Kinetics of cellular infiltration and cytokine production during the efferent phase of a delayed-type hypersensitivity reaction.

Cell-mediated immunity is a primary host resistance mechanism against many infectious organisms and is responsible for leucocyte recruitment to the infection site. Delayed-type hypersensitivity (DTH) reactions are in vivo correlates of cell-mediated immunity and have long been used to assess the level of cell-mediated immune (CMI) responsiveness to specific antigens. It has been difficult to study the kinetics of cellular influx and cytokine composition at the site of an on-going CMI reaction. Consequently, knowledge of the sequential events occurring during the efferent phase of a CMI response is incomplete. Here we report on the use of a gelatin sponge model for evaluating the progression of events during the effector phase of a DTH reaction to antigens of the mycotic organism Cryptococcus neoformans. Previously, we have shown that 24 hr after antigen injection into sponges in infected or immune mice, the leucocyte types infiltrating the sponges are consistent with a classical murine DTH reaction. Through kinetic studies, we show here that neutrophils are the first leucocytes to appear in DTH-reactive sponges, followed by increases in lymphocyte and then monocyte numbers. Tumour necrosis factor (TNF), interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and IL-5 were elevated in DTH-reactive sponges compared with control sponges, and each cytokine had a relatively unique temporal profile. IL-4 was not detectable in the sponges. Together our data indicate that the expression of a CMI response comprises a well-regulated sequential influx of leucocytes that contribute to the lymphokine composition of the reaction.     

Regulation of cytokine production during the expression phase of the anticryptococcal delayed-type hypersensitivity response.

Effects of both positive and negative regulatory T cells on cellular infiltration and cytokine production during the expression phase of the anticryptococcal immune response were examined. Tamp cells, which are induced by cryptococcal antigen, significantly amplify the anticryptococcal delayed-type hypersensitivity response, whereas a cascade of T suppressor (Ts) cells inhibits the response and decreases the clearance of Cryptococcus neoformans during an infection. By using the gelatin sponge implantation model, we found that Tamp cells do not stimulate a significant increase in cellular infiltration into the sponges in response to cryptococcal antigen compared with that into delayed-type hypersensitivity-reactive sponges in immune control mice. However, Tamp cells do stimulate significant increases in the production of gamma interferon and interleukin-2 (IL-2) in the antigen-injected sponges over the level of the representative cytokine in antigen-injected sponges from the immune control mice. Likewise, Ts1 cells, induced with cryptococcal antigen, do not significantly affect antigen-induced cellular infiltration into sponges in immune mice. In contrast, decreased levels of gamma interferon and IL-2 are observed in antigen-injected sponges from Ts1-cell-recipient, immunized mice compared with those of the positive immune controls. The presence of either Tamp or Ts1 cells in immunized mice stimulates increased production of IL-5 but not IL-4 over that of the positive immune controls.     

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.     

Effects of tumor necrosis factor alpha on dendritic cell accumulation in lymph nodes draining the immunization site and the impact on the anticryptococcal cell-mediated immune response

Cell-mediated immune (CMI) responses and tumor necrosis factor alpha (TNF-alpha) have been shown to be essential in acquired protection against Cryptococcus neoformans. Induction of a protective anticryptococcal CMI response includes increases in dendritic cells (DC) and activated CD4(+) T cells in draining lymph nodes (DLN). During the expression phase, activated CD4(+) T cells accumulate at a peripheral site where cryptococcal antigen is injected, resulting in a classical delayed-type hypersensitivity (DTH) reaction. Induction of a nonprotective anticryptococcal CMI response results in no significant increases in the numbers of DC or activated CD4(+) T cells in DLN. This study focuses on examining the role of TNF-alpha in induction of protective and nonprotective anticryptococcal CMI responses. We found that neutralization of TNF-alpha at the time of immunization with the protective immunogen (i) reduces the numbers of Langerhans cells, myeloid and lymphoid DC, and activated CD4(+) T cells in DLN and (ii) diminishes the total numbers of cells, the numbers of activated CD4(+) T cells, and amount of gamma interferon at the DTH reaction site. Although TNF-alpha neutralization during induction of the nonprotective CMI response had little effect on cellular and cytokine parameters measured, it did cause a reduction in footpad swelling when mice received challenge in the footpad. Our findings show that TNF-alpha functions during induction of the protective CMI response by influencing the accumulation of all three DC subsets into DLN. Without antigen stimulated DC in DLN, activated CD4(+) T cells are not induced and thus not available for the expression phase of the CMI response.     

