Cd45ro apc

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.

Transduced cells were stained with fluorochrome-conjugated monoclonal antibodies for 15 min at 4°C. The cells were washed twice with 1x PBS supplemented with 1% FBS. For cell surface marker analysis, the following antibodies were used: CD56-PE (HCD56/Cat#130-114-551), CD4-FITC (OKT4/130-114-531), CD8-APC (SK1/130-110-679), CD45RO-APC (UCHL1/130-113-556), CD62L-FITC (DREG-56/130-112-077), CD25-APC (BC96/130-113-284) (Miltenyi Biotec, Bergisch Gladbach, Germany), PD1-FITC (EH12.2H7/329904), LAG-3-FITC (11C3C65/369308), TIM-3-APC (F38-2E2/345012), and CD3-PerCP (clone OKT3/317336) (BioLegend, San Diego, CA, USA). The memory phenotypes were defined as naïve (TN; CD3⁺CD45RO⁻CD62L⁺), effector memory (TEM; CD3⁺CD45RO⁺CD62L⁻), central memory (TCM; CD3⁺CD45RO⁺CD62L⁺), and terminal effector (TE; CD3⁺CD45RO⁻CD62L⁻) T cells. For transduction efficiency of transduced cells, AF647-conjugated anti-human IgG (H+L) (Jackson ImmunoResearch/109-607-003) was used to detect CAR expression. Flow cytometry was performed using a BD Accuri™ C6 Plus Flow Cytometer (BD Bioscience), and data were analyzed by the FlowJo V10.7.1 software (FlowJo).