Cd3 pe

Nerve and peripheral blood samples were stained with pre-defined optimal concentration of antibodies and Dead cells stain dyes (LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, Life Technologies). The following anti mouse antibodies were used: CD45 APC-eFluor-780 conjugated (eBioscience) to select hematopoietic lineage cells; CD335 PE-eFluor 610 conjugated (eBioscience) characteristic of Natural Killer cells; CD3 PE conjugated (Miltenyi) to identified T lymphocytes; CD45R VioBlue conjugated (Miltenyi) to identified B lymphocytes; F4/80 PE Vio770 conjugated and MHC-II Fitc conjugated (Miltenyi) to select monocyte population. After 20 min of staining the cells were washed twice with Phosphate Buffered Saline (PBS) and then analyzed to flow cytometer equipped with 3 lasers (Cytoflex -Beckman Coulter). Cytoflorimetric analysis was performed with FlowJo software (FlowJo, LLC Company Ashland, OR, USA).

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.

Tumors were harvested, mechanically cut into small pieces, and then enzymatically treated with 1 mg/mL collagenase D (IV type, Clostridium histolyticum, Sigma) and 0.02 mg/mL DNase I (from bovine pancreas grade 2, Roche) at 37 °C for 30 min. The enzymatic reaction was stopped by adding EDTA (Sigma). The ensuing single-cell suspension was filtered by means of a 70-µm cell strainer and prepared for cytofluorimetric analysis. After Fc blocking, single-cell suspensions were stained with the following antibodies from Miltenyi Biotec: CD45-VioGreen (clone: REA737); NK1.1-APC (clone: PK136); CD11b-Vioblue (clone: REA592); CD3-PE (clone: REA641); CD69-PEVio770 (clone: REA937); Ly6C-PE (clone: 1G7.G10); CD4-PEVio770 (clone: REA604); CD8-Vioblue (clone: 53.6.7); and F4/80-PercPVio700 (clone: REA126). Subsequently, cell viability was determined by LIVE/Dead-633 nm (L10120) staining according to the manufacturer’s instructions (Invitrogen); negative cells were considered viable. Doublet exclusion and gating on live CD45⁺ cells were performed. The following subpopulations of CD45⁺ were identified: CD11b⁺ Ly6C⁺ (monocytes), CD11b⁺ F4/80⁺ (macrophages), CD3⁺CD4⁺ and CD3⁺C8⁺ (T cells) and NK1.1⁺CD3⁻ (NK cells). Cells were analyzed on a MACSQuant16 (Miltenyi) and analyzed with FlowJo software (RRID:SCR_008520, Treestar) [28 (link)].