Stuart M. Levitz

Dendritic cells (DC) have been shown to phagocytose and killCryptococcus neoformansin vitro and are believed to be important for inducing protective immunity against this organism. Exposure toC. neoformansoccurs mainly by inhalation, and in this study we examined the in vivo interactions ofC. neoformanswith DC in the lung. Fluorescently labeled liveC. neoformans and heat-killedC. neoformans were administered intranasally to C57BL/6 mice. At specific times postinoculation, mice were sacrificed, and lungs were removed. Single-cell suspensions of lung cells were prepared, stained, and analyzed by microscopy and flow cytometry. Within 2 h postinoculation, fluorescently labeledC. neoformanshad been internalized by DC, macrophages, and neutrophils in the mouse lung. Additionally, lung DC from mice infected for 7 days showed increased expression of the maturation markers CD80, CD86, and major histocompatibility complex class II. Finally, ex vivo incubation of lung DC from infected mice withCryptococcus-specific T cells resulted in increased interleukin-2 production compared to the production by DC from naïve mice, suggesting that there was antigen-specific T-cell activation. This study demonstrated that DC in the lung are capable of phagocytosingCryptococcusin vivo and presenting antigen toC. neoformans-specific T cells ex vivo, suggesting that these cells have roles in innate and adaptive pulmonary defenses against cryptococcosis.

T-cell-mediated immunity is necessary for effective host defenses against infections caused by Cryptococcus neoformans. Clinical and experimental stu

Cryptococcus neoformanscapsular glucuronoxylomannan (GXM) is shed during cryptococcosis and taken up by macrophages. The roles of the putative GXM receptors CD14, CD18, Toll-like receptor 2 (TLR2), and TLR4 in GXM clearance from serum and deposition in the liver and spleen in receptor-deficient mice were studied. While alterations in the kinetics of GXM redistribution were seen in the mutant mice, none of the receptors was absolutely required for serum clearance or hepatosplenic accumulation.

Summary: During the past two decades, invasive fungal infections have emerged as a major threat to immunocompromised hosts. Patients with neoplastic diseases are at significant risk for such infections as a result of their underlying illness and its therapy. Aspergillus, Candida, Cryptococcus and emerging pathogens, such as the zygomycetes, dark walled fungi, Trichosporon and Fusarium, are largely opportunists, causing infection when host defences are breached. The immune response varies with respect to the fungal species and morphotype encountered. The risk for particular infections differs, depending upon which aspect of immunity is impaired. This article reviews the current understanding of the role and relative importance of innate and adaptive immunity to common and emerging fungal pathogens. An understanding of the host response to these organisms is important in decisions regarding use of currently available antifungal therapies and in the design of new therapeutic modalities.

The encapsulated pathogenic yeastCryptococcus neoformansis poorly recognized by phagocytic cells in the absence of opsonins. Macrophages will bind and internalize complement- or antibody-opsonizedC. neoformans; however, less is known about the role of opsonins in dendritic cell (DC)-mediated recognition of the organism. Thus, we studied the opsonic requirements for binding toC. neoformansby cultured human monocyte-derived and murine bone marrow-derived DCs and whether binding leads to antifungal activity and cytokine release. Binding of unopsonizedC. neoformansto human and murine DCs was negligible. Opsonization with pooled human serum (PHS) increased binding, while heat treatment of PHS virtually abolished this binding, thus suggesting a role for heat-labile complement components. PHS plus a monoclonal anticapsular antibody, 3C2, had an additive effect on binding for most cryptococcal strains. Human and murine DCs exhibited pronounced anticryptococcal activity in the presence of the antibody at early (2-h) and late (24-h) time points; however, PHS opsonization did not supplement this anticryptococcal activity. Antifungal activity againstC. neoformansopsonized in PHS and/or antibody was partially reduced in the presence of inhibitors of the respiratory burst response. Human, but not murine, DCs released modest amounts of tumor necrosis factor alpha when stimulated withC. neoformansopsonized in PHS and/or antibody. However, opsonizedC. neoformansfailed to stimulate detectable release of interleukin 10 (IL-10) or IL-12p70 from either DC population. Thus, human and murine DCs show maximal binding to and antifungal activity againstC. neoformansvia a process highly dependent on opsonization.

