433 <i>Sleeping Beauty</i> mutagenesis uncovers crucial role of Bach2 in resisting chronic tumor stimulation in CD8⁺ T cells

<h3>Background</h3> Chronic stimulation of the T cell receptor (TCR) in the tumor microenvironment (TME) contributes to cytotoxic CD8⁺ T cell exhaustion and activation-induced cell death, which limits the effectiveness of immunotherapies. Therefore, enhancing CD8⁺ T cell fitness in the TME represents a promising opportunity for improving cancer therapy. While CRISPR screens have identified factors that enhance CD8⁺ T cell persistence, a genome-wide screen simultaneously encompassing both gain- (GOF) and loss-of-function (LOF) mutants has not been conducted. <h3>Methods</h3> We developed a quadruple (‘quad’) transgenic mouse model (hCD2-Cre; Col1a1-TRE-LSL-SB100X; T2/Onc2; Rosa26-CAGGS-LSL-rtTA). These mice were fed doxycycline to generate a genome-wide pool of T cell mutants driven by the bifunctional <i>Sleeping Beauty</i> (SB) transposon T2/Onc2, which can induce GOF and LOF, full-length and truncation mutations in endogenous genes. We isolated surviving quad CD8⁺ T cells after chronic stimulation with αCD3 for 16 days, using transiently activated and normally cultured cells as controls. Additionally, CD8⁺ T cells from C57BL/6J mice were included for comparison of viability and surface inhibitory receptor expression. We performed RNA sequencing (RNA-seq) and SB transposon insertion site recovery by hybrid capture on collected cells. To validate the candidate gene <i>Bach2,</i> we performed<i> in vitro</i> competition assays by transducing OT-I T cells with the retroviral vectors MSCV-mBach2-IRES-GFP (M-BIG) or empty vector (TagBFP⁺), mixing them at 1:1 ratio, and subjecting them to 3 rounds of αCD3 stimulation or exposure to irradiated B16-F10 melanoma cells pulsed with SIINFEKL peptide. Fluorescent population ratios were analyzed using flow cytometry post-stimulation. Co-cultures of OT-I cells transduced with MSCV-IRES-GFP and TagBFP served as controls. <h3>Results</h3> Under chronic αCD3 stimulation, quad CD8⁺ T cells showed increased viability and reduced PD-1 expression compared to C57BL/6 counterparts. RNA-seq of quad CD8⁺ T cells revealed an enrichment of T2/Onc2-driven <i>Bach2</i> transcripts in chronically stimulated cells over time but not in controls. In C57BL/6J chronically stimulated CD8⁺ T cells, <i>Bach2</i> expression remained low after day 10, whereas the insertion mutation in quad CD8⁺ T cells sustained <i>Bach2</i> expression similar to normally cultured controls. Additionally, in both <i>in vitro</i> competition assays, M-BIG OT-I T cells not only showed a selective survival advantage over the control TagBFP⁺ cells, but also exhibited a shift towards a TIM-3-negative population, suggesting a reduced T cell exhaustion phenotype. <h3>Conclusions</h3> SB-mutagenized CD8⁺ T cells exhibited prolonged persistence and reduced exhaustion under chronic αCD3 TCR stimulations. We highlighted the pivotal role of Bach2 in enhancing CD8⁺ T cell persistence amidst exhaustion cues from cancer.