Cd4 apc

Cells were stained according to the manufacturer’s recommendations with the following antibodies alone or in varying combinations, depending on the experiment: CD14-FITC, CD4-APC and CD45RA-PE (all Immunotools, Friesoythe, Germany), CD40-PE (AbD Serotec, Kidlington, UK), CD86-V450, HLA-DR-PerCPCy5.5, CD1a-PE and CD3-APC/Cy7 (all BD). For surface marker staining, PBS containing 1% FCS and 5mM EDTA (Sigma Aldrich) was used as staining buffer. For intracellular cytokine staining with IFNγ-PerCPCy5.5 (eBioscience), IL17A-PE and IL10-Brilliant Violet 421 (both BioLegend), monensin solution (BioLegend) was added to cell cultures for 6 hours before harvesting. Once harvested, cells were fixed and permeabilized using the fixation and permeabilization buffer set from BioLegend. Staining with FoxP3-Alexa Fluor 647 (BD) was performed with FoxP3 staining buffer set (eBioscience). The samples were analyzed by flow cytometry on a BD FACS Canto II.

The studied cells were freshly stained with anti-MUC1 antibody (EP1024Y) diluted to 1:50 in 2% FBS in PBS. Antibodies that were used to investigate the cell phenotypes including anti-CD3-FITC, CD4-APC, CD8-APC, CD19-APC, CD16-APC, CD69-APC, and CD62L-APC were purchased from ImmunoTools GmbH (Friesoythe, Germany), anti-CD45RA-PE-cyanine7 and PD-1-PE from Invitrogen (Carlsbad, USA), anti-CD56-PE, LAG-3-PE and TIM-3-PE from BioLegend (San Diego, USA), and anti-CD25-PerCP-cyanine5.5 from eBioscience, Inc. (California, USA).
To detect the CARs expression on transduced lymphocytes, the transduced cells were harvested, blocked with 1% BSA, and stained with biotin-conjugated protein-L (Thermo Fisher Scientific, Waltham, MA, USA). Alexa Fluor 488 dye-conjugated streptavidin was finally added. The data were acquired on a BD FACSVerse or BD Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with FlowJo software version 10.