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The role of nitric oxide in resistance to cryptococcal infection was investigated. Mice deficient in inducible nitric oxide synthase (INOS) did not survive a primary intratracheal infection as did INOS-replete control mice. Despite adequate recruitment of host cells and generation of interferon (IFN)-γ and tumor necrosis factor (TNF)-α at the site of infection, INOS-deficient mice failed to clear yeast from their lungs by five weeks of infection, in contrast to wild-type mice. INOS-deficient mice also had higher yeast brain burdens than did control mice after a primary intracerebral infection. Therefore, generation of nitric oxide is required for resistance to primary cryptococcal infection. However, INOS-deficient mice vaccinated subcutaneously and rechallenged intravenously had lung and brain yeast burdens equivalent to those of vaccinated controls, and therefore expressed effective acquired immunity to Cryptococcus neoformans. Cells harvested from infected INOS-deficient mice by bronchoalveolar lavage acted as anti-cryptococcal effectors in vitroat an effector:target ratio of 100:1, provided IFN-g was present, but did not inhibit yeast proliferation at a 10:1 effector:target ratio as cells from wild-type mice did. Therefore, INOS activity is important for anti-cryptococcal function of effectors of immunity during the primary response, but not for the generation or expression of secondary immunity to C. neoformans.

Although naive C.B-17 and BALB/cBy mice die of meningoencephalitis within 5 weeks of intravenous infection with an opportunistic strain of Cryptococcus neoformans, immunized mice express an acquired, CD4+ T-cell-dependent immunity and survive an intravenous infection. Infusion of lymphocytes from immune mice into severe combined immunodeficiency (SCID) mice renders these mice more resistant to cryptococcal brain infection than uninfused controls. We have investigated the role of gamma interferon (IFN-gamma) and tumor necrosis factor (TNF) in acquired resistance to C. neoformans. Neutralization of either IFN-gamma or TNF impaired resistance of immune BALB/cBy or C.B-17 mice to cryptococci. At 10 days postinfection, there were approximately 10 times as many yeast cells in the brains of mice treated with either anticytokine antibody as in the brains of mice treated with control antibody. Simultaneous neutralization of IFN-gamma and TNF further exacerbated infection. Neutralization of IFN-gamma or TNF also impaired resistance in immune lymphocyte-infused SCID mice, resulting in significantly higher yeast burdens in brains of cytokine-neutralized mice than in brains of controls. Concurrent neutralization of IFN-gamma and TNF rendered SCID recipients of immune cells equivalent to uninfused SCID mice with respect both to brain yeast burdens at 10 days and to survival. Anti-TNF treatment alone also curtailed survival. Histological examination of the brains of cytokine-neutralized mice revealed deficiencies in ability to focus inflammatory cells at brain lesions. These data demonstrate that both IFN-gamma and TNF are important mediators of acquired resistance to cryptococcal meningoencephalitis.

It is often stated that impaired immune functions in the aged underlie their greater susceptibility to infections. Indeed, in many experimental settings, T-cell responses in aged mice have been shown to be deficient compared with those from young adults. Nonetheless, there are very few examples where a greater susceptibility to infection in aged mice has been demonstrated to result from impaired T-cell function. The clinical importance of understanding the basis for increased susceptibility to infection that accompanies advanced age dictates a need for experimental models with which to study the effect that aging has on immunological resistance to infection. This study was undertaken to investigate whether aged mice were less resistant than young adult control mice to infection with the fungus Cryptococcus neoformans. After a primary intravenous challenge with yeast, aged mice died sooner and developed higher organ burdens of yeast than did young adults. Deficient in vitro responses were observed in T cells from aged mice; however, greater susceptibility to intravenous infection appeared not to result from less effective T-cell-dependent resistance in vivo. In fact, T-cell-replete aged mice were more susceptible to intravenous cryptococcal infection than were T-cell-depleted young adults. Furthermore, aged mice were as resistant to primary pulmonary challenge with Cryptococcus as were young adults. Similarly, vaccinated aged mice were as resistant to rechallenge as were young adult counterparts. Therefore, despite demonstrably deficient in vitro responses of T cells from aged mice, their T-cell-dependent resistance to C. neoformans is as effective as that of young adults.

Although Cryptococcus neoformans causes serious infections in AIDS patients, cryptococcosis in immunologically immature infants, as in immunocompetent adults, is rare. To investigate the resistance of neonates to C. neoformans and to determine whether they could be efficiently vaccinated as neonates against challenge with the yeast as adults, the course of infection was monitored in the lungs of mice infected intranasally with yeast cells. Neonates were less able than adults to reduce yeast burdens less than 24 h postinoculation and less able to control the progressive growth of yeast over several weeks. However, in both neonates and adults, yeasts were substantially eradicated by 6 to 8 weeks after infection. Numbers of all classes of leukocytes recovered from lung lavages of infected neonates and adults were similar. Significant differences appeared only on day 14, when neonates had more neutrophils and adults had significantly more CD4+ CD45RB cells with low fluorescence intensity. When vaccinated neonates were rechallenged after reaching adulthood, they expressed resistance to C. neoformans as effectively as did mice immunized as adults and survived an intravenous challenge that was lethal for unimmunized controls. Thus, exposure of neonatal mice to viable C. neoformans yeast, which persists in the lungs for many weeks, does not result in immunological tolerance to a yeast challenge in adult mice, as predicted by immunological dogma, but instead immunizes them. Therefore, even in immunologically immature individuals, the immune system serves to protect against pathogens rather than simply to distinguish self from nonself.

