Transcription factors (TFs) govern cell fate through coordinated gene-regulatory networks, yet the full potential of these networks to generate non-native, therapeutically advantageous cell states in vivo remains largely unexplored. We hypothesized that systematic gain-of-function (GOF) overexpression of TFs in CD8+ T cells, central mediators of immune protection, could reveal latent, or "hidden," regulatory programs capable of generating synthetic T cell states with therapeutic utility. To test this, we developed single-cell GOF sequencing (scGOF-seq), a multiplexed platform for unbiased, in vivo mapping of GOF effects on T cell fate in immunocompetent mouse models of infection and cancer. scGOF-seq uncovered unexpected regulators of T cell differentiation and accumulation, including SOX2, OCT4, and GATA2, which are normally silenced during T cell differentiation. Notably, outside its native regulatory context, supraphysiologic cMyc GOF reprogrammed CD8+ T cells into a synthetic stem-effector hybrid state, enabling >5,000-fold antigen-dependent expansion and antitumor activity, contrasting sharply with its native function in driving terminal differentiation. scGOF-seq further identified TF modules that cooperate with cMyc GOF to promote robust CD8+ T cell responses in solid tumors. Together, these findings establish GOF perturbation as a powerful strategy for revealing latent immune regulatory programs and engineering synthetic immune states with therapeutic potential.

CD8+ T cells differentiate into diverse states that shape immune outcomes in cancer and chronic infection1-4. To define systematically the transcription factors (TFs) driving these states, we built a comprehensive atlas integrating transcriptional and epigenetic data across nine CD8+ T cell states and inferred TF activity profiles. Our analysis catalogued TF activity fingerprints, uncovering regulatory mechanisms governing selective cell state differentiation. Leveraging this platform, we focused on two transcriptionally similar but functionally opposing states that are critical in tumour and viral contexts: terminally exhausted T (TEXterm) cells, which are dysfunctional5-8, and tissue-resident memory T (TRM) cells, which are protective9-13. Global TF community analysis revealed distinct biological pathways and TF-driven networks underlying protective versus dysfunctional states. Through in vivo CRISPR screening integrated with single-cell RNA sequencing (in vivo Perturb-seq) we delineated several TFs that selectively govern TEXterm cell differentiation. We also identified HIC1 and GFI1 as shared regulators of TEXterm and TRM cell differentiation and KLF6 as a unique regulator of TRM cells. We discovered new TEXterm-selective TFs, including ZSCAN20 and JDP2, with no previous known function in T cells. Targeted deletion of these TFs enhanced tumour control and synergized with immune checkpoint blockade but did not interfere with TRM cell formation. Consistently, their depletion in human T cells reduces the expression of inhibitory receptors and improves effector function. By decoupling exhaustion TEX-selective from protective TRM cell programmes, our platform enables more precise engineering of T cell states, accelerating the rational design of more effective cellular immunotherapies.

Tregs expressing forkhead box P3 (FOXP3) play crucial roles in maintaining immune tolerance and tissue integrity. EZH2, a histone H3 lysine 27 (H3K27) methyltransferase, is known as a key regulator of Treg identity and suppressive function upon activation. Here, we demonstrate that the H3K27 lysine demethylase KDM6B, which catalyzes the opposing reaction to EZH2, is also required for Treg identity and function after activation. Treg-specific deletion of Kdm6b impaired tissue Treg fate and function. KDM6B was upregulated after T cell antigen receptor signaling in Tregs and contributed to the regulation of Treg-associated gene expression through both direct and indirect mechanisms. A subset of Treg functional genes were direct targets of KDM6B and were co-occupied by FOXP3 at cis -regulatory regions, where KDM6B recruitment limited H3K27me3 accumulation. More broadly, KDM6B-dependent H3K27 demethylation facilitated Treg gene expression programs that supported tissue Treg homeostasis.

Regulatory T cells (Tregs) play a crucial role in mediating recovery from acute lung injury (ALI). However, the complex roles of functionally heterogeneous Treg subsets in the lung during the resolution of acute inflammation remain unclear. To investigate the role of peripherally induced Tregs, we utilized mice lacking conserved noncoding sequence 1 (CNS1) of the Foxp3 locus, a genetic deletion that impairs peripheral Treg induction. We found that CNS1-deficient mice exhibit greater mortality and delayed resolution during ALI. Tregs induced via CNS1 modulated antiviral and proinflammatory immune responses in the lung during influenza. Mechanistically, single-cell RNA sequencing reveals that CNS1-deficient Tregs fail to fully engage the Treg transcriptional program that supports optimal suppressive and reparative function in the lung. Our findings highlight a critical role for CNS1-dependent peripherally induced Tregs in determining ALI severity, providing insight into how distinct Treg subpopulations may be therapeutically harnessed to mitigate tissue damage during respiratory disease.

