Supplementary Table S3 shows UCSC-predicted transcription factors that bind to CKB promoters.

Supplementary Table S1 shows PCr IHC staining score in human glioma microarray.

BackgroundImmune checkpoint blockade therapy aims to restore T-cell function within the tumor microenvironment (TME), eliciting durable antitumor responses. However, clinical response rates to PD-1/PD-L1 inhibitors remain limited, particularly in immunologically "cold" tumors such as pancreatic cancer, underscoring the need for alternative immunotherapeutic strategies. V-set and immunoglobulin domain-containing 4 (VSIG4) has been implicated in tumor progression, but its functional role in T-cell regulation and mechanisms of tumor immune evasion remain unclear. METHODS:Syngeneic tumor models and human VSIG4 knock-in mice were employed to investigate the therapeutic effect of VSIG4 blockade. Mouse-specific and human-specific neutralizing antibodies against VSIG4 were administered across multiple tumor types, including pancreatic cancer. Tumor-infiltrating immune cells were analyzed by flow cytometry and functional assays. Mechanistic studies examined the interaction between VSIG4 and solute carrier family 3 member 2 (SLC3A2) and its impact on amino acid transport, ion flux, and T-cell activation. RESULTS:Therapeutic blockade of VSIG4 significantly suppressed tumor growth and prolonged survival in several cancer models, with particular efficacy in pancreatic cancer. VSIG4 expression correlated with tumor aggressiveness, and its blockade reactivated CD8+ T cells within the TME, enhancing intratumoral infiltration and effector function. Mechanistically, VSIG4 directly bound to SLC3A2, a chaperone for amino acid transporters, thereby impairing glutamine uptake, disrupting sodium and calcium flux, and ultimately suppressing T-cell activation. VSIG4 blockade restored nutrient and ion availability to CD8+ T cells, thereby reinvigorating antitumor immunity. CONCLUSIONS:These findings define a previously unrecognized metabolic-ionic checkpoint axis by which VSIG4 restricts T-cell activation through SLC3A2. Blockade of VSIG4 reprograms the TME and enhances antitumor immunity, highlighting VSIG4 as a promising therapeutic target, particularly in metabolically repressive tumors such as pancreatic cancer.

Cancer immunotherapy has achieved durable clinical benefit in a subset of patients; however, most solid tumors-particularly immunologically cold tumors-remain refractory due to profound T-cell exclusion and dysfunction within the tumor microenvironment (TME). Here, we identify CD209 (DC-SIGN), a C-type lectin receptor expressed by tumor-infiltrating myeloid cells, as a previously unrecognized myeloid-derived mechanical immune checkpoint that suppresses antitumor T-cell immunity. Using human CD209 knock-in mice, we demonstrate that therapeutic blockade of CD209 markedly enhances T-cell infiltration and activation, resulting in robust tumor regression across multiple models, including ovarian cancer and glioblastoma. Mechanistically, CD209 directly engages intercellular adhesion molecule 2 (ICAM-2) on T cells, triggering ezrin/radixin/moesin (ERM) phosphorylation and increasing T-cell cortical stiffness. This biophysical reprogramming destabilizes T cell-antigen-presenting cell conjugation and attenuates T-cell receptor signaling. Analyses of human cancer datasets and primary tumor specimens reveal that elevated CD209 expression inversely correlates with CD8 T-cell infiltration and is associated with reduced overall survival. Moreover, CD209 expression is significantly enriched in PD-1 therapy non-responders and positively correlates with TIDE (Tumor Immune Dysfunction and Exclusion) scores across multiple cancer types. Together, these findings establish CD209 as a central regulator of myeloid-driven T-cell mechanical dysfunction and highlight its potential as a therapeutic target and a candidate companion diagnostic biomarker for immunotherapy response. ### Competing Interest Statement The authors have declared no competing interest. the Key Project of the National Natural Science Foundation of China, 82430076 the National Science Fund for Distinguished Young Scholars, 82225029 the Special project of the National Natural Science Foundation of China, 82441031 the General Program of National Natural Science Foundation of China, 82572023 the Youth Fund of the National Natural Science Foundation of China, 82502806, 82302628, 82301989, 82301987, 82402704 the Postdoctoral Foundation of China, 2022M720658, 2022M720659 the Sichuan Postdoctoral Innovation Plan, BX202202 the Postdoctoral Foundation of Sichuan Province, TB2022086, TB2023092 The grants from the Department of Science and Technology of Sichuan Province, 2026NSFSC1929 the General Program of National Natural Science Foundation of China, 82572023 The Sichuan Province Innovative Talent Funding Project for Postdoctoral Fellows, BX202516 The General Program of Natural Science Foundation of Sichuan Province, 2026NSFSC0572

Supplementary Table S2 shows CKB IHC staining score in human glioma microarray.

