�����ƽ��

Highlights What are the main findings? Hypoxia, alone or combined with hyperglycemia, induces endothelial cell senescence without activating the classical pro-inflammatory SASP.This condition is associated with the upregulation of A20 and TNIP-3, suggesting a deviation from canonical senescence programs. What are the implications of the main findings? The non-canonical senescence profile observed under hypoxia indicates that endothelial senescence may be more heterogeneous than previously recognised.The functional significance of A20 and TNIP-3 upregulation in this context remains to be clarified and represents an important direction for future studies. AbstractBackground: Hypoxia and ageing both involve impaired oxygen delivery, leading to oxidative damage, and endothelial cell (EC) dysfunction. In the presence of chronic hyperglycemia, these effects are amplified, accelerating EC senescence and vascular impairment. Methods: We assessed key mediators of inflammatory signalling and senescence, as well as transcriptional regulators responsive to oxidative stress in ECs exposed to high glucose (30.5 mmol/L) for 72 h under either normoxia (21% O2) or prolonged (16 h) hypoxia (2% O2) followed by 2 h of reoxygenation. Results: ECs exposed to high glucose and hypoxia developed a senescent phenotype, as indicated by increased expression of p21 and p16, and elevated β-galactosidase staining. Interestingly, hypoxia-induced senescence did not coincide with the classical senescence-associated secretory phenotype (SASP). Compared to normoxia, ECs exposed to hypoxia, particularly under high-glucose conditions, showed reduced NF-κB-driven proinflammatory secretome (MCP-1, IL-6, IL-8), downregulation of the NF-κB p50 subunit, and simultaneous upregulation of the angiogenic factor VEGF-A with downregulation of YAP-1, a key regulator of cell survival. Notably, we observed a strong upregulation of A20 and TNIP-3, two well-characterized negative regulators of NF-κB signalling. Conclusions: Hypoxia-induced senescence did not trigger a typical inflammatory SASP. Although ECs enter a senescent state, they activate an anti-inflammatory response, suppressing NF-κB signalling and increasing the expression of its inhibitors, A20 and TNIP-3. This may reflect a non-canonical senescence response whose functional significance remains to be determined.

There is a subgroup of people infected with the SARS-CoV-2 virus who manifest lingering sequelae (LongC), with neurological symptoms (nLongC). We recruited 86 COVID-19 volunteers, 35 of whom were fully recovered (Cov) and 51 who had neurological symptoms (nLongC) 4–53 months after infection and compared them to 51 healthy pre-pandemic controls (HC). Thirty-five percent of nLongC individuals carried the apolipoprotein E4 (APOE4) gene, compared to 11% of Cov. Four plasma proteins, interleukin 1 beta (IL-1β), interleukin 8 (IL-8), glial fibrillary acidic protein (GFAP), and hemopexin, continued to be elevated in both Cov and nLongC compared to HC. Soluble CD14 was elevated in nLongC but not Cov. As a group, IL-1β decreased over time in Cov but not nLongC. Two of the elevated proteins, IL-8 and GFAP, correlated with age, with both Cov and nLongC showing higher levels than HC. Using a combination of four plasma proteins, along with age, body mass index, and APOE4 presence, we were able to achieve an area under the curve (AUC) of 0.81. These results suggest that SARS-CoV-2 infection causes a low-grade inflammatory process that, even months or years after infection, does not return to pre-COVID-19 levels, which may contribute to neurologic sequelae and accelerated aging.

