Cryopreserved GMP product cells were thawed at 37°C and washed. Cells were then stained with the CD19-CAR detection reagent (Miltenyi; cat. #130-115-965) for 10 minutes at room temperature followed by staining with an antibiotin antibody conjugated to APC (Miltenyi; cat. #130-110-952), a cocktail of antibodies (CD3, CD8, CD27, CD62L, CD25, and TIGIT), and a viability dye (Tonbo Biosciences; cat. #13-0870-T100). After staining for 10 minutes at room temperature, the cells were washed twice and resuspended in FACS buffer for sorting on the FACSAria Fusion (BD Biosciences). CD8+ CAR T cells with either the predicted effector surface profile (TIGIT+, CD62Llo, and CD27−) or the opposite, noneffector precursor profile (TIGIT−, CD62L+, and CD27+) were sorted into complete RPMI media. Cells were lysed with TRIzol for bulk TCR repertoire sequencing.
Antibodies used were anti-human CD3-APC-H7 (BD Pharmingen; cat. #560176), anti-human CD8-BV785 (BioLegend; cat. #344740), anti-human CD27-PE-CF594 (BD Horizon; cat. #562297), anti-human CD62L-BV421 (BioLegend; cat. #304828), anti-human CD25-VioBright FITC (Miltenyi Biotec; cat. #130-113-283), and anti-human TIGIT-PE (BioLegend; cat. #372703).
Antibodies used were anti-human CD3-APC-H7 (BD Pharmingen; cat. #560176), anti-human CD8-BV785 (BioLegend; cat. #344740), anti-human CD27-PE-CF594 (BD Horizon; cat. #562297), anti-human CD62L-BV421 (BioLegend; cat. #304828), anti-human CD25-VioBright FITC (Miltenyi Biotec; cat. #130-113-283), and anti-human TIGIT-PE (BioLegend; cat. #372703).