G9a/GLP Inhibition Promotes Derivation of Functional CAR T cells from Pluripotent Stem Cells

Elucidating mechanisms of T cell development can guide in vitro T cell differentiation from Induced Pluripotent Stem Cells (iPSCs) and facilitate off-the-shelf T cell-based immunotherapies. Using a stroma-free human iPSC-T cell differentiation platform, we screened for epigenetic modulators that influence T cell specification and identified the H3K9-directed histone methyltransferases G9a/GLP as repressors of T cell fate. We show that G9a/GLP inhibition during specific time windows of differentiation of hematopoietic stem and progenitor cells (HSPCs) skews cell fates towards lymphoid lineages. Inhibition of G9a/GLP promotes the production of lymphoid cells during zebrafish embryonic hematopoiesis, demonstrating the evolutionary conservation of G9a/GLP function. Importantly, G9a/GLP inhibition facilitates the generation of robust iPSC-T cells that bear transcriptional similarity to peripheral blood αβ T cells. When engineered to express chimeric antigen receptors, the epigenetically engineered iPSC-T cells exhibit enhanced effector functions in vitro and durable antitumor activity in a xenograft mouse model. Human iPSCs were differentiated into T cells and treated with DMSO or UNC0224 at different time windows (week 0-2 or week 0-4). Cell were collected on multiple time points (week2, week4, before and after T cell activation) and scRNA-seq experiments were performed.