Interferon-stimulated genes (ISGs) play a pivotal role in the innate immune response to viral infection. Among them, SAMD9 and its paralog SAMD9L have recently emerged as important antiviral effectors with translation-inhibitory activity. While both proteins restrict poxvirus, rotavirus and reovirus replication, only SAMD9L has been shown to inhibit HIV and other lentiviruses. In this study, we identify human SAMD9L as a potent and broad-spectrum restriction factor that targets multiple medically relevant flaviviruses, including West Nile virus (WNV), Zika virus (ZIKV), dengue virus (DENV), and Usutu virus (USUV). Exogenous expression of SAMD9L, but not SAMD9, efficiently suppressed replication of all tested flaviviruses. Furthermore, its knockdown in human myeloid cells, including microglial cells and primary macrophages, impaired the antiviral activity of type I interferon, identifying SAMD9L as a key antiviral ISG in primary target cells of flavivirus infection. Mechanistically, we demonstrate that SAMD9L inhibits viral replication by targeting the translation of flaviviral RNA, and that this activity depends on its Schlafen-like ribonuclease domain, previously implicated in the inhibition of HIV-1 translation. Interestingly, although SAMD9 does not inhibit flavivirus replication, it is able to repress the translation of flaviviral RNA outside the context of infection, suggesting that its activation may be virus-specific or that flaviviruses have evolved mechanisms to evade or counteract SAMD9’s antiviral activity. Finally, we confirm that SAMD9 and SAMD9L overexpression induces activation of the innate immune response. However, this immunostimulatory function is dispensable for SAMD9L-mediated antiviral activity, since SAMD9L is able to restrict flavivirus replication independently of innate immune activation. Together, our findings broaden the known antiviral repertoire of SAMD9L, establish its essential role in restricting flavivirus replication via translational repression, and highlight its function as a key component of the cellular defenses against flaviviruses in myeloid cells. Author summaryFlaviviruses are a group of viruses that include West Nile, Zika, dengue, Usutu, and tick-borne encephalitis viruses. They are transmitted by arthropods, either mosquitoes or ticks, and can cause serious and sometimes fatal illnesses as well as long-term complications. The body’s first line of defense against these infections is the innate immune system, which quickly expresses protective proteins in response to invading viruses. In this study, we investigated one of these proteins, called SAMD9L, and found that it plays a key role in protecting human cells from flavivirus infection. Human SAMD9L stops the virus from making its own proteins, which is a necessary step for the virus to multiply. This antiviral effect was especially strong in innate immune cells that are natural targets of flaviviruses. We also found that although human SAMD9L can trigger immune responses, its ability to block flavivirus replication does not rely on this function. These findings reveal a direct way in which human SAMD9L helps control flavivirus infections and highlight its importance in the body’s natural defense against viruses.

AbstractLynch syndrome (LS) is a colorectal cancer predisposition caused by an inherited heterozygous defect in any of four DNA mismatch repair (MMR) genes. MMR prevents nucleotide substitution mutations by correcting errors of replication opposite undamaged or subtly altered nucleotides. Here, we investigated whether dietary mutagens, which generally induce helix-distorting nucleotide lesions, affect LS-associated carcinogenesis. To this aim, we exposed mouse models of LS to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a food-derived heterocyclic amine that selectively adducts guanines. PhIP exposure induced loss of the wild-type MMR allele in heterozygous intestinal stem cells, leading to MMR deficiency and to impaired DNA damage signalling associated with the clonal expansion of MMR-deficient intestinal stem cells. Whole-genome sequencing revealed that PhIP becomes significantly more mutagenic in intestinal stem cells when MMR is lost, inducing not only PhIP-guanine adduct-mediated C:G>A:T transversions but also a broader substitution spectrum that resembles the spontaneous mutational signature of LS-associated colorectal cancer. Thus, MMR corrects PhIP-induced misincorporations outside of adducted guanines. Chronic PhIP exposure of intestine-specific MMR-deficient mice induced adenocarcinomas with histopathological features of LS-associated CRC. This study implicates food-derived mutagens in multiple stages of LS-associated carcinogenesis, including allelic loss, and defective DNA damage signalling and compound hypermutagenesis in the resulting MMR-deficient cells. Graphical Abstract Graphical Abstract

