Host defense in cryptococcosis. I. An in vivo model for evaluating immune response.

An inbred mouse model was used to evaluate in vivo host immune response to Cryptococcus neoformans. Within 1 week of immunization, mice developed delayed type hypersensitivity reactions (DTH) to cryptococcal extracts derived from either culture filtrates or disrupted cells. There was no significant cross reactivity with extracts of other fungi. Previous immunization provided considerable protection against subsequent challenge with multiple strains of cryptococci. DTH also developed after nonimmunized mice were challenged with C. neoformans; however, in this situation DTH was not associated with prolonged survival. These studies indicate that mice can be immunized and protected against cryptococcosis and that this protection is associated with acquisition of DTH to cryptococcal antigens.     

Polysaccharide antigens of the capsule of Cryptococcus neoformans.

The major significance of the capsular polysaccharide of C. neoformans is its role in potentiating opportunistic infections by the yeast. It has the ability to exert a broad spectrum of influences on the immune response, from activation of phagocytic cells and complement components of the alternative pathway, to the induction of specific antibody, T-suppressor cells, DTH responses, and cytokines (51). These biological properties along with the serotype specificities are all determined by the physical properties and chemical structures of the polysaccharide antigens that compose the capsule. There is evidence not only for an association of lethal infections with serotype A in patients with advanced AIDS (34, 56), but also for a role for the capsule in directly influencing the infection of CD4+ cells by HIV (57). Together, these phenomena raise intriguing questions about the possible connection between the chemistry of these capsular antigens and cryptococcal infections in AIDS patients. One speculation is that AIDS creates the optimal physiological conditions for the establishment and spread of cryptococcosis. It has been observed that during the progression of AIDS there is a shift towards a T-2 response (14). This could lead to conditions that would inhibit the cellular immune responses that block dissemination of cryptococcal infections. Thus, an important consideration in the application of vaccine or immune modulation therapies in the treatment of cryptococcosis in AIDS victims would be the design of vaccines that could boost the T-1 immune response. It has been shown that the form and dose of an antigenic challenge can influence the induction of a T-1 or T-2 immune response (61). Recently, Murphy has reported that gamma interferon and interleukin 2 are up-regulated in the spleens of mice that produce anticryptococcal TDH and TAMP cells in response to immunogenic doses of cryptococcal culture filtrate antigen given with Freund's complete adjuvant (49). Perhaps purified cryptococcal antigens (e.g., MP) conjugated to an appropriate carrier or adjuvant could be used in therapeutic strategies to limit cryptococcosis in immunocompromised individuals. Future investigations of virulence and pathogenicity in the context of defined polysaccharide antigens from encapsulated strains of C. neoformans will contribute to a better understanding of the regulation of cryptococcal infection and immunity at the cellular and molecular levels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Clearance of Cryptococcus neoformans from immunologically suppressed mice.

To assess the effects of cryptococcal antigen-induced immunosuppression on a Cryptococcus neoformans infection, CBA/J mice were injected intravenously with saline or suppressive doses of cryptococcal antigen (CneF) at weekly intervals and were then infected with viable C. neoformans cells. By the second week after infection, the cryptococcal antigen-injected mice had suppressed anticryptococcal delayed-type hypersensitivity (DTH) responses compared with the responses of the saline-treated, infected control mice. In addition, the immunosuppressed mice had higher numbers of cryptococcal CFU cultured from their lungs, livers, spleens, lymph nodes, and brains than did the control animals. A direct correlation of suppression of the anticryptococcal DTH response and reduced clearance of cryptococci from tissues was also observed after mice were given a single intravenous injection of CneF and infected. To determine whether or not the cryptococcal antigen was specifically reducing the clearance of C. neoformans or had a more generalized effect, mice were injected with saline or suppressive doses of CneF, infected with Listeria monocytogenes, and then followed daily for 7 days for the clearance of L. monocytogenes from spleens and on day 7 for DTH reactivity to Listeria antigen. There were no differences between the saline- and CneF-treated mice with respect to anti-Listeria DTH responses or clearance of L. monocytogenes from spleens, indicating that CneF was not altering natural resistance mechanisms responsible for early clearance of L. monocytogenes, nor was the CneF influencing the induction of the acquired immune response which was responsible for the late clearance of the bacteria. Together, these data indicate that the specific suppression of this cell-mediated immune response induced by cryptococcal antigen reduces the ability of the animals to eliminate the homologous organism (C. neoformans) but not a heterologous infectious agent, such as L. monocytogenes.     