Immune responses evolve across time and tissues through coordinated programs of proliferation, differentiation, and migration, yet most single-cell measurements capture only static molecular snapshots. As a result, reconstructing how immune cells transition between alternative fates remains challenging, particularly for CD8 T cells, whose differentiation is highly dynamic and shaped by rapid expansion, contraction, and tissue trafficking. Here, we introduce an optimal transport-based fate mapping framework that reconstructs continuous CD8 T cell trajectories across time and tissues. Applied to longitudinal single-cell RNA-seq data from CD8 T cells responding to acute viral infection in mice, this approach accurately recapitulates population dynamics and resolves coherent effector and memory T cell differentiation trajectories. Extending the model to multiple tissues, we identify and experimentally validate temporally distinct waves of migration into the small intestine that give rise to divergent tissue-resident memory (Trm) fates, long-lived T cells crucial in immunosurveillance. By integrating optimal transport inference with time-resolved in vivo labeling, we demonstrate that CD52 marks recent tissue entrants and distinguishes them from Trm precursors. Finally, trajectory-guided analysis of transcription factor regulons reveals both shared and context-specific gene regulatory programs and identifies AP4 as a key regulator of circulating versus tissue-resident specification. These results establish optimal transport as a principled framework for reconstructing immune cell fate dynamics and provide a quantitative map of early events governing antiviral CD8 T cell differentiation across tissues.

CD8+ T cells mediate host defense and tumor immunity through specialized differentiation states, yet the regulatory programs that guide these states may also limit their functional potential. Loss-of-function studies have defined many regulators required for T cell differentiation, but they do not readily reveal regulatory activities that emerge only when transcription factors are ectopically expressed outside their native lineage, dosage, or temporal context. Here, we developed single-cell gain-of-function (GOF) sequencing (scGOF-seq), a multiplexed platform for in vivo mapping of transcription factor overexpression in antigen-specific CD8+ T cells across immunocompetent models of infection and cancer. By enforcing expression of canonical T cell regulators, lineage-silenced developmental factors, and temporally restricted transcription factors, scGOF-seq uncovered unexpected in vivo activities. Developmental regulators normally silenced in T cells, including NANOG, SOX2, OCT4 and GATA2, reshaped T cell differentiation in context-dependent ways, with NANOG promoting stemness-associated phenotypes and accumulation during chronic infection. In parallel, sustained cMyc expression outside its native temporal window generated a stem-like, effector-featured state with enhanced metabolic fitness, reduced terminal exhaustion, and profound antigen-dependent expansion exceeding 5,000-fold. Importantly, cMyc GOF maintained cell-cycle checkpoint signatures and demonstrated a strong dependence on antigen presence for proliferation across the tested conditions. scGOF-seq further identified cooperating transcription factor modules that complemented cMyc-driven programs and improved T cell responses in solid tumors. These findings establish systematic GOF perturbation as a framework for uncovering latent and temporally constrained regulatory activities in CD8+ T cells and guiding immune-state engineering.

Systemic delivery of immunotherapy is dose-limited and often causes serious immune-related adverse events. Intratumoral injections can reduce systemic immunotoxicities and increase immunotherapy concentrations within a tumor. However, high pressures associated with direct tumor injection limits injectate retention, as low viscosity, saline-based solutions rapidly leak out of tumors. Viscoelastic solids, such as hydrogels, can improve local retention of co-formulated immunotherapies and provide sustained delivery. Here, we investigated the potential of a novel injectable hydrogel, called XCSgel, to localize immunotherapies within murine models of orthotopic triple-negative breast cancer (TNBC). Ex vivo and in vivo fluorescence imaging revealed enhanced intratumoral retention of immunotherapies co-formulated in XCSgel. Initial antitumor studies evaluated a range of antitumor cytokines co-formulated with XCSgel. Subsequent antitumor and rechallenge studies focused on the promising co-formulation of interleukin-12 (IL-12) and XCSgel (XCSgel-IL12). A single injection of XCSgel-IL12 eliminated 86 % of E0771 and 20 % of mWnt orthotopic primary TNBC tumors. Mice rendered tumor-free resisted a live tumor challenge. XCSgel-IL12 also eliminated untreated abscopal E0771 tumors in 67 % of mice. XCSgel-IL12 induced profound changes to the tumor-immune microenvironment, including a 3-fold reduction in the frequency of exhausted CD8+ T cells and a 3.2-fold increase in activated, proliferating CD8+ T cells. XCSgel and XCSgel-IL12 were well tolerated with no severe local or systemic side effects. Overall, XCSgel-IL12 is a promising localized immunotherapy capable of safely eliminating both primary treated and secondary abscopal tumors, indicating that systemic immunotherapy may not be required for systemic control of cancer.

