For some comparative studies, similar cultures

were performed using unpulsed or C. neoformans-pulsed Mφ as APC. Furthermore, the production of IFN-γ, TNF-α, IL-4, IL-13 and IL-10 by purified T cells was measured in supernatants of 96-hr cultures using anti-(rat IFN-γ), anti-(rat-TNF-α), anti-(rat-IL-4), anti-(rat-IL-13) and anti-(rat-IL-10) CytoSets (BioSource), as described above. For intracellular cytokine and surface-marker staining, C. neoformans-primed CD4+ and CD8+ T cells (1 × 106 cells) were co-cultured with unpulsed or C. neoformans-pulsed eosinophils (2 × 105 cells), or in medium alone, for 4 days. Then, T cells were stimulated with phorbol 12-myristate 13-acetate (PMA; 50 ng per 2 × 106 this website cells), ionomycin (500 ng per 1 × 106 cells) and brefeldin A (5 ng/1 × 106 cells) for 3 hr at 37° under a 5% CO2 humidified atmosphere. For cell-surface staining, cells were incubated for 30 min at 4° in the dark with FITC-conjugated mouse mAb specific to rat CD4 (0·5 mg per 106 cells) or CD8 (0·5 mg per 106 cells). Dactolisib The cells were then washed twice (30 min each wash, at room temperature) with PBS containing 3% FCS and then fixed overnight at 4° in PBS containing 1% paraformaldehyde. Before intracellular cytokine staining, the cells were washed with PBS containing 0·5% saponin. Cells

in this buffer were incubated with phycoerythrin (PE)-conjugated mouse anti-(rat IFN-γ) (0·125–0·5 μg per 106 cells) and anti-(rat IL-4) (0·1–0·5 μg per 106 cells), or appropriate controls (corresponding to IgG-negative isotypes) in the dark for 30 min at room temperature. Etomidate The cells were washed twice with PBS containing 0·5% saponin and finally resuspended in 0·2 ml of flow wash.29 The immunofluorescently stained cells were analyzed

using a Becton Dickinson FACS Canto II flow cytometer (San José, CA). The percentage of double-positive labelled cells was determined using dot-plot graphs. Data were expressed as means + standard errors of the mean (SEM) and analyzed statistically using the Student’s t-test. Statistical significance was taken to be a P-value of < 0·05. All experiments were repeated and equivalent results were obtained. First, we evaluated the ability of eosinophils to phagocytose live yeasts of C. neoformans. Purified eosinophils were exposed to yeasts of C. neoformans that were either opsonized with the mAb 3C2 which binds specifically to C. neoformans glucuronoxylomannan, or non-opsonized, at a ratio of 1:1, in the presence or absence of GM-CSF (5 ng/ml) for 24 hr. Eosinophils incubated with opsonized C. neoformans, non-opsonized C. neoformans or medium alone, showed more than 85% viability, as determined using the Trypan Blue dye-exclusion test, and < 10% apoptosis when tested by propidium iodide staining and flow cytometry (data not shown).