Host-Etiological Agent Interactions in Intranasally and Intraperitoneally Induced Cryptococcosis in Mice

Inbred CBA/J mice were used in developing a defined in vivo model for studying host-parasite relationships in cryptococcosis. Mice were infected either intranasally or intraperitoneally with 10³ viable Cryptococcus neoformans cells. At weekly intervals over a 92-day period, C. neoformans growth profiles in the lungs, spleens, livers, and brains of the infected animals were determined. In addition, humoral and delayed-type hypersensitivity responses and cryptococcal antigen levels were assayed in these mice. Intranasally infected mice developed strong delayed-type hypersensitivity reactions in response to cryptococcal culture filtrate (CneF) antigen, and there was good correlation between acquisition of delayed-type hypersensitivity and the reduction of C. neoformans cell numbers in infected tissues. In contrast, intraperitoneally infected mice displayed greater numbers of C. neoformans cells in tissues and had somewhat suppressed delayed-type hypersensitivity responses to CneF antigen. Anticryptococcal antibodies were not detected in intranasally or intraperitoneally infected mice, but cryptococcal polysaccharide antigen titers were relatively high in both groups. The transfer of sensitized spleen cells from intranasally infected mice to syngeneic naive recipient mice resulted in the transfer of delayed-type hypersensitivity responsiveness to cryptococcal antigen in the recipients. The intranasally induced infection in mice was similar to the naturally acquired infection in humans; therefore we are proposing that this murine-cryptococcosis model would be useful in gaining a greater understanding of host-etiological agent relationships in this disease.     

Effects of first-order Cryptococcus-specific T-suppressor cells on induction of cells responsible for delayed-type hypersensitivity.

Cell-mediated immunity is an important aspect of host resistance against Cryptococcus neoformans. Using a CBA/J murine model, we demonstrated that injection of cryptococcal antigen (CneF) at dosages sufficient to stimulate the antigenemia observed in cryptococcosis patients induces specific T-cell-mediated suppression of the cryptococcal delayed-type hypersensitivity response. The purpose of this study was to establish whether Lyt 1+, first-order T-suppressor (Ts1) cells block the induction of T cells responsible for delayed-type hypersensitivity (TDH cells) or whether they function by inducing Lyt 2+, efferent suppressor (Ts2) cells. In one set of experiments, suppression was observed when Ts1 cells were adoptively transferred to recipient animals the day before, the day of, or the day after immunization; however, when Ts1 cells were transferred after TDH cells were present, no suppression occurred. In other experiments, putative TDH cells from lymph nodes (LN) or spleens were adoptively transferred from mice after immunization or after a suppressive dose of CneF or adoptive transfer of Ts1 cells and immunization. Delayed-type hypersensitivity could not be transferred with LN or spleen cells from mice receiving the suppressive dose of CneF or the Ts1 cells, even when the LN or spleen cells were treated with anti-Lyt 2.1 antibody and complement to remove any Ts2 cells. Delayed-type hypersensitivity was readily transferred with LN or spleen cells from immunized mice whether the cells were or were not treated with anti-Lyt 2 and complement. Furthermore, the cells in the tolerized LN cell pools responsible for suppression of TDH cell induction were Lyt 1+ 2-, I-J+ cells, which is the phenotype of the Ts1 cells. Taken together, these data indicate that Ts1 cells inhibit the induction of TDH cells. This finding, coupled with the previous demonstration that Ts1 cells or a Ts1 cell-derived soluble factor (TsF1) induces Ts2 cells, establishes that the cryptococcal Ts1 cells are bifunctional in the suppressive pathway.     

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.     

Cryptococcal culture filtrate antigen for detection of delayed-type hypersensitivity in cryptococcosis.

