilutions of the isotype-variant antibodies. The initial concentration of all MAbs before dilution was 10 ,ug/ml. Goat anti-mouse kappa-chain antibodies (cat. no. OB114421; Fisher Scientific) were used to detect the isotype-variant antibodies. Anti-kappa-chain antibodies were selected as the second antibody, because the reactivity of this antibody is independent of the heavy-chain isotype. Horseradish peroxidase-conjugated rabbit anti-goat IgG (cat. no. 228072; Kent Laboratories, Redmond, Wash.) was used to detect the second antibody. A quantitative precipitin assay was used to measure the ability of isotype-switch antibodies to precipitate cryptococcal polysaccharide. Affinity-purified isotype-switch antibody (200 ,ug) was mixed with serotype A polysaccharide in amounts ranging from 1 to 128 p.g of polysaccharide in a total reaction volume of 0.6 ml. The tubes were incubated for 2 h at room temperature and for 48 h at 4°C. The precipitate was collected by centrifugation, washed twice with PBS, and dissolved in 1 N NaOH. The optical density at 280 nm was read to determine the amount of protein in the precipitate by using E17 = 14.6. Phagocytosis assays. Cells of C. neoformans 388, an encapsulated strain of serotype A that was provided by K. J. Kwon-Chung, were used for the phagocytosis experiments. Peritoneal macrophages were obtained from unstimulated Swiss Webster mice by using the procedure described by Cohn and Benson (5). Monolayers of macrophages (2.5 x 105 cells per chamber) were prepared in four-chamber culture slides (Miles Laboratories, Inc., Elkhart, Ind.). Phagocytosis assays were performed as described elsewhere (10). Briefly, cryptococcal yeast cells (5 x 106) were incubated with 1 ml of various dilutions of the isotype-switch antibodies for 1 h at 37°C. The initial concentration of all MAbs before dilution was 1.2 mg/ml. The opsonized yeast cells were collected by centrifugation and suspended in 5 ml of Hanks balanced salt solution. A portion (1 ml) of opsonized yeast cells (106 cells per ml) was incubated with the monolayer of murine macrophages for 1 h at 37°C under 6% CO2. Monocytes were isolated from human peripheral blood and cultured for 4 to 6 days in Teflon flasks, as described by Wright (29). Cultured human monocytes (250 cells per well) were prepared in Terasaki plates (Robbins Scientific, Mountain View, Calif.). Cryptococcal yeast cells were prepared as described above. A portion (10 ,ul) of opsonized yeast (105 cells per ml) was incubated with the monolayer of cultured human monocytes for 1 h at 37°C under 6% CO2. The slides and Terasaki plates were washed, fixed, stained with Giemsa stain, and examined by light microscopy. At least 200 phagocytes per monolayer of murine macrophages were examined. At least 50 human monocytes per Terasaki well were examined. Data are presented as the number of ingested yeast cells per 100 phagocytes (phagocytic index). Phagocytosis assays were repeated at least three times. The
1916
INFECT. IMMUN.
SCHLAGETER AND KOZEL
200 3
cz
FIG. 1. Reactivity of isotype-switch variant antibodies with polysaccharides of the four serotypes of C. neoformans. The center wells contain either MAb IgGl (a), IgG2a (b), or IgG2b (c). The outer wells contain polysaccharides of serotype A, B, C, or D.
results are reported as the mean of at least three replications for each experiment. RESULTS Isotype-switch variants were found at a frequency of one switch variant for every 500,000 cells of each parent line. The heavy-chain isotype of the class-switch antibodies was confirmed by double immunodiffusion against antisera with a known isotype specificity and by pH-dependent elution of the antibodies from protein A-Sepharose. Immunoaffinitypurified class-switch antibodies of the IgGl, IgG2a, and IgG2b isotypes were shown to be reactive with (respectively) heavy-chain-specific anti-IgGl, anti-IgG2a, and antiIgG2b. Analysis of the binding of each class-switch antibody to protein A-Sepharose showed that the IgGl antibody eluted from protein A at pH 6, IgG2a eluted at pH 5, and IgG2b eluted at pH 4, results consistent with the behavior of murine IgGl, IgG2a, and IgG2b, respectively (11). After electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the affinity-purified antibodies appeared as a single band (150 kilodaltons) under nonreducing conditions and produced two bands (50 and 25 kilodaltons) under reducing conditions. Parent cell line 471 secretes an IgGl that is reactive with an epitope found on polysaccharides from all four cryptococcal serotypes. Double immunodiffusion showed that the isotype variants retained the serotype specificity of the parent cell line (Fig. 1). However, it should be noted that the IgG2b antibody had a generally reduced ability to precipitate the polysaccharides, particularly serotype D. A quantitative precipitation assay was used to compare the abilities of the isotype-switch antibodies to precipitate cryptococcal polysaccharide. The results (Fig. 2) showed that the isotype variants differed in their abilities to form a precipitate. IgG2a had the greatest ability to form insoluble complexes; IgGl formed moderate amounts of precipitate; and IgG2b had a very limited ability to form a precipitate. The observed differences could not be attributed to differences in the amount of antibody added, because affinitypurified antibodies were used in all assays. Further evidence for retention of parental antigen-binding activity by the isotype variants was provided by an ELISA assay. Plates were coated with serotype A polysaccharide. The reactivity of each isotype variant was determined as described in Materials and Methods. The results showed that the three IgG isotypes were equally reactive with cryptococcal polysaccharide with regard to both the titer and the slope when antibody dilution was plotted against optical density at 490 nm (Fig. 3). This indicates that despite a reduced precipitating activity on the part of IgG2b, the antigenbinding activities of both IgG2a and IgG2b were not altered during the isotype selection.
