InVivoMAb anti-mouse CD8β (Lyt 3.2)

Enterovirus D68 (EV-D68) causes respiratory illness in children. It also causes severe paralysis called acute flaccid myelitis (AFM), which has become a global health threat. Here, we generated an mRNA vaccine expressing virus-like particles (VLPs) of EV-D68. We found that the mRNA vaccine elicited potent neutralizing antibodies against EV-D68 in the blood, and the neutralizing titer was superior to that of the inactivated whole virion (IWV) vaccine. The mRNA vaccine showed protective effects against intranasal challenge with EV-D68, and antisera from the vaccinated mice prevented the paralysis caused by EV-D68 infection in neonatal mice. Moreover, the mRNA vaccine induced neutralizing antibodies in the respiratory tract, which is the entry site for EV-D68. Additionally, it attenuated infection with coxsackievirus B3 (CVB3), which belongs to another enterovirus group, via CD8+ T cell responses. In conclusion, our results suggest that this mRNA vaccine is a promising candidate for EV-D68 prevention.

Host-pathogen interactions involve two critical strategies: resistance, whereby hosts clear invading microbes, and tolerance, whereby hosts carry high pathogen burden asymptomatically. Here, we investigate mechanisms by which Salmonella-superspreader (SSP) hosts maintain an asymptomatic state during chronic infection. We found that regulatory T cells (Tregs) are essential for this disease-tolerant state, limiting intestinal immunopathology and enabling SSP hosts to thrive, while facilitating Salmonella transmission. Treg depletion in SSP mice resulted in decreased survival, heightened gut inflammation, and impairment of the intestinal barrier, without affecting Salmonella persistence. Colonic Tregs from SSP mice exhibited a unique transcriptomic profile characterized by the upregulation of type 1 inflammatory genes, including the transcription factor T-bet. In the absence of Tregs, we observed robust expansion of cytotoxic CD4+ T cells, with CD4+ T cell depletion restoring homeostasis. These results uncover a critical host strategy to establish disease tolerance during chronic enteric infection, providing novel insights into mucosal responses to persistent pathogens and chronic intestinal inflammation.

Pancreatic ductal adenocarcinoma (PDAC) remains refractory to current immune checkpoint blockade (ICB) therapies, necessitating innovative therapeutic strategies. Emerging evidence implicates aberrant sialoglycan upregulation as a key mediator of immune evasion in PDAC. Herein, we report Y-320, a highly potent oral sialylation inhibitor discovered through high-throughput screening. Y-320 suppresses α-2,3/2,6-sialylation in PDAC cells (IC50 ≈ 200 nM) with >300-fold higher activity than the known pan-inhibitor P-3Fax-Neu5Ac. Structural analyses reveal competitive occupation of multiple sialyltransferases' substrate-binding pockets as Y-320's action mechanism. In vivo, Y-320 significantly inhibits tumor growth and remodels the tumor immune microenvironment. Mechanistic studies establish that the therapeutic efficacy of Y-320 depends on the coordinated engagement between CD8+ T cell and macrophage. Importantly, Y-320 synergizes with anti-PD-1 therapy to overcome ICB resistance in PDAC, demonstrating superior tumor suppression compared to monotherapies. Our findings demonstrate that Y-320 shows promise for use as a therapeutic agent for cancer and validates sialylation inhibition as a novel glycoimmune checkpoint strategy for PDAC and other immunotherapy-resistant malignancies.

Tumor immune microenvironment greatly influences triple-negative breast cancer (TNBC) progression. Identifying targets to convert "cold" tumors into "hot" tumors holds promise for improving treatment outcomes. Here, we show that high expression of NEDD4, an HECT-type E3 ubiquitin ligase, correlates with poor prognosis and reduced CD8+ T cell infiltration in TNBC patients. NEDD4 depletion in TNBC cells significantly inhibits tumor growth through enhancing CD8+ T cell-mediated cytotoxicity in immunocompetent hosts. Mechanistically, NEDD4 depletion stabilizes β-TrCP, leading to YAP ubiquitination and degradation. Downregulated YAP reprograms the immunosuppressive tumor extracellular matrix (ECM) to increase CD8+ T cell infiltration. Furthermore, a small-molecule inhibitor of NEDD4, XMU-MP-10, exhibits significant in vivo efficacy in inhibiting TNBC tumor growth by enhancing CD8+ T cell infiltration in mouse models. Collectively, our findings suggest that the genetic depletion or pharmacological inhibition of NEDD4 enhances antitumor immune responses via the β-TrCP/YAP/ECM cascades, offering a promising therapeutic strategy for TNBC treatment.

