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1.  Differential Regulation of Immune Responses by Highly and Weakly Virulent Cryptococcus neoformans Isolates 
Infection and Immunity  1999;67(7):3601-3609.
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.
PMCID: PMC116550  PMID: 10377145
2.  What makes Cryptococcus neoformans a pathogen? 
Emerging Infectious Diseases  1998;4(1):71-83.
Life-threatening infections caused by the encapsulated fungal pathogen Cryptococcus neoformans have been increasing steadily over the past 10 years because of the onset of AIDS and the expanded use of immunosuppressive drugs. Intricate host-organism interactions make the full understanding of pathogenicity and virulence of C. neoformans difficult. We discuss the current knowledge of the characteristics C. neoformans must possess to enter the host and establish progressive disease: basic growth requirements and virulence factors, such as the polysaccharide capsule; shed products of the organism; melanin production; mannitol secretion; superoxide dismutase; proteases; and phospholipases.
PMCID: PMC2627665  PMID: 9452400
3.  Direct interactions of human natural killer cells with Cryptococcus neoformans inhibit granulocyte-macrophage colony-stimulating factor and tumor necrosis factor alpha production. 
Infection and Immunity  1997;65(11):4564-4571.
Human natural killer (NK) cells and T lymphocytes can bind to and inhibit the growth of the yeast-like organism Cryptococcus neoformans. Binding of target cells to NK or T cells also has the potential to modulate cytokine production by the effector cells. In this study, we assessed the ability of C. neoformans to modulate NK cell production, or in some cases T-cell production, of granulocyte-macrophage colony-stimulating factor (GM-CSF) or tumor necrosis factor alpha (TNF-alpha). We found that freshly isolated human NK cells from most individuals make GM-CSF and TNF-alpha constitutively when cultured in vitro. The addition of C. neoformans to T-cell fractions which do not make GM-CSF constitutively did not affect GM-CSF production, but the addition of C. neoformans to NK cell fractions significantly reduced the amounts of GM-CSF produced in most NK cell samples. The reduction in the amount of GM-CSF in C. neoformans-NK cell cocultures could not be attributed to loss of lymphocyte viability or to C. neoformans adsorbing or degrading the cytokine and was dependent on direct contact between the NK cells and cryptococcal cells. GM-CSF was not the only cytokine to be down-regulated. TNF-alpha production was also diminished when NK cells were incubated with C. neoformans. The regulation of both cytokines was at the transcriptional level because GM-CSF and TNF-alpha mRNA levels were lower in NK cell samples incubated with C. neoformans than in NK cell samples incubated without C. neoformans. Diminished production of constitutively produced cytokines resulting from the interaction of NK cells with cryptococcal cells has the potential to affect phagocytic cells in the immediate regional environment and to damp the immune response.
PMCID: PMC175655  PMID: 9353034
4.  Secretion of the C3 component of complement by peritoneal cells cultured with encapsulated Cryptococcus neoformans. 
Infection and Immunity  1997;65(10):4114-4121.
Two isolates of Cryptococcus neoformans were identified as being widely divergent in pathogenic potential after intratracheal infection of mice. These isolates differed in their ability to upregulate capsule synthesis when grown under tissue culture conditions, and this property correlated with virulence. We postulated that differential capsule synthesis may cause differential stimulation of macrophages to produce products such as complement components. To test this hypothesis, heat-killed yeast cells were incubated with normal mouse peritoneal cells (PC) before the level of C3 secreted was determined. Cryptococcal stimulants were grown on mycological agar, which does not promote capsule synthesis, or in RPMI 1640 at 37 degrees C in an atmosphere of 5% CO2, which stimulates capsule synthesis, to determine the role that the capsule plays in the induction of C3 secretion. C3 levels were elevated in cultures containing cryptococci grown in RPMI 1640 at 37 degrees C in an atmosphere of 5% CO2, and the level of C3 detected was correlated with the amount of capsule expressed by the yeast cell stimulant. Nonencapsulated mutants of C. neoformans did not stimulate C3 secretion. Purified capsular polysaccharide (glucuronoxylomannan [GXM]) also stimulated the PC to secrete C3. Two signals were required before GXM stimulated C3 secretion. The second signal was identified as endotoxin present in small amounts (0.06 ng per ml) in tissue medium. Endotoxin may provide a priming stimulus for PC to express receptors or other cytokines needed for effective stimulation of C3. These experiments show that enhancement of C3 secretion by C. neoformans is due to GXM and is correlated with the virulence of the cryptococcal isolate.