CTLA-4 down-regulates the protective anticryptococcal cell-mediated immune response

Cell-mediated immune (CMI) responses defined by delayed-type hypersensitivity (DTH) reactivity to cryptococcal culture filtrate antigen (CneF) can be either protective or nonprotective against an infection with Cryptococcus neoformans. The protective and nonprotective anticryptococcal DTH responses are induced by different immunogens and have differing activated-T-cell profiles. This study examined the effects of blockade of the interaction between cytotoxic T lymphocyte antigen 4 (CTLA-4) and its ligands B7-1 (CD80) and B7-2 (CD86) on the anticryptococcal DTH responses and protection. We found that CTLA-4 blockade at the time of immunization with the immunogen that induces the protective response, CneF, in complete Freund's adjuvant (CFA) or the immunogen that induces the nonprotective response, heat-killed cryptococcal cells (HKC), enhanced anticryptococcal DTH reactivity. In contrast, blocking CTLA-4 after the immune response was induced failed to enhance responses. Blockade of CTLA-4 in an infection model resulted in earlier development of the anticryptococcal CMI response than in control mice. Concomitant with increases in DTH reactivity in mice treated with anti-CTLA-4 Fab fragments at the time of immunization, there were decreases in cryptococcal CFU in lungs, spleens, and brains compared to controls. Blockade of CTLA-4 resulted in long-term protection, as measured by significantly increased survival times, only in mice given the protective immunogen, CneF-CFA. Anti-CTLA-4 treatment did not shift the response induced by the nonprotective immunogen, HKC, to a long-term protective one. Our data indicate that blockade of CTLA-4 interactions with its ligands may be useful in enhancing host defenses against C. neoformans.     

Characterization of a cell population which amplifies the anticryptococcal delayed-type hypersensitivity response.

Cell-mediated immunity to Cryptococcus neoformans can be detected by delayed-type hypersensitivity (DTH) to a culture filtrate antigen of C. neoformans. Recently, we have identified a population of cells in spleens of mice immunized with cryptococcal antigen that, when transferred to recipient mice at the time of immunization, amplifies the anticryptococcal DTH response. If the cell donor mice are treated with cyclosporin A during induction of the anticryptococcal DTH response, the amplifier cells are not induced, whereas the cells which transfer DTH (TDH cells) are induced. The purpose of this study was to characterize the amplifier cells with respect to their surface and functional properties and, in so doing, determine whether or not the amplifier cells are analogous to long-lived memory cells. We demonstrated that the amplifier cells were nylon-wool-nonadherent, antigen-specific, CD4 (L3T4+ Lyt-2-) T lymphocytes which appear in the spleens of mice 5 days postimmunization with cryptococcal culture filtrate antigen in complete Freund adjuvant. The amplifier T (Tamp) cells are not considered to be memory cells because they are relatively short-lived, being present 14 but not 18 days after the stimulating immunization. Moreover, the amplified anticryptococcal DTH response does not fulfill the criteria of the typical secondary immune (anamnestic) response in that the amplified response does not appear early relative to the appearance of the primary anticryptococcal DTH response, and it does not persist longer than the primary DTH response. We speculate that Tamp cells are not long-lived memory cells but rather act in a T-helper cell capacity to amplify the anticryptococcal DTH response.     

