Alexander K. Tsai

Background: 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-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. Methods: We developed transgenic mice carrying D oxycycline (Dox)- i nducible 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 and RNA sequencing, respectively. Results: 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. T2/Onc2 insertions into Bach2 and Elmo1 were recurrently identified 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-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 Bach2 expression levels influence engineered T-cell differentiation lineage, as low Bach2 overexpression retained more functional progenitor exhausted T cells and exhibited improved therapeutic efficacy. Finally, in human CART19-28ζ cells, BACH2 overexpression enhanced cytotoxicity and tumor control following chronic cancer stimulation. Conclusions: Controllable SB 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.

Mechanisms driving immunotherapy resistance in pancreatic cancer are poorly defined. We demonstrate that programmed death-ligand 1 immune checkpoint blockade promoted immune evasion by epigenetic Tap1 ( transporter associated with antigen processing 1 ) silencing, increasing selection of metastatic tumor variants with defective interferon-γ (IFN-γ)–inducible class I major histocompatibility complex (MHC-I) expression. Unleashing CD4 conventional T cells by regulatory T cell (T _reg cell) depletion, transfer of tumor-reactive CD4 T cells, or anti–CTLA-4 prevented metastasis. Tumor-specific CD4 T cells adopted a TCF-1 ⁺ SLAMF6 ⁺ progenitor state in lymph nodes and differentiated in tumors. Anti–CTLA-4 increased intratumoral accumulation of CD4 T cells with stemness and tissue residency features, reduced metastasis, and induced gene signatures correlated with improved patient outcomes. MHC-I restoration with anti–CTLA-4 prolonged survival in murine models. In patient tumors, T _reg cells and CD4 T cells colocalized, and abundance correlated with survival. These findings identify targetable mechanisms of immune evasion and metastasis in immunotherapy-resistant cancer.

Adoptive cell therapy (ACT) has shown remarkable success in the treatment of some malignancies, particularly leukemia. However, there are multiple factors that limit the durability of ACT in solid tumors, including dose-limiting toxicities, the immunosuppressive tumor microenvironment, and T-cell exhaustion. As the manufacture and preparation of adoptive T-cell therapies allows time and adequate conditions for ex vivo T-cell engineering, forward genetic screens can identify novel genetic targets that could improve their effectiveness. CRISPR is a commonly used functional genomics tool that has been successfully used to both enhance our understanding of mechanisms of resistance and to discover potential genetic edits to improve ACT. A complementary approach, Sleeping Beauty transposon mutagenesis provides additional opportunities to identify novel genetic edits without being constrained by the annotated human genome. Here, we summarize forward genetic screens and their tools to uncover strategies to enhance ACT. Complementary approaches can be combined and improved on to identify translatable genetic editing strategies through studies that accurately recapitulate disease-specific challenges.

Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy resistant to therapy including immune checkpoint blockade (ICB). We previously showed that ICB selects for pancreatic tumor cells that are defective in IFN-γ–inducible MHC-I, prompting us to test the impact of IL-15 complex (IL-15C) in overcoming ICB resistance. Here, we show that IL-15C markedly expands circulating NK cells, CD8+ T cells, and CD4+Cxcr3+ T cells in an orthotopic pancreatic ductal adenocarcinoma (PDA) animal model. In tumors, IL-15C + anti-PD-L1 increased CD8+ T-cell effector cytokine production and interfered with T-cell exhaustion, including mitigating IL-10. In NK cells, IL-15C + anti-PD-L1 modulated NK cell IFN-γ production but did not alter Nkg2d, Nkg2a, Klrg1, IL-10, or granzyme B. IL-15C + anti-PD-L1 significantly prolonged animal survival, leading to tumor eradication in a subset of animals, whereas monotherapies only transiently prolonged survival. Therapeutic benefit was dependent on CD8+ T cells and independent of NK cells and Nkg2d. Together, our study supports that IL-15C improves anti-PD-L1 in PDA through sustaining antitumor T-cell function.

Background: Human leukocyte antigen (HLA) class I encompasses peptide-binding proteins that regulate T-cell interactions. We examined HLA class I expression in prostate cancers (PC), exploring associations with clinical outcomes, molecular features, and tumor immune microenvironment. Methods: We analyzed 8040 PC samples from the Caris Life Sciences database, stratifying them into HLA-high (upper quartile) and -low (lower quartile) groups. Genomic and transcriptomic alterations were compared. Immune cell fractions were inferred using quanTIseq, and overall survival (OS) data was obtained from insurance claims. Differences were computed with Cox proportional hazards. Results: Among 66 cancer types, PC ranked 3 ^rd -, 11 ^th -, and 19 ^th -lowest for HLA-A, -B, and -C expression, respectively. In PC, genes tied to androgen receptor ( AR ) signaling, immune checkpoint molecules ( CTLA4, PD-L1 ), and the epithelial-mesenchymal transition were significantly higher in HLA-high tumors. HLA-high status was linked to greater tumor immune activity, marked by higher T cell fractions and enhanced immune hallmarks. HLA-high tumors were less likely to possess alterations in AR, FOXA1 , and CDK12 , but harbored increased alterations in tumor suppressor gene ( RB1 , PTEN ) alterations. Tumors with high HLA-A and HLA-B had elevated TMB-H/MSI-H/dMMR status. Finally, shorter OS was observed in patients with high HLA-A or HLA-B expression, while longer OS was associated with high HLA-C expression. Conclusions: In PC, elevated HLA class I levels correlate with immune activity, molecular characteristics, and clinical outcomes. We suggest considering HLA expression as a supplementary marker of immune activity in PC, alongside genetic mutations and transcriptomic markers.

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.