Administration of IL-2 may promote the suppressive function and proliferation of Treg cells that cause immune tolerance in patients with cancer, which causes low-dose IL-2 to fail in achieving an optimal anti-tumor effect. Here, we designed an immunocytokine by fusing IL-2 and an anti-TIGIT monoclonal antibody, named αTIGIT-IL2, that targets Treg cells and promotes their fragility in the tumor milieu. These fragile-like Treg cells show impaired suppressive function and high IFN-γ production, triggering an immune-reactive tumor microenvironment. Such inflammation leads to the recruitment and functional reprogramming of intratumoral neutrophils, improving cross-talk between neutrophils and CD8+ T cells and enhancing the antitumor ability of CD8+ T cells. Combination therapy with αTIGIT-IL2 and PD-1 blocker could eliminate triple-negative breast cancer (TNBC) tumors resistant to immune checkpoint blockade (ICB) therapy. These findings provide the basis for developing a new generation of immunocytokines that target Treg cells and promote their fragility in the tumor milieu, resulting in robust antitumor immunity.

CTLA-4 is a promising target for immune checkpoint inhibition in cancer therapy, with CTLA-4 blockade achieving prolonged overall survival for responding patients. However, the progressively elevated doses of anti-CTLA-4 agents, aimed at achieving better efficacy, result in increased toxicities, limiting their clinical applications. Here, we generate a prodrug design of the anti-CTLA-4 antibody, named ProCTLA-4, by folding the Fab fragment of the antibody in a tumor-associated protease-based manner. In preclinical mouse models, ProCTLA-4 effectively depletes suppressive regulatory T cells within the tumor microenvironment and enhances tumor-associated antigen-specific CD8+ T cell responses, while exhibiting reduced toxicity compared to currently available CTLA-4 blockade approaches. Furthermore, compared to the currently used Probody therapeutics for anti-CTLA-4 (BMS986288), ProCTLA-4 has more advantages in efficacy amplification, such as in poor immunogenic melanoma. Our design establishes an alternative paradigm for antibody agents that limits the emergence of immune-related adverse events (irAE) while increasing therapeutic efficacy.

Long-term survival of glioblastoma multiforme (GBM) remains challenging, spurring the development of novel therapies such as oncolytic virus therapy. While oncolytic virus shows promise in clinical trials, many patients do not respond to this therapy. Here, we perform a CRISPR screening and identify the non-canonical BRG1/BRM-associated factor (ncBAF) complex as a pivotal tumor-intrinsic factor for oncolytic virotherapy resistance. Knocking out the ncBAF-specific subunit bromodomain-containing protein 9 (BRD9) markedly augments the oncolytic efficacy of oncolytic herpes simplex virus type 1 (oHSV1) and enhances antitumor immunity. Mechanistically, BRD9 binds to RELA and potentiates the expression of downstream antiviral genes. Notably, the application of BRD9 inhibitor (IBRD9) significantly enhances the oncolytic activity of oHSV1 in various GBM models. Moreover, reduced BRD9 levels strongly correlate with improved outcomes in clinical trials of oHSV1. These findings suggest that BRD9 is an attractive target for overcoming the resistance to oHSV1 in glioblastoma treatment.

Immunomodulatory agents benefit a small percentage of patients with oral cancer (OC), a subset of head and neck cancer. Cathepsin S (CTSS), a lysosomal protease, has been frequently associated with tumor immunity. This study aimed to investigate the mechanism by which tumor CTSS affects anti-tumor immunity through the regulation of interleukin-7 (IL-7) to overcome this obstacle.

CD38, an ecto-enzyme involved in NAD+ catabolism, is highly expressed in exhausted CD8+ T cells and has emerged as an attractive target to improve response to immune checkpoint blockade (ICB) by blunting T cell exhaustion. However, the precise role(s) and regulation of CD38 in exhausted T cells and the efficacy of CD38-directed therapeutic strategies in human cancer remain incompletely defined. Here, we show that CD38+CD8+ T cells are induced by chronic TCR activation and type I interferon stimulation and confirm their association with ICB resistance in human melanoma. Disrupting CD38 restores cellular NAD+ pools and improves T cell bioenergetics and effector functions. Targeting CD38 restores ICB sensitivity in a cohort of patient-derived organotypic tumor spheroids from explanted melanoma specimens. These results support further preclinical and clinical evaluation of CD38-directed therapies in melanoma and underscore the importance of NAD+ as a vital metabolite to enhance those therapies.

