Cd20 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.

The following mAbs were used for staining and flow cytometric measurements: CD3-VioGreen (BW264/56), CD4-allophycocyanin-Vio770 (VIT4), CD8-PE-Vio770 (BW135/80), CD14-peridinin chlorophyll protein (PerCP) (TÜK4), CD20-PerCP (LT20), CD27-FITC (M-T271), CD28-FITC (15E8), CD45RA-VioBlue (T6D11), CD45RO-FITC (UCHL1), CD62L-FITC (145/15), CD107a-PE (1D4B), CD127-FITC (MB15-18C9), CD137-allophycocyanin, CD137-PE (4B4-1), CD154-VioBlue (5C8), CD178-PE (NOK-1), CD197 (CCR7)-allophycocyanin, CD197 (CCR7)-PE (150503), CD279-allophycocyanin (PD1.3.1.3), anti-IL-2-PE-Vio770 (N7.48A), anti-IL-4-PE (7A3-3), anti-IL-17-FITC (CZ8-23G1), anti-IFN-γ-FITC, anti-IFN-γ-PE-Vio770 (45-15), anti-TNF-α-PE, and anti-TNF-α-VioBlue (cA2) (all Miltenyi Biotec).
Soluble biotinylated pHLA-A*0201 molecules loaded with WT1₃₇ (VLDFAPPGA), WT1₁₂₆ (RMFPNAPYL), WT1₁₈₇ (SLGEQQYSV), WT1₂₃₅ (CMTWNQMNL), or cytomegalovirus (CMV) pp65₄₉₅ (NLVPMVATV) were produced as described previously.14 (link) Tetramerization was achieved by binding to streptavidin-PE or streptavidin-allophycocyanin (both BioLegend, San Diego, CA). In each case, 2·5 × 10⁶ cells were stained with 1 µg/ml tetramer for 45 min at 4°C.
Data were acquired using a MACSQuant flow cytometer and analysed with MACSQuantify software (both Miltenyi Biotec).

K562-luc2 cells were thawed from cryopreservation 48h before tumor injections and maintained at 37°C, 5% CO₂ in Iscove’s DMEM supplemented with 10% FBS and 8µg/mL blasticidin for 2 days. On the day of tumor injections, K562-luc2 cells were removed from culture, washed three times with sterile PBS, and resuspended at a concentration of 1x10⁶ cells/200µL in sterile saline. Cryopreserved PBMCs were thawed in a 37°C water bath for two minutes or until a small ball of ice remained in solution. PBMCs were resuspended in 5mL RPMI + 10% FBS. A small aliquot (50 µL) was taken, and cells were labeled with monoclonal antibodies (CD8-VioBlue, CD14-VioGreen, CD3-FITC, CD4-PE, CD20-PerCP, CD45-APC, CD56-APC-Vio770 (Miltenyi Biotec) for surface staining prior to injection. The remaining cells were supplemented with IL-15 (0.1mg/mL) and incubated for 1 hour at 37°C, 5% CO₂ to improve activation and recovery of NK cells functions. After incubation, PBMCs were washed three times with PBS to remove all RPMI media and then resuspended at a concentration of 10x10⁶ PBMCs/200µL sterile filtered saline for injections.