Roles for CD40, B7 and major histocompatibility complex in induction of enhanced immunity by cryptococcal polysaccharide-pulsed antigen-presenting cells

Immunization of mice with activated antigen-presenting cells (APC) pulsed ex vivo with cryptococcal capsular polysaccharide, a glucuronoxylomannan (GXM-APC) results in prolongation of survival and delayed-type hypersensitivity (DTH) responsiveness following infection with Cryptococcus neoformans (NU-2). GXM-APC has both non-specific and GXM-specific effects that influence the immune responses that develop in mice after infection with NU-2. Type 1 cytokine responses are augmented after immunization with APC alone, while GXM must be present for the vaccine to influence survival and DTH reactions. This investigation evaluated the role that major histocompatibility complex (MHC) and co-stimulatory molecules play in the non-specific and GXM-specific responses induced by GXM-APC. APC from CD40 knockout mice were as effective as wild-type APC for the induction of non-specific and GXM-specific responses. Blocking activity of B7-1 and B7-2 by treatment of immunized mice with monoclonal antibodies specific for these molecules just before and for 6 days following GXM-APC immunization decreased the splenic interferon-gamma response of mice subsequently infected with NU-2, but only in mice that were treated with both antibodies. These antibody treatments had no effect on DTH reactivity in similarly treated animals. MHC class I molecules were not involved in the antigen non-specific or GXM-specific activities of the vaccine. MHC class II molecules were not required for augmentation of type 1 cytokine responses but were needed for induction of the GXM-specific response that regulates the expression of DTH reactivity. This investigation has shown that an MHC class II-restricted, GXM-specific response is responsible for altering DTH responsiveness which is the correlate of immunity in this model.     

Presentation of cryptococcal capsular polysaccharide (GXM) on activated antigen-presenting cells inhibits the T-suppressor response and enhances delayed-type hypersensitivity and survival.

A hallmark of infection with Cryptococcus neoformans is depression of the immune system characterized by poor inflammatory responses and loss of delayed-type hypersensitivity (DTH) and antibody responses. T-suppressor cell (Ts) responses, elicited by the capsular polysaccharide (GXM) of the organism, are known to develop during infection. This study was undertaken to develop a method to inhibit the anti-GXM Ts response and thereby study the influence of the Ts response on immune responsiveness and survival in cryptococcosis. Antigen-presenting cells (APC), elicited with complete Freund's adjuvant (CFA), were treated in vitro with GXM (GXM-APC). The GXM-APC were injected intravenously into normal mice. These mice were resistant to induction of anti-GXM Ts cells when soluble GXM was administered in tolerogenic doses or when animals were infected with C. neoformans. Inhibition of the anti-GXM Ts response was specific to GXM as levan-APC did not inhibit induction of anti-GXM Ts cells. Inhibition of the anti-GXM Ts response could not be attributed to increased clearance of GXM due to induction of anti-GXM antibodies or other mechanisms. Anti-cryptococcal DTH responses were lost in mice by the second week of infection. However, treatment with GXM-APC, but not levan-APC, allowed mice to maintain their DTH response. GXM-APC pretreatment enhanced survival of infected mice compared with mice pretreated with levan-APC. These results show that GXM-APC induces immune responses that inhibit the induction of Ts responses and enhances DTH responses in infected mice. These responses correlate with enhanced survival after cryptococcal infection.     

Effects of immunization with Cryptococcus neoformans cells or cryptococcal culture filtrate antigen on direct anticryptococcal activities of murine T lymphocytes.

