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.
When mice are vaccinated with a culture filtrate from Cryptococcus neoformans (CneF), they mount a protective cell-mediated immune response as detected by dermal delayed-type hypersensitivity (DTH) to CneF. We have identified a gene (DHA1) whose product accounts at least in part for the DTH reactivity. Using an acapsular mutant (Cap-67) of C. neoformans strain B3501, we prepared a culture filtrate (CneF-Cap67) similar to that used for preparing the commonly used skin test antigen made with C. neoformans 184A (CneF-184A). CneF-Cap67 elicited DTH in mice immunized with CneF-184A. Deglycosylation of CneF-Cap67 did not diminish its DTH activity. Furthermore, size separation by either chromatography or differential centrifugation identified the major DTH activity of CneF-Cap67 to be present in fractions that contained proteins of approximately 19 to 20 kDa. Using N-terminal and internal amino acid sequences derived from the 20-kDa band, oligonucleotide primers were designed, two of which produced a 776-bp amplimer by reverse transcription-PCR (RT-PCR) using RNA from Cap-67 to prepare cDNA for the template. The amplimer was used as a probe to isolate clones containing the full-length DHA1 gene from a phage genomic library prepared from strain B3501. The full-length cDNA was obtained by 5′ rapid amplification of cDNA ends and RT-PCR. Analysis of DHA1 revealed a similarity between the deduced open reading frame and that of a developmentally regulated gene from Lentinus edodes (shiitake mushroom) associated with fruiting-body formation. Also, the gene product contained several amino acid sequences identical to those determined biochemically from the purified 20-kDa peptide encoded by DHA1. Recombinant DHA1 protein expressed in Escherichia coli was shown to elicit DTH reactions similar to those elicited by CneF-Cap67 in mice immunized against C. neoformans. Thus, DHA1 is the first gene to be cloned from C. neoformans whose product has been shown to possess immunologic activity.
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.
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.
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.
Mice immunized with two different cryptococcal antigen preparations, one a soluble culture filtrate antigen (CneF) in complete Freund’s adjuvant (CFA) and the other heat-killed Cryptococcus neoformans cells (HKC), develop two different profiles of activated T cells. CneF-CFA induces CD4+ T cells responsible for delayed-type hypersensitivity (DTH) reactivity and for amplification of the anticryptococcal DTH response, whereas HKC induce CD4+ and CD8+ T cells involved in anticryptococcal DTH reactivity and activated T cells which directly kill C. neoformans cells. The main purpose of this study was to assess the level of protection afforded by each of the two different T-cell profiles against challenge with viable C. neoformans cells, thereby identifying which activated T-cell profile provides better protection. CBA/J mice immunized with CneF-CFA had significantly better protective responses, based on better clearance of C. neoformans from tissues, on longer survival times, and on fewer and smaller lesions in the brain, than HKC-immunized mice or control mice similarly infected with C. neoformans. Both immunization protocols induced an anticryptococcal DTH response, but neither induced serum antibodies to glucuronoxylmannan, so the protection observed in the CneF-CFA immunized mice was due to the activated T-cell profile induced by that protocol. HKC-immunized mice, which displayed no greater protection than controls, did not have the amplifier cells. Based on our findings, we propose that the protective anticryptococcal T cells are the CD4+ T cells which have been shown to be responsible for DTH reactivity and/or the CD4+ T cells which amplify the DTH response and which have been previously shown to produce high levels of gamma interferon and interleukin 2. Our results imply that there are protective and nonprotective cell-mediated immune responses and highlight the complexity of the immune response to C. neoformans antigens.
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.
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.
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.
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.
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.
The mobility of human neutrophils (PMN) in response to encapsulated or nonencapsulated Cryptococcus neoformans cells or cryptococcal culture filtrate (CneF) and its components was studied by using a 48-well modified Boyden chamber. Encapsulated C. neoformans (isolate 184A) cells and CneF-184A stimulated directed migration of human PMN in the absence of serum (direct chemotactic activity) and activated a heat-labile component(s) in fresh human serum to become a chemoattractant(s) for human PMN (indirect chemotactic activity). At a 1:8 dilution (0.25 mg of carbohydrate per ml), CneF-184A displayed chemokinetic activity when assessed with a checkerboard assay. Nonencapsulated C. neoformans isolate 602 cells did not have direct chemotactic activity but did have indirect chemotactic activity. The capsule of C. neoformans is composed predominantly of glucuronoxylomannan (GXM). Purified GXM displayed both direct and indirect chemotactic activity. CneF-184A contains, in addition to GXM, a concanavalin A-binding mannoprotein (MP), whereas CneF-602 contains no GXM but does contain MP. CneF-184A showed direct chemotactic activity and CneF-602 did not. Both CneF-184A and CneF-602 displayed indirect chemotactic activity for human PMN. In addition, purified MP from CneF-184A, like CneF-602, showed only indirect chemotactic activity. These results indicate that GXM contributes to the direct chemotactic activity of PMN observed with the whole encapsulated yeast cells and the unfractionated CneF derived from the encapsulated cells. Both MP and GXM from encapsulated C. neoformans cells mediate indirect chemotactic activity on human PMN.
