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Aging is associated with immune dysfunction, but long-term endurance training may confer protective effects on immune cell function. This study investigates how natural killer (NK) cell phenotypes, functional markers, and metabolism differ between endurance-trained and untrained older adults. Ex vivo expanded NK cells from endurance-trained (63.6 ± 2.1 years) and untrained (64.3 ± 3.3 years) males were exposed to adrenergic blockade (propranolol; 0–200 ng/mL) or mTOR inhibition (rapamycin; 10–100 ng/mL), both with or without PMA-induced inflammatory stimulation. Flow cytometry assessed NK subsets, activation (CD38, CD57, CD107a, NKG2D), senescence (KLRG1), and inhibitory markers (PD-1, LAG-3, TIM-3, NKG2A). Seahorse analysis measured metabolic parameters. Trained participants displayed healthier immune profiles (lower NLR, SII) and higher effector NK cells with lower cytotoxic subsets. Propranolol at 100 ng/mL blunted PMA-driven increases in CD57, CD107a, and NKG2D, while potentiating regulatory markers KLRG1, LAG-3, and PD-1 in the trained group, indicating stronger immunoregulation. With rapamycin, trained NK cells preserved NKG2D and CD107a at 10 ng/mL, maintaining cytotoxicity and degranulation. In contrast, at 100 ng/mL rapamycin plus PMA, trained NK cells shifted toward an effector phenotype with higher CD57 and CD107a, yet a blunted PMA-increased LAG-3 and TIM-3, suggesting resistance to exhaustion. PD-1 and KLRG1 remained elevated, reflecting balanced immune control. Mitochondrial analysis revealed that trained NK cells exhibited higher basal and maximal OCR, greater spare respiratory capacity, and OCR/ECAR ratio, reflecting superior metabolic fitness. These findings indicate that endurance-trained older adults have NK cells with greater functional adaptability, reduced senescence, and enhanced metabolism under inflammatory and pharmacological stress.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-06057-y.

ObjectiveBifidobacterium is known to be depleted in patients with atopic dermatitis (AD). This study aims to investigate the potential prophylactic effects of Bifidobacterium animalis subsp. lactis BX-BC08 (B. lactis BX-BC08) in a murine model of AD.DesignThe immunosuppressive and anti-inflammatory effects of BX-BC08 were evaluated in a MC903-induced AD mouse model. Gut microbiota composition was analyzed by metagenomic sequencing, while high-performance liquid chromatography-mass spectrometry (HPLC-MS) was employed to identify anti-inflammatory molecules produced by B. lactis BX-BC08.ResultsBX-BC08 significantly attenuated pro-inflammatory responses, scaling and swelling in the MC903-induced AD like murine model compared to controls. Fecal microbial profiling revealed an enrichment of probiotics and a reduction of pro-inflammatory bacteria in BX-BC08 treated mice. Metabolic analysis of BX-BC08 bacteria culture supernatant and treated mice identified a significant enrichment of alpha-Ketoglutaric acid (AKG). Functional validation in the murine AD model demonstrated that AKG strongly suppressed T helper 2 (Th2)-driven pro-inflammatory responses.ConclusionBX-BC08 mitigates AD-like inflammation by producing the anti-inflammatory metabolite AKG. BX-BC08 could serve as a novel prophylactic agent for AD prevention.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-025-06769-9.

The long-term maintenance of hematopoietic stem cells (HSCs) relies on the regulation of endoplasmic reticulum (ER) stress at a low level, but the underlying mechanism remains poorly understood. Here, we demonstrate that suppression of ER stress improves the functions of HSCs and protects HSCs against ionizing radiation (IR)-induced injury. We identify epithelial membrane protein 1 (EMP1) as a key regulator that mitigates ER stress in HSCs. Emp1 deficiency leads to the accumulation of protein aggregates and elevated ER stress, ultimately resulting in impaired HSC maintenance and self-renewal. Mechanistically, EMP1 is located within the ER and interacts with ceramide synthase 2 (CERS2) to limit the production of a class of sphingolipids, dihydroceramides (dhCers). DhCers accumulate in Emp1-deficient HSCs and induce protein aggregation. Furthermore, Emp1 deficiency renders HSCs more susceptible to IR, while overexpression of Emp1 or inhibition of CERS2 protects HSCs against IR-induced injury. These findings highlight the critical role played by the EMP1-CERS2-dhCers axis in constraining ER stress and preserving HSC potential. A new study shows EMP1 protects hematopoietic stem cells by suppressing sphingolipid metabolism and ER stress. EMP1 interacts with CERS2 to limit dihydroceramide production, which causes protein aggregation when elevated.

