<i>Sleeping Beauty</i> mutagenesis identifies <i>BACH2</i> and other regulators of CD8 <sup>+</sup> T cell exhaustion, persistence <i>in vivo</i> , and CAR-T function under tumor-associated chronic antigen stimulation
Bibliographic record
Abstract
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 Sleeping Beauty ( SB ) insertional mutagenesis, which induces both gain-(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 D oxycycline (Dox)-inducible SB mutag e nesis s y 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 SB transposon genomic insertion sites and corresponding fusion transcripts from the persistent DiSBey CD8⁺ T cells using enhanced-specificity tagmentation sequencing (esTag-seq) and RNA-seq, respectively. Under chronic stimulation, SB -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 SB transposon T2/Onc2 and differentially expressed under chronic anti-CD3 stimulation stress. Among these, T2/Onc2 insertions into Bach2 and Elmo1 were repeatedly found at the genomic level and were associated with altered nascent transcript expression. Bach2 , known as a key regulator of T cell memory formation and resistance to chronic viral infection but less characterized in engineered T cells for cancer therapy, was found to enhance in vivo tumor persistence in the B16-Ova tumor model. We showed that ectopic Bach2 expression levels influence engineered T cell differentiation lineage. A Bach2 low signature allowed differentiation into both KLRG1⁺ and CD62L⁺ phenotypes, whereas Bach2 high restricted differentiation predominantly to the CD62L⁺ subset. Finally, in human CART19-28ζ cells, BACH2 overexpression enhanced cytotoxicity and improved tumor control following chronic cancer stimulation. Controllable SB mutagenesis using DiSBey mice provides a novel platform for functional screening of genes that improve T cell therapeutic phenotypes. Our findings highlight a dose-dependent role of BACH2 in enhancing the function of engineered T cells under conditions of chronic antigenic stimulation.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.002 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".