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.
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.
Cryptococcus neoformans var. gattii (serotype B and C) isolates have a relative predilection for immunocompetent hosts, and C. neoformans var. neoformans (serotype A and D) isolates have a relative predilection for immunocompromised hosts, suggesting that normal host resistance to the former may be relatively inefficient compared with that to the latter variety. In order to assess the possibility that normal cellular host defense is inadequate in protecting against C. neoformans var. gattii, we compared the two varieties of C. neoformans cells and their culture filtrate antigens (CneF) with respect to effects on neutrophil (polymorphonuclear leukocyte [PMN]) locomotion. In a 48-well modified Boyden chamber, the cells and CneF of C. neoformans var. neoformans (serotype A and D) isolates stimulated chemotaxis and chemokinesis of human PMN and activated a complement component(s) in pooled human serum to become a chemoattractant(s) for human PMN. In contrast, the cells and CneF of C. neoformans var. gattii (serotype B and C) isolates did not stimulate chemotaxis or chemokinesis in human PMN but rather inhibited chemokinesis and chemotactic responses of PMN to pooled human serum and formylmethionyl leucyl phenylalanine. Neither of the CneF from the C. neoformans var. gattii isolates was cytotoxic to PMN. Furthermore, with the mouse model, we found that CneF from C. neoformans var. neoformans caused migration of PMN into gelatin sponges implanted in naive and immunized mice, whereas CneF from C. neoformans var. gattii inhibited PMN migration into sponges. Our results, combined with findings of others showing reduced PMN infiltration in lungs of mice infected with C. neoformans var. gattii compared with PMN infiltration in lungs of mice infected with C. neoformans var. neoformans, indicate that the relative inadequacy of normal host resistance mechanisms to prevent infection with C. neoformans var. gattii results, in part, from inhibition of PMN migration to the site of the organism.
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.
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.
We have shown previously that CBA/J mice immunized with Candida albicans developed delayed hypersensitivity (DH) demonstrable with mannan (MAN) extracted from the same organism and that the intravenous (i.v.) injection of MAN prior to or during the immunization phase resulted in the suppression of the MAN-specific DH response. In this study, we demonstrate that MAN-induced suppression of DH is a T-lymphocyte-mediated phenomenon. Suppressor cells induced in vivo by the i.v. injection of MAN into naive mice 1 to 7 days prior to harvest were passaged through nylon wool, treated with various surface-specific antibodies and complement, and then injected i.v. into immunized syngeneic recipients. Enrichment of splenic T cells by passage over nylon wool and transfer of the nylon-wool-nonadherent populations to immunized recipient mice suppressed DH in a dose-dependent manner. Depletion of Thy+ or Lyt-2+ cells from nylon-wool-nonadherent populations regularly ablated the ability of such suspensions to transfer suppression. Treatment of the same transfer suspensions with anti-Lyt-1 had variable effects, suggesting that the surface density of the Lyt-1 antigen was not as constant from population to population as was the Lyt-2 antigen. In addition, C. albicans MAN-induced suppressor cells were able to suppress DH demonstrable with Candida tropicalis MAN in animals immunized with C. tropicalis. Suppression of DH by MAN in this model, therefore, is mediated by Thy+ Lyt-2+ lymphocytes.
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.
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.
The functional role of the T cell (Tv) which can replicate vesicular stomatitis virus (VSV) on activation by the antigen was investigated in antibody response in vitro. By the inoculation of VSV into the culture, marked augmentation of antibody response to sheep erythrocytes (SRBC) was observed in the culture of spleen cells taken more than 3 days after the immunization with SRBC, suggesting that the VSV-susceptible suppressor cells were included in these spleen cells and the activity was eliminated by the effect of VSV. Development of two distinct types of suppressor T cells was revealed in the spleen of mice after the priming with SRBC. First, nylon wool nonadherent (NAd) suppressor T cells found in the spleen cells taken 3 days after immunization, and second, nylon wool adherent (Ad) suppressor T cells found in the spleen cells taken approximately 1 wk after immunization. The activity of nylon Ad suppressor T cells was completely abolished by VSV- preinfection, whereas that of nylon NAd suppressor T cells was unaffected. It was also shown that the helper T-cell activity was not influenced by VSV-preinfection. These results provided direct evidence that nylon Ad suppressor T cell but not nylon NAd suppressor T cell nor helper T cell can actually replicate VSV after antigenic stimulation. Thus it was strongly suggested that Tv represents the nylon Ad suppressor T cells.