Metabolic stress in the tumor microenvironment (TME) promotes T cell dysfunction and immune checkpoint inhibitor (ICI) resistance. We examined the contribution of activating transcription factor 4 (ATF4), the central node of the integrated stress response (ISR), to T cell dysfunction in tumors. CD8+ tumor-infiltrating lymphocytes (TILs) in patient samples exhibited chronic ATF4 activity, which was reflected across various tumor models. Hypoxia in the TME imposed chronic ATF4 activity via the ISR kinases. ATF4 overexpression in CD8+ T cells induced metabolic polarity, mitochondrial oxidative stress, and cell death, impairing antitumor immunity. Chronic ATF4 transcriptional activity replicated the terminal exhaustion CD8+ T cell state independent of T cell receptor (TCR) stimulation. Genetic or pharmacologic attenuation of ATF4 reduced mitochondrial oxidative stress and promoted CD8+ TIL viability, enabling response to programmed cell death protein-1 (PD-1) inhibitor therapy and conferring protection from re-emergent disease. Thus, the ISR converges on chronic ATF4 activity in CD8+ TILs as a barrier to ICI response, positioning ISR therapeutics as candidates for immunotherapy.

The abscopal effect, in which local radiotherapy (RT) drives regression of distant tumors, remains unpredictable and mechanistically elusive. Using a panel of p53-null murine breast cancer models, we identified tumor-intrinsic determinants of abscopal competence to RT and immune checkpoint inhibitors (ICI). Abscopal-competent tumors exhibited heightened type I interferon stimulated gene (ISG) expression, induction of the necroptosis mediator ZBP1, and recruitment of antigen-presenting cells (APCs) and effector T cells in distant tumors. Transcriptomic analyses revealed PARP7 as a tumor-intrinsic suppressor of RT-induced ISGs and necroptosis. Pharmacologic PARP7 inhibition amplified RT-driven ISGs and ZBP1-dependent necroptosis. In vivo, PARP7 blockade combined with RT + ICI conferred abscopal competency to resistant tumors, improving distant tumor control, systemic immune activation, and survival. Notably, ZBP1 loss abrogated these effects, preventing APC recruitment and T cell priming. These findings establish PARP7 and necroptosis as opposing regulators of abscopal responses and nominate PARP7 inhibition as a strategy to overcome RT + ICI resistance.

Chimeric antigen receptor T cells (CAR T cells) with T stem (TSCM) cell-like phenotypic characteristics promote sustained antitumor effects. We performed an unbiased and automated high-throughput screen of a kinase-focused compound set to identify kinase inhibitors (KIs) that preserve human TSCM cell-like CAR T cells. We identified three KIs, UNC10225387B, UNC10225263A and UNC10112761A, that combined in vitro increased the frequency of CD45RA+CCR7+TCF1hi TSCM cell-like CAR T cells from both healthy donors and patients with cancer. KI-treated CAR T cells showed enhanced antitumor effects both in vitro and in vivo in mouse tumor models. The KI cocktail maintains TSCM cell-like phenotype preferentially in CAR T cells originating from naive T cells and causes transcriptomic changes without arresting T cell activation or modulating the chromatin organization. Specific kinases, ITK, ADCK3, MAP3K4 and CDK13, targeted by the KI cocktail in a dose-dependent manner are directly associated with the preservation of TSCM cell-like CAR T cells. Knockdown of these kinases individually or in combination enriches for TSCM cell-like CAR T cells, but only CAR T cells generated in the presence of the KI cocktail show robust expansion and differentiation on stimulation with tumor cells. Overall, transient pharmacological inhibition of strategically targeted kinases maintains stem-like features in CAR T cells and improves their antitumor activity. Dotti and colleagues performed an unbiased, high-throughput screen of kinase inhibitors to identify a three-kinase inhibitor cocktail capable of preserving a stem-cell-like subset in chimeric antigen receptor T cells.