Despite harboring the highest tumor mutational burden of all cancers, basal cell carcinoma (BCC) has low immunogenicity. Here, we demonstrate that BCC's low immunogenicity is associated with epigenomic suppression of antigen presentation machinery reminiscent of its cell of origin. Primary BCC had low T cell infiltrates and low human leukocyte antigen class I (HLA-I) expression compared with cutaneous squamous cell carcinoma (SCC) and normal keratinocytes. Forkhead box C1 (Foxc1), a regulator of quiescence in hair follicle stem cells, was expressed in BCC. Foxc1 bound to promoter of interferon regulatory factor 1 and HLA-I genes, leading to their deacetylation and reduced expression. A histone deacetylase inhibitor, entinostat, overcame Foxc1's effect and upregulated HLA-I in BCC. Topical entinostat plus imiquimod immunotherapy blocked BCC development in mice. Collectively, our findings demonstrate that low BCC immunogenicity is associated with a stem-like quiescent program preserved in the tumor cells, which can be blocked to enable BCC immunotherapy.

Trastuzumab deruxtecan (T-DXd) is an antibody-drug conjugate (ADC) targeting HER2, exhibiting significant clinical efficacy in breast cancer (BC) with varying HER2 expression, including HER2-low and HER2-ultralow. However, the precise mechanism underlying its efficacy and the contribution of immune activation in these settings remain unclear. Here, we demonstrate that T-DXd efficacy in HER2-low and HER2-negative BC is independent of HER2 engagement and ADC internalization. Instead, its activity relies on extracellular proteases, such as cathepsin L (CTSL), within the tumor microenvironment. Irrespective of their HER2 status, tumor and stromal compartments of invasive BC abundantly express CTSL, which efficiently cleaves the specialized linker of T-DXd, facilitating payload release and inducing cytotoxicity against HER2-low/negative tumors. In HER2-positive BC, the antibody backbone of T-DXd engages Fcγ-receptors and drives antibody-dependent cellular phagocytosis (ADCP). Concurrently, its cytotoxic payload (DXd) induces immunogenic cell death, further activating myeloid cells via TLR4 and STING pathways to enhance tumor antigen presentation to CD8+ T cells. Notably, T-DXd cytotoxicity also upregulates tumor CD47 expression, dampening immune activation. Combining T-DXd with CD47 checkpoint blockade significantly enhances anti-tumor immune responses in a HER2-transgenic BC mouse model, while also inducing durable CD8+ T cell memory to prevent tumor recurrence after therapy cessation.

The impact of amino acids on tumor immunotherapy is gradually being uncovered. In this study, we screened various essential and non-essential amino acids and found that methionine enhances mRNA methylation and reduced the activation of Type I interferon pathway in bladder cancer. Through RNA sequencing, point mutations, MB49 mouse tumor models, and single-cell RNA sequencing, we demonstrated that high methionine levels elevate the expression of m6A reader YTHDF1, promoting the degradation of RIG-I, thereby inhibiting the RIG-I/MAVS-mediated IFN-I pathway and reducing the efficacy of tumor immunotherapy. Additionally, immunoprecipitation and mass spectrometry revealed that YTHDF1 binds to the eukaryotic translation initiation factor eIF5B, which acts on PD-L1 mRNA to enhance its translation and promote immune evasion. By intravesical administration of oncolytic bacteria VNP20009, we effectively depleted methionine locally, significantly prolonging mouse survival and enhancing immune cell infiltration and differentiation within tumors. Multiplex immunofluorescence assays in bladder cancer immunotherapy patients confirmed our findings. Our research elucidates two mechanisms by which methionine inhibits bladder cancer immunotherapy and proposes a targeted methionine depletion strategy that advances research while minimizing nutritional impact on patients.

Clinically available KRAS* inhibitors and IO agents alleviated the immunosuppressive tumor microenvironment in PDAC. Profound tumor regression and prolonged survival in an autochthonous PDAC model provide a compelling rationale for combining KRAS* inhibition with IO agents targeting multiple arms of the immunity cycle to combat PDAC.