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.     

Differences in components at delayed-type hypersensitivity reaction sites in mice immunized with either a protective or a nonprotective immunogen of Cryptococcus neoformans.

Cell-mediated immunity is the major protective mechanism against Cryptococcus neoformans. Delayed swelling reactions, i.e., delayed-type hypersensitivity (DTH), in response to an intradermal injection of specific antigen are used as a means of detecting a cell-mediated immune (CMI) response to the antigen. We have found previously that the presence of an anticryptococcal DTH response in mice is not always indicative of protection against a cryptococcal infection. Using one immunogen that induces a protective anticryptococcal CMI response and one that induces a nonprotective response, we have shown that mice immunized with the protective immunogen undergo a classical DTH response characterized by mononuclear cell and neutrophil infiltrates and the presence of gamma interferon and NO. In contrast, immunization with the nonprotective immunogen results in an influx of primarily neutrophils and production of tumor necrosis factor alpha (TNF-alpha) at the DTH reaction site. Even when the anticryptococcal DTH response was augmented by blocking the down-regulator, CTLA-4 (CD152), on T cells in the mice given the nonprotective immunogen, the main leukocyte population infiltrating the DTH reaction site is the neutrophil. Although TNF-alpha is increased at the DTH reaction site in mice immunized with the nonprotective immunogen, it is unlikely that TNF-alpha activates the neutrophils, because the density of TNF receptors on the neutrophils is reduced below control levels. Uncoupling of DTH reactivity and protection has been demonstrated in other infectious-disease models; however, the mechanisms differ from our model. These findings stress the importance of defining the cascade of events occurring in response to various immunogens and establishing the relationships between protection and DTH reactions.     

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.     

Cytokine and chemokine expression in the central nervous system associated with protective cell-mediated immunity against Cryptococcus neoformans.

Cryptococcus neoformans is a yeast that causes cryptococcosis, a life-threatening disease that develops following inhalation and dissemination of the organisms. C. neoformans has a predilection for the central nervous system (CNS) and mortality is most frequently associated with meningoencephalitis. Susceptibility to cryptococcosis is increased in patients with deficiencies in cell-mediated immunity (CMI). Because cryptococcal CNS infections are associated with mortality and diagnosis of cryptococcosis is often not made until after dissemination to the CNS, a better understanding of host defense mechanisms against C. neoformans in the CNS is needed to design improved therapies for immunocompromised individuals suffering from cryptococcosis. Using a mouse model, we previously described a protective cell-mediated immune response induced in the periphery that limited the growth of C. neoformans in the CNS. In the current investigation, we examined cytokine and chemokine expression in the CNS to identify factors important in achieving protective immunity. We observed increased expression of IL-1beta, TNF-alpha, IFN-gamma, MCP-1, RANTES, and IP-10 in C. neoformans-infected brains of immune mice compared to control mice suggesting that these cytokines and chemokines are associated with the protective immune response. Furthermore, the Th1-type cytokines TNF-alpha and IFN-gamma, but not the Th2 cytokines IL-4 and IL-5, were secreted at significantly higher levels in C. neoformans-infected brains of immune mice compared to control mice. Our results demonstrate that cytokines and chemokines associated with CMI are produced following infection in the CNS of immunized mice, and the expression of these factors correlates with protection against C. neoformans in the CNS.     