Previous studies on a cryptococcal culture filtrate (CneF) antigen have shown that the antigen is useful in detecting delayed-type hypersensitivity and that it is specific for Cryptococcus. This study further defined one more parameter of specificity, showing that the CneF antigen does not elicit delayed-type hypersensitivity responses in Cryptococcus albidus-sensitized guinea pigs. When the crude CneF antigen was subjected to ultrafiltration fractionation, the skin test active components were found to be in the 50,000 or greater molecular weight range fraction. The concentrated retentates of the XM50 ultrafiltration membrane were more sensitive antigens than the crude CneF antigens. Further fractionation of the XM50 retentate using 3% acrylamide gel electrophoresis separated the antigen into two bands. One band, the P fraction, migrated only a short distance into the gel; the fraction was carbohydrate-like and did not elicit significant skin test responses in sensitized guinea pigs. The other band, G fraction, appeared with the tracking dye, was glycoprotein-like, and elicited significantly positive skin tests in sensitized guinea pigs. G fractions prepared using three different serotypes of Cryptococcus neoformans elicited similar size indurations when used in skin testing guinea pigs sensitized with either the homologous serotype isolated of C. neoformans or the heterologous serotype isolate.     

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.     

In vitro interactions of immune lymphocytes and Cryptococcus neoformans.

CBA/J mice immunized subcutaneously with emulsions of heat-killed Cryptococcus neoformans in complete Freund adjuvant displayed delayed-type hypersensitivity to cryptococcal culture filtrate antigen and developed sensitized splenic lymphoid cells which inhibited the growth of C. neoformans in vitro. The in vitro assay of growth inhibition served to investigate further the kinetics of the effect of sensitized lymphoid cells on the pathogen. There was a close correlation between the delayed-type hypersensitivity response in mice and inhibition of growth of C. neoformans by lymphoid cells. Sensitized splenic lymphocytes capable of inhibiting the growth of the cryptococci were detected at day 6 after immunization and reached maximum levels by days 8 through 16. Inhibition of growth was highest with effector-to-target cell ratios of 300:1 or greater. Inhibition of growth of C. neoformans by sensitized lymphoid cells was detectable as early as 4 h after effector and target cells were mixed and increased gradually, reaching a maximum at 24 h, but dropped significantly by 48 h. By supplementing the reaction mixtures with fresh medium or additional sensitized effector cells during incubation, the inhibition of growth of C. neoformans could be maintained through 48 h. C. neoformans-sensitized effector lymphoid populations not only inhibited the growth of the pathogen in vitro but also restricted C. neoformans proliferation in various vital organs upon transfer to naive recipient animals, indicating that the in vitro growth inhibition assay may be a means of assessing the resistance of animals to C. neoformans. The effector cells from sensitized animals were nylon wool-nonadherent Thy-1+ and Ia+ lymphocytes.     

Serological, electrophoretic, and biological properties of Cryptococcus neoformans antigens.

We compared a cryptococcal culture filtrate antigen referred to as CneF with chemically defined cryptococcal antigen fractions isolated by Cherniak and co-workers by using double immunodiffusion gels, polyacrylamide gel electrophoresis, immunoblots, and footpad reactivity of immunized mice. The three previously described components of cryptococcal culture filtrates are a high-molecular-weight glucuronoxylo-mannan (GXM), which is the major constituent, a galactoxylomannan (GaIXM), and a mannoprotein (MP). In this study we demonstrated that CneF contained components which were serologically and electrophoretically similar to the three previously described cryptococcal culture filtrate fractions. The MP fraction elicited significantly stronger delayed-type hypersensitivity responses than did the GXM or GaIXM fraction when used in mice immunized either with the CneF in complete Freund adjuvant or whole heat-killed Cryptococcus neoformans yeast cells. These findings were confirmed when the footpads of immunized mice were challenged with GaIXM and MP preparations from a culture filtrate of a C. neoformans acapsular mutant that does not produce GXM. Thus, we concluded that the MP was the primary component recognized by the anticryptococcal cell-mediated immune response in mice.     