Loss of IFNγ-sensitivity by tumours is thought to be a mechanism enabling evasion, but recent studies suggest that IFNγ-resistant tumours can be sensitised for immunotherapy, yet the underlying mechanism remains unclear. Here, we show that IFNγ receptor-deficient B16-F10 mouse melanoma tumours are controlled as efficiently as WT tumours despite their lower MHC class I expression. Mechanistically, IFNγ receptor deletion in B16-F10 tumours increases IFNγ availability, triggering a remodelling of the immune landscape characterised by inflammatory monocyte infiltration and the generation of 'mono-macs'. This altered myeloid compartment synergises with an increase in antigen-specific CD8+ T cells to promote anti-tumour immunity against IFNγ receptor-deficient tumours, with such an immune crosstalk observed around blood vessels. Importantly, analysis of transcriptomic datasets suggests that similar immune remodelling occurs in human tumours carrying mutations in the IFNγ pathway. Our work thus serves mechanistic insight for the crosstalk between tumour IFNγ resistance and anti-tumour immunity, and implicates this regulation for future cancer therapy.

Radiotherapy (RT) is commonly used to try to eliminate any remaining tumor cells following surgical resection of glioma. However, tumor recurrence is prevalent, highlighting the unmet medical need to develop therapeutic strategies to enhance the efficacy of RT in glioma. Focusing on the radiosensitizing potential of the currently approved drugs known to cross the blood-brain barrier can facilitate rapid clinical translation. Here, we assessed the role of catechol-O-methyltransferase (COMT), a key enzyme to degrade catecholamines and a drug target for Parkinson's disease, in glioma treatment. Analysis of The Cancer Genome Atlas data showed significantly higher COMT expression levels in both low-grade glioma and glioblastoma compared to normal brain tissues. Inhibition of COMT by genetic knockout or FDA-approved COMT inhibitors significantly sensitized glioma cells to RT in vitro and in vivo. Mechanistically, COMT inhibition in glioma cells led to mitochondria dysfunction and increased mitochondrial RNA release into the cytoplasm, activating the cellular antiviral double-stranded RNA sensing pathway and type I interferon (IFN) response. Elevated type I IFNs stimulated the phagocytic capacity of microglial cells, enhancing RT efficacy. Given the long-established safety record of the COMT inhibitors, these findings provide a solid rationale to evaluate them in combination with RT in patients with glioma. Significance: Inhibition of catechol-O-methyltransferase, a well-established drug target in Parkinson's disease, interferes with mitochondrial electron transport and induces mitochondrial double-stranded RNA leakage, activating type I interferon signaling and sensitizing glioma to radiotherapy.

Progressive decline of the adaptive immune system with increasing age coincides with a sharp increase in cancer incidence. In this study, we set out to understand whether deficits in antitumor immunity with advanced age promote tumor progression and/or drive resistance to immunotherapy. We found that multiple syngeneic cancers grew more rapidly in aged versus young adult mice, driven by dysfunctional CD8+ T-cell responses. By systematically mapping immune cell profiles within tumors, we identified loss of tumor antigen-specific CD8+ T cells as a primary feature accelerating the growth of tumors in aged mice and driving resistance to immunotherapy. When antigen-specific T cells from young adult mice were administered to aged mice, tumor outgrowth was delayed and the aged animals became sensitive to PD-1 blockade. These studies reveal how age-associated CD8+ T-cell dysfunction may license tumorigenesis in elderly patients and have important implications for the use of aged mice as preclinical models of aging and cancer.

Immunotherapy successfully complements traditional cancer treatment. However, primary and acquired resistance might limit efficacy. Reduced antigen presentation by MHC-I has been identified as potential resistance factor. Here we show that the epigenetic regulator ubiquitin-like with PHD and ring finger domains 1 (UHRF1), exhibits altered expression and aberrant cytosolic localization in cancerous tissues, where it promotes MHC-I ubiquitination and degradation. Cytoplasmic translocation of UHRF1 is induced by its phosphorylation on a specific serine in response to signals provided by factors present in the tumor microenvironment (TME), such as TGF-β, enabling UHRF1 to bind MHC-I. Downregulation of MHC-I results in suppression of the antigen presentation pathway to establish an immune hostile TME. UHRF1 inactivation by genetic deletion synergizes with immune checkpoint blockade (ICB) treatment and induces an anti-tumour memory response by evoking low-affinity T cells. Our study adds to the understanding of UHRF1 in cancer immune evasion and provides a potential target to synergize with immunotherapy and overcome immunotherapeutic resistance.