Requirement for CD4(+) T lymphocytes in host resistance against Cryptococcus neoformans in the central nervous system of immunized mice

The importance of cell-mediated immunity (CMI) and CD4(+) T lymphocytes in host resistance against Cryptococcus neoformans is well documented and is exemplified by the high susceptibility to progressive infection with this pathogen of AIDS patients with reduced CD4(+) T-cell numbers. Although much has been learned about the role of CMI in the clearance of C. neoformans from the lungs and other internal organs, less is known about the protective mechanisms in the brain, the organ most frequently involved with a fatal outcome of cryptococcosis. We hypothesized that host resistance mechanisms against C. neoformans in the central nervous system (CNS) were similar to those outside the CNS (i.e., gamma interferon [IFN-gamma], CD4(+) T cells, and others). To test this hypothesis, we used a murine model of cryptococcal meningitis whereby cryptococci are introduced directly into the CNS. In experiments where mice were immunized to mount an anticryptococcal CMI response, our results indicate that immunization induced protective mechanisms that could be detected in the CNS by inhibition of the growth of viable yeast cells. Flow cytometric analyses of leukocytes in brain and spinal cord homogenates revealed that T lymphocytes, macrophages, and neutrophils accumulated in C. neoformans-infected brains of immune mice. In vivo depletion of CD4(+) T cells, but not CD8(+) T cells, resulted in significantly reduced leukocyte accumulation in the brains of immune mice. Furthermore, depletion of CD4(+) T cells or neutralization of IFN-gamma exacerbated CNS infection in immune mice, suggesting a critical role for CMI mechanisms in acquired protection in the CNS.     

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.     

Effects of cyclosporin A on the cells responsible for the anticryptococcal cell-mediated immune response and its regulation.

Cyclosporin A (CsA), a potent immunosuppressive drug, was used to explore further the induction, expression, and regulation of lymphoid cells involved in the delayed-type hypersensitivity (DTH) response to cryptococcal antigen(s). We found that the induction of the cells responsible for DTH (TDH cells) was not affected by CsA, but their expression was inhibited in CsA-treated mice. The inhibition of expression of the TDH cells could not be attributed to the Cryptococcus neoformans-specific suppressor T (Ts) cells, even though the Ts cells were induced in CsA-treated mice. Instead, the suppressed expression of the TDH cells in CsA-treated mice was a direct effect of CsA or its products. Our studies with CsA also resulted in the first identification of a population of cells that significantly amplify the anticryptococcal DTH response. The amplifier cells were induced in mice that were given a primary immunizing dose of cryptococcal antigen in complete Freund adjuvant, and they amplified the anticryptococcal DTH response in recipient mice when they were transferred at the time of immunization of the recipient. The amplifier cell population was distinct from the TDH cells in that CsA inhibited the production of the amplifying cells but did not affect the induction of TDH cells. Amplification of the DTH response was a cell-mediated event, since cells but not serum from immunized mice mediated the amplified response in recipient mice. Thus, CsA enabled us to characterize anticryptococcal TDH and Ts cells further and to add to the immune cell circuit of the cryptococcal system a distinct population of cells that amplifies the anticryptococcal DTH response.     

Effects of Cryptococcus neoformans-specific suppressor T cells on the amplified anticryptococcal delayed-type hypersensitivity response.

Cell-mediated immunity is an important host resistance mechanism against Cryptococcus neoformans, the etiological agent of cryptococcosis. Previous studies from our laboratory have shown that the anticryptococcal cell-mediated immune response as measured by delayed-type hypersensitivity (DTH) is down-regulated by a cascade of antigen-specific T suppressor (Ts) cells. Recently, we have identified a population of CD4 T cells that up-regulate the anticryptococcal DTH response (Tamp cells). The Tamp cells are found in the spleens of donor mice at 6 days after immunization with cryptococcal antigen, and they amplify the anticryptococcal DTH response when transferred to syngeneic recipients at the time of immunization of the recipients. In this study, we determined the effects of C. neoformans-specific Ts cells on the induction of the Tamp cells in the Tamp cell-donor mice and on the induction and expression of the amplified anticryptococcal DTH response in the Tamp cell-recipient mice. When cryptococcal-specific Ts1 cells were given at the time of immunization of the Tamp cell-donor mice, induction of Tamp cells was inhibited. In contrast, when Ts1 cells were given at the time of adoptive transfer of Tamp cells, the recipients displayed amplified DTH responses, indicating that Ts1 cells do not affect the Tamp cells' function once the Tamp cells have been produced. C. neoformans-specific Ts2 cells given at the time of either immunization or footpad challenge of the Tamp cell-recipient mice did not alter, to any measurable extent, the amplified DTH response. These results indicate that in addition to amplifying the anticryptococcal DTH response, Tamp cells may protect the anticryptococcal TDH cells from suppression by C. neoformans-specific Ts cells, much like contrasuppressor cells do in other systems. However, further characterization of the Tamp cells revealed that they are not adherent to Viscia villosa lectin, indicating that the anticryptococcal Tamp cells do not have this characteristic in common with contrasuppressor cells of other antigen systems.     