Cyclic GMP–AMP synthase (cGAS) is a key cytosolic DNA sensor that triggers antiviral immunity by generating the second messenger cyclic GMP–AMP (cGAMP) upon binding to double-stranded DNA. While numerous mechanisms have been shown to restrain cGAS activity, its posttranslational regulation remains incompletely understood. Here, we identify the receptor tyrosine kinase Eph receptors B2 (EPHB2) as a negative regulator of DNA virus–induced immune responses. During viral infection, phosphorylation of EPHB2 by spleen-associated tyrosine kinase (SYK) promotes the direct phosphorylation of cGAS at Tyr483 and Tyr510, thereby facilitating the recruitment of the E3 ubiquitin ligase MARCH8 and the autophagy adaptor p62, which mediate selective autophagic degradation of cGAS. Myeloid-specific EphB2 deletion enhances HSV-1 clearance and markedly improves mouse survival. Given its high expression in the nervous system, we further show that neuronal EPHB2 limits antiviral immunity, as its loss in human neuronal cells or neuron-specific knockout mice enhances cGAS–STING signaling and protects against herpes simplex virus 1 (HSV-1) infection. Moreover, a newly identified small-molecule EPHB2 inhibitor, Q-1-28, restores cGAS levels, amplifies antiviral cytokine production, and protects mice from HSV-1–induced lethality. Together, our findings reveal a phosphorylation-dependent mechanism governing cGAS degradation and establish EPHB2 as a potential therapeutic target for host-directed antiviral intervention.

Immune checkpoint inhibitors (ICIs) targeting PD-1/PD-L1 have achieved clinical success, yet most patients fail to respond and many develop immune-related adverse events (irAEs). Although interferon gamma (IFN-γ) is considered the canonical driver of PD-L1 expression, regulation of PD-L1 in myeloid cells within the tumor microenvironment (TME) remains poorly defined. Here, we identify human epididymis protein 4 (HE4), a tumor-secreted glycoprotein overexpressed in multiple cancers, as an unrecognized inducer of myeloid PD-L1 transcription. HE4 directly binds IFN-γ receptors, activates JAK-STAT3 signaling, and upregulates PD-L1. Neutralization of mouse or human HE4 with monoclonal antibodies reduced myeloid PD-L1 expression, restored CD8+ T cell activity, and suppressed tumor growth in syngeneic and humanized models, while inducing fewer irAEs than PD-1 blockade. Clinically, high HE4 expression predicts poor prognosis but correlates with improved response to PD-1 inhibitors in lung adenocarcinoma, highlighting HE4 as both a therapeutic target and predictive biomarker.

The function of innate immune sensors is intricately shaped by their spatial distribution within cells. cGAS (cyclic GMP-AMP synthase), a key cytosolic DNA sensor, illustrates this principle through its unexpected localisation to diverse organelles-including the nucleus, micronuclei, mitochondria, and plasma membrane. In these compartments, cGAS assumes distinct regulatory states and executes specialised functions. For instance, chromatin-bound nuclear cGAS remains inactive under homeostasis but contributes to genome maintenance during genotoxic stress, whereas mitochondrial or micronuclear cGAS links damage signals to inflammation and cell death. This review synthesises recent advances in the spatial regulation of cGAS, focusing on mechanisms such as membrane interactions and post-translational modifications. We further reframe cGAS as a multifunctional regulator in infection, cancer, autoimmunity, and ageing, and introduce a unifying 'location code' framework. This framework proposes that the combined influence of PTMs, protein interactions, and membrane affinities dictates cGAS localisation, functional output, and pathological outcomes, thereby paving the way for spatially informed therapeutic interventions.

