Cd14 percp

Up to 4 biopsies per patient were pooled and digested in 1 mL of Hanks’ balanced salt solution Ca²⁺Mg²⁺ (Gibco) supplemented with 0.5% (v/v) human AB-serum, 100 IU ml⁻¹ of penicillin, 100 µg ml⁻¹ of streptomycin, 0.25 µg ml⁻¹ of amphotericin B, 10 IU ml⁻¹ of DNase I (CellSystems) and 1 mg ml⁻¹ of collagenase type CLS IV (Biochrom) for 30 min at 37 °C while shaking. Cells were filtered through a 40-µm cell strainer to remove aggregates and stained with fluorochrome-conjugated antibodies to CD4-VioBlue, CD3-PE, CD8-PerCP, CD14-PerCP, CD20-PerCP, CD45RO-APC and CCR7-PE-Vio770 (all Miltenyi Biotec). Each 200 CD3⁺CD4⁺CD45RO⁺ cells were sorted in multiple wells of a 96-well plate containing irradiated allogeneic feeder cells in TexMACS medium, supplemented with 5% (v/v) human AB-serum, 200 U ml⁻¹ of IL-2 and 100 IU ml⁻¹ of penicillin, 100 µg ml⁻¹ of streptomycin, 0.25 µg ml⁻¹ of amphotericin B at a density of 2 × 10⁵ cells cm⁻². Cells were polyclonally expanded for 4 weeks in the presence of 30 ng ml⁻¹ of anti-CD3 (OKT-3; Miltenyi Biotec). Restimulation with different fungal lysates was performed as described above. To calculate the frequencies of reactive T cells, the mean values were calculated for each antigen and divided by the number of input wells.

Human monocytes were isolated from mononuclear fractions of peripheral human blood by monocyte enrichment with (RossetteSep, Cat. no. 15028) for 20 min, then monocyte separation carried out by density Ficoll (GE Healthcare, Cat. no. 17-1440-03). Cells were cultured in the presence of GM-CSF (1000 unit/ml, Gemini Bio-Product, Cat. no. 300-124P) and IL-4 (1000 unit/ml, Gemini Bio-Product, Cat. no. 300-154P) at a concentration (3–4 × 105 cells/ml) for 5-6 days. Flow cytometry analyses were carried out to verify the immature DC phenotype (CD1a+, CD83−, CD14−, DC-SIGN+). Cell surface markers of DCs were evaluated by four-color immunofluorescence staining with the following antibodies: CD1a-PE (Miltenyi, Cat. no. 120-000-889), DC-SIGN-FITC (Miltenyi, Cat. no. 130-092-873), CD14-PerCP (Miltenyi, Cat. no. 130-094-969) and CD83-APC (Miltenyi, Cat. no. 130-094-186).

Intracellular cytokine staining was used to assess IFN-α-producing pDCs, monocytes and mDCs after RV16 (multiplicity of infection of 1) stimulation. PBMCs (2 × 10⁶) per well were seeded in a 96-well U-bottom plate and stimulated with or without RV16 at 37 °C with 5% CO₂ for 18 h, and further incubated with Brefeldin A (eBioscience, San Diego, CA, USA) for 4 h. Cells were washed with FACs buffer (1% heat-inactivated fetal calf serum in phosphate-buffered saline; fetal calf serum; Bovogen Biologicals, Keilor East, VIC, Australia) and incubated with normal goat IgG (Sigma-Aldrich) at 4 °C for 15 min to block non-specific Fc binding. The cells then were surface stained with CD303-PE, CD14-PerCP and CD1c-FITC (Miltenyi Biotec, Bergisch Gladbach, Germany) for 30 min at 4 °C, then fixed and permeabilized before antigen-presenting cell conjugated anti-IFN-α (Miltenyi Biotec) intracellular staining for 30 min at 4 °C. The cells were then washed twice with the FACs buffer, finally fixed in 0.5% paraformaldehyde before analysis. A total of ~500 000 gated events per sample were collected using FACS Canto (BD Biosciences, Franklin Lakes, NJ, USA), and the results were analyzed using the FlowJo Tree Star software (version 7.6.1; Flowjo LLC, Ashland, OR, USA). Unstimulated background was subtracted from the data.