Previous data from this laboratory indicate that normal murine nylon wool nonadherent splenic cells with characteristics of natural killer (NK) cells effectively inhibit in vitro growth of Cryptococcus neoformans, a yeastlike pathogen. Since NK cells have been shown to be involved in antibody-dependent, cell-mediated cytotoxicity against immunoglobulin G (IgG)-coated tumor cells and xenogenic erythrocytes, we were interested in assessing the effects of the IgG fraction of rabbit anticryptococcal serum on NK cell-mediated inhibition of C. neoformans growth. Early in the study it became apparent that the conventional method of determining the numbers of CFU that was used previously for assessment of viable cryptococci at the end of the growth inhibition assay was not reliable for these studies, owing to minor clumping of the organisms in the presence of anticryptococcal antibody. Therefore, the BACTEC radiometric system was evaluated and determined to be a reliable replacement for the CFU count method. Using the BACTEC methodology, we showed that the anticryptococcal antibody significantly augmented the in vitro ability of NK cells to inhibit the growth of C. neoformans compared with normal rabbit serum or tissue culture medium. Furthermore, the antibody alone did not have an adverse effect on the organism, confirming that reduced growth indices obtained from test wells containing antibody, NK cells, and cryptococci were due to the effects of the NK cells. Maximum anticryptococcal activity of the NK cells was observed in the presence of 16 micrograms of IgG per ml; however, significant augmentation of anticryptococcal activity was seen with antibody concentrations as low as 3 micrograms/ml. Using different populations of murine splenic cells which had varying degrees of NK cell activity, we were able to show that NK cell activities, as determined by 51Cr release from YAC-1 targets, directly correlated with antibody-dependent, cell-mediated growth inhibition against cryptococci, suggesting that NK cells were effector cells in the antibody-dependent assays. Furthermore, in every case, the antibody-dependent activity of NK cells against C. neoformans was higher than the spontaneous activity of NK cells against the organism, emphasizing that NK cell activity against cryptococci can be augmented by specific antibody. When NK cell numbers were enriched by Percoll fractionation of nylon wool nonadherent splenic cells, antibody-dependent and spontaneous growth inhibitory activities of the effector cells were concomitantly augmented, confirming that NK cells were the effector cells in antibody-dependent growth inhibition of cryptococci.(ABSTRACT TRUNCATED AT 400 WORDS)
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.
Antigen-specific suppressor factor produced by metabolically active in vitro-induced suppressor cells, upon further antigenic stimulation, act on nylon wool nonadherent, Ly-2-negative target cells within helper cell population, resulting in suppression of both the IgM and IgG antibody responses. Thus the target is an Ly-1+ T cell, possibly the helper cell. All the mouse strains tested so far have been able to produce the factor, and when tested in CBA or B10 mice, there seems to be no genetic restriction involved e.g., nonsyngeneic suppressor factors suppress as well as do the syngeneic factors. Comparison of the properties of suppressor factor with those of extracts of suppressor cells yield differences in origin, target of action and effect, indicating that these are different molecules. The heterogeneity of suppressor pathways is discussed.
Previous studies in our laboratory and others have demonstrated that T and/or NK cells can directly bind to and inhibit the growth of the medically important fungal pathogens Cryptococcus neoformans and Candida albicans by apparently non-major histocompatibility complex-restricted mechanisms. Here, we examined whether this direct interaction between lymphocytes and fungi also results in cytokine gene expression and release. Nonadherent lymphocytes (NAL), isolated from human peripheral blood mononuclear cells by depletion of cells adherent to plastic and nylon wool, released gamma interferon (IFN-gamma), but not interleukin-4 (IL-4) and IL-10, following stimulation with C. neoformans yeast cells and C. albicans yeast cells, hyphae, and supernatants. The fungal stimuli also induced IFN-gamma mRNA, with peak gene expression seen at or after 18 h. IFN-gamma release was still seen even when either NK cells or T lymphocytes were depleted by negative selection, suggesting that both cell types can be stimulated by fungi to produce IFN-gamma. Release of IFN-gamma from fungus-stimulated NAL occurred in the absence of an intact complement system and was not especially enhanced by culture with IL-2 or IL-12. These data expand the mechanisms by which the direct interaction of NAL with fungal targets can lead to immune activation. Moreover, to our knowledge, this is the first demonstration of direct stimulation of T-cell cytokine release by microbial pathogens.