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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

The ovary is one of the first organs to exhibit signs of aging,characterized by reduced tissue function,chronic inflammation,and fibrosis. Multinucleated giant cells (MNGCs),formed by macrophage fusion,typically occur in chronic immune pathologies,including infectious and non-infectious granulomas and the foreign body response,but are also observed in the aging ovary. The function and consequence of ovarian MNGCs remain unknown as their biological activity is highly context-dependent,and their large size has limited their isolation and analysis through technologies such as single-cell RNA sequencing. In this study,we define ovarian MNGCs through a deep analysis of their presence across age and species using advanced imaging technologies as well as their unique transcriptome using laser capture microdissection. MNGCs form complex interconnected networks that increase with age in both mouse and nonhuman primate ovaries. MNGCs are characterized by high Gpnmb expression,a putative marker of ovarian and non-ovarian MNGCs. Pathway analysis highlighted functions in apoptotic cell clearance,lipid metabolism,proteolysis,immune processes,and increased oxidative phosphorylation and antioxidant activity. Thus,MNGCs have signatures related to degradative processes,immune function,and high metabolic activity. These processes were enriched in MNGCs compared to primary ovarian macrophages,suggesting discrete functionality. MNGCs express CD4 and colocalize with T-cells,which were enriched in regions of MNGCs,indicative of a close interaction between these immune cell types. These findings implicate MNGCs in modulation of the ovarian immune landscape during aging given their high penetrance and unique molecular signature that supports degradative and immune functions. Ovarian multinucleated giant cells are a unique macrophage population that arise within the aging mammalian ovary. This study characterizes their transcriptome in mice,uncovering a potential role in degradation of cellular debris and immune signaling,suggesting a potential contribution to ovarian inflammation during aging. View Publication

BackgroundIn persons living with HIV,antiretroviral therapy (ART) reduces HIV RNA in their plasma and increases CD4 + T lymphocytes,thus restoring their immune function and reducing mortality rates.MethodsThe heavy and light chains of B cell receptor (BCR) were amplified,sequenced,analyzed,and determined to be anti-CD4 mAb. The cytotoxicity of NK cells mediated by the anti-CD4 mAb was assessed using CCK-8,flow cytometry,ELISA,and western blotting. Detecting the viability/regulation of CD4 cells involved inhibiting the attachment of autoantibodies against CD4 to crucial receptors and detecting the inhibition of key molecules in B cells to produce anti-CD4 mAb in patients with immune non-responders (INR). Furthermore,through Phage Random Peptide Library Screening,we discovered that the AAPMFHSSVQLP-CD4 peptide has an affinity for the anti-CD4 mAb.ResultsAdministering anti-CD4 mAb enhanced NK cytotoxicity. The simultaneous administration of anti-CD4 mAb alongside GST-CD4 alleviated the harmful impacts of anti-CD4 mAb on the CD3 + population in humanized mice,and HIV virus (p24). Individuals diagnosed with INR displayed abnormal B cell activity,particularly with elevated BAFFR expression and increased levels of anti-CD4 mAb. Nevertheless,suppression of BAFFR hindered B cell function and decreased the production of anti-CD4 mAb. In HIV-infected individuals,the dysregulation of B-cells led to the production of anti-CD4 mAb,which in turn facilitated NK cell cytotoxicity and the CD4 + T effect by upregulating the expression of BAFFR.ConclusionThe dysregulation of B-cells in person living with HIV increased the production of anti-CD4 mAb,which in turn promoted NK cell cytotoxicity and the CD4 + T effect.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10020-025-01286-3. Highlights1) B-cell dysregulation increased anti-CD4 mAb levels.2) B cells are abnormally active in patients with INR.3) Knockdown of BAFFR obviously reduced the secretion of anti-CD4 mAb.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10020-025-01286-3. View Publication

Post-transplant complications reduce allograft and recipient survival. Current approaches for detecting allograft injury non-invasively are limited and do not differentiate between cellular mechanisms. Here,we monitor cellular damages after liver transplants from cell-free DNA (cfDNA) fragments released from dying cells into the circulation. We analyzed 130 blood samples collected from 44 patients at different time points after transplant. Sequence-based methylation of cfDNA fragments were mapped to an atlas of cell-type-specific DNA methylation patterns derived from 476 methylomes of purified cells. For liver cell types,DNA methylation patterns and multi-omic data integration show distinct enrichment in open chromatin and functionally important regulatory regions. We find that multi-tissue cellular damages post-transplant recover in patients without allograft injury during the first post-operative week. However,sustained elevation of hepatocyte and biliary epithelial cfDNA within the first month indicates early-onset allograft injury. Further,cfDNA composition differentiates amongst causes of allograft injury indicating the potential for non-invasive monitoring and intervention. Current approaches to detect allograft damages non-invasively are limited and do not differentiate between cellular mechanisms. Here,the authors show that the composition of cell-free DNA in blood samples can reveal cellular causes of allograft injury after liver transplant. View Publication