PMCID: PMC175592  PMID: 9317016
5.  Cryptococcal polysaccharides bind to CD18 on human neutrophils. 
Infection and Immunity  1997;65(2):557-563.
CD18, the beta chain of the beta 2 integrin family of adhesion molecules, is associated with three different alpha chains (CD11a, -b, and -c) and is expressed on the surface of all types of leukocytes. CD18-containing molecules are up-regulated on the surface of neutrophils (polymorphonuclear cells [PMN]) in response to chemotactic agents and are implicated in mediating adhesion to an inflamed endothelium, which is a prerequisite to migration of PMN into infected tissues. In a previous study, we found that a cryptococcal culture filtrate (CneF), when injected into the bloodstream of mice to simulate the antigenemia in cryptococcosis, inhibits PMN accumulation at the site of an inflammatory stimulus. In the present study, we assessed the ability of CneF and its individual components, i.e., glucuronoxylomannan (GXM), galactoxylomannan (GalXM), and mannoprotein (MP), to interact with CD18 on human PMN. CneF labeled with 14C was shown to bind to human PMN in a dose-dependent manner. Pretreatment of PMN with anti-CD18, but not an isotype-matched control monoclonal antibody (MAb) or anti-CD11a MAb, blocked the binding of 14C-labeled CneF to PMN. In addition, CneF, GXM, and GalXM but not MP significantly blocked the binding of the anti-CD18 MAb to CD18 on the surface of unactivated and formyl methionyl leucyl phenylalanine-activated PMN as determined by indirect immunofluorescence staining and flow cytometric analysis. In the same experiments, the cryptococcal polysaccharides did not affect the binding of an anti-CD11a or anti-L-selectin MAb to the surface of PMN at 4 degrees C. The results suggest that CneF and its components GXM and GalXM bind to CD18 on human PMN. Based on our findings, we propose that CD18 is a possible molecular target of cryptococcal polysaccharides and that binding of the polysaccharides to CD18 has the potential to inhibit leukocyte infiltration into inflammatory sites.
PMCID: PMC176096  PMID: 9009313
6.  Cryptococcal polysaccharides induce L-selectin shedding and tumor necrosis factor receptor loss from the surface of human neutrophils. 
Journal of Clinical Investigation  1996;97(3):689-698.
High titers of cryptococcal polysaccharides in the serum and spinal fluid and the lack of cellular infiltrates in the infected tissues are hallmarks of disseminated cryptococcosis. Cryptococcal polysaccharides given intravenously to mice inhibit the influx of leukocytes into sites injected with inflammatory mediators. The purpose of this investigation was to determine if cryptococcal polysaccharides, i.e., glucuronoxylomannan (GXM), galactoxylomannan, and mannoprotein, affect expression of molecules on the surface of neutrophils that are important in extravasation. GXM in the absence of serum was shown to induce human neurophils to shed L-selectin, a molecule needed in the first step of neutrophil movement into tissues. In the presence of serum, GXM caused a further shedding of L-selectin. Shedding of L-selectin was evident by reduced amounts of L-selectin on the neutrophils treated with GXM and by increased levels of soluble L-selectin in the GXM-treated neutrophil supernatants. GXM also stimulated neutrophils to have reduced expression of TNF receptor. In contrast, GXM-treated neutrophils showed increased levels of CD15 and CD11b, and unchanged CD16 expression. In the absence of serum, galactoxylomannan and mannoprotein did not affect L-selectin, TNF receptor, CD15, CD11b, or CD16 on neutrophils but did induce loss of L-selectin in the presence of serum. Our results indicate that cryptococcal polysaccharides, especially GXM, can cause shedding of L-selectin from the surface of neutrophils, and this may prevent neutrophils from attaching to the endothelial cell surfaces. Blockage of this early step in cell migration from the vessels into tissues may be responsible in part for reduced cellular infiltration into infected tissues of individuals with disseminated cryptococcosis.
PMCID: PMC507105  PMID: 8609224
7.  Effects of the two varieties of Cryptococcus neoformans cells and culture filtrate antigens on neutrophil locomotion. 
