Immunomodulation with CD40 stimulation and interleukin-2 protects mice from disseminated cryptococcosis

Abstract

Cryptococcus neoformans is a ubiquitous fungus that can cause life-threatening infections during immunosuppressive states such as AIDS and after bone marrow transplantation. In this study we investigated the antifungal efficacy of an agonist antibody to CD40, an important costimulator of immune function, in combination with interleukin 2 (IL-2) in a murine model of disseminated cryptococcosis. Only the combination of anti-CD40 and IL-2 significantly prolonged the survival time of infected mice. This protection was correlated with decreased yeast burdens in the brain and kidney. Increased immune cell populations in the spleens, as well as increased serum gamma interferon (IFN-gamma) and tumor necrosis factor alpha levels were observed in infected mice treated with anti-CD40 and IL-2. Further experiments with IFN-gamma knockout mice demonstrated that the protection induced by anti-CD40 and IL-2 treatment was dependent on IFN-gamma. Depletion of CD4+ T cells did not affect the increased serum IFN-gamma levels induced by anti-CD40 and IL-2 treatment and, importantly, did not affect the antifungal effect of combination therapy. These studies indicate that immunotherapy using anti-CD40 and IL-2 has therapeutic potential in augmenting host resistance to disseminated cryptococcosis and that IFN-gamma is essential for efficacy.

FIG. 1.

Anti-CD40 and IL-2 provide antifungal effects in mice previously infected with C. neoformans. One day after infection, C57BL/6 mice (n = 6 to 8/group) were treated with anti-CD40 (▴) or recombinant human IL-2 alone (▾) or in combination (×) or were given rat IgG and PBS as controls (▪). Anti-CD40 or isotype control rat IgG was administered at 100 μg/dose i.p. once a day for 4 days. IL-2 was administered at 500,000 IU/dose i.p. twice a day twice a week, for a total of eight injections. DPBS (vehicle control for IL-2, 0.2 ml/dose) was given on the same schedule as IL-2. The regimen was completed 10 days after C. neoformans infection. The combination of anti-CD40 and IL-2 resulted in significantly (by log rank test; *, P < 0.005) prolonged survival time compared with that for control mice or mice receiving anti-CD40 or IL-2 alone. Survival analysis was plotted according to the Kaplan-Meier method. Representative data from one of five similar experiments are shown, and statistical differences were determined with the log rank test.

FIG. 2.

Anti-CD40 and IL-2 treatment decreases the cryptococcal burdens in the brains and kidneys of mice previously infected with C. neoformans. One day following completion of treatment (day 11), three mice per group were sacrificed and quantitative organ cultures of brains (A) and kidneys (B) were prepared. Statistical differences were determined by one-way ANOVA with post hoc comparisons with the Tukey test. A P value of <0.05 is considered significant. Representative data from one of three similar experiments are shown. Error bars indicate standard deviations.

FIG. 3.

Effects of anti-CD40 and IL-2 treatment on immune cell parameters in the spleens of mice. Mice were i.v. injected with C. neoformans or PBS. One day following completion of treatments (day 11), three mice/group were sacrificed and flow cytometry was used to assess the effect of anti-CD40 and IL-2 on numbers of whole splenocytes (A), CD8⁺ T cells (B), CD4⁺ T cells (C), and dendritic cells (D) in the spleens. Three mice were analyzed per group in each experiment. The results are representative of three independent experiments. Statistical differences are indicated (one-way ANOVA with post hoc comparisons with Tukey test; P < 0.05). Error bars indicate standard deviations.

FIG. 4.

Effects of anti-CD40 and IL-2 treatment on anti-GXM-specific IgG levels in the sera of C. neoformans infected mice. One day following completion of treatments (day 11), mice were bled for sera. Anti-GXM IgG was assessed by ELISA. Optical densities (O.D.) at a 1/40 dilution of sera are shown. Three mice per group were analyzed in each experiment. The results are a compilation of two independent experiments (experiment 1, open symbols; experiment 2, closed symbols). Statistical differences are indicated (one-way ANOVA with post hoc comparison with Bonferroni test; *, P < 0.05).

FIG. 5.

Effects of anti-CD40 and IL-2 treatment on serum IFN-γ and TNF-α levels. Mice were injected i.v. with C. neoformans or PBS (Naive α-CD40/IL-2). One day following completion of anti-CD40 and IL-2 treatments (day 11), three mice per group were bled for sera. Serum IFN-γ (A) and TNF-α (B) levels were then tested by mouse IFN-γ and TNF-α ELISA. Representative data from four similar experiments are shown. Statistical differences are indicated (by one-way ANOVA with post hoc comparisons with Tukey test; P < 0.05). Error bars indicate standard deviations.

FIG. 6.

Requirement for IFN-γ in the antifungal effects of anti-CD40 and IL-2 treatment. C57BL/6J WT and GKO mice (eight mice per group) received either control injections or anti-CD40 and IL-2. The combination of anti-CD40 and IL-2 had no protective effects in GKO mice (▵ and ▴), while anti-CD40 and IL-2 prolonged the survival of WT mice (□ versus ▪, P < 0.0002). GKO mice were more susceptible to C. neoformans infection than were WT controls (▵ versus □, P < 0.0002). Survival analysis was plotted according to the Kaplan-Meier method, and statistical differences were determined with the log rank test.

FIG. 7.

CD4⁺ T cells are not required for the antifungal effect of anti-CD40 and IL-2 treatment. (A) C57BL/6 mice (eight mice per group) received either isotype control rat IgG or anti-CD4 antibody starting 1 day prior to C. neoformans injection and every 4 days during the anti-CD40 and IL-2 treatment. The combination of anti-CD40 and IL-2 treatments significantly prolonged the survival time of CD4⁺ T-cell-depleted mice with disseminated cryptococcosis (▵versus▴, P < 0.0001), as well as WT mice (□ versus ▪, P < 0.002), although WT treated mice survived significantly longer than CD4⁺ T-cell-depleted and treated mice (▪ versus ▴, P < 0.05). CD4⁺ T-cell-depleted mice were significantly more susceptible to disseminated cryptococcosis (▵ and □, P < 0.0001). Survival analysis was plotted according to the Kaplan-Meier method, and statistical differences were determined with the log rank test. Representative data from one of two similar experiments are shown. (B) Sera were taken 1 day after completion of anti-CD40 and IL-2 treatments (day 11), and IFN-γ levels were determined by ELISA. Anti-CD40 and IL-2 were able to increase serum IFN-γ levels with or without the presence of CD4⁺ T cells. Representative data from one of two similar experiments are shown, and statistical differences are indicated (one-way ANOVA with post hoc comparisons with Tukey test; P < 0.05). Error bars indicate standard deviations.