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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. View Publication

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. View Publication

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. View Publication

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. View Publication

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. View Publication

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. View Publication

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 View Publication

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. View Publication

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. View Publication

AbstractBackgroundWhile recent clinical trials of combination immunotherapies for hepatocellular carcinoma (HCC) have shown promising clinical efficacy and survival improvements breakthroughs,there is still much room for further improvement. A key limiting factor for HCC immunotherapy is the intrinsic immunosuppression within the liver microenvironment,resulting in suboptimal priming of tumor-specific CD8 cytotoxic T cells and thus immune evasion by the tumor. Hence,identifying new key molecular pathways suppressing T-cell responses within the liver is critical for the rational design of more effective combination immunotherapies for HCC.MethodsWe identified the 5-HT2A serotonin receptor as a potential target for HCC immunotherapy in a chemical screening approach and validated that targeting 5-HT2A signaling could be a viable approach for HCC immunotherapy via in vitro and in vivo studies.ResultsDisruption of 5-HT2A signaling using either a selective antagonist small molecule,ketanserin,or by knockout of its coding gene Htr2a augments the cytotoxic effector phenotype of mouse CD8 T cells activated in vitro with immunosuppressive liver non-parenchymal cells. Ketanserin treatment of in vitro activated human CD8 T cells also increased expression of the cytotoxic effector molecules granzyme B and perforin. Abrogation of 5-HT2A signaling was associated with increased expression of cytotoxicity-related genes such as granzyme B and reduced expression of transcription factors downstream of MAP kinase signaling. In vivo,systemic ketanserin treatment significantly prolonged survival of HCC tumor-bearing mice and was non-inferior to α-programmed death ligand 1 (PD-L1)+α-vascular endothelial growth factor A (VEGFA) combination antibody treatment. Combining ketanserin with αPD-L1+αVEGFA antibodies also significantly prolonged survival relative to control-treated mice while preserving the occurrence of complete tumor regression observed with αPD-L1+αVEGFA treatment alone.ConclusionsTogether,our data describe a role for 5-HT2A as a negative regulator of the cytotoxic effector phenotype in CD8 T cells and highlight the therapeutic potential of targeting 5-HT2A for HCC immunotherapy. View Publication