Infection and Immunity  1995;63(7):2632-2644.
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.
PMCID: PMC173353  PMID: 7790079
8.  Effects of immunization with Cryptococcus neoformans cells or cryptococcal culture filtrate antigen on direct anticryptococcal activities of murine T lymphocytes. 
Infection and Immunity  1995;63(5):1645-1651.
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.
PMCID: PMC173204  PMID: 7729868
9.  Direct anticryptococcal activity of lymphocytes from Cryptococcus neoformans-immunized mice. 
Infection and Immunity  1995;63(5):1637-1644.
Assessment of the direct anticryptococcal activity of murine lymphocytes from both Cryptococcus neoformans-immunized and control mice was the focus of this investigation. We demonstrate that at a 2:1 effector cell-to-cryptococcal target cell ratio, effector cell populations comprised of alpha beta T-cell receptor-positive T lymphocytes (98 to 99% CD3+) from C. neoformans-immunized mice inhibited the growth of cryptococcal cells better than similar populations of lymphocytes from nonimmunized control mice. Almost immediately after mixing of cryptococci with the effector cells, C. neoformans-lymphocyte conjugates were observed. The percentage of conjugates increased over the first 30 min of incubation and then remained constant over the next 1.5 h. T-lymphocyte-enriched populations from C. neoformans-immunized mice formed significantly greater percentages of conjugates with cryptococci than control T lymphocytes at each time period that assessment was made. For growth inhibition to occur, direct contact between the effector and target cells was necessary, as evidenced by abrogation of cryptococcal growth inhibition when lymphocyte and cryptococcal cell populations were separated by a porous membrane during the growth inhibition assay. Vital staining of cryptococci after incubation with the T-cell-enriched populations showed that the T lymphocytes killed the cryptococcal cells.
PMCID: PMC173203  PMID: 7729867
10.  Intravascular cryptococcal culture filtrate (CneF) and its major component, glucuronoxylomannan, are potent inhibitors of leukocyte accumulation. 
Infection and Immunity  1995;63(3):770-778.
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.
PMCID: PMC173069  PMID: 7868246
11.  Murine natural killer cell interactions with a fungal target, Cryptococcus neoformans. 
Infection and Immunity  1989;57(7):1990-1997.
Earlier investigations have shown that murine natural killer (NK) cells bind to and inhibit the growth of the fungal pathogen Cryptococcus neoformans in vitro and in vivo. To define the stages of NK cell-mediated inhibition of C. neoformans growth and the requirements for the completion of these stages, the events which lead to cryptococcal growth inhibition were compared with those previously elucidated for NK cell-mediated tumor cell lysis. Our data indicate that NK cell-cryptococci binding is a distinct event that precedes inhibition; is temperature independent, although it is slowed at 4 degrees C; and is Mg2+ dependent. In contrast to binding, NK cell-mediated cryptococcal growth inhibition is temperature, Mg2+, and Ca2+ dependent. The removal of Ca2+ by EDTA addition within 3 h after maximal NK cell-cryptococci binding significantly reduced cryptococcal growth inhibition, indicating that Ca2+ is required either late in the NK cell trigger stage or early in the inhibitory stage. These stages and requirements are similar to those previously demonstrated for the model of NK cell-mediated tumor cell lysis; however, the NK cell-cryptococci interactions are somewhat slower than the interactions which culminate in the lysis of the YAC-1 tumor cell targets. These results suggest that C. neoformans cells, although structurally distinct from the standard tumor cell targets, are capable of similar cell-to-cell interactions with NK effector cells as the tumor cell targets.
PMCID: PMC313832  PMID: 2659531
12.  Regulation of cytokine production during the expression phase of the anticryptococcal delayed-type hypersensitivity response. 
Infection and Immunity  1994;62(7):2930-2939.
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.
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PMCID: PMC302900  PMID: 7911788
13.  Mobility of human neutrophils in response to Cryptococcus neoformans cells, culture filtrate antigen, and individual components of the antigen. 
Infection and Immunity  1993;61(12):5067-5077.
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.
PMCID: PMC281285  PMID: 8225584
14.  Cytokine profiles associated with induction of the anticryptococcal cell-mediated immune response. 
Infection and Immunity  1993;61(11):4750-4759.
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.