Immunizing CBA/J mice with intact Cryptococcus neoformans cells or with a cryptococcal culture filtrate antigen (CneF) induces an anticryptococcal delayed-type hypersensitivity response. Recently, it has been shown that two phenotypically different T-cell populations are responsible for delayed-type hypersensitivity reactivity in mice immunized with intact cryptococcal cells, whereas only one of those populations is present in mice immunized with soluble cryptococcal antigens in complete Freund's adjuvant (CFA). The purpose of this study was to determine if differences occur with regard to direct anticryptococcal activity between T-lymphocyte-enriched populations from mice immunized with intact viable or dead cryptococcal cells and similar cell populations from mice immunized with the soluble cryptococcal culture filtrate antigen, CneF, emulsified in CFA. The percentage of lymphocytes which form conjugates with C. neoformans and the percentage of cryptococcal growth inhibition in vitro are greater with T-lymphocyte-enriched populations from mice sublethally infected with C. neoformans or from mice immunized with intact heat-killed cryptococcal cells in the presence or absence of CFA than with lymphocyte populations from mice immunized with CneF-CFA. Enhanced anticryptococcal activity of T lymphocytes could be induced by immunizing mice with heat-killed C. neoformans cells of serotype A, B, C, or D as well as by immunizing with a similar preparation of an acapsular C. neoformans mutant but not by immunizing with CFA emulsified with CneF prepared from any one of the C. neoformans isolates. These data indicate that the soluble cryptococcal culture filtrate antigens do not induce the same array of functional T lymphocytes as whole cryptococcal cells.     

Transfer of immunity to cryptococcosis by T-enriched splenic lymphocytes from Cryptococcus neoformans-sensitized mice.

Splenic enriched T-cells and sera were obtained from inbred CBA/J mice injected 7 or 35 days earlier with either 10(3) viable Cryptococcus neoformans or sterile physiological saline. The transfer of enriched T-cells collected 7 days after immunization or of normal enriched T-cells did not transfer immunity to C. neoformans or delayed-type hypersensitivity responsiveness to cryptococcal culture filtrate (CneF) antigen to the recipients. However, enriched T-cells harvested 35 days after immunization, when transferred to recipient mice, were able to confer immunity as indicated by the reduction in numbers of C. neoformans cells in the tissues, and they also transferred delayed-type hypersensitivity responsiveness to CneF antigens. Sera from either sensitized or normal mice were unable to transfer immunity to recipient animals. These results suggested that there was a time requirement for development of the immune response in the donor mice and that T-cells were crucial in the host defense against a cryptococcal infection. Culturing of day-35 C. neoformans-sensitized T-cells in the presence of homologous antigen (CneF) but not in the presence of heterologous antigen (purified protein derivative or 2, 4-dinitro-1-fluorobenzene) induced the production of migration inhibition factor, thus indicating that lymphocytes from C. neoformans-injected mice were specifically sensitized to CneF antigen.     

Protection against cryptococcosis by using a murine gamma interferon-producing Cryptococcus neoformans strain

We evaluated cell-mediated immune (CMI) responses in mice given a pulmonary infection with a Cryptococcus neoformans strain engineered to produce the Th1-type cytokine gamma interferon (IFN-gamma). Mice given a pulmonary infection with an IFN-gamma-producing C. neoformans strain were able to resolve the primary infection and demonstrated complete (100%) protection against a second pulmonary challenge with a pathogenic C. neoformans strain. Pulmonary cytokine analyses showed that Th1-type/proinflammatory cytokine and chemokine expression were significantly higher and Th2-type cytokine expression was significantly lower in mice infected with the IFN-gamma-producing C. neoformans strain compared to wild-type-infected mice. This increased pulmonary Th1-type cytokine expression was also associated with significantly lower pulmonary fungal burden and significantly higher pulmonary leukocyte and T-lymphocyte recruitment in mice infected with the IFN-gamma-producing C. neoformans strain compared to wild-type-infected mice. Our results demonstrate that pulmonary infection of mice with a C. neoformans strain expressing IFN-gamma results in the stimulation of local Th1-type anti-cryptococcal CMI responses and the development of protective host immunity against future pulmonary cryptococcal infections. The use of fungi engineered to produce host cytokines is a novel method to study immune responses to infection and may be useful in developing vaccine strategies in humans.     