Response of Congenitally Athymic (Nude) and Phenotypically Normal Mice to Cryptococcus neoformans Infection

A Cryptococcus neoformans infection in congenitally athymic (nude) mice and phenotypically normal heterozygote BALB/c mice was used to determine how T lymphocyte-deficient mice compared with normal mice in restricting proliferation of C. neoformans and to determine whether a correlation exists between delayed-type hypersensitivity and resistance to C. neoformans. Although nude mice displayed the ability to maintain cryptococcal population levels lower than did the phenotypically normal animals during the first 14 days of infection, the resistance was not sufficient to control the infection during the remainder of the 35-day experimental period. Heterozygote mice began to demonstrate positive delayed-type hypersensitivity responses by day 14 postinfection; however, nude mice were unable to mount delayed-type hypersensitivity responses. The appearance of the delayed-type hypersensitivity response in the heterozygote mice was concomitant with the reduced rate of proliferation of C. neoformans observed in those animals from days 14 to 35. Because anticryptococcal antibody titers and cryptococcal antigen levels were equivalent in both groups of mice, T-lymphocyte function was considered to be responsible for the resistance observed in the heterozygote mice. The mechanism by which cryptococcal populations were reduced was not addressed; however, the mouse model system used in these studies would be an ideal tool for studying those mechanisms. Nude mice were able to produce antibodies against cryptococcal cells, indicating that at least one component of C. neoformans is a T-independent antigen. The antibody response was predominantly immunoglobulin M in nude and heterozygote mice. Cryptococcal antigen levels were extremely high in both groups of animals and appeared to increase as C. neoformans cell numbers increased.     

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 third-order suppressor T cell (Ts3) induced by cryptococcal antigen(s).

Previous studies from our laboratory have shown that a high dose of cryptococcal culture filtrate antigen (CneF) administered intravenously induces a complex suppressor cell cascade which down-regulates the cell-mediated immune response to Cryptococcus neoformans antigens. The primary objective of this investigation was to determine whether a suppressor cell induced by immunization is required for efferent suppression of the cryptococcal delayed-type hypersensitivity (DTH) response. Our approach to this problem was to immunize CBA/J mice with CneF emulsified in complete Freund adjuvant and then 6 days later to collect spleen cells from the immunized mice and adoptively transfer these cells along with C. neoformans-specific second-order suppressor T cells (Ts2) to naive syngeneic recipients at the time of footpad challenge of the recipients with CneF. To establish which populations of cells in the spleens of immunized mice play a suppressive role, mass cytolysis with specific antibodies and complement was performed before the spleen cells were transferred to naive animals. Since the phenotype of the cells responsible for the transfer of the cryptococcal DTH response had not been completely determined, we first demonstrated that the cells responsible for DTH were L3T4+ Lyt-2- cells. Subsequently, we established that a Thy-1+ L3T4- Lyt-2+ I-J+ cell population induced by immunization was required along with C. neoformans-specific Ts2 cells for efferent suppression of the cryptococcal DTH response. In addition, we demonstrated that the suppressor cells in the immune cell population were derived from cyclophosphamide-sensitive precursors. These data indicate that a third suppressor cell population is required for efferent suppression of the cryptococcal DTH response. As in the azobenzenearsonate and 4-hydroxy-3-nitrophenyl acetyl hapten suppressor models, the Ts2 cells in the circuit mediate their effects through this third suppressor component. Since the mode of induction and the phenotype of the third C. neoformans-specific suppressor cells are similar to those reported for Ts3 cells in other antigen-specific suppression models, we referred to this third suppressor cell in the C. neoformans-specific suppressor cell cascade as a Ts3 cell.     

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.     

Cytokine profiles associated with induction of the anticryptococcal cell-mediated immune response.

Previous studies with a murine model have shown that immunization with cryptococcal culture filtrate antigen (CneF) emulsified in complete Freund adjuvant (CFA) induces two populations of anticryptococcal reactive CD4+ T cells. One population (TDH cells) transfers anticryptococcal delayed-type hypersensitivity (DTH), and the other population (Tamp cells) amplifies the anticryptococcal DTH response of given to recipient mice at the time of immunization of the recipient. Treatment of mice with cyclosporin A (CsA) ablates the induction of Tamp cells but not TDH cells. The present study focused on assessing the cytokines produced by spleen cells taken from CsA-treated and control (solvent-treated) mice at days 1, 2, 4, and 6 after immunization. Supernatants from the spleen cells cultured in vitro for 24 or 48 h in medium alone or with CneF, concanavalin A, or phorbol 12-myristate 13-acetate plus calcium ionophore were assessed for the presence of interleukin-2 (IL-2), gamma interferon (IFN-gamma), IL-4, IL-5, and tumor necrosis factor. Spleen cells from CneF-CFA-treated mice produced IL-2 and IFN-gamma, but not IL-4 or IL-5, constitutively and in response to CneF, indicating that CneF-CFA induces a Th1 response. Tumor necrosis factor was not produced. Anticryptococcal TDH cells developed in spleens in which there were low levels of IFN-gamma and IL-2 (CsA-treated, immunized mice), whereas anticryptococcal Tamp cells along with TDH cells matured in spleens in which production of IFN-gamma and IL-2 was high (solvent-treated, immunized mice). The data also suggest that IL-2 and IFN-gamma produced by Tamp cells early after adoptive transfer are influential in the development of the amplified anticryptococcal DTH response that has been observed in Tamp cell-recipient mice.     