PMCID: PMC281230  PMID: 8406874
15.  Characterization of cellular infiltrates and cytokine production during the expression phase of the anticryptococcal delayed-type hypersensitivity response. 
Infection and Immunity  1993;61(7):2854-2865.
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.
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PMCID: PMC280931  PMID: 8514388
16.  Direct interactions of human lymphocytes with the yeast-like organism, Cryptococcus neoformans. 
Journal of Clinical Investigation  1993;91(4):1553-1566.
Lymphocytes, especially CD4+ T cells, are essential for clearance of the yeast-like organism Cryptococcus neoformans from the infected host. The mechanism(s) by which the lymphocytes facilitate elimination of cryptococci has not been elucidated. It is generally thought, however, that lymphocytes reactive with C. neoformans indirectly function by production of lymphokines to enhance clearance of the organism by natural effector cells such as macrophages. In the present study, we assessed the ability of freshly isolated human lymphocytes to interact directly with C. neoformans and to limit the growth of the organism in vitro. We found that large granular lymphocytes (LGL) as well as T cells bound to cryptococcal cells when the lymphocytes were mixed with the cryptococcal cells at a 2:1 ratio. The physical binding interactions of the two lymphocyte populations were different. LGL attached to the cryptococcal cells by many microvilli; T lymphocytes associated with the yeast through broad areas of membrane attached to the cryptococcal cell surface. The two types of lymphocyte interactions did not result in phagocytosis but resulted in direct inhibition of cryptococcal growth, making these lymphocyte interactions with cryptococci distinctly different from interactions of monocytes with cryptococci. With the human natural killer (NK) cell line, NK 3.3, we confirmed that NK cells that were present in the LGL population were capable of limiting the growth of C. neoformans. Through immunoelectron microscopy, human CD3+ lymphocytes were seen attached to cryptococcal cells and by mass cytolysis, human CD3+ lymphocytes were shown to be responsible for inhibition of C. neoformans growth. The direct inhibitory interactions of NK cells and T lymphocytes with cryptococcal cells may be important means of host defense against this ubiquitous organism that frequently causes life-threatening disease in AIDS patients.
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PMCID: PMC288131  PMID: 8473499
17.  Early events in initiation of alternative complement pathway activation by the capsule of Cryptococcus neoformans. 
Infection and Immunity  1991;59(9):3101-3110.
The capsule of Cryptococcus neoformans is a powerful activator of the alternative complement pathway. This study examined the manner in which the cryptococcal capsule influences initiation of and early events in complement activation by C. neoformans. These studies examined the effects of the classical and alternative pathways on the kinetics and early sites for deposition of C3 fragments on encapsulated cryptococci, nonencapsulated cryptococci, and zymosan. The results showed that nonencapsulated cryptococci and zymosan are qualitatively and quantitatively similar in the manner in which they initiate complement activation. Both utilize the classical and alternative pathways. Initiation via the classical pathway occurs suddenly and simultaneously at sites distributed over the entire cell surface. Initiation of the alternative pathway by zymosan and nonencapsulated cryptococci is characterized by a lag of 6 to 8 min before appreciable amounts of C3 accumulate on the cells. Alternative pathway initiation by zymosan and nonencapsulated cryptococci occurs at a limited number of focal initiation sites that expand with alternative pathway amplification to cover the cell surface. Presence of the cryptococcal capsule blocks classical pathway initiation, which would normally occur at the cryptococcal cell wall, and produces an initiation that is dependent solely on the alternative pathway. Initiation of the alternative pathway by the cryptococcal capsule is characterized by a lag in C3 accumulation and the appearance of a limited number of focal initiation sites which resemble those observed when the alternative pathway is activated by zymosan and nonencapsulated cryptococci.
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PMCID: PMC258140  PMID: 1831795
18.  Murine natural killer cells are fungicidal to Cryptococcus neoformans. 
Infection and Immunity  1991;59(5):1747-1754.