Age-related resistance of C57BL/6 mice to Cryptococcus neoformans is dependent on maturation of NKT cells

Conflicting results have been reported regarding the ability of C57BL/6 mice to clear infections due to Cryptococcus neoformans. Examination of the various experimental protocols used suggested that C57BL/6 mice might develop the ability to resist infection as they mature. We analyzed the ability of C57BL/6 mice of different ages to respond to immunization with cryptococcal antigen or to clear a cryptococcal infection. Mice were immunized with a soluble cryptococcal culture filtrate antigen (CneF) emulsified in complete Freund's adjuvant (CneF-CFA). Delayed-type hypersensitivity (DTH) reactions elicited by the immunization were significantly stronger in 15-week-old C57BL/6 mice than in 7-week-old mice. Analysis of cryptococcal CFU 8 weeks following intratracheal infection of 7-week-old mice or 15-week-old mice revealed a relative inability of the younger animals to control the infection. Six-week-old immunized and infected mice cleared cryptococci from brain, spleen, and liver in a manner similar to that of immunized and infected 15-week-old mice. However, the older mice cleared cryptococci much more efficiently from the lungs. The possible role for NKT cells was determined by passive transfer of thymocytes from 10-week-old mice (containing mature NKT cells) or 2-week-old mice (containing immature NKT cells) to 6-week-old mice. The 10-week-old thymocytes significantly enhanced the ability of the mice to develop a DTH response after immunization with CneF-CFA, while animals treated with 2-week-old thymocytes did not improve their DTH response after immunization. The cells in the 10-week-old thymocyte population responsible for improvement of DTH responses were identified as being NK1.1 positive.     

Induction of antigen-specific suppression by circulating Cryptococcus neoformans antigen.

Immunoaffinity chromatography of sera from mice infected with Cryptococcus neoformans (Inf-MS) on a column with rabbit anti-cryptococcal antibody as the ligand resulted in the adsorption of the component(s) that induce suppression of the cryptococcal delayed-type hypersensitivity (DTH) response. In contrast, immunoaffinity chromatography of Inf-MS on columns coupled with cryptococcal antigen or goat anti-mouse IgM, IgG, and IgA did not adsorb the suppressive component(s). Quantification of cryptococcal antigen and anti-cryptococcal antibody in Inf-MS and column fractions established a direct correlation between cryptococcal antigen levels and suppressive activity; no correlation was observed between anti-cryptococcal antibody levels and suppressive activity. The suppression induced by Inf-MS was shown to be specific in that suppressive sera did not affect the induction of DTH responses to Listeria monocytogenes or dinitrofluorobenzene. These collective results provide evidence that cryptococcal antigen is the component in Inf-MS that induces antigen-specific suppression of the cell-mediated immune response to C. neoformans.     

Cellular Immunity in a Cutaneous Model of Cryptococcosis

The temporal development of cellular immune responses in mice inoculated cutaneously with viable Cryptococcus neoformans 145 was determined in vivo and in vitro by comparing several antigen preparations for their efficacy in the assays selected. Three antigens derived from C. neoformans 145, viz., a culture filtrate preparation (CneF-145), a membrane extract (B-HEX), and soluble cytoplasmic substances (SCS), were compared for their ability to detect delayed hypersensitivity (DH) in vivo in a footpad assay or to stimulate lymphocytes in vitro in a thymidine incorporation assay. DH to B-HEX could be demonstrated as early as 1 week after infection, whereas significant responses to SCS and CneF-145 were not regularly detected until 3 weeks after infection. Substantial reactions were observed to all three antigens up to 12 weeks, although they peaked at 2 to 3 weeks. Reactions to B-HEX and SCS were somewhat better than those to CneF. Differences in the efficacies of the three antigens were not obvious after the sixth week of infection, however. In vitro, lymph node cells from infected animals were stimulated significantly with all three antigens beginning at week 1. As with DH, however, responses to CneF-145 were usually less than those to SCS and B-HEX. In vitro lymphocyte responses waned after approximately 6 weeks, whereas DH responses were clearly positive through 12 weeks. In addition to the studies in infected animals, animals immunized with heat-killed cells of C. neoformans 145 or 184 were tested 6 to 8 days later for DH with CneF-145, CneF-184, or B-HEX derived from C. neoformans 145. The CneF-145 and CneF-184 were equally effective for detecting DH, regardless of the cryptococcal strain used for immunization. Likewise, the B-HEX detected equivalent responses in mice sensitized with each cryptococcal strain. Since all three antigens were soluble and easily extracted and since each elicited significant cellular immune responses in infected animals, further studies involving their specificity and the nature of their reactive components seems warranted as they may help evaluate immune responses in humans infected with this fungus.     

