Cd4 vioblue

For the analysis of DC surface molecules, DCs were stained with APC-conjugated human anti-CD80, PEVio770-CD83, FITC-CD86, PE-CD14, and VioBlue-HLA-DR monoclonal antibodies (Miltenyi Biotech, Bergisch Gladbach, Germany). T cell surface molecules were analyzed with APC-conjugated human anti-CD8 and VioBlue-CD4 (Miltenyi Biotech, Germany), while proliferation was measured with CFSE. In order to avoid unspecific antibody binding, all cytometry samples were previously blocked using FcR Blocking Reagent (Miltenyi Biotech, Germany). Flow cytometry was conducted on a MACSQuant cytometer, and data analysis was performed using the MACSQuantify software v2.13 (Miltenyi Biotech, Germany). The gating strategy is presented in Figure 3 and Figure 5.

Cells were stained according to standard procedures using the following antibodies (clone, manufacturer): PerCP/APC-CD3 (SK7, BD), Vioblue-CD4 (VIT4, Miltenyi Biotec Germany), APC-H7-CD8α (SK1, BD), FITC/APC-CD62L (LT-TD180, ImmunoTools), APC-CD25 (2A3, BD Pharmingen), PE-CF594-A-CCR7 (150503, BD), CD45RO, PE-Cy7-CD45RA (H/100, BD), CD27, UV1-A-CD25, APC-CD69 (FN50, Biolegend), CD276, UV3-CD28, CD95. Intracellular fixation/permeabilization kit (eBioscience) and Brilliant-Violet 785-TNF-α (MAb11), PE-IFN-γ (B27) were used for intracellular cytokine staining according to the manufacturer’s instructions. Dead cells were excluded via Alexa Fluor 350 (Invitrogen) or via Zombie Aqua™ (Biolegend). PBMCs were pretreated with FcR Blocking Reagent (Miltenyi Biotec) according to the manufacturer’s recommendations. Samples were analyzed on a LSR II or FACS Canto II with FACS Diva software (BD Biosciences).

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.