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Sleeping Beauty mutagenesis identifies BACH2 and other regulators of CD8 + T-cell exhaustion, persistence in vivo, and CAR-T cell function under tumor-associated chronic antigen stimulation
Journal article   Open access   Peer reviewed

Sleeping Beauty mutagenesis identifies BACH2 and other regulators of CD8 + T-cell exhaustion, persistence in vivo, and CAR-T cell function under tumor-associated chronic antigen stimulation

Chang-Jung Lee, Alex T Larsson, Tyler A Jubenville, Wendy A Hudson, Zach J Seeman, Carli Stewart, Alexander K Tsai, Adam Burrack, Brooke L Kimball, Elizabeth L Siegler, …
Journal for immunotherapy of cancer, Vol.14(7), e013536
07/15/2026
DOI: 10.1136/jitc-2025-013536
PMID: 42457339
url
https://doi.org/10.1136/jitc-2025-013536View
Published (Version of record) Open Access

Abstract

Genes that enhance T-cell function represent promising targets for improving engineered T-cell therapies for cancer. While extensive CRISPR knockout screens have identified key genes enhancing T-cell persistence, employing ( ) insertional mutagenesis, which induces both gain-of-function (GOF) and loss-of-function (LOF) mutations via the generation of fusion transcripts with endogenous genes, may uncover additional critical factors that previous approaches have overlooked. We developed transgenic mice carrying oxycycline (Dox)- nducible mutag nesis s stem (DiSBey) in primary T cells. Using DiSBey, we conducted screens for genetic alterations enhancing T-cell persistence under chronic antigen exposure. Specifically, CD8 T cells from Dox-fed DiSBey mice were subjected to repeated anti-CD3 stimulation over 18 days to mimic chronic antigenic stimulation. We then identified transposon genomic insertion sites and corresponding fusion transcripts from the persistent DiSBey CD8 T cells using enhanced-specificity tagmentation sequencing and RNA sequencing, respectively. Under chronic stimulation, -mutagenized CD8 T cells exhibited improved persistence and reduced terminal exhaustion phenotype. Across six independent screens, we identified 38 genes that were recurrently targeted by the transposon T2/Onc2 and differentially expressed under chronic anti-CD3 stimulation. T2/Onc2 insertions into and were recurrently identified at the genomic level and were associated with altered nascent transcript expression. , known as a key regulator of T-cell memory formation and resistance to chronic viral infection-induced exhaustion but less characterized in engineered T cells for cancer therapy, was found to counteract exhaustion in vitro and enhance in vivo tumor persistence in the B16-Ova tumor model. Further, we showed that ectopic expression levels influence engineered T-cell differentiation lineage, as low overexpression retained more functional progenitor exhausted T cells and exhibited improved therapeutic efficacy. Finally, in human CART19-28ζ cells, overexpression enhanced cytotoxicity and tumor control following chronic cancer stimulation. Controllable mutagenesis using DiSBey mice provides a novel platform for functional screening of genes that improve T-cell phenotypes important for their use as therapies. Our findings highlight a dose-dependent role of BACH2 in enhancing the function of engineered T cells under conditions of chronic antigenic stimulation.
 Mutagenesis Animals Antigens, Neoplasm - immunology Basic-Leucine Zipper Transcription Factors - genetics Basic-Leucine Zipper Transcription Factors - metabolism CD8-Positive T-Lymphocytes - immunology CD8-Positive T-Lymphocytes - metabolism Humans Immunotherapy, Adoptive - methods Mice Mice, Transgenic Mutagenesis, Insertional Receptors, Chimeric Antigen - immunology Receptors, Chimeric Antigen - metabolism T-Cell Exhaustion

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