Specific antibody can prevent fungal biofilm formation and this effect correlates with protective efficacy

One of the most troublesome medical problems today is infection of prosthetic devices with organisms that form polysaccharide biofilms. This combined with increasing antimicrobial drug resistance is making many infectious diseases incurable. Cryptococcus neoformans is a human-pathogenic fungus that has a polysaccharide capsule and can form biofilms in prosthetic medical devices. We developed a system to study cryptococcal biofilm formation in vitro and studied the effect of antibody to the C. neoformans capsular polysaccharide on this process. C. neoformans biofilm formation was dependent on the presence of a polysaccharide capsule and correlated with the ability of capsular polysaccharide to bind the polystyrene solid support. Protective antibodies prevented biofilm formation whereas nonprotective antibodies were not effective. The mechanism of antibody action involved interference with capsular polysaccharide release from the fungal cell. In contrast, lactoferrin, an effector molecule of innate immune mechanisms, was unable to prevent fungal biofilm formation despite its efficacy against bacterial biofilms. Our results suggest a new role of adaptive humoral immunity whereby some antibodies can inhibit biofilm formation by encapsulated organisms. Vaccines that elicit antibody responses to capsular antigens and/or passive transfer of antibodies to microbial polysaccharides may be useful in preventing biofilm formation.     

Production and characterization of monoclonal antibodies specific for Cryptococcus neoformans capsular polysaccharide.

Cryptococcus neoformans is surrounded by a capsular polysaccharide. There are at least four known serotypes of the polysaccharide. The objective of this study was to produce monoclonal antibodies (MAbs) that could be used to study the distribution of epitopes among the serotypes of C. neoformans. BALB/c mice were immunized with cryptococcal polysaccharides of serotype A or D that were coupled to sheep erythrocytes. Splenocytes were isolated, and hybridomas secreting MAbs specific for cryptococcal polysaccharides were isolated. Two hybridomas, designated MAbs 439 and 1255, were produced from mice immunized with serotype A polysaccharide. One hybridoma, designated MAb 302, was produced from mice immunized with serotype D polysaccharide. All three antibodies were of the immunoglobulin G1 isotype. MAb 302 showed a specificity for serotypes A and D in Ouchterlony diffusion, agglutination, and opsonophagocytosis assays. MAb 1255 was reactive with polysaccharides and cells of serotypes A, B, and D. MAb 439 was reactive with polysaccharides and cells of serotypes A, B, C, and D. The reactivity of these MAbs closely matched the distribution of epitopes among cryptococcal polysaccharides predicted in previous studies of polyclonal antibodies reactive with cryptococcal polysaccharides. The ability to produce a MAb against an epitope shared by all four serotypes may have value for the detection of cryptococcal antigens in body fluids.     

Evaluation of host immune responses to pulmonary cryptococcosis using a temperature-sensitive C. neoformans calcineurin A mutant strain