Murine natural killer (NK) cells have been shown to bind to and inhibit the growth of Cryptococcus neoformans in vitro and to contribute to clearance of the organism in vivo. However, it is unclear whether NK cells actually kill cryptococci or simply inhibit proliferation of the fungal target. Therefore, the studies presented here were designed to determine whether NK cells are fungicidal to C. neoformans targets. C. neoformans viability was determined on the basis of the metabolic function of two different enzyme systems, as measured by the two vital stains MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and fluorescein diacetate. Cryptococcal viability, as determined by vital stains, was compared with cryptococcal proliferation, as measured by microcolony formation in agarose at the individual cell level and by CFU counts or extinction dilution analysis in the total cell suspension. Initial comparisons of the vital stains and proliferation assays indicated that these methods effectively distinguished between live and heat-killed cryptococci at the individual cell level and in the total cell suspensions. After cryptococci were incubated with murine NK cells for 18 h, vital stains demonstrated that at the single conjugate level and in the total cell suspension, NK cells kill bound C. neoformans target cells. In addition, the numbers of dead cryptococci in the NK cell-C. neoformans suspensions as determined by the vital stains were comparable to the numbers of cryptococci that were unable to proliferate. Kinetics of NK cell-mediated C. neoformans binding and killing at the single conjugate level and in the total cell suspension were assessed by MTT staining at 2-h intervals after mixing effector and target cells, and the data support the concept that NK cell-C. neoformans binding precedes cryptococcal death. Furthermore, unbound, dead fungal cells were observed in the NK cell-C. neoformans suspensions after 18 h, suggesting that NK cell-C. neoformans interactions may involve both effector cell recycling and killing of unbound cryptococci by soluble cytotoxic factors. In conclusion, the results of these studies firmly establish that NK cells kill C. neoformans.
PMCID: PMC257911  PMID: 1708359
19.  Binding interactions of murine natural killer cells with the fungal target Cryptococcus neoformans. 
Infection and Immunity  1991;59(4):1476-1488.
Murine natural killer (NK) cells have been shown to inhibit the growth of the yeastlike organism Cryptococcus neoformans both in vivo and in vitro. An essential first step in NK cell-mediated damage of cryptococcal cells is the binding of the NK cell to the cryptococcal cell. The studies presented here focused on the binding event. Electron photomicrographs and three-dimensional reconstructions of NK cell-C. neoformans conjugates show that NK cells bind to cryptococci through many microvilli. This is in contrast to the broad membrane-membrane interactions which form the binding site of NK cell-YAC-1 tumor cell conjugates. NK cell binding to cryptococci is much slower than NK cell binding to YAC-1 targets. Maximal conjugate formation with cryptococcal targets is reached after 2 h, whereas maximal conjugate formation with YAC-1 targets is obtained after 20 min. Once maximum NK cell-C, neoformans conjugate formation is obtained, another 4 h is required before damage to the cryptococcal cells can be detected with the CFU assay. These data indicate that the binding and action of NK cells on C. neoformans cells requires considerably more time than is necessary for similar events to occur in the NK cell-tumor cell model. NK cell membrane integrity is necessary for NK cells to bind to tumor targets, since some disruption of membrane integrity with 0.1 M dimethyl sulfoxide reduces conjugate formation and tumor cell lysis. In contrast, 0.1 M dimethyl sulfoxide did not diminish NK cell binding to cryptococcal targets; however, it significantly reduced cryptococcal growth inhibition. Although we have observed several differences in NK cell binding to the cryptococcal target compared with NK cell binding to tumor cell targets, there are some similarities in binding interactions of NK cells with the two different targets. Disulfide bonding appears to play a role in the binding of NK cells to both targets, since 5 mM 2-mercaptoethanol, a reagent that reduces disulfide bonds, prevented NK cells from binding to the tumor targets as well as the cryptococcal targets. Actin filaments, components of the cytoskeletal network, must be intact for NK cells to bind to YAC-1 cells or cryptococci. Taken together, our data confirm that binding of NK cells to the cryptococcal target is prerequisite to the stages that result in damage to the cryptococcal cell and that there are similarities and differences in NK cell-binding interactions with structurally different target cells.
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PMCID: PMC257866  PMID: 2004826
20.  Responses of murine natural killer cells to binding of the fungal target Cryptococcus neoformans. 
Infection and Immunity  1991;59(4):1489-1499.