150±
LIJ 0
L C,
0'
l00t 50-
20 30 40 50 10 CRYPTOCOCCAL POLYSACCHARIDE ADDED (pg) FIG. 2. Quantitative precipitin curves showing precipitation of serotype A polysaccharide by antibodies of the IgGl, IgG2a, and IgG2b isotypes. 0
Phagocytosis assays were used to compare the opsonic activities of the isotype-switch antibodies. Yeast cells precoated with dilutions of the isotype-switch antibodies were incubated with monolayers of murine macrophages. Encapsulated cryptococci were opsonized by all three antibodies (Fig. 4); however, there was less ingestion of yeast cells opsonized with IgG2b antibody than of yeast cells opsonized by antibody of the IgGl or IgG2a isotype. Nonopsonized yeast cells were not ingested (data not shown). The opsonic activity of the isotype-switch antibodies for phagocytosis by cultured human monocytes was also determined. Yeast cells precoated with dilutions of the antibodies were incubated with cultured human monocytes. The results (Fig. 5) showed that encapsulated cryptococci were opsonized by each of the isotype-switch antibodies. As with murine macrophages, IgG2b was less opsonic than IgGl or IgG2a. DISCUSSION sib selection was used to isolate a family of isotype subclass switch variants. We sequentially isolated cell lines producing IgG2a and IgG2b from a hybridoma which produced an IgGl antibody specific for the capsular polysac-
0 0
0
0.0
8 10
100 RECIPROCAL ANTIBODY DILUTION
FIG. 3. Reactivity of the isotype-switch variant antibodies with serotype A polysaccharide in an ELISA. OD490, Optical density at 490 nm.
ANTICRYPTOCOCCAL ISOTYPE-SWITCH VARIANTS
VOL. 58, 1990
200
ai e fr oto FIG 4 Copaisn o te psoicaciviie o IgG1,Ig2, n
I
o/0
140
/0
ANTIBODY DILUTION
Comparison of the opsonic activities of IgGl, IgG2a, and IgG2b antibodies for phagocytosis of encapsulated cryptococci by FIG. 4.
murine peritoneal macrophages. Data shown are the means standard errors.
+
charide of C. neoformans. All members of the isotypeswitch family retained reactivity with the four cryptococcal serotypes, as shown by the double immunodiffusion assay. All three antibodies formed precipitin bands with polysaccharides of serotypes A, B, and C and weak precipitin bands with serotype D polysaccharide. The IgG2b antibody was less reactive with serotype D polysaccharide in the double immunodiffusion assay and showed a substantially reduced ability to precipitate serotype A polysaccharide in the quantitative precipitin assay. The reduced precipitating activity of the IgG2b antibody could be due to a mutation in the variable region. However, it is unlikely that a mutation affected the antigen-binding region for two reasons. First, the IgG2a-secreting cell line was derived from the IgG2bsecreting clone. It is unlikely that a mutation would occur during switching from IgGl to IgG2b and then correct itself when another shift occurred from IgG2b to IgG2a. Second, ELISA results for the isotype-switch antibodies were identical with regard to both titer and slope. The ELISA is independent of the secondary effects of the heavy chain that might influence precipitation. The identical slopes of the three IgG isotypes in the ELISA suggest a similar affinity for the polysaccharide (3).
x
IgG2a
m1 IgGl
200
Li
IgG2b
0
z
a0 c]
100
I
100
1/200 1/400 ANTIBODY DILUTION
1/800
FIG. 5. Comparison of the opsonic activities of IgGl, IgG2a, and IgG2b antibodies for phagocytosis of encapsulated cryptococci by cultured human monocytes. Data shown are the means + standard errors.
1917
Several studies have shown that the precipitating activity of IgG is markedly influenced by the Fc portion of the antibody (18, 24). The poor precipitating activity of antibodies of the IgG2b isotype has been noted in previous reports (6, 12). These studies used MAbs whose affinities did not differ significantly. Our results with a class-switch family of antibodies having identical epitope specificities provides additional evidence that IgG2b antibody has poor precipitating activity. This observation has important implications for the possible use of passive immunization in cryptococcosis. The high levels of antigenemia that occur in cryptococcosis raise the likelihood that passive immunization with antibody having strong precipitating activity will lead to one or more forms of immune complex disease (1). The poor precipitating activity of the IgG2b antibody would make that immunoglobulin isotype a candidate for passive immunization if it is shown to have in vivo efficacy. Antibodies of the IgGl, IgG2a, and IgG2b isotypes were opsonic for the yeast; however, there were consistent differences in the relative opsonic activities of the three antibodies. The IgG2a antibody was most opsonic, followed in order by IgGl and IgG2b. This relative opsonic activity was observed with both cultured human monocytes and murine peritoneal macrophages. These results are in partial agreement with those of a previous analysis of the role of isotype in opsonization of erythrocytes. As with our study, Ralph et al. found that IgG2a was a potent opsonin but that IgGl and IgG2b also had substantial opsonic activities (23). The main difference between our results and those of Ralph et al. is the relative ability of IgGl and IgG2b to facilitate phagocytosis. A direct comparison of our results with those reported by Ralph et al. is difficult, because their study utilized antibodies of different isotypes that were obtained from fractionated antiserum or hybridoma cell lines rather than a family of isotype-switch variants. Thus, there is no assurance that observed differences were independent of the antibodycombining site. Our results are also in good agreement with those of previous studies that compare the biological activities of families of isotype-switch variants. These studies examined the ability of the antibodies to mediate antibody-directed cell-mediated cytotoxicity. Effector cells that have been examined include peripheral blood mononuclear cells (19), isolated human monocytes (2, 17, 27), and nonadherent K cells (13), while target cells have been sheep erythrocytes (2) or tumor cells (13, 17, 19, 27). A consistent pattern has emerged in all of these studies, in which IgG2a is the most effective facilitator of the interaction between target cells and effector cells. This result is identical with our observation that IgG2a is the most effective opsonin of encapsulated cryptococci. There is no consistent pattern for the relative efficacy of IgGl and IgG2b. Some studies have found IgGl to be a more effective mediator of antibody-directed cellmediated cytotoxicity than IgG2b (2, 17, 27), whereas others have found IgG2b to be more active (13, 19). Such differences may be due to differences in effector cells, levels of activation of the effector cell, target cells, or densities of epitopes on target cells. Our study was initiated in an attempt to develop MAbs with the potential for prophylactic or therapeutic use with cryptococcosis. The rationale for the development of a class-switch family is predicated on the known variation in biological activities of the different IgG subclasses. Our results have shown that IgGl, IgG2a, and IgG2b are all potent opsonins for encapsulated cryptococci, but they differ in their relative ability to opsonize the yeast. We describe
1918
SCHLAGETER AND KOZEL
the prophylactic and therapeutic efficacy of these antibodies in an accompanying paper (25). ACKNOWLEDGMENTS This work was supported by Public Health Service grants A114209 and A124357 from the National Institutes of Health.
