GSM5709965: ND566.Day24_Continuous.ID3_KO; Homo sapiens; OTHER
For flow cytometry and sorting assays of CAE, M5 CAR T cells were stained in fluorescence-activated cell sorting (FACS) buffer consisting of PBS (Gibco), 0.5% bovine serum albumin (BSA) (GEMINI), 2 mM EDTA (Invitrogen), and 100ug/ml DNase (Roche). CountBrightTM Absolute Counting Beads, (ThermoFisher) were used as an internal standard according to the manufacturer's instructions to calculate absolute cell counts in cell suspensions.  Antibodies specific for human CD45 (ref 304032, clone HI30), CD3 (317322, OKT3), CD4 (357412, A161A1 / 317440), CD56 (304608, MEM-188), EpCAM (324226/324238, 9C4), CD94 (305520, DX22), KLRB1 (339918, HP-3G10), TIGIT (372716, A15153G), TCR Va24-Ja18 (342922, 6B11), PD-1 (329928, EH12.2H7) were purchased from BioLegend. Antibodies specific for human CD8 (560179, SK1) was purchased from BD Pharmingen. Antibodies specific for human NKG2A (FAB1059P, 131411) and Methothelin (FAB32652P, 420411) were purchased from R&D Systems. Antibodies specific for human CTLA-4 (12-1529-42, 14D3) was purchased from eBioscience. Antibody specific for human NKG2C (130-103-636, REA205) was purchased from Miltenyi.  M5 CAR expression was assessed using biotinylated goat anti-human IgG F(ab')2 (Jackson ImmunoResearch, 109-066-006) followed by streptavidin (FITC or APC) (BioLegend) or using an anti-idiotype antibody provided by Novartis Pharmaceuticals. Live/dead staining was performed using a Live/Dead Fixable Aqua Dead Cell Stain Kit (Life Technologies following manufacturer's protocol followed by cell surface staining for 20 minutes at room temperature in the dark. Intracellular staining was performed with the Foxp3/Transcription Factor Staining Buffer set (Thermo Fisher) according to the manufacturer's instructions. Samples were acquired on an LSRII Fortessa Cytometer (BD Bioscience) and analyzed with FlowJo v10 software (FlowJo, LLC). Sorting assays were performed using a FACS Aria Cytometer (BD Bioscience). Donors ND566 and ND539: scRNA-seq libraries were generated using a Chromium Single Cell 3' Library and Gel Bead Kit v3.1 (10x Genomics) according to the manufacturer's protocol. Briefly, 16,000 CD8+ T cells were sorted by flow cytometry and washed with ice cold PBS + 0.04% BSA. After washing, cells were used to generate single cell gel beads in emulsion. Following reverse transcription, gel beads in emulsion were disrupted and barcoded complementary DNA was isolated and amplified by PCR for 12 cycles. After fragmentation, end repair, and poly A tailing, samples indexes were added and amplified following the manufacturer's protocol.