Dendritic cell-based immunization ameliorates pulmonary infection with highly virulent Cryptococcus gattii

Abstract

Cryptococcosis due to a highly virulent fungus, Cryptococcus gattii, emerged as an infectious disease on Vancouver Island in Canada and surrounding areas in 1999, causing deaths among immunocompetent individuals. Previous studies indicated that C. gattii strain R265 isolated from the Canadian outbreak had immune avoidance or immune suppression capabilities. However, protective immunity against C. gattii has not been identified. In this study, we used a gain-of-function approach to investigate the protective immunity against C. gattii infection using a dendritic cell (DC)-based vaccine. Bone marrow-derived dendritic cells (BMDCs) efficiently engulfed acapsular C. gattii (Δcap60 strain), which resulted in their expression of costimulatory molecules and inflammatory cytokines. This was not observed for BMDCs that were cultured with encapsulated strains. When Δcap60 strain-pulsed BMDCs were transferred to mice prior to intratracheal R265 infection, significant amelioration of pathology, fungal burden, and the survival rate resulted compared with those in controls. Multinucleated giant cells (MGCs) that engulfed fungal cells were significantly increased in the lungs of immunized mice. Interleukin 17A (IL-17A)-, gamma interferon (IFN-γ)-, and tumor necrosis factor alpha (TNF-α)-producing lymphocytes were significantly increased in the spleens and lungs of immunized mice. The protective effect of this DC vaccine was significantly reduced in IFN-γ knockout mice. These results demonstrated that an increase in cytokine-producing lymphocytes and the development of MGCs that engulfed fungal cells were associated with the protection against pulmonary infection with highly virulent C. gattii and suggested that IFN-γ may have been an important mediator for this vaccine-induced protection.

FIG 1

The C. gattii acapsular Δcap60 strain efficiently activates BMDCs. (A) Capsule formation was assessed using the conventional India ink method. Parental strain PNG18, the Δcap60 strain, and the revertant (CAP60C) were grown in YPD medium at 30°C overnight. BMDCs were incubated with heat-killed C. gattii cells for 24 h, and the phagocytosis rate (B), costimulatory molecule expression (C), and cytokine production (D) were evaluated by fluorescence microscopy, flow cytometry, and ELISA, respectively. For flow cytometry analysis, gates were set for CD11c⁺ CD11b⁺ cells. Representative data (means ± SDs) from 2 or 3 independent experiments are shown. *, P < 0.05 versus PNG18 by unpaired t test; #, P < 0.05 versus PNG18 by Mann-Whitney U test.

FIG 2

Transferring CAP60Δ/DCs ameliorates pulmonary infection with highly virulent C. gattii strain R265. CAP60Δ/DCs or unloaded DCs were injected into tail veins of mice at 14 days and 1 day prior to intratracheal infection with 3 × 10³ CFU of strain R265. (A and B) Fungal burdens (n = 5) in the lungs were determined at day 14 (A) and day 3 (B). Pooled data from two independent experiments are plotted, and median values are shown by short horizontal lines. *, P < 0.05 by analysis of variance with Dunn's post hoc test (A) or P < 0.05 by Mann-Whitney U test (B). (C) Survival curves (n = 8) were compared by log rank test. Representative data from two independent experiments are shown. *, P < 0.01. Median survival times were 28 days in the case of nontransfer and 238 days in the case of CAP60Δ/DC transfer.

FIG 3

Multinucleated giant cells (MGCs) engulf fungal cells in the lungs of immunized mice. Lung sections obtained from three mice, at 13 days after infection with strain R265, were stained with hematoxylin-eosin (HE) or alcian blue (AB). C. gattii cells were stained with alcian blue. Each section was observed at magnifications of ×40 (A), ×200 (B), and ×1,000 (C).

FIG 4

Transferring CAP60Δ/DCs induces cytokine-producing CD4 T cells in spleen. Splenocytes were obtained from three mice at 14 days after infection and cultured with (+Ag) or without (−Ag) antigen and with or without heat-killed Δcap60 cells (MOI = 0.1) for 5 to 6 days. For flow cytometry analysis, gates were set for CD4⁺ cells. Representative flow cytometry profiles (A) and histograms for statistical analysis (B) are shown. Pooled data from two separate experiments were used to prepare histograms (means ± SEMs). *, P < 0.05 by analysis of variance with Dunnett's post hoc test.

FIG 5

Transferring CAP60Δ/DCs induces IL-17A-producing cells in lungs. Lung leukocytes were obtained from three mice at 1 day after infection and were stimulated with PMA and ionomycin for 3 h. For flow cytometry analysis, gates were set for CD3⁺ Thy1.2⁺ cells or CD3⁻ Thy1.2⁺ cells. Representative flow cytometry profiles (A) and histograms for statistical analysis (B) from two independent experiments are shown. Results in histograms are means ± SDs. *, P < 0.05 versus control by unpaired t test; #, P < 0.05 versus control by unpaired t test with Welch's correction.

FIG 6

DC-based vaccine amelioration of the fungal burden is partially reduced in IFN-γ KO mice. (A) Fungal burdens in the lungs of C57BL/6J (WT) mice and IFN-γ KO mice were determined on days 9 to 14 postinfection. Data from six independent experiments were combined. (B and C) Vaccination and infection were performed as described in the legend to Fig. 2, and the fungal burden in lungs was determined on day 14 postinfection. Data from three independent experiments were combined. Vaccine effects between WT and KO mice were compared based on the reduced fungal burden rate (C). Panels A and B show dot plots with geometric means, and panel C shows means ± SEMs. *, P < 0.05 by Mann-Whitney U test.