Characterization of anticapsular monoclonal antibodies that regulate activation of the complement system by the Cryptococcus neoformans capsule

Abstract

Incubation of the encapsulated yeast Cryptococcus neoformans in human serum leads to alternative pathway-mediated deposition of C3 fragments in the capsule. We examined the ability of monoclonal antibodies (MAbs) specific for different epitopes of the major capsular polysaccharide to alter the kinetics for classical and alternative pathway-mediated deposition of C3 onto a serotype A strain. We studied MAbs reactive with capsular serotypes A, B, C, and D (MAb group II); serotypes A, B, and D (MAb group III); and serotypes A and D (MAb group IV). The MAb groupings are based on antibody variable region usage which determines the antibody molecular structure. When both the classical and alternative pathways were operative, group II MAbs induced early classical pathway-mediated binding of C3 but reduced the overall rate of C3 accumulation and the amount of bound C3. Group III MAbs closely mimicked the effects of group II MAbs but exhibited reduced support of early classical pathway-facilitated accumulation of C3. Depending on the antibody isotype, group IV MAbs slightly or markedly enhanced early binding of C3 but had no effect on either the rate of C3 accumulation or the amount of bound C3. When the classical pathway was blocked, group II and III MAbs markedly suppressed C3 binding that normally would have occurred via the alternative pathway. In contrast, MAbs of group IV had no effect on alternative pathway-mediated C3 binding. These results indicate that anticapsular antibodies with different epitope specificities may have distinct regulatory effects on activation and binding of C3.

FIG. 1

Kinetics of activation and binding of C3 fragments to C. neoformans cells incubated with 40% NHS in the presence or absence of anti-GXM MAbs. The indicated antibody concentrations are micrograms of MAb per milliliter of reaction mixture volume. Binding of C3 fragments was determined by incorporation of trace amounts of ¹²⁵I-labeled C3 into the reaction mixture.

FIG. 2

Immunofluorescence analysis of the sites for binding of C3 to C. neoformans cells incubated for 2, 4, 8, and 16 min with 40% NHS in the presence (50 μg/ml) or absence of anti-GXM MAbs. Sites of C3 deposition were determined by use of FITC-labeled antiserum to C3. All images were collected under identical conditions of image acquisition, including the number of image integrations (five) and camera gain (−3 db), with the exception of selected (∗) cells incubated with NHS for 2 min, in which case the number of image integrations was increased to 20. The fluorescence found with some samples was so intense that digital deconvolution of the images could not completely remove haze found in the center of the cell, e.g., cells incubated with NHS in the presence of MAb 386.

FIG. 3

Kinetics of activation and binding of C3 fragments to C. neoformans cells incubated with 40% NHS in the absence of anti-GXM MAbs or in the presence of an isotype switch (IgG1→IgG2b→IgG2a) family of MAbs derived from MAb 439 (top row) and MAb 471 (bottom row). The indicated antibody concentrations are micrograms of MAb per milliliter of reaction mixture volume. Binding of C3 fragments was determined by incorporation of trace amounts of ¹²⁵I-labeled C3 into the reaction mixture.

FIG. 4

Kinetics of alternative pathway-mediated activation and binding of C3 fragments to C. neoformans cells incubated with 40% NHS containing 10 mM Mg-EGTA in the presence (50 μg/ml) or absence of anti-GXM MAbs. Binding of C3 fragments was determined by incorporation of trace amounts of ¹²⁵I-labeled C3 into the reaction mixture.

FIG. 5

Immunofluorescence analysis of the sites for binding of C3 to C. neoformans cells incubated for 2, 4, 8, and 16 min with 40% NHS containing 10 mM Mg-EGTA in the presence (50 μg/ml) or absence of anti-GXM MAbs. Sites of C3 deposition were determined by use of FITC-labeled antiserum to C3. Unless otherwise indicated, images were collected under identical conditions of image acquisition, including the number of image integrations (five) and camera gain (−3 db). ∗, 20 image integrations; ∗∗, 50 image integrations. As in the case with images shown in Fig. 2, the fluorescence found with some samples was so intense that digital deconvolution of the images could not completely remove haze found in the center of the cell.

FIG. 6

Stereoscopic images of sites for binding of C3 to C. neoformans cells incubated for 4 min with 40% NHS containing 10 mM Mg-EGTA in the presence (50 μg/ml) or absence of anti-GXM MAb 3C2. Sites of C3 deposition were determined by use of Oregon Green 514-labeled antiserum to C3. Conditions for collection of the immunofluorescence image were optimized for the intensity of fluorescence exhibited by each cell type (NHS-Mg-EGTA, 10 image integrations and camera gain of 0 db; NHS-Mg-EGTA plus MAb 3C2, 30 image integrations and camera gain of 9 db).