Protective immunity against experimental pulmonary cryptococcosis in T cell-depleted mice

Abstract

Individuals with defects in T cell-mediated immunity (CMI) are highly susceptible to infection with Cryptococcus neoformans. The purpose of these studies was to determine if protection against experimental pulmonary cryptococcosis can be generated in T cell-deficient hosts. BALB/c mice were depleted of CD4⁺and/or CD8⁺ T cells or given an isotype control antibody prior to vaccination with a C. neoformans strain, designated H99γ, previously shown to induce protection against C. neoformans infection in immunocompetent mice. Mice depleted of CD4⁺ or CD8⁺ T cells, but not both subsets, survived an acute pulmonary infection with C. neoformans strain H99γ and a subsequent second challenge with wild-type C. neoformans strain H99. We observed a significant increase in the percentage of CD4⁺ and CD8⁺ T cells expressing the activation marker CD69 in the lungs of mice immunized with C. neoformans strain H99γ prior to a secondary challenge with wild-type cryptococci. CD4⁺ T cells within the lungs of immunized mice also appeared to acquire a predominantly activated effector memory cell phenotype (CD69⁺ CD44⁺ CCR7⁻ CD45RB⁻ CD62L⁻) following a second pulmonary challenge with wild-type C. neoformans, compared to CD4⁺ T cells from naïve mice. Lastly, immunization of immunocompetent mice with C. neoformans strain H99γ prior to depletion of CD4⁺ and/or CD8⁺ T cells resulted in significant protection against a second challenge with wild-type C. neoformans. Our studies demonstrate that protective immunity against pulmonary cryptococcosis can be generated in immunosuppressed hosts, thus supporting the development of cryptococcal vaccines.

Fig. 1.

Role of CD4⁺ T cells and CD8⁺ T cells during the induction of protective immunity by C. neoformans strain H99γ. (A) BALB/c mice were treated with isotype control antibodies or depleted of CD4⁺ and/or CD8⁺ T cells prior to inoculation with C. neoformans strain H99γ. Depletion was maintained throughout the observation period. The survival data shown are from one experiment using 10 mice per group. (B) Alternatively, BALB/c mice were treated with isotype control antibodies or depleted of CD4⁺ or CD8⁺ T cells prior to immunization with C. neoformans strain H99γ or HK C. neoformans (C.n.) and allowed 70 days to resolve the infection. Depletion was ceased at day 70, and mice were subsequently given a second intranasal challenge with wild-type C. neoformans strain H99. The survival data shown are from one experiment using 10 mice per group.

Fig. 2.

Activation marker expression on CD4⁺ and CD8⁺ T cells prior to and following a secondary challenge. BALB/c mice received an intranasal inoculation with either sterile PBS (white bars) or C. neoformans strain H99γ and were allowed 70 days to resolve the infection. C. neoformans strain H99γ-immunized mice were subsequently given a second intranasal challenge with sterile PBS (light gray bars) or C. neoformans strain H99 (dark gray bars), and the cell surface expression of CD3, CD4, CD8, CD25, and CD69 was determined by flow cytometry analysis of whole lung dispersions. Shown are cumulative data from four experiments using 5 mice per group. Results are expressed as means ± the standard errors of the means. Asterisks indicate where significant differences (P < 0.05) from naïve mice were observed.

Fig. 3.

Memory marker expression on CD4⁺ and CD8⁺ T cells prior to and following a secondary challenge. BALB/c mice received an intranasal inoculation with either sterile PBS (white bars) or C. neoformans strain H99γ and were allowed 70 days to resolve the infection. C. neoformans strain H99γ-immunized mice were subsequently given a second intranasal challenge with sterile PBS (light gray bars) or C. neoformans strain H99 (dark gray bars), and the expression of CD3, CD4, CD8, CD44, CD45B, CD62, and CCR7 was determined by flow cytometry analysis of whole lung digests. Shown are cumulative data from four experiments using 5 mice per group. Results are expressed as means ± the standard errors of the means. Asterisks indicate where significant differences (P < 0.05) from naïve mice were observed.

Fig. 4.

Role of CD4⁺ and CD8⁺ T cells during the efferent response to pulmonary C. neoformans infection. BALB/c mice received an initial immunization with HK C. neoformans (C.n.) or C. neoformans strain H99γ in 50 μl of sterile PBS and were allowed 70 days to resolve the infection. Mice were then treated with isotype control antibodies or depleted of CD4⁺ T cells and/or CD8⁺ T cells 48 h prior to a challenge with C. neoformans strain H99 in 50 μl of sterile PBS. Depletion was maintained throughout the observation period. (A) Lungs were excised on days 7 and 14 after a secondary inoculation, and the cryptococcal burden was quantified. Pulmonary fungal burden data are cumulative for three experiments using 5 mice per time point. Separate mice were used for each time point. Results are expressed as the mean log CFU per milliliter ± the standard error of the mean. Asterisks indicate where significant decreases (P < 0.05) from mice immunized with HK C. neoformans were observed. The symbol τ indicates where significant increases (P < 0.05) from immunocompetent mice immunized with C. neoformans strain H99γ were observed. (B) Alternatively, the survival of challenged mice was monitored twice daily and mice that appeared moribund or did not maintain normal habits (grooming) were sacrificed by CO₂ inhalation. The survival data shown are from one experiment using 10 mice per experimental group.