Cryptococcus neoformans is an opportunistic fungal pathogen that threatens individuals with impaired cell-mediated immunity (CMI). Presently, there are no standardized vaccines available to prevent cryptococcal infections and conventional anti-fungal drug therapy does not induce host immune reactivity and thus cannot efficiently resolve C. neoformans infections in immunocompromised individuals. The present study was designed to characterize pulmonary immune responses following infection with an avirulent temperature-sensitive (ts) mutant, calcineurin A1 (cna1) compared to the pathogenic C. neoformans strain H99 and its potential to induce protective anti-cryptococcal immunity. Host CMI responses in cna1-inoculated mice were observed to be dose-dependent, and comprise increases in pulmonary macrophages and CD4(+) T lymphocytes. However, cytokine analysis demonstrated a mixed pulmonary cytokine response (increases in IL-4, and MCP-1) with no induction of IFN-gamma. Also, pre-immunization with the ts cna1 mutant did not result in protection from a subsequent secondary pulmonary infection with the pathogenic C. neoformans strain H99. Taken together, these results suggest that host pulmonary CMI responses to the ts cna1 mutant that is eventually eliminated from the host without the induction of IFN-gamma appear to be dose-dependent, diverse, and require further stimulation to induce C. neoformans-specific Th1-type cytokine responses to resolve subsequent experimental pulmonary cryptococcal infections.     

Characterization of cellular infiltrates and cytokine production during the expression phase of the anticryptococcal delayed-type hypersensitivity response.

Cryptococcosis, an increasingly important opportunistic infection caused by the encapsulated yeast-like organism Cryptococcus neoformans, is limited by an anticryptococcal cell-mediated immune (CMI) response. Gaining a thorough understanding of the complex anticryptococcal CMI response is essential for developing means of controlling infections with C. neoformans. The murine cryptococcosis model utilizing footpad swelling to cryptococcal antigen (delayed-type hypersensitivity [DTH]) has proven to be a valuable tool for studying the induction and regulation of the anticryptococcal CMI response, but this technique has limitations with regard to evaluating the role of the final effector cells recruited by an ongoing CMI response. The purpose of this study was to assess the types of cells and cytokines induced into the site of cryptococcal antigen deposition in C. neoformans-infected and -immunized mice compared with those for control mice. We used a gelatin sponge implant model to examine the cells and cytokines present at the site of an anticryptococcal DTH response. Sponges implanted in infected mice and injected with cryptococcal culture filtrate antigen (CneF) 24 h before assessment had significantly increased numbers of infiltrating leukocytes compared with saline-injected sponges in the same animals. Exaggerated influxes of neutrophils and mononuclear cells were the major contributors to the increase in total numbers of cells in the DTH-reactive sponges. The numbers of CD4+ and LFA-1+ cells were found to be significantly increased in the CneF-injected sponges of infected and immunized mice over the numbers in control sponges. The numbers of large granular lymphocytes were also increased in DTH-reactive sponges compared with control sponges. Gamma interferon, interleukin 2 (IL-2), and IL-5 are clearly relevant cytokines in the anticryptococcal CMI response, since they were produced in greater amounts in the CneF-injected sponges from C. neoformans-infected and -immunized mice than in control sponges. IL-4 was not associated with the expression of DTH to cryptococcal antigen. The gelatin sponge model is an excellent tool for studying cells and cytokines involved in specific CMI responses.     

Protective efficacy of antigenic fractions in mouse models of cryptococcosis

Infections due to the encapsulated fungus Cryptococcus neoformans are 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 with C. neoformans strain 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 of C. neoformans contain components that protect mice from disseminated cryptococcosis, and this protection appears to be T-cell mediated.     

Kinetics of lymphocyte transformation in mice immunized with viable avirulent forms of Cryptococcus neoformans.

A murine model was developed to study the cell-mediated immune response of mice immunized with one of two live, avirulent forms of Cryptococcus neoformans: a nonencapsulated mutant and a thinly encapsulated pseudohyphal variant. A lymphocyte transformation assay was used to evaluate the cellular response of control and sensitized spleen cells after in vitro incubation with three merthiolate-killed whole-cell antigens of C. neoformans. An antigen-to-spleen cell ratio of 10:1 and 5 days of incubation of antigen-spleen cell mixtures were established as optimal conditions for maximum lymphocyte transformation. Maximum responses occurred from 2 to 3 weeks after the last of eight weekly intraperitoneal inoculations of C. neoformans. This assay provided an accurate, reproducible method of studying cell-mediated immunity to C. neoformans, and applications to the study of cryptococcal pathogenesis are proposed.