Skin Testing of Guinea Pigs and Footpad Testing of Mice With a New Antigen for Detecting Delayed Hypersensitivity to Cryptococcus neoformans

This study was undertaken to evaluate the potential of a cryptococcal culture filtrate antigen, cryptococcin C184, for detecting delayed hypersensitivity in Cryptococcus neoformans-injected animals. The antigen was tested on guinea pigs which had received saline or C. neoformans and on animals sensitized to Histoplasma capsulatum, Blastomyces dermatitidis, Candida albicans, or Sporothrix schenckii. A delayed-type hypersensitivity response was elicited by cryptococcin C184 in C. neoformans-injected guinea pigs, whereas no indurations or erythemas were seen at 48 h after skin testing of saline controls or heterologously sensitized guinea pigs. Besides being specific for Cryptococcus, the antigen showed a high degree of sensitivity and was reproducible. Footpad tests were conducted with the antigen on mice which had previously received either 10(5) viable C. neoformans cells or saline. Delayed hypersensitivity was indicated in the C. neoformans-injected mice by the increase in thickness of antigen-injected footpads when compared with the saline-injected footpads. In control mice, antigen- and saline-injected footpads were comparable in thickness 24 h after injection. Mice sensitized to B. dermatitidis were footpad tested with C184, and no cross-reactivity was demonstrated.     

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.     

Intravascular cryptococcal culture filtrate (CneF) and its major component, glucuronoxylomannan, are potent inhibitors of leukocyte accumulation.

Disseminated cryptococcosis is characterized by high titers of cryptococcal polysaccharides in serum and minimal cellular infiltrates in infected tissues of patients. The main objective of this study was to determine whether the circulating cryptococcal polysaccharides could contribute to the lack of cellular infiltration into infected tissues. To assess this possibility, a gelatin sponge implantation model was used. We found that intravenous (i.v.) injection of mice with cryptococcal culture filtrate antigen (CneF) inhibited migration of leukocytes (neutrophils, lymphocytes, and monocytes) into the intrasponge sites of acute inflammation induced by CneF, tumor necrosis factor alpha, or formylmethionyl leucyl phenylalanine. In addition, i.v. administration of CneF inhibited leukocyte migration into the intrasponge sites of a cell-mediated immune reaction irrespective of whether the delayed-type hypersensitivity response was to CneF or the mycobacterial antigen purified protein derivative. Glucuronoxylomannan, a major constituent of CneF and a major cryptococcal antigen detected in the sera of patients with disseminated cryptococcosis, when given i.v. to mice, inhibited leukocyte migration into the sponges. Our results suggest that the minimal cellular infiltrates observed in infected tissues of cryptococcosis patients may be due, in part, to the circulating cryptococcal polysaccharide functioning as we have demonstrated in the mouse model. Furthermore, the high titers of cryptococcal antigen in the sera of patients may diminish leukocyte migration in response to stimuli other than Cryptococcus neoformans, a point that may be relevant in AIDS patients with cryptococcosis.     

