Fc-dependent and Fc-independent opsonization of Cryptococcus neoformans by anticapsular monoclonal antibodies: importance of epitope specificity

Abstract

Monoclonal antibodies (MAbs) reactive with glucuronoxylomannan (GXM), the major capsular polysaccharide of the yeast Cryptococcus neoformans, produce distinct capsular reactions when viewed by differential interference contrast microscopy. These reactions depend on the epitope specificity of the antibody. Opsonic activities of immunoglobulin G1 (IgG1) MAbs that produce patterns termed rim and puffy were examined. Rim-pattern MAbs are reactive with an epitope shared by GXM serotypes A, B, C, and D. Puffy-pattern MAbs are reactive only with serotypes A and D. In phagocytosis assays, using serotype A cells and resident murine peritoneal macrophages, rim-pattern MAbs were markedly more opsonic than puffy-pattern MAbs. F(ab')(2) fragments of rim-pattern MAbs were synergistic with heat-labile factors in normal human serum for opsonization of the yeast. F(ab')(2) fragments of puffy-pattern MAbs were also synergistic with normal serum in opsonization but at a much lower level than fragments of rim-pattern MAbs. Normal serum alone was not opsonic. F(ab')(2) fragments of rim-pattern MAbs, but not puffy-pattern MAbs, stimulated phagocytosis of encapsulated cryptococci in the absence of serum. This serum-independent opsonic action of F(ab')(2) fragments was abrogated by pretreatment of macrophages with purified GXM, suggesting the involvement of a phagocyte GXM receptor. The results indicate that (i) there are multiple mechanisms by which anticapsular IgG MAbs facilitate phagocytosis of encapsulated cryptococci, (ii) some anti-GXM antibodies are opsonic in an Fc-independent manner, and (iii) opsonic activity correlates with the capsular reaction and occurs in an epitope-specific manner.

FIG. 1.

Capsular binding patterns produced by anti-GXM MAbs. MAbs 3C2, 471, 1326, and 302 [IgG, F(ab′)₂, and Fab; 50 μg/ml] incubated with CN6 serotype A yeast cells produce two distinct capsular binding patterns, rim and puffy. Bivalent molecules of MAbs 3C2 and 471 [IgG and F(ab′)₂] produce the rim pattern while the Fab fragments produce a form of the puffy pattern. MAbs 1326 and 302 produce variations of the puffy pattern with all antibody molecules. Images were acquired with a 100× oil immersion objective and DIC optics.

FIG. 2.

Phagocytosis of encapsulated cryptococci treated with MAbs (50 μg/ml) that produce rim (MAbs 3C2 and 471) and puffy (MAbs 1326 and 302) capsular reactions. Serotype A cryptococci were treated with (i) IgG alone (ii) IgG and NHS, or (iii) IgG and heat-inactivated NHS (ΔNHS). Data are reported as the mean phagocytic index (number of ingested yeast cells per macrophage) from three independent experiments ± SEM.

FIG. 3.

Phagocytosis of cryptococci treated with intact IgG MAbs or the F(ab′)₂ fragments of MAbs (50 μg/ml) that produce rim (MAbs 3C2 and 471) or puffy (MAbs 1326 and 302) capsular reactions. Cryptococci were treated with intact IgG or F(ab′)₂ alone or in combination with NHS. Data are reported as the mean phagocytic index from three independent experiments ± SEM.

FIG. 4.

Inhibition of F(ab′)₂-mediated phagocytosis by soluble GXM. GXM (20 or 100 μg in 150 μl of medium), was added to macrophages and left for 5 min prior to addition of cryptococci opsonized with F(ab′)₂ fragments of MAbs that produce the rim capsular reaction. Data are reported as the mean phagocytic index from three independent experiments ± SEM.

FIG. 5.

Inhibition of Fc- and C3-mediated phagocytosis by soluble GXM. GXM (100 μg in 150 μl of medium) was added to macrophages 5 min before addition of cryptococci treated with (i) intact MAb 3C2, (ii) F(ab′)₂ fragments of MAb 3C2, or (iii) both F(ab′)₂ fragments of MAb 3C2 and NHS. Data are reported as the mean phagocytic index from three independent experiments ± SEM.

FIG. 6.

Effect of C. albicans mannan on Fc-independent opsonization of encapsulated cryptococci. Macrophages were untreated or preincubated for 5 min with Candida mannan (100 μg) or GXM (100 μg) prior to the addition of cryptococci opsonized with F(ab′)₂ fragments of MAb 3C2. Data are reported as the mean phagocytic index from three independent experiments ± SEM.