LITERATURE CITED 1. Agodoa, L. Y. C., V. J. Gauthier, and M. Mannik. 1988. Precipitating antigen-antibody systems are required for the formation of subepithelial electron-dense immune deposits in rat glomeruli. J. Exp. Med. 158:1259-1271. 2. Boot, J. H. A., M. E. J. Geerts, and L. A. Aarden. 1989. Functional polymorphisms of Fc receptors in human monocytemediated cytotoxicity towards erythrocytes induced by murine isotype switch variants. J. Immunol. 142:1217-1223. 3. Butler, J. E., T. L. Feldbush, P. L. McGivern, and N. Stewart. 1978. The enzyme-linked immunosorbent assay (ELISA): a measure of antibody concentration or affinity? Immunochemistry 15:131-136. 4. Cherniak, R., E. Reiss, M. E. Slodki, R. D. Plattner, and S. 0. Blumer. 1980. Structure and antigenic activity of the capsular polysaccharide of Cryptococcus neoformans serotype A. Mol. Immunol. 17:839-854. 5. Cohn, Z. A., and B. Benson. 1965. The differentiation of mononuclear phagocytes. Morphology, cytochemistry, and biochemistry. J. Exp. Med. 121:153-161. 6. Cosio, F. G., D. J. Birmingham, D. J. Sexton, and L. A. Hebert. 1987. Interactions between precipitating and nonprecipitating antibodies in the formation of immune complexes. J. Immunol. 138:2587-2592. 7. Dangl, J. L., D. R. Parks, V. T. Oi, and L. A. Herzenberg. 1982. Rapid isolation of cloned isotype switch variants using fluorescence activated cell sorting. Cytometry 2:395-401. 8. Diamond, R. D., and J. E. Bennett. 1974. Prognostic factors in cryptococcal meningitis. Ann. Intern. Med. 80:176-181. 9. Dromer, F., J. Charreire, A. Contrepois, C. Carbon, and P. Yeni. 1987. Protection of mice against experimental cryptococcosis by anti-Cryptococcus neoformans monoclonal antibody. Infect. Immun. 55:749-752. 10. Eckert, T. F., and T. R. Kozel. 1987. Production and characterization of monoclonal antibodies specific for Cryptococcus neoformans capsular polysaccharide. Infect. Immun. 55:18951899. 11. Ey, P. L., S. J. Prowse, and C. R. Jenkin. 1978. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-Sepharose. Immunochemistry 15:429-436. 12. Hirayama, N., T. Hirano, G. Kohler, A. Kurata, K. Okumura, and Z. Ovary. 1982. Biological activities of antitrinitrophenyl and antidinitrophenyl mouse monoclonal antibodies. Proc. Natl. Acad. Sci. USA 79:613-615. 13. Kipps, T. J., P. Parham, J. Punt, and L. A. Herzenberg. 1985. Importance of immunoglobulin isotype in human antibodydependent, cell-mediated cytotoxicity directed by murine
INF'ECT. IMMUN.
monoclonal antibodies. J. Exp. Med. 161:1-17. 14. Kozel, T. R., and J. L. Follette. 1981. Opsonization of encapsulated Cryptococcus neoformans by specific anticapsular antibody. Infect. Immun. 31:978-984. 15. Kozel, T. R., and C. A. Hermerath. 1988. Benzoquinone activation of Cryptococcus neoformans capsular polysaccharide for construction of an immunoaffinity column. J. Immunol. Methods 107:53-58. 16. Liesegang, B., A. Radbruch, and K. Rajewsky. 1978. Isolation of myeloma variants with predefined variant surface immunoglobulin by cell sorting. Proc. Natl. Acad. Sci. USA 75:3901-3905. 17. Lubeck, M. D., Y. Kimoto, Z. Steplewski, and H. Koprowski. 1988. Killing of human tumor cell lines by human monocytes and murine monoclonal antibodies. Cell. Immunol. 111:107117. 18. M0ller, N. P. H. 1979. Fc-mediated immune precipitation I. A new role of the Fc-portion of IgG. Immunology 38:631-640. 19. Mujoo, K., T. J. Kipps, H. M. Yang, D. A. Cheresh, U. Wargalla, D. J. Sander, and R. A. Reisfeld. 1989. Functional properties and effect on growth suppression of human neuroblastoma tumors by isotype switch variants of monoclonal antiganglioside GD2 antibody 14.18. Cancer Res. 49:2857-2861. 20. Oi, V. T., T. M. Vuong, R. Hardy, J. Reidler, J. Dangl. L. A. Herzenberg, and L. Stryer. 1984. Correlation between segmental flexibility and effector function of antibodies. Nature (London) 307:136-140. 21. Preud'homme, J. L., B. K. Birshtein, and M. D. Scharff. 1975. Variants of a mouse myeloma cell line that synthesize immunoglobulin heavy chains having an altered serotype. Proc. Natl. Acad. Sci. USA 72:1427-1430. 22. Radbruch, A., B. Liesegang, and K. Rajewsky. 1980. Isolation of variants of mouse myeloma X63 that express changed immunoglobulin class. Proc. Natl. Acad. Sci. USA 77:2909-2913. 23. Ralph, P., I. Nakoinz, B. Diamond, and D. Yelton. 1980. All classes of murine IgG antibody mediate macrophage phagocytosis and lysis of erythrocytes. J. Immunol. 125:1885-1888. 24. Rodwell, J. D., L. H. Tang, and V. N. Schumaker. 1980. Antigen valence and Fc-localized secondary forces in antibody precipitation. Mol. Immunol. 17:1591-1597. 25. Sanford, J. E., D. M. Lupan, A. M. Schlageter, aind T. R. Kozel. 1990. Passive immunization against Cryptococcus neoformans with an isotype-switch family of monoclonal antibodies reactive with cryptococcal polysaccharide. Infect. Immun. 58:19191923. 26. Spira, G., A. Bargellesi, J. L. Teillaud, and M. D. Scharif. 1984. The identification of monoclonal class switch variants by sib selection and an ELISA assay. J. Immunol. Methods 74: 307-315. 27. Steplewski, Z., G. Spira, M. Blaszczyk, M. D. Lubeck, A. Radbruch, H. Illges, D. Herlyn, K. Rajewsky, and M. Scharff. 1985. Isolation and characterization of anti-monosialoganglioside monoclonal antibody 19-9 class-switch variants. Proc. Natl. Acad. Sci. USA 82:8653-8657. 28. Vieira, P., and K. Rajewsky. 1988. The half-lives of serum immunoglobulins in adult mice. Eur. J. Immunol. 18:313-316. 29. Wright, S. D. 1986. Methods for the study of receptor-mediated phagocytosis. Methods Enzymol. 132:204-221.
