Induction of T helper type 1 responses by a polysaccharide deacetylase from Cryptococcus neoformans

Abstract

A 25-kDa cryptococcal deacetylase (d25) was found here to induce cell proliferation, as well as secretion of interleukin 2 and gamma interferon, but not interleukin 4, in spleen cells from d25-immunized or Cryptococcus neoformans-infected mice. The gamma interferon, but not the interleukin 2, response was required for the protective activities of d25 immunization in a murine cryptococcosis model.

FIG. 1.

Proliferation of splenocytes from C. neoformans-infected (left) or rd25-immunized (right) mice after rd25 stimulation. Upper panels, spleens were collected at 14 days after immunization or infection, and splenocytes (6 × 10⁵/well) were cultured for 72 h in the presence of various concentrations of rd25 before the addition of [³H]TdR. Eighteen hours later the cells were collected onto filter paper, and the dried filters were counted in a liquid scintillation β-counter. Middle panels, spleens were collected at 14 days after immunization or infection, and various numbers of splenocytes were cultured in the presence of rd25 (10 μg/ml). Lower panels, spleens were collected at 7, 14, and 28 days after immunization or infection, and splenocytes (6 × 10⁵/well) were cultured in the presence of rd25 (10 μg/ml). Negative controls consisted of splenocytes from mice injected with PBS-CFA (white bars). Values represent the means ± SEM from three different experiments, each performed in triplicate. Asterisk, P < 0.05 relative to results for cells from PBS-CFA-injected mice by one-way analysis of variance and the Student-Keuls-Newman test.

FIG. 2.

rd25-induced IFN-γ and IL-2 production in cultures of splenocytes from rd25-immunized or C. neoformans-infected mice. Spleens were collected at 7, 14, and 28 days after immunization or infection (see the legend to Fig. 1), and splenocytes (5 × 10⁶/ml) were stimulated with CneF (10 μg/ml), rd25 (10 μg/ml), or PBS. Cytokine levels were assayed at 24 h (IL-2) or 48 h (IFN-γ) after the addition of the stimuli. Data are expressed as means ± SEM for three experiments, each performed in triplicate. Asterisk, P < 0.05, relative to results with cells from PBS-CFA-injected mice by one-way analysis of variance and the Student-Keuls-Newman test.

FIG. 3.

rd25-induced IFN-γ and IL-2 production in cultures of CD4⁺ or CD8⁺ cells from C. neoformans-infected mice. Spleens were collected at 14 days after infection, and CD4⁺ or CD8⁺ T cells (5 × 10⁶/ml), separated by means of a panning procedure (16), were cultured in the presence of splenic adherent cells (5 × 10⁵/ml) and CneF (10 μg/ml), d25 (10 μg/ml), or PBS. Cytokine levels were assayed as indicated in the legend to Fig. 2. Data are expressed as means ± SEM for three experiments, each performed in triplicate. Asterisk, P < 0.05, relative to results with cells from PBS-CFA-injected mice by one-way analysis of variance and the Student-Keuls-Newman test.

FIG. 4.

Role of IL-12 and TNF-α in the development of cytokine responses to d25. Spleens were collected at 7 and 14 days after immunization with rd25 (100 μg per mouse in CFA), and cultures of splenocytes (5 × 10⁶/ml) were stimulated with rd25 (10 μg/ml). Cytokine levels were assayed at 24 h (IL-2) or 48 h (IFN-γ) after the addition of the stimulus. Experimental groups included immunized mice that were injected i.p. with 0.2 mg of goat polyclonal anti-mouse TNF-α IgG, goat anti-IL-12 IgG, or normal goat IgG at 1 h prior to infection. Data are expressed as means ± SEM for three experiments, each performed in triplicate. Asterisk, significantly (P < 0.05) different from normal IgG control group by one-way analysis of variance and the Student-Keuls-Newman test.

FIG. 5.

Role of IL-2 and IFN-γ in the protective effects of d25 immunization. Panels A and B show the effects of, respectively, IL-2 blockade and IFN-γ deficiency on C. neoformans-induced lethality in d25-immunized and unimmunized mice. (A) Eight-week-old female BALB/c mice were immunized s.c. with rd25 (100 μg per mouse in CFA) or injected with PBS-CFA. Mice belonging to each of these two subgroups were injected i.p. at the time of immunization and 15 days later with 0.5 mg of goat polyclonal anti-mouse IL-2 IgG or normal goat IgG. All mice were challenged i.v. with 7.5 × 10³ viable C. neoformans cells (strain H99) at 1 week after immunization, and lethality was observed daily for 30 days. (B) Eight-week-old female IFN-γ^−/− mice on a C57BL/6 background and control WT C57BL/6 mice were immunized with d25 in CFA or injected with PBS-CFA as described above. After 1 week, mice were challenged i.v. with 3 × 10⁴ viable C. neoformans cells (strain H99), and lethality was observed daily for 30 days. Survival data were analyzed with Kaplan-Meier survival plots followed by the log rank test (JMP Software; SAS Institute, Cary, N.C.) on an Apple Macintosh computer.