Epigenetic sequencing of allogeneic HSC-derived CAR-engineered NKT cells

CAR-T cell therapy has achieved remarkable clinical outcomes, yet the autologous nature of FDA-approved CAR-T products present significant challenges in manufacturing, cost, and patient selection. Therefore, there is a growing demand for off-the-shelf cell therapy. Here we introduce an ex vivo feeder-free culture to differentiate gene-engineered HSCs into allogeneic NKT cells, as well as their CAR-armed and IL-15-enhanced derivatives (Allo15CAR-NKT cells). In order to study the epigenetic regulation of the generated cells, we performed DNA methylation sequencing on both IL-15-enhanced and non-IL-15-engineered AlloCAR-NKT cells. Conventional CAR-T cells were included as a control. Genomic DNA from samples was isolated with QIAGEN DNeasy Blood & Tissue kit following the corresponding manufacturer’s protocols. The isolated genomic DNA was sonicated by Covaris system. Size selection by Ampure XP beads (Beckman-Coulter) was performed to enrich DNA fragments of around 250 bp in average sizes before library construction. In brief, 0.7 volume beads were added to the fragmented genomic DNA. After incubation, the supernatant was transferred to a new tube and an additional 1.3 volume beads were added. After washing two times with 80% ethanol, genomic DNA was eluted with 40 µl EB buffer. Subsequently, 200 ng genomic DNA was used for DNA library preparation with NEBNext Ultra II DNA library prep Kit (Cat#E7645). DNA library preparation has the following steps: DNA fragments end prep, adaptor ligation, size selection or purification of Adaptor-ligated DNA with Ampure XP beads, bisulfite conversion by Epitect Fast DNA Bisulfite Kits (QIAGEN, Cat#59826), PCR enrichment of Adaptor-ligated DNA, and purification of PCR product with 0.9 x Ampure XP beads. The average size of resulting DNA fragments was around 480 bp, validated by Tapestation. The libraries were then subjected to sequencing on Illumina NovaSeq sequencer with 2 x 150 bp configuration (Azenta). The raw bisulfite sequencing reads were trimmed by cutadapt to remove sequencing adapters. The trimmed reads were then aligned to hg19 reference genome by Bismark. Then duplicated reads from PCR amplification were identified and removed by Bismark. The deduplicated reads were then sorted and indexed using Samtools. After that, the methylated and unmethylated cytosines were counted at every CpG site by Bismark. The methylation at a gene promoter region was quantified as the beta value, i.e., the ratio between the number of methylated cytosines and the total number of cytosines mapped to the region. The promoter region of a gene was identified by GeneHancer with the highest confidence score. The beta values were then used for heatmap visualization.