INFECTION AND IMMUNITY, June 1990, p. 1914-1918 0019-9567/90/061914-05$02.00/0 Copyright © 1990, American Society for Microbiology
Opsonization of Cryptococcus neoformans by a Family of IsotypeSwitch Variant Antibodies Specific for the Capsular Polysaccharide ANNETTE M. SCHLAGETER AND THOMAS R. KOZEL* Department of Microbiology and Cell and Molecular Biology Program, School of Medicine, University of Nevada, Reno, Nevada 89557 Received 30 January 1990/Accepted 27 March 1990
A family of immunoglobulin isotype-switch variants was isolated by sib selection from a murine hybridoma which produced an immunoglobulin G subclass 1 (IgGl) antibody specific for the capsular polysaccharide of Cryptococcus neoformans. Antibodies of the IgGl, IgG2a, and IgG2b isotypes had similar serotype specificity patterns in double immunodiffusion assays which used polysaccharides of the four cryptococcal serotypes as antigens. A quantitative difference in the ability of the isotypes to form a precipitate with the polysaccharide was observed in a double immunodiffusion assay and confirmed in a quantitative precipitin assay. The relative precipitating activity of the antibodies was IgG2a > IgGl >> IgG2b. Analysis by enzyme-linked immunosorbent assay of the reactivity of the three isotypes with cryptococcal polysaccharide showed identical titers and slopes, suggesting that the variable region of the class-switch antibodies was unaltered. This system allowed us to examine the effect of the Fc portion of the antibody on opsonization of encapsulated cryptococci. Yeast cells were precoated with antibodies of each isotype and incubated with murine macrophages or cultured human monocytes. Antibodies of all three isotypes exhibited a dose-dependent opsonization for phagocytosis by both human and murine phagocytes. The relative opsonic activity of the antibodies was IgG2a > IgGl > IgG2b.
the affinity or epitope specificity of the antibody (7, 16, 21, 22). In a previous report, we described the production and characterization of MAbs reactive with cryptococcal polysaccharide (10). One of these antibodies, designated MAb 471, is reactive with an epitope shared by all four serotypes of C. neoformans. The broad specificity of this antibody makes it an ideal candidate for use in passive immunization. The objectives of this study were (i) to develop a family of isotype-switch variant antibodies from MAb 471 which differ only in their heavy-chain isotype, (ii) to verify that the different isotypes had identical reactivity with the capsular polysaccharide, and (iii) to compare the ability of the isotype-switch variants to opsonize encapsulated cryptococci for phagocytosis by murine macrophages and cultured human monocytes. Cryptococcosis frequently produces high levels of antigenemia, particularly in the patient with acquired immunodeficiency syndrome. Passive immunization has the possibility for producing in vivo immune complexes in patients with antigenemia, with accompanying pathology. As a consequence, our examination of the in vitro properties of the family of isotype-switch variants included an evaluation of their ability to produce precipitating antigen-antibody complexes. Our results indicate that IgG2b has a markedly weaker ability to precipitate cryptococcal polysaccharide than IgGl or IgG2a. Our results also indicate that the isotype-switch variants can opsonize cryptococci and that the opsonic activity can be ordered IgG2a > IgGl > IgG2b.