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.     

Differential Regulation of Immune Responses by Highly and Weakly Virulent Cryptococcus neoformans Isolates

Early inflammatory responses, delayed-type hypersensitivity (DTH) responses, and cytokine profiles were studied in mice infected by the pulmonary route with either a highly virulent isolate (NU-2) or a weakly virulent isolate (184A) of Cryptococcus neoformans. After infection, NU-2 remained in the lungs and the capsule became more pronounced during the first 24 h, whereas 184A induced an immediate inflammatory reaction and was rapidly cleared from the lungs. Cryptococcal antigen (GXM) appeared in sera early after infection with NU-2 and increased over the entire observation period. There was no detectable GXM in sera from 184A-infected mice. Both C. neoformans isolates induced anticryptococcal cell-mediated immune responses, but the responses had different profiles. DTH in NU-2-infected mice appeared at day 15 after infection and waned by day 21, whereas DTH in 184A-infected mice was present by day 5 and continued to increase. T helper 1 (Th1) cytokines (interleukin 2 [IL-2] and gamma interferon) were made by spleen cells early after infection with either isolate. NU-2-infected mice lost their ability to produce these cytokines, but 184A-infected mice retained it. IL-4, a Th2 cytokine, was not detected in infected mice. The regulatory cytokine IL-10 was made by spleen cells early but not later after infection with the highly virulent isolate and was not produced by spleen cells from 184A-infected mice. IL-10-deficient mice survived an NU-2 infection significantly longer than wild-type mice, suggesting that IL-10 is important in down-regulating the protective immune response. The induction of anergy appears to be responsible for the inability of NU-2-infected mice to control a C. neoformans infection.     

Role of CD4+ T cells in a protective immune response against Cryptococcus neoformans in the central nervous system.

Cryptococcosis is a life-threatening disease caused by the encapsulated yeast, Cryptococcus neoformans. Although infection with C. neoformans is initiated in the lungs, morbidity and mortality is mostly associated with infections of the central nervous system (CNS). Individuals with deficiencies in cell-mediated immunity, such as patients with AIDS, are more susceptible to disseminated cryptococcosis, highlighting the importance of cell-mediated immunity and CD4+ T cells in host resistance against C. neoformans. Using a mouse model of cryptococcal meningoencephalitis, we have shown that immunization of mice with a cryptococcal antigen induced a protective immune response that crossed the blood-brain barrier and initiated an immune response directly in the CNS if C. neoformans was present. The regional protective response was characteristic of a Type-1 (Th1) response in the types of cells present at the site of infection and in the cytokines and chemokines expressed. Here, we extend those findings and report that CD4+ T cells are required for survival of immune mice infected directly in the brain with C. neoformans and sensitized CD4 + T cells can transfer partial protection to naive mice infected intracerebrally with C. neoformans. Furthermore, CD4 + T cells were also important for optimal infiltration of inflammatory cells at the site of infection and in the expression of cytokines and chemokines associated with protection in the brain. Lastly, CD4+ T cells were required for optimal regional production and secretion of IFNgamma and in the significantly increased expression of iNOS in C. neoformans-infected brains of immune 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.     

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.     

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.     

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.     

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)