AbstractAcquired resistance presents a major challenge for targeted therapies, with initially responsive tumors eventually reverting underlying vulnerabilities. Our group recently reported that cancers harboring isocitrate dehydrogenase 1/2 (IDH1/2) mutations have defective recruitment of homology-directed repair (HDR) factors to sites of DNA damage and consequent sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPi), a vulnerability that is being tested in clinical trials. To probe potential mechanisms by which resistance to PARPi might arise in this setting, we modeled PARPi resistance in IDH-mutant tumors via serial transplantation of patient-derived xenografts in mice treated with PARPi. An analysis of candidate DNA repair factors in these resistant tumor populations identified downregulation of two end protection factors that are negative regulators of HDR, 53BP1, and REV7. Knockout of these factors by CRISPR–Cas9 in IDH1-mutant cancer cells conferred robust resistance to PARPi and restored HDR capacity. To overcome this resistance, we found that treatment with the receptor tyrosine kinase inhibitor, cediranib, previously reported to suppress expression of downstream HDR factors, resensitizes 53BP1 and REV7-knockout cells to PARPi treatment. Our findings identify key pathways driving PARPi resistance in IDH1-mutant cancers and highlight potential therapeutic strategies to overcome this resistance. Graphical Abstract Graphical Abstract

Rhodehouse et al. characterize a nonhuman primate model of natural control of SIV infection in which the immune system blocks new infection events with an efficiency approaching that of antiretroviral therapy. Insights into HIV-1 pathogenesis have come from studies of viral dynamics. However, there is little information on viral dynamics in lentiviral infections in which viral replication is naturally controlled in a subset of infected individuals. We evaluated the decay of simian immunodeficiency virus (SIV) RNA and cell-associated SIV genomes in a nonhuman primate (NHP) model in which replication of an engineered SIV variant is naturally controlled by cellular immune responses in most infected animals. This variant lacks a trafficking motif in the gp41 cytoplasmic tail. A trajectory of control was evident by 21 days after infection. In animals with natural control, we observed similar biphasic decay of intact proviruses in blood and lymph nodes, at rates close to those in animals that failed to control the virus and were put on antiretroviral therapy (ART). Both natural control and ART effectively blocked viral evolution, but not persistence. Thus, in this NHP model, natural control can be nearly as effective as ART in controlling viral replication.

BackgroundAtherosclerotic cardiovascular disease (ASCVD) pathogenesis is closely associated with macrophages. This study sought to explore the role of phagocytosis by monocyte-derived macrophages (MDMs) in the blood in the context of coronary heart disease (CHD) and acute coronary syndromes (ACSs).MethodsThis study employed a matched case–control design. Individuals with suspected CHD were recruited and allocated to a control cohort or a CHD cohort, with the latter further stratified into stable angina pectoris and ACS subgroups according to clinical diagnoses. Clinical data were collected, MDMs were isolated, and macrophage phagocytic activity was evaluated using fluorescent-labeled latex microspheres.ResultsMacrophage phagocytic rates were significantly reduced in the CHD group relative to the control group, with further decreases observed in the ACS subgroup. Multivariable linear regression revealed that age, low-density lipoprotein cholesterol (LDL-C), high-sensitivity C-reactive protein (hs-CRP), and fibrinogen were independently and negatively correlated with macrophage phagocytic rates. Multivariable analyses suggested that diminished macrophage phagocytic rates were linked to an elevated risk of both CHD and ACS. Receiver operating characteristic (ROC) curve analysis identified the optimal cutoff values of macrophage phagocytic rates for predicting CHD and ACS as 62.6% and 63.4%, respectively, with the area under the curves (AUCs) measured at 0.679 and 0.669.ConclusionsMacrophage phagocytic activity is reduced in CHD patients, particularly in those with ACS. Diminished macrophage phagocytic function is linked to CHD and ACS. Macrophage phagocytosis could act as a protective biomarker in CHD and ACS, providing new insights into the pathophysiology of ASCVD.