Cryptococcus neoformans is a pathogenic yeast surrounded by an antiphagocytic capsule. Nonencapsulated cryptococci are readily engulfed by phagocytes, but encapsulated yeast cells are resistant to phagocytosis. Previous studies have demonstrated that anticapsular immunoglobulin G (IgG) can opsonize the yeast for ingestion by phagocytes (14), suggesting a role for antibodies in host defense. The possible importance of antibody is supported by clinical evidence that circulating antibodies to the capsular polysaccharide are correlated with a positive prognosis (8). Direct evidence for the protective role of antibody was provided by Dromer et al., who demonstrated that passive immunization with a monoclonal antibody (MAb) specific for the capsular polysaccharide protected mice against the lethal acute pulmonary infection that occurs in complement component 5 (C5)-deficient mice (9). Since antifungal therapy against cryptococcosis is difficult in immunocompromised patients, particularly patients with acquired immunodeficiency syndrome, antibodies may have potential value in immunotherapy. Many biological properties of antibodies are related to the structure of the Fc portion of the molecule. The heavy-chain isotype of IgG determines biological activities such as complement fixation (20), serum half-life (28), antibody-directed cell-mediated cytotoxicity (13), and binding to monocyte receptors (2). Given the importance of the immunoglobulin isotype in determining the opsonic activity of an antibody, it is likely that the efficacy of an antibody for passive immunization is also influenced by the heavy-chain isotype. In a rare event, variant cells arise in antibody-producing hybridoma cell lines. These variant cells produce a different heavy-chain isotype than the parent cell line. The switch involves only the heavy-chain constant region, leaving the light chains and the heavy-chain variable region unaltered. Therefore, variation in the isotype occurs with no change in *
MATERIALS AND METHODS Cell lines and selection of class-switch variants. The parent murine hybridoma cell line 471 produces an IgGl antibody reactive with an epitope shared by polysaccharides of C. neoformans serotypes A, B, C, and D. Procedures for production and characterization of this MAb have been described elsewhere (10). We used sib selection to sequentially isolate spontaneously produced isotype-switch vari-
Corresponding author. 1914
VOL. 58, 1990
ants secreting IgG2b and IgG2a (26). Cells of hybridoma line 471 were cultured in a 96-well flat-bottomed plate at a density of 1,000 cells per well and grown to confluency. All wells were screened by enzyme-linked immunosorbent assay (ELISA), as described below, for the presence of immunoglobulins of the IgG2b subclass. Cells from wells which contained class-switch antibodies were placed in 1-ml cultures and grown to confluency. The supernatant fluid was screened to ensure that class-switch antibodies were present, and positive wells were subcultured at a density of 20 cells per well. After 10 days, the supernatant fluid from the wells was screened for the presence of the class-switch variant. Wells containing IgG2b-secreting cells were further cloned at least twice by limiting dilution. A cell line producing antibody of the IgG2a isotype was isolated from the IgG2b-secreting line in an identical manner. The lineage of the complete family of isotype-switch variants was IgGl
IgG2b -+ IgG2a. ELISA for detection of class-switch antibodies. A sandwich ELISA assay was used to screen supernatant fluids for the presence of IgG2b or IgG2a antibodies. Microtiter plates (Immunol 1; Dynatech Laboratories, Inc., Chantilly, Va.) were coated with affinity-purified goat anti-mouse IgG2b or IgG2a (cat. no. OB1174-21 or OB1165-21; Fisher Scientific Co., Orangeburg, N.Y.) at 0.5 ,ug per well in 50 mM phosphate buffer, pH 7.0, for 1.5 h at 37°C. The plates were washed with phosphate-buffered saline (PBS; pH 7.0) containing 0.05% Tween 20 and 0.5% gelatin (PBS-Tweengelatin). Supernatant fluid from cell cultures was diluted 1:2 in PBS-Tween-gelatin, and 200 ,ul of diluted sample was added to the wells and incubated for 1.5 h at 37°C. After washing, 200 pI of peroxidase-labeled goat anti-mouse IgG (heavy and light chains) (cat. no. 1030-05; Southern Biotechnology Associates, Birmingham, Ala.) diluted 1:5,000 in PBS-Tween-gelatin was added to the wells. The plates were incubated for 1.5 h at 37°C and washed, and the substrate solution (40 mg of o-phenylenediamine and 40 ,ul of 30% H202 in 100 ml of 0.1 M phosphate-citrate, pH 5.0) was added. The reaction was stopped after 30 min with 4 N H2SO4, and the optical density at 490 nm was read on an enzyme immunoassay plate reader (Bio-Tek Instruments, Inc., Burlington, Vt.). Purification of antibodies. BALB/c mice were primed with pristane and injected intraperitoneally with 3 x 10' hybridoma cells secreting antibody of the IgGl, IgG2a, or IgG2b isotype. Ascites fluid was collected, and the antibody was affinity purified on a column in which cryptococcal polysaccharide serotype A was coupled to AH-Sepharose (15). A stepwise pH gradient was used with a protein ASepharose CL-4B (Pharmacia, Uppsala, Sweden) column to further purify the antibodies and to ensure the absence of antibody of a contaminating subclass. Antibodies were equilibrated in binding buffer (3.0 M NaCl, 1.5 M glycine, pH 8.9), bound to protein A-Sepharose, and eluted sequentially with citric acid buffers (0.1 M citric acid) at pH values of 6.0, 5.0, and 4.0. Antibodies were assessed for purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The purified antibodies were dialyzed against PBS, filter sterilized, and frozen at -700C. Immunochemical analysis of class-switch antibodies. The isotypes of the class-switch antibodies was confirmed by double immunodiffusion against antiserum specific for the various murine IgG isotypes (IgGl, cat. no. 1070-01; IgG2b, cat. no. 1090-01; IgG2a, cat. no. 1080-01; Fisher Scientific). The serotype specificity of the isotype-variant antibodies (2
ANTICRYPTOCOCCAL ISOTYPE-SWITCH VARIANTS
1915
mg/ml) was determined by double immunodiffusion against polysaccharides (2 mg/ml) of the four cryptococcal serotypes. Cryptococcal polysaccharides of serotypes A, B, C, and D were isolated (respectively) from culture supernatant fluids of cryptococcal strains ATCC 24064, ATCC 24065, ATCC 24066, and ATCC 24067 (American Type Culture Collection, Rockville, Md.). Polysaccharides were purified by sequential precipitation with ethanol and cetyltrimethylammonium bromide (4). Reactivity of the antibodies with cryptococcal polysaccharide was further demonstrated by a variation of the ELISA assay described by Eckert and Kozel (10). Microtiter plates were coated with serotype A polysaccharide and incubated with dilutions of the isotype-variant antibodies. The initial concentration of all MAbs before dilution was 10 ,ug/ml. Goat anti-mouse kappa-chain antibodies (cat. no. OB114421; Fisher Scientific) were used to detect the isotype-variant antibodies. Anti-kappa-chain antibodies were selected as the second antibody, because the reactivity of this antibody is independent of the heavy-chain isotype. Horseradish