Role of interleukin-4 in resistance to Cryptococcus neoformans infection

The role of interleukin (IL)-4 in cryptococcal disease was studied in IL-4 knockout (IL-4KO) and wild-type (WT) mice infected with Cryptococcus neoformans isolates that vary widely in their virulence. Delayed-type hypersensitivity responses were reduced in IL-4KO mice following primary infection with either isolate. Splenic T helper 1 (Th1) cytokine responses were increased in the IL-4KO mice infected with the weakly virulent isolate (184A) but did not change during infection with the highly virulent isolate (NU-2). Th2 cytokine responses (IL-5, IL-10) were downregulated in the IL-4KO mice infected with either isolate. Survival after primary infection with either isolate was not influenced by the absence of IL-4. Fewer colony-forming units were found in the lungs of 184A-infected, IL-4KO mice as compared to WT mice, suggesting that some immunity had developed. IL-4KO mice, primed with small doses of cryptococcal antigen (CneF), had significantly enhanced delayed-type hypersensitivity responses after intravenous infection with 184A and were more resistant to infection compared with WT mice. Increased expression of IL-5 with decreased interferon-gamma contributed to the inability of primed WT mice to resist infection with 184A. Enhanced immunity in the primed IL-4KO mice was reflected in a more moderate increase in IL-5 and IL-10 with maintenance of interferon-gamma levels.     

Delayed-type hypersensitivity responses in infected mice elicited by cytoplasmic fractions of Cryptococcus neoformans.

Four subcellular fractions of Cryptococcus neoformans prepared by differential centrifugation of disrupted whole yeast and a 3-day culture filtrate were examined for their ability to elicit delayed-type hypersensitivity in sensitized animals. The methods used to detect sensitization were (i) the footpad swelling test and inhibition of peritoneal macrophage migration in mice and (ii) skin testing in guinea pigs. Two entities, the post-mitochondrial supernatant and the culture filtrate, showed considerable activity in the footpad test, with 26- and 30-microliter 24-h swellings, respectively, at 6 weeks after infection. With the latter there was interference from a strong antibody-mediated 4-h skin reaction. The post-mitochondrial supernatant produced strong delayed-type hypersensitivity in guinea pigs at a dose of 69 microgram, and there was no demonstrable cross-reactivity in animals sensitized with heterologous fungi. The footpad swelling in mice correlated well with the macrophage migration inhibition test, with 71% inhibition in mice infected subcutaneously with C. neoformans at 6 weeks. However, mice infected intravenously developed poorer cell-mediated immunity than the subcutaneously infected mice. The post-mitochondrial supernatant was found to contain detectable amounts of cryptococcal capsular polysaccharide.     

Immunoadsorption of Cryptococcus-specific suppressor T-cell factors.

In the murine cryptococcal suppressor cell circuit, two different T-cell suppressor factors, TsF1 and TsF2, have been identified which specifically suppress the delayed-type hypersensitivity (DTH) response to cryptococcal culture filtrate antigen (CneF). TsF1 is produced by a first-order T suppressor (Ts1) cell population and suppresses the afferent limb of the DTH response, whereas TsF2 is produced by a second-order T suppressor (Ts2) cell population and suppresses the efferent limb of the cryptococcal DTH response. The objective of this study was to ascertain whether TsF1 or TsF2 could bind to cryptococcal antigen. To assess this, adsorption of TsF1 and TsF2 was performed with heat-killed Cryptococcus neoformans cells and by solid-phase immunoadsorption (SPIA) on columns containing cryptococcal antigens, i.e., CneF covalently bound to Sepharose 4B. The suppressive effect of TsF1 was removed by adsorption with intact heat-killed cryptococci and by SPIA on CneF-Sepharose 4B. The binding of cryptococcal TsF1 to the cryptococcal SPIA column was shown to be specific since Sepharose 4B columns either coupled with Saccharomyces cerevisiae mannan or blocked with glycine did not adsorb the suppressor activity. In contrast, the suppressive component of TsF2 did not bind to heat-killed cryptococci, CneF-Sepharose 4B, S. cerevisiae mannan-Sepharose 4B, or glycine-Sepharose 4B columns. These results, together with the finding that cryptococcal antigen, anticryptococcal antibody, and C1q-binding immune complexes were not demonstrated in either TsF1 or TsF2, establish that TsF1 and TsF2 can be differentiated on the basis of their affinity for cryptococcal antigen.     

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.     

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.