Stop codon readthrough is widespread across eukaryotes and often dismissed as translational noise, yet its tissue/stage-specific occurrence suggests adaptive roles in proteome tuning. We asked whether readthrough-related mechanisms can mitigate stage-specific pleiotropic trade-offs without genomic change. In the filamentous ascomycete Fusarium graminearum, the functional solution relies on developmentally programmed A-to-I "stop-loss" RNA editing of the terminal NDR kinase gene FgDBF2 (UAG→UIG, read as UGG), instead of stochastic readthrough. This edit adds a short, intrinsically disordered C-terminal extension acting as a cis-encoded destabilizing element, lowering FgDbf2 dosage during ascospore formation. Genetic and cell biological analyses show meiosis proceeds independently of FgDbf2, but accurate one-nucleus/one-spore encapsulation is promoted by the edited, destabilized isoform. Blocking editing (stop retained) or increasing unedited FgDbf2 yields malformed, multinucleate spores despite normal nuclear counts, establishing ascospore morphogenesis as dosage-sensitive rather than isoform-specific. Conversely, constitutive production of the edited, destabilized isoform impairs vegetative growth and hyphal septation, suggesting stage-specific antagonism with mitotic functions. Mechanistically, the edited tail destabilizes Dbf2 and GFP, likely via nonclassical proteostasis pathways. Epistasis analysis indicates the CDK Cdc2A also restrains FgDbf2 and elevated Cdc2A partially suppresses defects caused by excess unedited FgDbf2. Comparative and transcriptomic analyses reveal conservation of DBF2 stop-loss editing across Sordariomycetes and identify many stop-loss edits encoding destabilizing tails consistent with positive genome-wide selection. We propose that stage-specific stop-loss editing is a developmentally gated dosage-buffering mechanism that transiently reduces NDR kinase abundance during ascospore formation, thereby alleviating growth-reproduction trade-offs without requiring gene duplication.

Supplementary Table S6 shows the sequences of shRNAs used in this study.

Supplementary Table S4 shows PCr and BRD2 IHC staining scores in the serial sections of human glioma microarrays.

Supplementary Figures S1 shows that GSCs produce high levels of phosphocreatine through upregulating CKB. Supplementary Figures S2 shows that ZEB1 promotes CKB transcription in GSCs. Supplementary Figures S3 shows that knockdown of CKB impedes GBM growth. Supplementary Figures S4 shows that disruption of phosphocreatine production impedes GBM growth. Supplementary Figures S5 shows that cCr treatment shows no side effect on mice. Supplementary Figures S6 shows that phosphocreatine binds to BRD2 and inhibits its ubiquitin mediated proteasomal degradation. Supplementary Figures S7 shows that phosphocreatine promotes chromosome segregation and GSC proliferation through BRD2 mediated transcription. Supplementary Figures S8 shows that disruption of phosphocreatine biosynthesis by cCr improves JQ1 therapeutic efficacy in GBM.

Supplementary Table S5 shows the detailed genetic mutations of four GBM cell lines.

Supplementary Table S7 shows the primer sequences used for Real-Time PCR.

How viral envelope sensing is coupled to intracellular DNA sensing remains poorly defined. Here, we identify the receptor tyrosine kinase EPHA5 as a membrane-associated immune sensor that directly recognizes herpes simplex virus type 1 (HSV-1) glycoprotein gD, triggering receptor oligomerization and autophosphorylation. Activated EPHA5 phosphorylates both cGAS and STING, thereby enhancing cGAS DNA binding, cGAMP production, and downstream type I interferon (IFN-I) responses. Genetic ablation of EPHA5 in macrophages or mice markedly impairs antiviral cytokine induction and viral clearance in vivo. Conversely, genetic deletion or pharmacological inhibition of EPHA5 attenuates aberrant cGAS–STING activation and ameliorates lupus-like pathology, highlighting EPHA5 as a druggable upstream regulator of IFN-I responses. Together, our findings establish a receptor-kinase signaling axis that links viral envelope recognition to cytosolic DNA sensing and innate immune activation.

Systematic identification of prebiotic-microbe interactions is essential for developing precision microbiome-targeted interventions to improve human health. In this study, we developed an in vivo systematic screening platform to evaluate microbiota-prebiotic crosstalk and applied it to identify a synbiotic combination effective against dextran sulfate sodium (DSS)-induced colitis in mice. Specifically, we first established a humanized gut microbiota mouse model by colonizing mice with 73 microbial strains, which showed highly abundant and prevalent in the human gut. Concurrently, we administered the mice with 28 different prebiotic or prebiotic candidates, including polyphenols, polysaccharides, vitamins, and minerals common in the market. Following the DSS-induced colitis, we evaluated the protective effects of each microbiota-prebiotic pairing. Fourteen prebiotic or prebiotic candidates, designated as the ESS group, significantly alleviated colitis, partly by enriching specific beneficial microbes such as Bacteroides thetaiotaomicron, Akkermansia muciniphila, and Erysipelatoclostridium ramosum prior to disease onset. Further experiments revealed two symbiotic combinations with the strongest anti-inflammatory effects: calcium-magnesium tablets (CMT) combined with either B. thetaiotaomicron or A. muciniphila. Mechanistically, CMT promoted the growth of B. thetaiotaomicron and alleviated inflammation by upregulating genes associated with probiotic activity. Finally, in an intervention trial involving healthy human volunteers, CMT selectively increased B. thetaiotaomicron abundance without altering the overall gut microbiota composition. Together, our study presents a systematic framework for elucidating microbe-prebiotic interactions, identifying synbiotic combinations with therapeutic potential, and advancing precision microbiome-based strategies for disease prevention and treatment.

Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) detects viral or endogenous DNA, activating the innate immune response to infections and autoimmune diseases. Upon binding to double-stranded DNA, cGAS synthesizes 2030 cGMP-AMP, which triggers type I interferon production. Besides its presence in the cytosol and nucleus, cGAS is found at the plasma membrane, although its significance remains unclear. Here, we report that cGAS associates with myosin 1F (MYO1F) at the plasma membrane of human and mouse macrophages. During viral infection, phosphorylation of MYO1F by spleen-associated tyrosine kinase (SYK) facilitates the recruitment of lysine acetyltransferase 2A (KAT2A), which acetylates cGAS at lysine residues 421, 292, and 131, essential for its activation. Moreover, membrane-localized cGAS is crucial for signaling activation and type I interferon production triggered by virus- cell fusion due to Mn2+ release from organelles. Our results highlight the importance of MYO1F-mediated cGAS localization for its full activation in response to viral infection.

Many virus species, including Ebola virus, Marburg virus, SARS-CoV-2, dengue virus (DENV) and Zika virus (ZIKV), exploit CD209 and CD209L as alternative or attachment receptors for viral cis- or trans-infection. Thus, CD209 and CD209L may be critical targets for the development of therapeutic monoclonal blocking antibody drugs to disrupt the infection process caused by multiple viruses. Here, we produced a human chimeric monoclonal blocking antibody that simultaneously blocks CD209 and CD209L, namely 7-H7-B1. We show that 7-H7-B1 effectively blocks multiple pseudotyped or live viral infections in vitro, including SARS-CoV, SARS-CoV-2, Ebola virus, Marburg virus, ZIKV and DENV infections. However, the 7-H7-B1 mAb does not provide favourable protection against Zaire Ebola virus or ZIKV infection in hCD209 knock-in mice in vivo. Thus, our findings indicate that although CD209 and CD209L are critical for multiple viral infections in vitro, they may play only a partial role in viral infections in vivo.

Tumor-associated neutrophils (TANs) represent a significant barrier to the effectiveness of immune checkpoint blockade (ICB) therapy. A comprehensive understanding of TANs' regulatory mechanisms is therefore essential for predicting ICB efficacy and improving immunotherapy strategies. Our study reveals that MYO1F is selectively downregulated in neutrophils within both human cancers and murine tumor models, showing a negative correlation with ICB response. Mechanistically, MYO1F normally inhibits neutrophil immunosuppression and proliferation by restraining STAT3 activity. However, during tumorigenesis, tumor-derived TGF-β1 disrupts the binding of SPI1 to intron 8 of Myo1f via DNA methylation, thereby suppressing Myo1f transcription. The resultant decrease in MYO1F reprograms neutrophils into an immunosuppressive state through the STAT3-dependent signaling pathways. This immunosuppressive state further contributes to tumor microenvironment (TME) remodeling by inducing CTL exhaustion. These findings establish MYO1F as a critical regulator within TANs, highlighting its significant role in modulating ICB therapy efficacy.

As a common disease in human life, fungal infection poses a serious threat to human life and health. Moreover, owing to the rapid development of the immune escape mechanisms of fungi and the emergence of new drug-resistant fungi, existing therapeutic drugs are no longer able to meet the treatment needs of patients. In particular, the World Health Organization (WHO) published the first Fungal Priority Pathogens List (FPPL) in 2022, further emphasizing that we need to pay more attention to invasive fungal infections. In addition, the WHO has called for increased global investment in fungal infections and the development of antifungal drugs. In this review, we introduce the mechanism by which innate immune cell PRRs recognize fungal pathogen PAMPs, the role of adaptive immune cells in antifungal immunity, fungal infections caused by immune deficiencies, and the latest research progress in immune-based fungal therapies.