IntroductionCD8+ T cells are vital in the immune control of cancer and a key player in cell-based cancer immunotherapy. Recent studies have shown that microbial short-chain fatty acids (SCFA) can promote both effector and memory phenotypes in CD8+ T cells and may thereby enhance protection against cancer.MethodsIn this study, we determined the effect of SCFA butyrate on mouse CD8+ T cell function in vitro and in vivo, using the OT-I model.ResultsButyrate co-culture with anti-CD3 + anti-CD28 activated T cells in vitro enhanced the frequency of effector CD8+ IFN-γ-producing cells, and the amount of cytokine produced per cell. Culture with butyrate also enhanced the activation, TCR expression, and levels of phosphorylated mTOR proteins within CD8+ T cells but reduced proliferation rate and increased apoptosis. Butyrate-treated activated cells conferred tumor protection after adoptive transfer. Butyrate-treated cells were present at higher frequencies within the tumor compared to non-butyrate treated cells, and expressed IFN-γ. When analyzed using high dimensional cytometry, the tumors of mice that received butyrate-treated cells were enriched in clusters displaying an effector memory phenotype with high expression of IL-15Rβ and T-bet.DiscussionOur findings show that butyrate promotes the effector activity of CD8+ T cells in culture, which can persist in vivo while also stimulating memory phenotypes. Consequently, butyrate treatment may have strong application in T cell-based immunotherapies to improve protective cell functions and patient outcomes.

Neutrophils are highly abundant in the oral mucosal tissues, and their balanced activation and clearance are essential for immune homeostasis. Here, we demonstrate that neutrophils infected with the bacterial pathogen Porphyromonas gingivalis (Pg) are captured alive by macrophages in a manner that bypasses all known receptor-ligand interactions involved in the phagocytosis of either live or dead cells. Mechanistically, upon interaction with Pg, or its protease RgpB (gingipains), live neutrophils undergo rapid remodeling of their proteomes, generating neoepitopes. N-terminomics-based proteomic profiling identified multiple RgpB cleavage sites on several azurophilic granule proteins that are translocated to the surface of live neutrophils via low-level degranulation and activate macrophage αMβ2 integrin receptors, thus mediating internalization of non-apoptotic neutrophils within macrophage phagosomes. Macrophages with entrapped live neutrophils exhibit phenotypic and transcriptional reprogramming, consistent with inflammatory outcomes in vitro and in vivo. In contrast to the immunosuppressive outcomes associated with efferocytosis of apoptotic neutrophils, live neutrophil entrapment failed to fully activate several catabolic and metabolic processes and exhibited a defective activation of PPAR-γ mediated pro-resolution pathways, thereby promoting bacterial persistence and hindering the resolution of inflammation. Thus, our data demonstrate a novel immune subversion strategy unique to Pg and reveal a previously unknown mode of live neutrophil sequestration into macrophages during an infection.

The human bone marrow (BM) microenvironment involves hematopoietic and non-hematopoietic cell subsets organized in a complex architecture. Tremendous efforts have been made to model it in order to analyze normal or pathological hematopoiesis and its stromal counterpart. Herein, we report an original, fully-human in vitro 3D model of the BM microenvironment dedicated to study interactions taking place between mesenchymal stromal cells (MSC) and hematopoietic stem and progenitor cells (HSPC) during the hematopoietic differentiation. This fully-human Artificial Marrow Organoid (AMO) model is highly efficient to recapitulate MSC support to myeloid differentiation and NK cell development from the immature CD34 + HSPCs to the most terminally differentiated CD15 + polymorphonuclear neutrophils, CD64 + monocytes or NKG2A-KIR2D + CD57 + NK subset. Lastly, our model is suitable for evaluating anti-leukemic NK cell function in presence of therapeutic agents. Overall, the AMO is a versatile, low cost and simple model able to recapitulate normal hematopoiesis and allowing more physiological drug testing by taking into account both immune and non-immune BM microenvironment interactions.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-07717-9.

Myelodysplastic syndrome disease (MDS) is caused by the successive acquisition of mutations and thus displays a variable risk for progression to AML. Mutations in CEBPA are commonly associated with a high risk of disease progression, but whether they are causative for AML development is unclear. To analyse the molecular basis of disease progression we generated MDS patient-derived induced pluripotent stem cells from a low risk male patient harbouring RUNX1/SRSF2 mutations. This experimental model faithfully recapitulates the patient disease phenotypes upon hematopoietic differentiation. Introduction of a frameshift mutation affecting the C/EBPα bZIP domain in cells from low-risk stages mimicks disease progression by reducing clonogenicity of myeloid cells, blocking granulopoiesis and increasing erythroid progenitor self-renewal capacity. The acquisition of this mutation reshapes the chromatin landscape at distal cis-regulatory regions and promotes changes in cellular composition as observed by single cell RNAseq. Mutant C/EBPα is therefore causative for MDS disease progression. Our work identifies mutant CEBPA as causative for MDS disease progression, providing a new isogenic MDS experimental model for drug screening to improve diagnostic and therapeutic strategies. In Myeloiddysplastic syndromes, CEBPA mutations are linked to disease progression and AML. Here, the authors use somatic reprogramming and genome editing to generate isogenic cell lines from an MDS patient, identifying CEBPA bZIP domain disruption as causative for disease progression.

