Cd3 allophycocyanin apc

PBMC were plated in a 96-well plate (10⁶ cells per well). PBMC stimulation was performed in 10% FBS/RPMI media in the presence of 1 μg/ml anti-CD28 and anti-CD49d and Brefeldin A (BD Biosciences, San Diego, CA) and stimulated with HIV peptides (New England Peptide, Gardner, MA) of 15-mer overlapping by 11 amino acids representing HIV subtype E-Env (TH023; 162 peptides) and HIV subtype B-Gag (LAI; 120 peptides). PBMC supplemented with DMSO was used as a negative control. After 6 h of stimulation at 37 °C, 5% CO₂ EDTA (20 mM, Sigma) was added and incubated for 15 min. Subsequently, PBMC were fixed and permeabilized using FACS lysing solution and FACS permeabilizing solution 2 (BD) according to the manufacturer’s instructions. The following antibodies were added for 60 min at room temperature in the dark: CD4-fluorescein isothiocyanate (FITC), CD3-allophycocyanin (APC), IFNγ-phycoerythrin (PE), Il-2-phycoerythrin (PE), and CD8-PerCP-Cy5.5 (all BD Biosciences). PBMC were washed and fixed with 1% paraformaldehyde. The analysis was performed using a FACSCalibur flow cytometer (BD Immunocytometry Systems). ICS data were provided to us by the trial investigators in either an ICS-positivity score (call) format and aggregate value format^{2 (link)}. ICS analytes included CD154, IFNγ, IL-4, IL-2, IL-17α, and TNFα.

Cell surface proteins on (CAR-)T cells were stained with the following antibodies: annexin V-PE, CCR7-PerCP/Cy5.5, CD3-allophycocyanin (APC), CD3-BV421, CD4-APC/H7, CD8a-PE/Cy7, CD25-PE/Cy7, CD69-fluorescein isothiocyanate (FITC), CD95-PE, CD127-PerCP/Cy5.5, CXCR3-AF488 (all from BD Biosciences, San Jose, CA); CD8a-APC, CD8a-FITC, CD27-PE, CD28-FITC, CD45RA-APC, LAG-3-PE, TIGIT-PerCP/Cy5.5, TIM-3-FITC (all from BioLegend, San Diego, CA); and PD-1-APC (eBioscience, Thermo Fisher Scientific). Monocytes were stained with CD14-PE (eBioscience). To determine CAR expression, cells were labeled with a recombinant Fc-tagged BCMA protein (Enzo Life Sciences, Farmingdale, NY) followed by a BV421-conjugated anti-Fc antibody (BioLegend). Samples prepared from mouse organs were treated with Fc block (BD Biosciences) prior to staining. Cells were washed and resuspended in fluorescence-activated cell sorting (FACS) buffer (1% FCS in PBS) or 1% paraformaldehyde prior to acquisition. In experiments to assess viability and/or apoptosis, cells were resuspended in FACS buffer containing 1 μg/mL 7-aminoactinomycin D (7-AAD). In all other experiments, live cells were gated based on forward and side scatter.
All flow cytometry data were acquired on a FACSCanto II (BD Biosciences) and analyzed using FlowJo software version X.0.7 (Treestar, Ashland, OR).