The role of B cells in immunity to Cryptococcus neoformans was investigated. Genetically targeted, B-cell-deficient mice (mu Mt) examined at various times after intravenous infection with C. neoformans 184 had lung and brain yeast burdens that were equivalent to tissue burdens in control B-cell-sufficient mice. Both B-cell-deficient and B-cell-sufficient control mice were effectively vaccinated by a sublethal intratracheal instillation of strain 184 yeast against a systemic infection with the C. neoformans strain carrying ura5; vaccinated control and vaccinated B-cell-deficient mice had equivalent brain and lung burdens of the ura5 strain 10 days after intravenous rechallenge. Additionally, B-cell-deficient and B-cell-sufficient vaccinated mice survived an intravenous rechallenge with a dose of yeast cells which is normally lethal for unimmunized mice. In further studies of the role of B cells in murine cryptococcosis, SCID mice were reconstituted with lymphocytes from B-cell-deficient and B-cell-sufficient mice. SCID mice reconstituted with lymphocytes from vaccinated B-cell-deficient animals failed to express effective adoptive immunity to C. neoformans brain infection. In contrast, SCID mice reconstituted with lymphocytes from vaccinated B-cell-sufficient mice had 10-fold fewer yeast cells in their brains than did uninfused SCID controls. However, SCID mice given lymphocytes from B-cell-deficient immune donors had fewer yeast cells in their lungs than did uninfused controls. Fewer CD4+ lymphocytes were recovered at 7 and 11 days after infection from the peripheral blood and spleens of SCID mice reconstituted with lymphocyte suspensions from B-cell-deficient animals than from the peripheral blood and spleens of SCID mice reconstituted with suspensions from B-cell-sufficient control donors. These data suggest that B cells can play an important role in host defense against Cryptococcus in the brain under conditions in which T-cell-mediated immunity is impaired.

We have studied the resistance of Toxoplasma gondii-infected mice to subsequent infection with Cryptococcus neoformans. Mice infected with the moderately virulent ME49 strain of T. gondii are resistant to proliferation of yeast cells in their brains after intravenous inoculation of the serotype A C. neoformans strain 184. The resistance serves to limit proliferation of yeast cells that colonize the brain. Maximal levels of resistance correlate not with maximal systemic specific anti-Toxoplasma resistance but rather with high levels of inflammatory response, presumably to parasites released from cysts in the brain. Resistance is localized, as mice infected with ME49 show only limited resistance in their lungs after intratracheal instillation of yeast cells, but there is substantial protection against development of cerebral cryptococcosis.

Although naive C.B-17 and BALB/cBy mice die of meningoencephalitis within 5 weeks of intravenous infection with an opportunistic strain of Cryptococcus neoformans, immunized mice express an acquired, CD4+ T-cell-dependent immunity and survive an intravenous infection. Infusion of lymphocytes from immune mice into severe combined immunodeficiency (SCID) mice renders these mice more resistant to cryptococcal brain infection than uninfused controls. We have investigated the role of gamma interferon (IFN-gamma) and tumor necrosis factor (TNF) in acquired resistance to C. neoformans. Neutralization of either IFN-gamma or TNF impaired resistance of immune BALB/cBy or C.B-17 mice to cryptococci. At 10 days postinfection, there were approximately 10 times as many yeast cells in the brains of mice treated with either anticytokine antibody as in the brains of mice treated with control antibody. Simultaneous neutralization of IFN-gamma and TNF further exacerbated infection. Neutralization of IFN-gamma or TNF also impaired resistance in immune lymphocyte-infused SCID mice, resulting in significantly higher yeast burdens in brains of cytokine-neutralized mice than in brains of controls. Concurrent neutralization of IFN-gamma and TNF rendered SCID recipients of immune cells equivalent to uninfused SCID mice with respect both to brain yeast burdens at 10 days and to survival. Anti-TNF treatment alone also curtailed survival. Histological examination of the brains of cytokine-neutralized mice revealed deficiencies in ability to focus inflammatory cells at brain lesions. These data demonstrate that both IFN-gamma and TNF are important mediators of acquired resistance to cryptococcal meningoencephalitis.