peroxidase-conjugated rabbit anti-goat IgG (cat. no. 228072; Kent Laboratories, Redmond, Wash.) was used to detect the second antibody. A quantitative precipitin assay was used to measure the ability of isotype-switch antibodies to precipitate cryptococcal polysaccharide. Affinity-purified isotype-switch antibody (200 ,ug) was mixed with serotype A polysaccharide in amounts ranging from 1 to 128 p.g of polysaccharide in a total reaction volume of 0.6 ml. The tubes were incubated for 2 h at room temperature and for 48 h at 4°C. The precipitate was collected by centrifugation, washed twice with PBS, and dissolved in 1 N NaOH. The optical density at 280 nm was read to determine the amount of protein in the precipitate by using E17 = 14.6. Phagocytosis assays. Cells of C. neoformans 388, an encapsulated strain of serotype A that was provided by K. J. Kwon-Chung, were used for the phagocytosis experiments. Peritoneal macrophages were obtained from unstimulated Swiss Webster mice by using the procedure described by Cohn and Benson (5). Monolayers of macrophages (2.5 x 105 cells per chamber) were prepared in four-chamber culture slides (Miles Laboratories, Inc., Elkhart, Ind.). Phagocytosis assays were performed as described elsewhere (10). Briefly, cryptococcal yeast cells (5 x 106) were incubated with 1 ml of various dilutions of the isotype-switch antibodies for 1 h at 37°C. The initial concentration of all MAbs before dilution was 1.2 mg/ml. The opsonized yeast cells were collected by centrifugation and suspended in 5 ml of Hanks balanced salt solution. A portion (1 ml) of opsonized yeast cells (106 cells per ml) was incubated with the monolayer of murine macrophages for 1 h at 37°C under 6% CO2. Monocytes were isolated from human peripheral blood and cultured for 4 to 6 days in Teflon flasks, as described by Wright (29). Cultured human monocytes (250 cells per well) were prepared in Terasaki plates (Robbins Scientific, Mountain View, Calif.). Cryptococcal yeast cells were prepared as described above. A portion (10 ,ul) of opsonized yeast (105 cells per ml) was incubated with the monolayer of cultured human monocytes for 1 h at 37°C under 6% CO2. The slides and Terasaki plates were washed, fixed, stained with Giemsa stain, and examined by light microscopy. At least 200 phagocytes per monolayer of murine macrophages were examined. At least 50 human monocytes per Terasaki well were examined. Data are presented as the number of ingested yeast cells per 100 phagocytes (phagocytic index). Phagocytosis assays were repeated at least three times. The
1916
INFECT. IMMUN.
SCHLAGETER AND KOZEL
200 3
cz
FIG. 1. Reactivity of isotype-switch variant antibodies with polysaccharides of the four serotypes of C. neoformans. The center wells contain either MAb IgGl (a), IgG2a (b), or IgG2b (c). The outer wells contain polysaccharides of serotype A, B, C, or D.
results are reported as the mean of at least three replications for each experiment. RESULTS Isotype-switch variants were found at a frequency of one switch variant for every 500,000 cells of each parent line. The heavy-chain isotype of the class-switch antibodies was confirmed by double immunodiffusion against antisera with a known isotype specificity and by pH-dependent elution of the antibodies from protein A-Sepharose. Immunoaffinitypurified class-switch antibodies of the IgGl, IgG2a, and IgG2b isotypes were shown to be reactive with (respectively) heavy-chain-specific anti-IgGl, anti-IgG2a, and antiIgG2b. Analysis of the binding of each class-switch antibody to protein A-Sepharose showed that the IgGl antibody eluted from protein A at pH 6, IgG2a eluted at pH 5, and IgG2b eluted at pH 4, results consistent with the behavior of murine IgGl, IgG2a, and IgG2b, respectively (11). After electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the affinity-purified antibodies appeared as a single band (150 kilodaltons) under nonreducing conditions and produced two bands (50 and 25 kilodaltons) under reducing conditions. Parent cell line 471 secretes an IgGl that is reactive with an epitope found on polysaccharides from all four cryptococcal serotypes. Double immunodiffusion showed that the isotype variants retained the serotype specificity of the parent cell line (Fig. 1). However, it should be noted that the IgG2b antibody had a generally reduced ability to precipitate the polysaccharides, particularly serotype D. A quantitative precipitation assay was used to compare the abilities of the isotype-switch antibodies to precipitate cryptococcal polysaccharide. The results (Fig. 2) showed that the isotype variants differed in their abilities to form a precipitate. IgG2a had the greatest ability to form insoluble complexes; IgGl formed moderate amounts of precipitate; and IgG2b had a very limited ability to form a precipitate. The observed differences could not be attributed to differences in the amount of antibody added, because affinitypurified antibodies were used in all assays. Further evidence for retention of parental antigen-binding activity by the isotype variants was provided by an ELISA assay. Plates were coated with serotype A polysaccharide. The reactivity of each isotype variant was determined as described in Materials and Methods. The results showed that the three IgG isotypes were equally reactive with cryptococcal polysaccharide with regard to both the titer and the slope when antibody dilution was plotted against optical density at 490 nm (Fig. 3). This indicates that despite a reduced precipitating activity on the part of IgG2b, the antigenbinding activities of both IgG2a and IgG2b were not altered during the isotype selection.
150±
LIJ 0
L C,
0'
l00t 50-
20 30 40 50 10 CRYPTOCOCCAL POLYSACCHARIDE ADDED (pg) FIG. 2. Quantitative precipitin curves showing precipitation of serotype A polysaccharide by antibodies of the IgGl, IgG2a, and IgG2b isotypes. 0
Phagocytosis assays were used to compare the opsonic activities of the isotype-switch antibodies. Yeast cells precoated with dilutions of the isotype-switch antibodies were incubated with monolayers of murine macrophages. Encapsulated cryptococci were opsonized by all three antibodies (Fig. 4); however, there was less ingestion of yeast cells opsonized with IgG2b antibody than of yeast cells opsonized by antibody of the IgGl or IgG2a isotype. Nonopsonized yeast cells were not ingested (data not shown). The opsonic activity of the isotype-switch antibodies for phagocytosis by cultured human monocytes was also determined. Yeast cells precoated with dilutions of the antibodies were incubated with cultured human monocytes. The results (Fig. 5) showed that encapsulated cryptococci were opsonized by each of the isotype-switch antibodies. As with murine macrophages, IgG2b was less opsonic than IgGl or IgG2a. DISCUSSION sib selection was used to isolate a family of isotype subclass switch variants. We sequentially isolated cell lines producing IgG2a and IgG2b from a hybridoma which produced an IgGl antibody specific for the capsular polysac-
0 0
0
0.0
8 10
100 RECIPROCAL ANTIBODY DILUTION
FIG. 3. Reactivity of the isotype-switch variant antibodies with serotype A polysaccharide in an ELISA. OD490, Optical density at 490 nm.