DEAD-box helicase 41 (DDX41) is implicated in germline (GL)-predisposed myeloid neoplasms, where pathogenic GL variants often lead to disease following the acquisition of a somatic variant in trans, most commonly p.R525H. However, the precise molecular mechanisms by which DDX41 variants contribute to the pathogenesis of myeloid neoplasms remain poorly understood, partly due to challenges in establishing cellular and animal models that faithfully recapitulate the human disease phenotype. This limitation highlights the necessity of directly analyzing primary human disease cells. In this case report, conducted to pursue this objective, we implemented single-cell RNA sequencing integrated with genotyping at the p.R525 locus in a myelodysplastic neoplasm (MDS) harboring both germline and somatic DDX41 variants, leveraging highly efficient Terminator-Assisted Solid-phase cDNA amplification and sequencing. We found that acquiring p.R525H induced G2/M cell cycle arrest selectively in colony-forming unit-erythroid cells, accompanied by R-loop accumulation, which impaired erythropoiesis through DNA damage. In hematopoietic stem and myeloid progenitor populations, gene expression profiles were largely similar between p.R525H-positive and -negative cells. However, ligand-receptor interaction and transcriptional regulation analyses suggested a non-cell-autonomous influence from p.R525H-expressing cells on GL variant-only cells. This interaction drove convergence toward a shared expression profile, highlighting an intricate interplay shaping the patient’s MDS phenotype.

AbstractBackgroundThe tumor microenvironment (TME) poses challenges that limit the efficacy of conventional CAR-T cell therapies. Homing barriers, immunosuppressive factors, and target antigen heterogeneity can impair CAR-T cell functional activity within the TME. Alternative strategies have contemplated incorporating the use of gamma delta (γδ) T cells as a CAR-T cell approach to potentially overcome these limitations. γδ T cells possess both innate and adaptive immunity to facilitate broad tumor recognition, and their natural propensity for tissue tropism may allow for more effective tumor infiltration. Reported here is the preclinical characterization of ADI-270, an allogeneic γδ CAR-T cell product targeting CD70+ cancers, engineered with a third-generation CAR based on the natural CD27 receptor. ADI-270 is also double-armored to mitigate the immunosuppressive effects of TGFβ and reduce the potential for allogeneic rejection.MethodsVδ1 T cells engineered to express an anti-CD70 CAR and dominant negative TGFβ receptor II (dnTGFβRII) were expanded from healthy donor human PBMCs. The phenotype and functional characterization of ADI-270 were assessed with in vitro cell culture assays and in vivo tumor xenograft models.ResultsADI-270 exhibited high levels of in vitro cytotoxicity against a panel of cancer cell lines and displayed a favorable inflammatory cytokine profile compared with reference scFv-based anti-CD70 CAR αβ T cells. Cytotoxicity remained potent despite low CD70 expression observed in multiple solid and hematologic tumor cell models. When armored with dnTGFβRII, ADI-270 exhibited functional resilience to TGFβ-mediated inhibition of T cell effector activity. In addition, the incorporation of potent and sensitive CD70-targeting decreased T cell-mediated alloreactive killing against ADI-270 in vitro without evidence of fratricide. Finally, ADI-270 displayed robust tumor tropism and control of primary and secondary tumor challenges in xenograft mouse models.ConclusionsThese results demonstrate the robust potency and capacity of ADI-270 to extend antitumor activity to cancers with heterogeneous antigen expression. The functional armoring incorporated into ADI-270 provides a mechanism to overcome the limitations of reduced efficacy and persistence within the TME. ADI-270 has the potential to target multiple CD70+ cancers with initial clinical evaluation proceeding in relapsed/refractory clear cell renal cell carcinoma.Trial registration numberNCT06480565.