The role of B cells in immunity to Cryptococcus neoformans was investigated. Genetically targeted, B-cell-deficient mice (mu Mt) examined at various times after intravenous infection with C. neoformans 184 had lung and brain yeast burdens that were equivalent to tissue burdens in control B-cell-sufficient mice. Both B-cell-deficient and B-cell-sufficient control mice were effectively vaccinated by a sublethal intratracheal instillation of strain 184 yeast against a systemic infection with the C. neoformans strain carrying ura5; vaccinated control and vaccinated B-cell-deficient mice had equivalent brain and lung burdens of the ura5 strain 10 days after intravenous rechallenge. Additionally, B-cell-deficient and B-cell-sufficient vaccinated mice survived an intravenous rechallenge with a dose of yeast cells which is normally lethal for unimmunized mice. In further studies of the role of B cells in murine cryptococcosis, SCID mice were reconstituted with lymphocytes from B-cell-deficient and B-cell-sufficient mice. SCID mice reconstituted with lymphocytes from vaccinated B-cell-deficient animals failed to express effective adoptive immunity to C. neoformans brain infection. In contrast, SCID mice reconstituted with lymphocytes from vaccinated B-cell-sufficient mice had 10-fold fewer yeast cells in their brains than did uninfused SCID controls. However, SCID mice given lymphocytes from B-cell-deficient immune donors had fewer yeast cells in their lungs than did uninfused controls. Fewer CD4+ lymphocytes were recovered at 7 and 11 days after infection from the peripheral blood and spleens of SCID mice reconstituted with lymphocyte suspensions from B-cell-deficient animals than from the peripheral blood and spleens of SCID mice reconstituted with suspensions from B-cell-sufficient control donors. These data suggest that B cells can play an important role in host defense against Cryptococcus in the brain under conditions in which T-cell-mediated immunity is impaired.

We have studied the resistance of Toxoplasma gondii-infected mice to subsequent infection with Cryptococcus neoformans. Mice infected with the moderately virulent ME49 strain of T. gondii are resistant to proliferation of yeast cells in their brains after intravenous inoculation of the serotype A C. neoformans strain 184. The resistance serves to limit proliferation of yeast cells that colonize the brain. Maximal levels of resistance correlate not with maximal systemic specific anti-Toxoplasma resistance but rather with high levels of inflammatory response, presumably to parasites released from cysts in the brain. Resistance is localized, as mice infected with ME49 show only limited resistance in their lungs after intratracheal instillation of yeast cells, but there is substantial protection against development of cerebral cryptococcosis.

Although Cryptococcus neoformans causes serious infections in AIDS patients, cryptococcosis in immunologically immature infants, as in immunocompetent adults, is rare. To investigate the resistance of neonates to C. neoformans and to determine whether they could be efficiently vaccinated as neonates against challenge with the yeast as adults, the course of infection was monitored in the lungs of mice infected intranasally with yeast cells. Neonates were less able than adults to reduce yeast burdens less than 24 h postinoculation and less able to control the progressive growth of yeast over several weeks. However, in both neonates and adults, yeasts were substantially eradicated by 6 to 8 weeks after infection. Numbers of all classes of leukocytes recovered from lung lavages of infected neonates and adults were similar. Significant differences appeared only on day 14, when neonates had more neutrophils and adults had significantly more CD4+ CD45RB cells with low fluorescence intensity. When vaccinated neonates were rechallenged after reaching adulthood, they expressed resistance to C. neoformans as effectively as did mice immunized as adults and survived an intravenous challenge that was lethal for unimmunized controls. Thus, exposure of neonatal mice to viable C. neoformans yeast, which persists in the lungs for many weeks, does not result in immunological tolerance to a yeast challenge in adult mice, as predicted by immunological dogma, but instead immunizes them. Therefore, even in immunologically immature individuals, the immune system serves to protect against pathogens rather than simply to distinguish self from nonself.

The role of nitric oxide in resistance to cryptococcal infection was investigated. Mice deficient in inducible nitric oxide synthase (INOS) did not survive a primary intratracheal infection as did INOS-replete control mice. Despite adequate recruitment of host cells and generation of interferon (IFN)-? and tumor necrosis factor (TNF)-\ga at the site of infection, INOS-deficient mice failed to clear yeast from their lungs by five weeks of infection, in contrast to wild-type mice. INOS-deficient mice also had higher yeast brain burdens than did control mice after a primary intracerebral infection. Therefore, generation of nitric oxide is required for resistance to primary cryptococcal infection. However, INOS-deficient mice vaccinated subcutaneously and rechallenged intravenously had lung and brain yeast burdens equivalent to those of vaccinated controls, and therefore expressed effective acquired immunity to Cryptococcus neoformans. Cells harvested from infected INOS-deficient mice by bronchoalveolar lavage acted as anti-cryptococcal effectors in vitro at an effector:target ratio of 100:1, provided IFN-g was present, but did not inhibit yeast proliferation at a 10:1 effector:target ratio as cells from wild-type mice did. Therefore, INOS activity is important for anti-cryptococcal function of effectors of immunity during the primary response, but not for the generation or expression of secondary immunity to C. neoformans.