BackgroundBispecific antibodies that redirect T cells or NK cells to tumors have demonstrated substantial therapeutic efficacy, but their broader application is often constrained by immune-related toxicities, limited effector cell availability, and suboptimal access to tumor sites. These challenges have prompted efforts to identify alternative effector cell types that are more abundant in circulation, readily accessible, and capable of cytotoxic activity in the tumor microenvironment. Neutrophils, which constitute the most prevalent circulating leukocyte population, represent a promising yet underutilized target for immune cell engager design. However, efforts to exploit neutrophil-mediated tumor killing through CD89 (FcαRI) have been limited by the inherent drawbacks of IgA-based formats, including poor stability, short serum half-life, and reduced developability.ResultsTo address these challenges, we established an engineered bispecific antibody platform that incorporates CD89 engagement into an IgG1 scaffold. This design enables neutrophil redirection while preserving the favorable pharmacokinetic and manufacturing profiles of IgG-based therapeutics. The resulting bispecific architecture allows for programmable neutrophil engagement alongside tumor antigen recognition, offering a clinically viable strategy for innate immune activation. Among the bispecific designs evaluated, ZT-8, a humanized CD89 × HER2 bispecific antibody, demonstrated potent neutrophil-mediated cytotoxicity against tumor cells even in the absence of cytokine priming, suggesting a distinct activation mechanism that operates within the tumor microenvironment. Compared to IgA-based antibodies, ZT-8 exhibited superior immune effector engagement, enhanced tumoricidal activity, and substantially prolonged in vivo half-life through FcRn-mediated recycling.ConclusionThese findings define IgG-based CD89 bispecifics as a next-generation neutrophil engager platform and exemplify how antibody engineering and synthetic immunology can be leveraged to expand the effector landscape of bispecific immunotherapies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13036-025-00580-2.

PurposeCancer immunotherapy aims to enhance the immune system’s ability to recognize and eliminate cancer cells, providing a sustained and effective immune response. However, the tumor microenvironment (TME), characterized by an abundance of tumor-associated M2 macrophages and the presence of exhausted or naïve T cells (non-effector T cells), remains a major barrier to effective immunotherapy. Herein, inflammatory M1 macrophage-derived extracellular vesicles (M1EV) were surface-modified to display interleukin-2 (M1EV_IL2), aiming to develop a multifunctional cancer immunotherapeutic agent capable of modulating both innate and adaptive immune responses.MethodsWe engineered M1EV to label the surface with azide groups through metabolic glycoengineering and developed M1EV_IL2 that displayed IL-2 via bioorthogonal chemistry. M1EV_IL2 were purified by size-exclusion chromatography (SEC) and characterized through comprehensive analyses, including nanoparticle tracking analysis (NTA). In vitro macrophage repolarization and T cell activation were evaluated at the gene-expression level, followed by ex vivo assays assessing T-cell proliferation, cytokine secretion, and activation marker expression.ResultsM1EV_IL2 effectively retained the intrinsic physicochemical properties of EVs while displaying IL-2 stably on its surface. It upregulated M1 macrophage markers, IL-1β and CXCL10, while downregulating the M2 macrophage marker CD206, thereby inducing M2-to-M1 macrophage repolarization. In addition, M1EV_IL2 also activated CD4+ T cells and induced the activation of naïve CD8+ T cells to effector T cells, leading to enhanced cell proliferation and secretion of antitumor cytokines.ConclusionThese results indicate that M1EV_IL2 has the potential to reshape the tumor immune landscape by simultaneously activating macrophages and T cells, thereby enhancing both innate and adaptive immune responses. Unlike conventional cancer therapies, which directly target tumor cells, M1EV_IL2 is expected to enhance immune responses, potentially mitigating adverse effects while improving therapeutic efficacy. Graphical Abstract

AbstractThe large-scale production of mammalian cells, particularly stem cells for clinical applications, remains challenging with existing cell culture technologies such as two-dimensional cell culture flasks or three-dimensional stirred tank bioreactors. Current methods have issues such as excessive cell aggregation and significant shear stress-induced cell death, resulting in low cell yield, unacceptable batch-to-batch variation, high production costs, and difficulties in scaling up. We hypothesize that creating a cell-friendly microenvironment that has efficient mass transport and minimized shear stress can enhance cell culture efficiency. In this study, we developed a novel hydrogel tube microbioreactor using collagen proteins (ColTubes) to test this hypothesis. First, we designed an innovative micro-extruder for fabricating ColTubes loaded with cells. Our results show that collagen proteins form a dense and robust nanofiber network capable of shielding cells from hydrodynamic stress while maintaining cell mass below 400 µm in diameter. The tube shell contains abundant nanopores that allow the cell culture medium to permeate and nourish the cells. Additionally, the collagen fibers serve as a substrate for cell adhesion. We show that ColTubes support high cell viability, rapid expansion, and impressive volumetric yields, offering substantial improvements over current methods. To our knowledge, ColTubes is a novel approach that has not been previously reported for cell manufacturing. ColTubes represents a scalable, cost-effective, and efficient solution for large-scale cell production.