ANTICRYPTOCOCCAL ISOTYPE-SWITCH VARIANTS
VOL. 58, 1990
200
ai e fr oto FIG 4 Copaisn o te psoicaciviie o IgG1,Ig2, n
I
o/0
140
/0
ANTIBODY DILUTION
Comparison of the opsonic activities of IgGl, IgG2a, and IgG2b antibodies for phagocytosis of encapsulated cryptococci by FIG. 4.
murine peritoneal macrophages. Data shown are the means standard errors.
+
charide of C. neoformans. All members of the isotypeswitch family retained reactivity with the four cryptococcal serotypes, as shown by the double immunodiffusion assay. All three antibodies formed precipitin bands with polysaccharides of serotypes A, B, and C and weak precipitin bands with serotype D polysaccharide. The IgG2b antibody was less reactive with serotype D polysaccharide in the double immunodiffusion assay and showed a substantially reduced ability to precipitate serotype A polysaccharide in the quantitative precipitin assay. The reduced precipitating activity of the IgG2b antibody could be due to a mutation in the variable region. However, it is unlikely that a mutation affected the antigen-binding region for two reasons. First, the IgG2a-secreting cell line was derived from the IgG2bsecreting clone. It is unlikely that a mutation would occur during switching from IgGl to IgG2b and then correct itself when another shift occurred from IgG2b to IgG2a. Second, ELISA results for the isotype-switch antibodies were identical with regard to both titer and slope. The ELISA is independent of the secondary effects of the heavy chain that might influence precipitation. The identical slopes of the three IgG isotypes in the ELISA suggest a similar affinity for the polysaccharide (3).
x
IgG2a
m1 IgGl
200
Li
IgG2b
0
z
a0 c]
100
I
100
1/200 1/400 ANTIBODY DILUTION
1/800
FIG. 5. Comparison of the opsonic activities of IgGl, IgG2a, and IgG2b antibodies for phagocytosis of encapsulated cryptococci by cultured human monocytes. Data shown are the means + standard errors.
1917
Several studies have shown that the precipitating activity of IgG is markedly influenced by the Fc portion of the antibody (18, 24). The poor precipitating activity of antibodies of the IgG2b isotype has been noted in previous reports (6, 12). These studies used MAbs whose affinities did not differ significantly. Our results with a class-switch family of antibodies having identical epitope specificities provides additional evidence that IgG2b antibody has poor precipitating activity. This observation has important implications for the possible use of passive immunization in cryptococcosis. The high levels of antigenemia that occur in cryptococcosis raise the likelihood that passive immunization with antibody having strong precipitating activity will lead to one or more forms of immune complex disease (1). The poor precipitating activity of the IgG2b antibody would make that immunoglobulin isotype a candidate for passive immunization if it is shown to have in vivo efficacy. Antibodies of the IgGl, IgG2a, and IgG2b isotypes were opsonic for the yeast; however, there were consistent differences in the relative opsonic activities of the three antibodies. The IgG2a antibody was most opsonic, followed in order by IgGl and IgG2b. This relative opsonic activity was observed with both cultured human monocytes and murine peritoneal macrophages. These results are in partial agreement with those of a previous analysis of the role of isotype in opsonization of erythrocytes. As with our study, Ralph et al. found that IgG2a was a potent opsonin but that IgGl and IgG2b also had substantial opsonic activities (23). The main difference between our results and those of Ralph et al. is the relative ability of IgGl and IgG2b to facilitate phagocytosis. A direct comparison of our results with those reported by Ralph et al. is difficult, because their study utilized antibodies of different isotypes that were obtained from fractionated antiserum or hybridoma cell lines rather than a family of isotype-switch variants. Thus, there is no assurance that observed differences were independent of the antibodycombining site. Our results are also in good agreement with those of previous studies that compare the biological activities of families of isotype-switch variants. These studies examined the ability of the antibodies to mediate antibody-directed cell-mediated cytotoxicity. Effector cells that have been examined include peripheral blood mononuclear cells (19), isolated human monocytes (2, 17, 27), and nonadherent K cells (13), while target cells have been sheep erythrocytes (2) or tumor cells (13, 17, 19, 27). A consistent pattern has emerged in all of these studies, in which IgG2a is the most effective facilitator of the interaction between target cells and effector cells. This result is identical with our observation that IgG2a is the most effective opsonin of encapsulated cryptococci. There is no consistent pattern for the relative efficacy of IgGl and IgG2b. Some studies have found IgGl to be a more effective mediator of antibody-directed cellmediated cytotoxicity than IgG2b (2, 17, 27), whereas others have found IgG2b to be more active (13, 19). Such differences may be due to differences in effector cells, levels of activation of the effector cell, target cells, or densities of epitopes on target cells. Our study was initiated in an attempt to develop MAbs with the potential for prophylactic or therapeutic use with cryptococcosis. The rationale for the development of a class-switch family is predicated on the known variation in biological activities of the different IgG subclasses. Our results have shown that IgGl, IgG2a, and IgG2b are all potent opsonins for encapsulated cryptococci, but they differ in their relative ability to opsonize the yeast. We describe
1918
SCHLAGETER AND KOZEL
the prophylactic and therapeutic efficacy of these antibodies in an accompanying paper (25). ACKNOWLEDGMENTS This work was supported by Public Health Service grants A114209 and A124357 from the National Institutes of Health.