Natural killer (NK) cells bind to and inhibit the growth of the fungal target Cryptococcus neoformans. Since C. neoformans is structurally and chemically distinct from the standard tumor cell target used in the model of NK cell-mediated cytotoxicity, this study was designed to investigate the NK cell response after binding to cryptococci. Transmission electron micrographs and three-dimensional reconstructions of NK cell-cryptococci conjugates demonstrated focusing of the NK cell centrioles and Golgi apparatus toward the cryptococcal attachment site. NK cell cytoskeletal changes after cryptococcal binding were confirmed by immunofluorescence studies in which NK cells were allowed to bind to cryptococci in Mg2(+)-containing, Ca2(+)-free medium. One hour after the addition of Ca2+ to the preformed conjugates, the bound NK cells demonstrated a significant increase in the percentage of microtubule organizing centers focused toward the cryptococcal binding site. Colchicine, a drug that inhibits microtubule assembly, did not affect NK cell-cryptococci binding but abrogated NK cell-mediated cryptococcal growth inhibition, indicating that microtubule assembly, an important prerequisite for the secretory process, is not required for NK cell-cryptococci binding but is essential for inhibition of cryptococcal growth. In addition, the Ca2+ channel-blocking reagents, lidocaine and verapamil, did not affect NK cell-cryptococci binding but blocked the NK cell-mediated anticryptococcal activity, suggesting that a Ca2+ flux is essential for inhibition of cryptococcal growth. Considered together, these data indicate that NK cells respond to binding of a target cell that has a capsule and cell wall, in addition to a cell membrane, in a manner similar to that seen following binding to target cells that are surrounded by only a cell membrane; however, the response of the NK cells to the binding of C. neoformans is slower and possibly less efficient than the response after tumor cell binding.
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PMCID: PMC257867  PMID: 2004827
21.  Effects of Cryptococcus neoformans-specific suppressor T cells on the amplified anticryptococcal delayed-type hypersensitivity response. 
Infection and Immunity  1991;59(1):29-35.
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.
PMCID: PMC257701  PMID: 1824761
22.  Characterization of a cell population which amplifies the anticryptococcal delayed-type hypersensitivity response. 
Infection and Immunity  1990;58(2):393-398.
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.
PMCID: PMC258468  PMID: 2105273
23.  Clearance of Cryptococcus neoformans from immunologically suppressed mice. 
Infection and Immunity  1989;57(7):1946-1952.
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.
PMCID: PMC313825  PMID: 2499541
24.  Effects of cyclosporin A on the cells responsible for the anticryptococcal cell-mediated immune response and its regulation. 
Infection and Immunity  1989;57(4):1158-1164.
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.
PMCID: PMC313245  PMID: 2522419
25.  Characterization of an in vitro-stimulated, Cryptococcus neoformans-specific second-order suppressor T cell and its precursor. 
Infection and Immunity  1988;56(5):1267-1272.
Using a cryptococcal culture filtrate antigen (CneF) in a murine model, we have demonstrated previously that a cascade of Cryptococcus neoformans-specific suppressor T cells and soluble factors function in suppressing the cryptococcal delayed-type hypersensitivity (DTH) response. In addition, we have successfully hybridized the C. neoformans-specific, first-order T-suppressor (Ts1) cell and have established that the culture supernatant (hTsF1) from this hybridoma induces second-order T-suppressor (Ts2) cells in vivo. Here we report the in vitro induction of expression-phase suppressor cells. The suppressor cells were induced by culturing nylon wool-nonadherent splenic cells from naive mice with hTsF1 in the absence of CneF. Nylon wool-nonadherent splenic cells similarly cultured with supernatants from the BW5147 thymoma cells, the fusion partners of the hybridoma, did not significantly suppress the cryptococcal DTH response. The suppressor cells were designated Ts2 cells based on their similarities in function, specificity, and phenotype, i.e. L3T4-, Lyt-2+, and I-J+, to the in vivo-induced Ts2 cells. By employing the in vitro culture technique, we demonstrated that the precursors of the functional Ts2 cells were L3T4- Lyt-1-2+ I-J- cells. The induction of Ts2 cells was not associated with [3H]thymidine incorporation; therefore, we concluded that hTsF1 induces the Lyt-2+ I-J- cells to differentiate into Lyt-2+ I-J+ functional Ts2 cells without a significant amount of proliferation. From the results of this study, a better understanding of the processes involved in the regulation of the DTH response to CneF was achieved. The in vitro culture technique will allow for further detailed studies of the interactions between the various cell populations and the Ts1 cell-derived soluble factor during the induction of Ts2 cells.
PMCID: PMC259805  PMID: 2965681

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