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

Abstract

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

FIG. 1

CD4⁺ but not CD8⁺ T lymphocytes are required for growth inhibition of C. neoformans in the brains of immune mice. Brains of immune mice depleted of CD4⁺ T lymphocytes contained significantly more cryptococcal CFU than those of immune mice and CD8-depleted immune mice. Depletion of specific T-cell subsets by antibodies was confirmed by flow cytometric analysis of spleen cells from the treated mice. The data are representative of one of three experiments that yielded similar results. Ab, antibody.

FIG. 2

CD4⁺ T lymphocytes are required for optimal leukocyte accumulation in C. neoformans-infected brains of immune mice. Brains and spinal cords of immune mice depleted of CD4⁺ T cells contained significantly fewer leukocytes 6 days after i.c. infection with C. neoformans than those of CD8⁺ T-cell-depleted immune mice and immune mice with both CD4⁺ and CD8⁺ T cells. Leukocytes from five or six mice from each group were obtained as described in Materials and Methods and counted. Depletion of specific T-cell subsets by antibodies was confirmed by flow cytometric analysis of spleen cells from treated mice. The experiment was repeated once, with similar results.

FIG. 3

IFN-γ is required for optimal host resistance in the CNS of immune mice. Cryptococcal CFU in brains of i.c. infected immune mice given neutralizing antibodies to IFN-γ were significantly higher than those in i.c. infected immune mice given no antibody or control antibody. Five mice were used per group.