Ingunn M. Stromnes

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers, and current therapeutics provide limited benefits to patients. Desmoplasia and the highly immunosuppressive tumor microenvironment (TME) play a significant role in PDAC being elusive to current therapeutics. In this study, we strategically selected four transgenes to express from adenovirus (Ad) vectors to target desmoplasia and the highly immunosuppressive TME in PDAC: interferon γ (IFNγ), the extracellular (soluble) domain of transforming growth factor beta receptor 2 (sTGFβRII), interleukin 7 (IL7), and the extracellular (soluble) domain of T cell immunoglobulin and mucin domain–containing protein 3 (sTIM3). We used an immunocompetent syngeneic mouse model harboring tumors derived from KrasG12D/+;Trp53R172H/+; p48-Cre (KPC) cells, a common mouse pancreatic cancer cell line, to test the antitumor effects of our transgene-expressing Ad vectors. We showed that all four transgenes elicited significant antitumor effects and shifted the tumors to a more immunogenic profile by increasing intratumoral expression of immune activation proteins and infiltration of antitumor immune cells. Furthermore, inhibition of desmoplasia was observed in three of the four proteins. Of the four transgenes analyzed, IFNγ and sTGFβRII induced the greatest antitumor results and were combined in a single Ad vector. This dual-expressing Ad vector elicited the greatest antitumor effect, an increase in immunogenicity, and a decrease in fibrosis and showed abscopal effects. These results provide insight into how the analyzed transgenes can favorably alter the TME in patients with pancreatic cancer and show encouraging potential when combined with other therapeutic regimens.

Pancreatic ductal adenocarcinoma (PDA) is profoundly immunosuppressive. To help define this behavior, we present integrated experimental and computational frameworks to elucidate therapeutic T cell dynamics. Through the development of TME-CARTographer (TME-CART), a computational pipeline integrating high-dimensional data, graph theory, behavior analysis, and deep learning (DL), we present quantitative insights on 4D T cell-TME interactions in live PDA tumors. Mapping physical immunosuppression demonstrates that collagen fiber architectures direct migration while concomitantly limiting off-axis movement, creating immune exclusion zones. Expanding these findings, we establish that the collagen matrix harbors and spatially organizes immunosuppressive myeloid cells to serve as cooperative co-modulators of T cell behaviors, including migration, sampling, repulsion, and sequestration. Consistent with these findings, DL defines both linear and nonlinear collagen matrix and cellular neighborhood interactions as drivers of T cell behavior. The TME-CART DL framework also accurately predicts shifts in immunosuppression following depletion of myeloid cells. Overall, we identify synergistic barriers impeding anti-tumor T cell behaviors and present TME-CART as a discovery platform for interpreting complex 4D data to enhance the understanding and design of immunotherapies.

We interrogate antigen-specific CD4 T cells during immunotherapy in pancreatic ductal adenocarcinoma. Vaccination with MHC-II-restricted tumor epitopes impart superior protection compared to an immunodominant MHC-I epitope, prompting development of MHC-II affinity-enhanced tetramers to track tumor-specific CD4 T cells. As tumors progress, tumor-specific CD4 T cells decline, and remaining cells acquire features of regulation. Agonistic anti-CD40 increases Th1 cell clonal expansion and transiently decreases Tregs. Anti-PD-L1 promotes Tfh clonal expansion in draining lymph nodes and tumor while preventing Treg rebound after anti-CD40. Treatment with anti-CD40 promotes intratumoral Stat1 ⁺ macrophages, tertiary lymphoid structures (TLS), and immune triads. MHC-II on myeloid cells but not B cells is required for immunotherapy-induced TLS formation and antitumor effects. IL-15 complex enhances immunotherapy-induced Th1 effectors without promoting Tregs. Human immunotherapy transcriptomics shows conserved Th1 programming and Treg destabilization as a feature of response. Thus, tumor-specific CD4 T cells are central mediators of effective immunotherapy in solid tumors.

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.

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.