Short-interfering RNAs (siRNAs) can treat a wide range of human diseases from viral outbreaks to cancer, however, targeted siRNA delivery remains a significant challenge. Recently, significant efforts have focused on developing novel delivery technologies for siRNAs, targeting tumors and other extrahepatic tissues. B7-H3 (CD276), a B7 family of immune checkpoint proteins, is highly expressed in cancer cells and tumor endothelial cells (TECs). We employed an mRNA display campaign against the hB7-H3 protein and discovered a macrocyclic binding peptide, JP5. Using surface plasmon resonance, we confirmed specific binding of JP5 to both human and murine B7-H3 with a KD of approximately 770nM and 600nM, respectively. Characterization of protease stability showed that greater than 80% of the peptide remains intact after incubation with 50% serum for 24h. Further, our team conjugated JP5 to chemically stabilized siRNAs labeled with a Cy5 fluorophore to observe biodistribution of siRNAs, including cancer cells and and TECs. Mice were implanted with A431 WT or B7H3 KO cells, and upon xenograft tumor formation, mice were given PBS (control) or JP5-siRNA-Cy5 (n=5 mice/group). Gross biodistribution by IVIS imaging was found in the tumors, skin, bowel, liver and kidney, with minimal accumulation in the heart and lungs. Flow cytometry and immunofluorescence revealed that JP5 conjugation directed siRNAs to ∼80% of TECs and to 40-60% of cancer cells, with a significant reduction in B7H3 KO cells. In conclusion, the JP5 macrocycle delivers siRNAs to B7H3-expressing cancer cells and TECs in the tumor microenvironment, paving a path forward for sophisticated cancer therapeutics. Danyan Li, Lincy Edatt, Jillian Perry, Avery Huber, William David Green, Jarred Michael Green, Justin Milner, Albert Bowers, Chad Pecot. A novel B7H3-targeting macrocyclic peptide conjugate efficiently delivers siRNAs to cancer cells and tumor endothelium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6690.

Non-small cell lung cancer (NSCLC) largely consists of lung squamous carcinoma (LUSC) and lung adenocarcinoma (LUAD). Alterations in the tumor protein p53 (TP53) and phosphatase and tensin homolog (PTEN) tumor suppressors are common in both subtypes, but their relationship with SOX2 is poorly understood. We deleted Trp53 or Pten in a C57BL/6 Sox2hi Nkx2-1-/-Lkb1-/- (SNL) genetic background and generated a highly metastatic LUSC cell line (LN2A; derived from a Sox2hi mouse model, followed by Trp53, Pten, and cyclin dependent kinase inhibitor 2A [Cdkn2a] deletion). Histologic and single-cell RNA-Seq analyses corroborated that SNL mice developed mixed tumors with both LUAD and LUSC histopathology while SNL-Trp53 and SNL-Pten mice developed LUAD and LN2A tumors that retained LUSC morphology. Compared with SNL mice, additional loss of Trp53 or Pten resulted in significantly reduced survival, increased tumor burden, and altered tumor mucin composition. We identified a subcluster of CD38+ tumor-associated inflammatory monocytes in the LN2A model that was significantly enriched for activation of the classical and alternative complement pathways. Complement factor B (CFB) is associated with poor survival in patients with LUSC, and we observed the LN2A model had significantly improved survival on a Cfb-/- background. Our findings demonstrate a cooperative role of Trp53 and Pten tumor suppressors in Sox2-mediated NSCLC tumor progression, mucin production, and remodeling of the immune tumor microenvironment.

Tumor-infiltrating lymphocytes (TILs) with a tissue-resident memory CD8+ T cell (Trm) phenotype are associated with improved patient outcomes in solid malignancies. To define programs governing the formation of Trm-like TIL, we performed paired single-cell RNA sequencing and single-cell ATAC sequencing of T cell receptor (TCR)-matched CD8+ T cells in models of infection and cancer. Enhancer-driven regulons assembled from multiomic profiling data revealed epigenetic and transcriptional programs regulating the formation of Trm-like TIL in relation to canonical exhausted and memory T cell states. The transcriptional regulator KLF2 repressed the formation of CD69+CD103+ Trm-like TIL and limited anti-tumor activity. Conversely, sustained expression of the transcription factor BATF enhanced formation of CD69+CD103+ TIL, contingent upon downregulation of KLF2. Transforming growth factor β (TGF-β) signaling and CD103 expression were necessary for Trm-like TIL formation, but BATF overexpression was sufficient to drive formation of CD69+CD103+ TIL in TGFBR2-silenced cells. These findings reveal mechanisms of Trm-like TIL differentiation and provide a framework for considering tissue residency in the context of CD8+ T cell heterogeneity in the tumor microenvironment.

