Single cell RNA sequencing monitoring the development 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.