The ion channel-kinase TRPM7 maintains intracellular Mg2+ homeostasis, affects early Ca2+ signals, influences downstream Ca2+-dependent signaling pathways and thus is crucial for T-cell activation and differentiation. By modulating AKT/SMAD2 signaling, TRPM7 acts as a molecular switch to balance Treg/TH17 polarization. T-lymphocyte activation is a crucial process in the regulation of innate and adaptive immune responses. The ion channel-kinase TRPM7, transient receptor potential cation channel subfamily M, member 7, has previously been implicated in cellular Mg2+ homeostasis, proliferation, and immune cell modulation. Here, we show that pharmacological and genetic silencing of TRPM7 leads to diminished activation and influences signaling pathways that guide human TH17 or Treg cell differentiation, following TCR-mediated stimulation. In primary human CD4 T cells and CRISPR-Cas9-engineered Jurkat T cells, inactivation or loss of TRPM7 led to distorted Mg2+ homeostasis and Ca2+ signaling, reduced NFAT translocation, decreased IL-2 secretion and altered TH cell differentiation. While the activation of primary human CD4 T cells, as well as in vitro polarization into pro-inflammatory TH17 cells was critically dependent on TRPM7, the polarization of naïve CD4 T cells into FOXP3+ regulatory T cells was not. Taken together, these results highlight TRPM7 as molecular switch in lymphocyte activation and polarization. Thus, suggesting a therapeutic potential for TRPM7 in numerous T-cell mediated diseases.

Key Points•The costimulatory profile of AML cells determines the quality and persistence of T-cell responses to BiTE molecules.•CAR T cells demonstrate improved long-term fitness via intrinsic costimulation, reducing dependence on AML-derived signals. Visual Abstract AbstractT-cell–based immunotherapies have revolutionized treatment paradigms in B-cell malignancies, yet their translation to acute myeloid leukemia (AML) has been hindered by a scarcity of tumor-restricted antigens and the risk of on-target off-leukemia toxicity. FLT3 has emerged as a promising therapeutic target with limited expression in healthy hematopoietic tissues. Here, we performed a head-to-head preclinical comparison of an FMS-like tyrosine kinase 3 (FLT3)-directed bispecific T-cell engager (BiTE) molecule and second-generation FLT3-specific chimeric antigen receptor (CAR) T cells. Both approaches induced potent cytotoxicity against AML cell lines and primary patient-derived cells but spared healthy hematopoietic stem and progenitor cells in vitro. Despite similar short-term efficacy, prolonged antigen exposure demonstrated progressive functional decline and metabolic exhaustion; however, CAR T cells maintained cytotoxic capacity and proliferative potential over time. In AML xenograft models, CAR T cells achieved superior tumor control, prolonged survival, and greater T-cell infiltration than BiTE molecule–treated counterparts. Transcriptomic profiling of T cells recovered from the bone marrow further revealed a distinct exhaustion-associated gene signature in samples from mice that had been treated with the FLT3 BiTE molecule. Importantly, provision of CD86-mediated costimulation enhanced antitumor activity of BiTE-redirected T cells in vitro and in vivo. These findings establish FLT3 as a viable and selective immunotherapeutic target in AML and underscore the functional and transcriptional differences between BiTE molecule–redirected T cells and CAR T cells. Moreover, they reveal a critical role for costimulatory signaling in sustaining the efficacy of T-cell–based therapies in vivo, offering a rationale for improving T cell–redirection strategies in myeloid malignancies.