Maturation and persistence of CAR T cells derived from human pluripotent stem cells via chemical inhibition of G9a/GLP

Cell Stem Cell. 2025 Jan 2;32(1):71-85.e5. doi: 10.1016/j.stem.2024.10.004. Epub 2024 Nov 5.

Abstract

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 toward 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, chemical inhibition of G9a/GLP facilitates the generation of mature 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, persistent antitumor activity in a xenograft tumor-rechallenge model.

Keywords: CAR-T cells; G9a/GLP; T cell differentiation; cancer immunotherapy; chemical screen; epigenetic regulation; hematopoiesis; lymphoid development; pluripotent stem cells.

MeSH terms

  • Animals
  • Cell Differentiation* / drug effects
  • Histocompatibility Antigens / metabolism
  • Histone-Lysine N-Methyltransferase* / antagonists & inhibitors
  • Histone-Lysine N-Methyltransferase* / metabolism
  • Humans
  • Induced Pluripotent Stem Cells / cytology
  • Induced Pluripotent Stem Cells / metabolism
  • Mice
  • Pluripotent Stem Cells / cytology
  • Pluripotent Stem Cells / metabolism
  • Receptors, Chimeric Antigen / metabolism
  • T-Lymphocytes* / cytology
  • T-Lymphocytes* / immunology
  • T-Lymphocytes* / metabolism
  • Zebrafish*

Substances

  • Histone-Lysine N-Methyltransferase
  • EHMT2 protein, human
  • Histocompatibility Antigens
  • Receptors, Chimeric Antigen
  • EHMT1 protein, human