The major capsular polysaccharide ofCryptococcus neoformans, glucuronoxylomannan (GXM), is recognized by Toll-like receptor 2 (TLR2), TLR4, and CD14. In these studies, mice deficient in CD14, TLR2, TLR4, and the TLR-associated adaptor protein, MyD88, were utilized to investigate the contribution of TLRs and CD14 to in vivo host defenses againstC. neoformans. MyD88^−/−mice had significantly reduced survival compared with wild-type C57BL/6 mice after intranasal (i.n.) and intravenous (i.v.) infection with liveC. neoformans. CD14^−/−mice had reduced survival when infected i.v., while TLR2^−/−mice died significantly earlier after i.n. infection. Mortality was similar comparing TLR4 mutant C3H/HeJ mice and control C3H/HeOuJ mice following i.v. or i.n. challenge withC. neoformans. The course of pulmonary cryptococcosis was studied in more detail in the CD14^−/−, TLR2^−/−, and MyD88^−/−mice. MyD88^−/−mice infected i.n. had higher numbers of CFU in the lungs as well as higher GXM levels in the sera and lungs 7 days after infection than wild-type mice did. Surprisingly, there were no major differences in the levels of tumor necrosis factor alpha, interleukin-4 (IL-4), IL-10, IL-12p70, or gamma interferon in the lungs ofC. neoformans-infected knockout mice compared with wild-type mice. Histopathologic analysis of the lungs on day 7 postinfection revealed minimal inflammation in all mouse groups. These studies demonstrate a major role for MyD88 and relatively minor roles for CD14 and TLR2 in the response to cryptococcal infection, with the decreased survival of MyD88^−/−mice correlating with increased numbers of lung CFU and serum and lung GXM levels.

Infections due to the encapsulated fungusCryptococcus neoformansare a significant cause of morbidity and mortality in patients with impaired T-cell function, particularly those with AIDS. Presumably then, T-cell responses to cryptococcal antigens are critical for protection against this ubiquitous fungus. To test the protective efficacy of these antigens as vaccine candidates, secreted cryptococcal antigens were separated by concanavalin A affinity chromatography into adherent (mannoprotein [MP]) and nonadherent (flowthrough [FT]) fractions, and the fractions were tested in murine models of disseminated cryptococcosis. Compared with adjuvant alone, C57BL/6 mice that received two inoculations of MP and FT exhibited prolonged survival and reduced brain and kidney fungal loads following intravenous challenge withC. neoformansstrain B3501. MP-immunized animals had increased brain levels of tumor necrosis factor alpha, gamma interferon, and interleukin-2. Histopathologic examination revealed that compared with organs from mice that received only adjuvant, MP-immunized mice were able to recruit a stronger cellular infiltrate in brain, kidney, and liver in response to cryptococcal infection. Conjugated O-linked glycans were necessary for optimal MP-mediated protection, because chemical O deglycosylation reduced the protective efficacy of MP immunization. FT and MP immunization protected B-cell-deficient, but not T-cell-deficient mice, suggesting that protection was T-cell mediated. CBA/J mice also benefited from immunization with FT and MP, although the benefits were more modest than those seen with C57BL/6 mice. Thus, both MP and FT fractions ofC. neoformanscontain components that protect mice from disseminated cryptococcosis, and this protection appears to be T-cell mediated.

Although T-cell responses are known to be critical for effective host defenses against the fungal pathogenCryptococcus neoformans, the antigens that stimulate protective responses are poorly characterized but are thought to be comprised, at least in part, of mannoproteins. Recently, we created a panel of murine CD4⁺-T-cell hybridomas that react withC. neoformansantigens. A mannoprotein antigen, MP98, that stimulated one of the hybridomas was purified, and the gene encoding MP98 was cloned. In the present study, the cryptococcal antigen, MP88, that stimulated a second T-cell hybridoma, X5A3, to secrete interleukin-2 was characterized. MP88 was purified from supernatants of glass bead-disruptedC. neoformansby anion-exchange and hydrophobic interaction chromatography. A single band with an apparent molecular mass of 88 kDa was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to partial internal amino acid sequencing. The gene encoding MP88 was cloned and sequenced. MP88 features a C-terminal serine/threonine-rich region, which presumably serves as a site for extensive O glycosylation, followed by a putative glycosylphosphatidylinositol anchor site. A search ofC. neoformansgenomic databases revealed thatMP88shares this feature with at least 11 other genes, includingMP98. The mannoprotein nature of MP88 was established based upon the capacity of (i) the mannoprotein fraction ofC. neoformanssupernatants to stimulate X5A3 and (ii) mannosylated ligands to competitively inhibit this stimulation. Thus, a second cryptococcal mannoprotein has been identified which stimulates T-cell responses and is a vaccine candidate.