ObjectiveB lymphocytes play a crucial role in immunity but also contribute to the pathogenesis of various diseases. Cannabis plants produce numerous biologically active compounds, including cannabinoids. The two most studied phytocannabinoids are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). These cannabinoids exert diverse and potent biological effects primarily through the endocannabinoid system (ECS), which also plays a key role in mature B-cell function. Both the immune system and the ECS undergo age-related changes that lead to a clinically significant decline in function.MethodsThis study compares the effects of THC and CBD on B-cell activity in young and aged mice. Murine B lymphocytes were activated using lipopolysaccharide (LPS) and interleukin-4 (IL-4), and the impact of cannabinoid treatments was assessed in terms of cell phenotype, proliferation, antibody secretion, tumor necrosis factor-alpha (TNFα) secretion, extracellular signal-regulated kinase (ERK) phosphorylation, and the cellular metabolome.ResultsBoth THC and CBD exhibited dose-dependent inhibitory effects on B-cell activation in young and aged mice. However, we show here, for the first time, that the treatments induce distinct metabolic profiles. Although some metabolites, such as glucose-6-phosphate, pentose phosphate pathway (PPP) and nucleotide metabolites, were reduced by both cannabinoids, THC selectively reduced the levels of a distinct set of amino acids, while only CBD increased the levels of Citrulline and Allantoin. Additionally, the effects of THC and CBD differed between young and aged B cells, suggesting that age-related changes in the ECS may influence cannabinoid sensitivity.ConclusionsThese findings provide insights into the distinct mechanisms by which THC and CBD regulate immune activation and may open the door for investigating the mechanisms behind cannabinoids effects on the immune system. They also highlight the need for further research into phytocannabinoid-based therapies, particularly in age-specific contexts. Given the immunoregulatory properties of cannabinoids, especially CBD, tailored therapeutic strategies may enhance their clinical applications

BackgroundCNS stromal cells, especially fibroblasts and endothelial cells, support leukocyte accumulation through upregulation of adhesion molecules and lymphoid chemokines. While chronically activated fibroblast networks can drive pathogenic immune cell aggregates known as tertiary lymphoid structures (TLS), early stromal cell activation during CNS infection can support anti-viral T cells. However, the cell types and factors driving early stromal cell activation is poorly explored.AimsA neurotropic murine coronavirus (mCoV) infection model was used to better characterize signals that promote fibroblast networks supporting accumulation of antiviral lymphocytes. Based on the early appearance of IgD+ B cells with unknown functions during several CNS infections, we probed their potential to activate stromal cells through lymphotoxin β (LTβ), a molecule critical in maintaining fibroblast-networks in lymphoid tissues as well as promoting TLS in autoimmunity and cancers.ResultsKinetic analysis of stromal cell activation in olfactory bulbs and brains revealed that upregulation of adhesion molecules and lymphoid chemokines Ccl19, Ccl21 and Cxcl13 closely tracked viral replication. Immunohistochemistry revealed that upregulation of the fibroblast marker podoplanin (PDPN) at meningeal and perivascular sites mirrored kinetics of RNA expression. Moreover, both B cells and T cells colocalized to areas of PDPN reactivity, supporting a potential role in regulating stromal cell activation. However, specific depletion of LTβ from B cells using Mb1-creERT2 x Ltβfl/fl mice had no effect on T or B cell recruitment or viral replication. B cell depletion by anti-CD20 antibody also had no adverse effects. Surprisingly, LTβR agonism reduced viral control and parenchymal T cell localization despite increasing stromal cell lymphoid chemokines and PDPN. Additional assessment of direct stromal cell activation by the viral RNA mimic poly I:C showed induction of Pdpn and Ccl19 preceding Ltb.ConclusionsNeither B cell-derived LTβ or B cells are primary drivers of stromal cell activation networks in the CNS following mCoV infection. Although supplementary agonist mediated LTβR engagement confirmed a role for LTβ in enhancing PDPN and lymphoid chemokine expression, it impeded T cell migration to the CNS parenchyma and viral control. Our data overall indicate that stromal cells can integrate LTβR signals to tune their activation, but that LTβ is not necessarily essential and can even dysregulate protective antiviral T cell functions.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12974-025-03491-7.

Autoantibodies targeting cytosolic 5′-nucleotidase 1A (cN1A) are found in several autoimmune diseases, including inclusion body myositis (IBM), Sjögren’s syndrome, and systemic lupus erythematosus. While they have diagnostic relevance for IBM, little is known about the autoreactive B cells that produce these antibodies. To address this, we developed a robust protocol for the expression and site-specific biotinylation of recombinant human cN1A in Escherichia coli. The resulting antigen is suitable for generating double-labelled fluorescent baits for the isolation and characterisation of cN1A-specific B cells by flow cytometry. Site-specific biotinylation was achieved using the AviTag and BirA ligase, preserving the protein’s structure and immunoreactivity. Western blot analysis confirmed that the biotinylated cN1A was recognised by both human and rabbit anti-cN1A antibodies. Compared to conventional chemical biotinylation, this strategy minimises structural alterations that may affect antigen recognition. This approach provides a reliable method for producing biotinylated antigens for use in immunological assays. While demonstrated here for cN1A, the protocol can be adapted for other autoantigens to support studies of antigen-specific B cells in autoimmune diseases.