Patient 6 and patient 10 CAR-NK study

To evaluate whether the findings of our pre-clinical study hold true in patients treated with CAR19/IL-15 NK cells, we isolated and profiled CAR-NK cells and B cells from peripheral blood collected at multiple timepoints from two patients diagnosed with CD19+ relapse/refractory hematological malignancies and treated with CAR19/IL-15 NK cells (11). Each patient received one infusion of CAR-NK cells Single cells were sorted into 96 well plates with lysis buffer (10 l of TCL buffer, Qiagen and 1% 2-mercaptoethanol) stored at -80°C immediately until library preparation. cDNA was synthesized as described by the Smart-Seq2 protocol from Picelli et al.1 Briefly, RNA was purified using RNAClean XP beads (Beckman Coulter) and reverse transcribed to cDNA using Maxima RNAse H-minus (ThermoFisher) in the presence of biotinylated oligo-dT30VN, biotinylated template-switching oligonucleotides, and betaine. Next, a PCR preamplification step was done with KAPA HiFi HotStart Ready Mix (Roche Diagnostics) and biotinylated ISPCR primers. DNA products were further purified with Agencourt XP DNA beads, and the concentration of each sample was quantified by Qubit dsDNA HS Assay kits (Invitrogen). Quality of samples was analyzed on a 2100 Bioanalyzer using a High Sensitivity DNA Kit (Agilent Technologies). 0.15 ng of each sample were tagmented and Nextera index adapters were used for barcoding according to the Illumina Nextera XT DNA sample preparation kit (Illumina). For sequencing, single cell libraries were pooled equally, purified using AMPure XP beads and analyzed on a 2100 Bioanalyzer. Pooled libraries were denatured and diluted to 1.8pM prior to sequencing using 75 cycle paired-end kit on a NextSeq 500 (Illumina) with an average sequencing depth of 1 million reads per cell. The obtained mRNA sequencing data was demultiplexed into individual FASTQ files followed by alignment to the human reference genome hg19.