Immune evasion by tumors is promoted by low T cell infiltration, ineffective T cell activity directed against the tumor, and reduced tumor antigen presentation. The TET2 DNA dioxygenase gene is frequently mutated in hematopoietic malignancies and loss of TET enzymatic activity is found in a variety of solid tumors. We showed previously that vitamin C (VC), a cofactor of TET2, enhances tumor-associated T cell recruitment and checkpoint inhibitor therapy responses in a TET2-dependent manner. Using single-cell RNA sequencing (scRNA-seq) analysis performed on B16-OVA melanoma tumors, we have shown here that an additional function for TET2 in tumors is to promote expression of certain antigen presentation machinery genes, which is potently enhanced by VC. Consistently, VC promoted antigen presentation in cell-based and tumor assays in a TET2-dependent manner. Quantifying intercellular signaling from the scRNA-seq dataset showed that T cell-derived IFN-γ-induced signaling within the tumor and tumor microenvironment requires tumor-associated TET2 expression, which is enhanced by VC treatment. Analysis of patient tumor samples indicated that TET activity directly correlates with antigen presentation gene expression and with patient outcomes. Our results demonstrate the importance of tumor-associated TET2 activity as a critical mediator of tumor immunity, which is augmented by high-dose VC therapy.

Systemic delivery of immunotherapy is dose-limited and often causes serious immune-related adverse events. Intratumoral injections can reduce systemic immunotoxicities and increase immunotherapy concentrations within a tumor. However, high pressures associated with direct tumor injection limits injectate retention, as low viscosity, saline-based solutions rapidly leak out of tumors. Viscoelastic solids, such as hydrogels, can improve local retention of co-formulated immunotherapies and provide sustained delivery. Prior work demonstrated that a chitosan-based hydrogel, XCSgel, was shear-thinning, self-healing, injectable, biocompatible, and clinically imageable. Here, we investigated XCSgel as a localized intratumoral delivery platform in the context of murine models of orthotopic triple-negative breast cancer. The intratumoral retention of immunotherapeutics co-formulated in XCSgel was characterized both ex vivo and in vivo via fluorescence imaging. Histopathological responses to intratumoral injections of XCSgel alone were scored by a veterinary pathologist. Initial antitumor studies evaluated a range of antitumor cytokines co-formulated with XCSgel. Subsequent antitumor and rechallenge studies evaluated the efficacy of a single intratumoral injection of interleukin-12 (IL-12) co-formulated in XCSgel (XCSgel-IL12) to control the growth of primary and abscopal tumors while inducing protective immunity. Pharmacokinetics studies quantified the systemic dissemination of IL-12 and consequent production of interferon-gamma following intratumoral injection with XCSgel co-formulation. Spectral flow cytometry was used to document changes in the tumor-immune microenvironment (TIME). XCSgel resisted tumor leakage and slowly released three model cytokines. XCSgel could be tuned for faster or slower release of embedded therapeutics. XCSgel-IL12 outperformed XCSgel formulations with other commonly used antitumor cytokines. A single injection of XCSgel-IL12 eliminated 86% E0771 and 20% mWnt orthotopic primary TNBC tumors. Mice rendered tumor-free resisted a live tumor challenge. XCSgel-IL12 also eliminated 67% untreated abscopal E0771 tumors. XCSgel-IL12 induced profound changes to the TIME, including a 3-fold reduction in the frequency of exhausted CD8+ T cells and a 3.2-fold increase in activated, proliferating CD8+ T cells. XCSgel is a promising localized delivery platform well-suited to enhance the retention and antitumor activity of potent immunotherapeutics. A single injection of XCSgel-IL12 can eliminate both primary and abscopal solid tumors, indicating that systemic immunotherapy may not be required for systemic control of cancer. ### Competing Interest Statement The authors have declared no competing interest.