GSM9038873: postInfusion_ADT2; Homo sapiens; OTHER
Cyropreserved post-infusion CAR-T cells were thawed and rested overnight at 2x10^6 cells/mL in cRPMI (RPMI 1640 Medium + 10% heat-inactivated FCS, 0.05 mM β-mercaptoethanol, 0.05 mg/mL gentamicin, 1.1915 g/L HEPES, 0.2 g/L L-glutamine, 100 U/mL Penicillin-Streptomycin). After manual counting, all cells (1.35x106 C14, 3.55x106 C16 and 3.1x106 C20 cells) were collected and washed twice in 200 µL sterile-filtrated Wash buffer (PBS, pH 7.4, with 1% filtered FCS) and then resuspended in the 50 µL (C14) or 100 µL (C16, C20) sterile-filtrated Stain buffer (PBS, pH 7.4 containing 1% FSC, 0.1 g/L Herring sperm DNA). 4.4 µL (for C14) or 8.8 µL (for C16, C20) of dCODE reagent mastermix (2.0 μL of CD19-dCODE® Dextramer (Immudex, DS005), 2.0 μL of control-dCODE® Dextramer (Immudex, WI03233), and 0.4 μL 100 μM d-Biotin for C14. For C16 and C20 these volumes were doubled.) were added and incubated for 15 min in the dark at room temperature. Mastermixes comprising a unique DNA-barcoded hashtag antibody (0.5 µL per 1x106 PBMCs of TotalSeq-C anti-human hashtag antibodies 1–3 (BioLegend: for C14: 394661, for C16: 394663, for C20: 394665)), a CITEseq-antibody mix of DNA-barcoded CITE-Seq TotalSeq C antibodies (0.25 µg per 5x10^6 PBMCs of anti-human CD4 (BioLegend, 300567), 0.25 µg per 5x106 PBMCs of anti-human CD8 (BioLegend, 344753), 0.078 µg per 5x106 PBMCs of anti-human CD45RA (BioLegend, 304163), 0.078 µg per 5x10^6 PBMCs of anti-human CD62L (BioLegend, 304851), 0.3125 µg per 5x10^6 PBMCs of anti-human CD95 (BioLegend, 305651), 0.277 µg per 5x10^6 PBMCs of anti-human CCR7 (BioLegend, 353251) in Stain buffer (PBS, pH 7.4, with 1% FCS and 0.1 g/L Herring sperm DNA) and a mix surface markers and viability dye (anti-CD56-FITC (1:200, Invitrogen, 11-0566-42), anti-CD19-FITC (1:200, BioLegend, 302205), anti-CD4-BV711 (1:400, BD Horizon, 568371), anti-CD3-PE/Cy7 (1:200, Beckman Coulter, 737657), anti-CD8a-APC (1:400, BioLegend, 301014), anti-CD14-APC/Cy7 (1:200, BD Pharmingen, 557831) and ZombieAqua (1:500, BioLegend, 423102) in Stain buffer) was centrifuged (14,000 xg, 10 min, room temperature) and then added to the respective cells suspension. After 20 min incubation in the dark on ice, samples were washed 4 times with Wash buffer (300 xg, 5 min, 4°C) and then resuspended in 500 µL sterile-filtered FACS buffer (PBS, 0.5% BSA). Sort enrichment was performed for single, live, CD56-, CD19-, CD14-, CD3+ dCODE-Dextramer+ cells on a MoFlo Astrios EQ sorter (Beckman). In total, 5,670 C14 cells, 69,925 C16 cells and 11,560 C20 cells were sorted into pre-coated 1.5 mL tubes containing FACS-buffer. Immediately after sorting, cells were loaded onto a Chromium Next GEM Chip K (10X Genomics) and Chromium Next GEM Single-Cell 5′ kits v.2 to generate gene expression (GEX), T cell receptor (VDJ) and cell surface (CITEseq) libraries according to the manufacturer's instructions (10X Genomics, 1000263, 1000256, 1000252, 1000286, 1000250, 1000215, 1000190). Libraries were sequenced at Novogene (Cambridge, UK) on an Illumina NovaSeq platform with PE150 strategy. Library construction was performed according to manufacturer's instructions (10x Genomics) for 5' GEX, VDJ and CITEseq, V2 chemistry dual index