Enhancers and transcription factors (TFs) are crucial in regulating cellular processes. Current multiomic technologies to study these elements in gene regulatory mechanisms lack multiplexing capability and scalability. Here we present single-cell ultra-high-throughput multiplexed sequencing (SUM-seq) for co-assaying chromatin accessibility and gene expression in single nuclei. SUM-seq enables profiling hundreds of samples at the million cell scale and outperforms current high-throughput single-cell methods. We demonstrate the capability of SUM-seq to (1) resolve temporal gene regulation of macrophage M1 and M2 polarization to bridge TF regulatory networks and immune disease genetic variants,(2) define the regulatory landscape of primary T helper cell subsets and (3) dissect the effect of perturbing lineage TFs via arrayed CRISPR screens in spontaneously differentiating human induced pluripotent stem cells. SUM-seq offers a cost-effective,scalable solution for ultra-high-throughput single-cell multiomic sequencing,accelerating the unraveling of complex gene regulatory networks in cell differentiation,responses to perturbations and disease studies. This work presents SUM-seq,an ultra-high-throughput method for co-profiling chromatin accessibility and gene expression in single nuclei across multiplexed samples,advancing the study of gene regulation in diverse biological systems. View Publication

IntroductionImmune resilience is the capacity of the immune system to recover from sepsis-induced organ injury and reestablish host defense. While sepsis survivors are often viewed as immunocompromised,recent studies suggest that some may acquire adaptive immune traits that enhance resistance to secondary infections.MethodsWe employed a murine cecal ligation and puncture (CLP) model to study polymicrobial sepsis and subsequent immune responses. Listeria monocytogenes was used as a model intracellular pathogen to assess immune protection. We analyzed myeloid cell phenotypes using single-cell RNA sequencing and evaluated lipid metabolic changes through quantitative RT-PCR,immunohistochemistry,serum cytokine assays,and plasma lipidomics.ResultsSepsis-surviving mice showed enhanced resistance to Listeria infection. Single-cell RNA sequencing revealed transcriptional reprogramming in splenic CD11b+Ly6Chigh myeloid cells,including downregulation of lipoprotein lipase and lipid efflux genes. CD11b+ myeloid cells exhibited increased lipid droplet accumulation,accompanied by elevated serum interferon-gamma (IFN-γ) levels. Plasma lipidomics identified systemic lipid remodeling,with increased phosphatidylserine and decreased phosphatidylinositol and phosphatidylglycerol levels.DiscussionOur findings suggest that sepsis survival induces lipid metabolic reprogramming in myeloid cells,contributing to enhanced immunity against intracellular pathogens. These insights reveal potential therapeutic targets within lipid metabolic pathways to improve host defense in sepsis survivors. View Publication

AbstractObjectiveNatural killer (NK) cells are the largest innate lymphocyte subset with potent antitumour and antiviral functions. However,clinical utilisation of human NK cells is hampered due to a lack of reliable methods to augment their antitumour potential. We demonstrated technology in which human NK cells were cocultured with osteoclasts in the presence of probiotic bacteria. This approach significantly augmented the antitumour cytotoxicity and polyfunctionality of human NK cells,resulting in the generation of supercharged NK (sNK) cells.Methods and analysisWe explored the proteomic,transcriptomic and functional characterisation of sNK cells using cell imaging,flow cytometric analysis,51-chromium release cytotoxicity assay,ELISA,ELIspot,IsoPLexis single-cell secretome analysis,proteomic analysis,RNA analysis,western blot and enzyme kinetics.ResultsWe found that sNK cells were less susceptible to split anergy and tumour-induced exhaustion. Proteomic analyses revealed that sNK cells significantly increased their cell motility and proliferation. Single-cell transcriptomes uncovered sNK cells undertaking a unique differentiation trajectory and turning on STAT1,JUN,BHLHE40,ELF1,MAX and MYC regulons essential for augmenting antitumour effector functions and proliferation,respectively. Both proteomic and single-cell transcriptomes revealed that an increase in Cathepsin C helped to augment the quantity and function of Granzyme B.ConclusionsThese results support that this unique method produces potent NK cells for clinical utilisation and delineate the molecular mechanisms associated with this process. View Publication

BackgroundMonocytes have been confirmed to increase in persistently food-allergic children. A phenomenon of innate immune memory,called trained immunity,has also been observed in monocytes from allergic children. However,the underlying mechanism remains poorly understood.MethodsWe enrolled a cohort of HDM-allergic children alongside age-matched healthy controls and established an HDM-sensitized allergic mouse model. Flow cytometric analyses were conducted to quantify monocyte frequencies in clinical cohorts and experimental animals. We performed integrated transcriptomic profiling via RNA-seq combined with chromatin occupancy analysis using CUT&Tag technology in parallel human and murine samples to elucidate the molecular mechanisms.ResultsIn our study,we demonstrated a reduced H3K27me3 methylation level accompanied by an increased proportion and a proinflammatory transcriptional memory in monocytes from house dust mite (HDM)-allergic human subjects. The same transcriptional and epigenetic phenotype was also confirmed in HDM-sensitized mice. Finally,the administration of GSK-J4,which upregulates H3K27me3 level in murine monocytes,attenuated the inflammatory response in vitro and in vivo.ConclusionsOur study confirms that H3K27me3 methylation modulates the trained immunity in monocytes and regulates HDM-allergic diseases through an inflammatory-dependent mechanism. View Publication