LITERATURE CITED 1. Agodoa, L. Y. C., V. J. Gauthier, and M. Mannik. 1988. Precipitating antigen-antibody systems are required for the formation of subepithelial electron-dense immune deposits in rat glomeruli. J. Exp. Med. 158:1259-1271. 2. Boot, J. H. A., M. E. J. Geerts, and L. A. Aarden. 1989. Functional polymorphisms of Fc receptors in human monocytemediated cytotoxicity towards erythrocytes induced by murine isotype switch variants. J. Immunol. 142:1217-1223. 3. Butler, J. E., T. L. Feldbush, P. L. McGivern, and N. Stewart. 1978. The enzyme-linked immunosorbent assay (ELISA): a measure of antibody concentration or affinity? Immunochemistry 15:131-136. 4. Cherniak, R., E. Reiss, M. E. Slodki, R. D. Plattner, and S. 0. Blumer. 1980. Structure and antigenic activity of the capsular polysaccharide of Cryptococcus neoformans serotype A. Mol. Immunol. 17:839-854. 5. Cohn, Z. A., and B. Benson. 1965. The differentiation of mononuclear phagocytes. Morphology, cytochemistry, and biochemistry. J. Exp. Med. 121:153-161. 6. Cosio, F. G., D. J. Birmingham, D. J. Sexton, and L. A. Hebert. 1987. Interactions between precipitating and nonprecipitating antibodies in the formation of immune complexes. J. Immunol. 138:2587-2592. 7. Dangl, J. L., D. R. Parks, V. T. Oi, and L. A. Herzenberg. 1982. Rapid isolation of cloned isotype switch variants using fluorescence activated cell sorting. Cytometry 2:395-401. 8. Diamond, R. D., and J. E. Bennett. 1974. Prognostic factors in cryptococcal meningitis. Ann. Intern. Med. 80:176-181. 9. Dromer, F., J. Charreire, A. Contrepois, C. Carbon, and P. Yeni. 1987. Protection of mice against experimental cryptococcosis by anti-Cryptococcus neoformans monoclonal antibody. Infect. Immun. 55:749-752. 10. Eckert, T. F., and T. R. Kozel. 1987. Production and characterization of monoclonal antibodies specific for Cryptococcus neoformans capsular polysaccharide. Infect. Immun. 55:18951899. 11. Ey, P. L., S. J. Prowse, and C. R. Jenkin. 1978. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-Sepharose. Immunochemistry 15:429-436. 12. Hirayama, N., T. Hirano, G. Kohler, A. Kurata, K. Okumura, and Z. Ovary. 1982. Biological activities of antitrinitrophenyl and antidinitrophenyl mouse monoclonal antibodies. Proc. Natl. Acad. Sci. USA 79:613-615. 13. Kipps, T. J., P. Parham, J. Punt, and L. A. Herzenberg. 1985. Importance of immunoglobulin isotype in human antibodydependent, cell-mediated cytotoxicity directed by murine
INF'ECT. IMMUN.
monoclonal antibodies. J. Exp. Med. 161:1-17. 14. Kozel, T. R., and J. L. Follette. 1981. Opsonization of encapsulated Cryptococcus neoformans by specific anticapsular antibody. Infect. Immun. 31:978-984. 15. Kozel, T. R., and C. A. Hermerath. 1988. Benzoquinone activation of Cryptococcus neoformans capsular polysaccharide for construction of an immunoaffinity column. J. Immunol. Methods 107:53-58. 16. Liesegang, B., A. Radbruch, and K. Rajewsky. 1978. Isolation of myeloma variants with predefined variant surface immunoglobulin by cell sorting. Proc. Natl. Acad. Sci. USA 75:3901-3905. 17. Lubeck, M. D., Y. Kimoto, Z. Steplewski, and H. Koprowski. 1988. Killing of human tumor cell lines by human monocytes and murine monoclonal antibodies. Cell. Immunol. 111:107117. 18. M0ller, N. P. H. 1979. Fc-mediated immune precipitation I. A new role of the Fc-portion of IgG. Immunology 38:631-640. 19. Mujoo, K., T. J. Kipps, H. M. Yang, D. A. Cheresh, U. Wargalla, D. J. Sander, and R. A. Reisfeld. 1989. Functional properties and effect on growth suppression of human neuroblastoma tumors by isotype switch variants of monoclonal antiganglioside GD2 antibody 14.18. Cancer Res. 49:2857-2861. 20. Oi, V. T., T. M. Vuong, R. Hardy, J. Reidler, J. Dangl. L. A. Herzenberg, and L. Stryer. 1984. Correlation between segmental flexibility and effector function of antibodies. Nature (London) 307:136-140. 21. Preud'homme, J. L., B. K. Birshtein, and M. D. Scharff. 1975. Variants of a mouse myeloma cell line that synthesize immunoglobulin heavy chains having an altered serotype. Proc. Natl. Acad. Sci. USA 72:1427-1430. 22. Radbruch, A., B. Liesegang, and K. Rajewsky. 1980. Isolation of variants of mouse myeloma X63 that express changed immunoglobulin class. Proc. Natl. Acad. Sci. USA 77:2909-2913. 23. Ralph, P., I. Nakoinz, B. Diamond, and D. Yelton. 1980. All classes of murine IgG antibody mediate macrophage phagocytosis and lysis of erythrocytes. J. Immunol. 125:1885-1888. 24. Rodwell, J. D., L. H. Tang, and V. N. Schumaker. 1980. Antigen valence and Fc-localized secondary forces in antibody precipitation. Mol. Immunol. 17:1591-1597. 25. Sanford, J. E., D. M. Lupan, A. M. Schlageter, aind T. R. Kozel. 1990. Passive immunization against Cryptococcus neoformans with an isotype-switch family of monoclonal antibodies reactive with cryptococcal polysaccharide. Infect. Immun. 58:19191923. 26. Spira, G., A. Bargellesi, J. L. Teillaud, and M. D. Scharif. 1984. The identification of monoclonal class switch variants by sib selection and an ELISA assay. J. Immunol. Methods 74: 307-315. 27. Steplewski, Z., G. Spira, M. Blaszczyk, M. D. Lubeck, A. Radbruch, H. Illges, D. Herlyn, K. Rajewsky, and M. Scharff. 1985. Isolation and characterization of anti-monosialoganglioside monoclonal antibody 19-9 class-switch variants. Proc. Natl. Acad. Sci. USA 82:8653-8657. 28. Vieira, P., and K. Rajewsky. 1988. The half-lives of serum immunoglobulins in adult mice. Eur. J. Immunol. 18:313-316. 29. Wright, S. D. 1986. Methods for the study of receptor-mediated phagocytosis. Methods Enzymol. 132:204-221.
