技术资料

Current clinical strategies for the delivery of pulmonary therapeutics to the lung are primarily targeted to the upper portions of the airways. However,targeted delivery to the lower regions of the lung is necessary for the treatment of parenchymal lung injury and disease. Here,we have developed an mRNA therapeutic for the lower lung using one-component Ionizable Amphiphilic Janus Dendrimers (IAJDs) as a delivery vehicle. We deliver an anti-inflammatory cytokine mRNA,transforming growth factor-beta (TGF-β),to produce transient protein expression in the lower regions of the lung. This study highlights IAJD’s potential for precise,effective,and safe delivery of TGF-β mRNA to the lung. This delivery system offers a promising approach for targeting therapeutics to the specific tissues,a strategy necessary to fill the current clinical gap in treating parenchymal lung injury and disease. View Publication

Trained immunity may play a role in vaccine-induced protection against infections. We showed that the highly efficacious recombinant VZV-gE zoster vaccine (RZV) generated trained immunity in monocytes,natural killer (NK) cells,and dendritic cells (DCs) and that the less efficacious live zoster vaccine did not. RZV stimulated ex vivo gE-specific monocyte,DC and NK cell responses that did not correlate with CD4 + T-cell responses. These responses were also elicited in purified monocyte and NK cell cocultures stimulated with VZV-gE and persisted above prevaccination levels for ≥ 4 years post-RZV administration. RZV administration also increased ex vivo heterologous monocyte and NK cell responses to herpes simplex and cytomegalovirus antigens. ATAC-seq analysis and ex vivo TGFβ1 supplementation and inhibition experiments demonstrated that decreased tgfβ1 transcription resulting from RZV-induced chromatin modifications may explain the development of monocyte trained immunity. The role of RZV-trained immunity in protection against herpes zoster and other infections should be further studied. View Publication

Background & AimsThe immune tolerance induced by hepatitis B virus (HBV) is a major challenge for achieving effective viral clearance,and the mechanisms involved are not well-understood. One potential factor involved in modulating immune responses is mesencephalic astrocyte-derived neurotrophic factor (MANF),which has been reported to be increased in patients with chronic hepatitis B. In this study,our objective is to examine the role of MANF in regulating immune responses to HBV.MethodsWe utilized a commonly used HBV-harboring mouse model,where mice were hydrodynamically injected with the pAAV/HBV1.2 plasmid. We assessed the HBV load by measuring the levels of various markers including hepatitis B surface antigen,hepatitis B envelope antigen,hepatitis B core antigen,HBV DNA,and HBV RNA.ResultsOur study revealed that following HBV infection,both myeloid cells and hepatocytes exhibited increased expression of MANF. Moreover,we observed that mice with myeloid-specific MANF knockout (ManfMye-/-) displayed reduced HBV load and improved HBV-specific T cell responses. The decreased HBV-induced tolerance in ManfMye-/- mice was associated with reduced accumulation of myeloid-derived suppressor cells (MDSCs) in the liver. Restoring MDSC levels in ManfMye-/- mice through MDSC adoptive transfer reinstated HBV-induced tolerance. Mechanistically,we found that MANF promoted MDSC expansion by activating the IL-6/STAT3 pathway. Importantly,our study demonstrated the effectiveness of a combination therapy involving an hepatitis B surface antigen vaccine and nanoparticle-encapsulated MANF siRNA in effectively clearing HBV in HBV-carrier mice.ConclusionThe current study reveals that MANF plays a previously unrecognized regulatory role in liver tolerance by expanding MDSCs in the liver through IL-6/STAT3 signaling,leading to MDSC-mediated CD8+ T cell exhaustion. Graphical abstract View Publication

BackgroundDiffuse large B-cell lymphoma (DLBCL) represents a prevalent malignant tumor,with approximately 40% of patients encountering treatment challenges or relapse attributed to rituximab resistance,primarily due to diminished or absent CD20 expression. Our prior research identified PDK4 as a key driver of rituximab resistance through its negative regulation of CD20 expression. Further investigation into PDK4’s resistance mechanism and the development of advanced exosome nanoparticle complexes may unveil novel resistance targets and pave the way for innovative,effective treatment modalities for DLBCL.MethodsWe utilized a DLBCL-resistant cell line with high PDK4 expression (SU-DHL-2/R). We infected it with short hairpin RNA (shRNA) lentivirus for RNA sequencing,aiming to identify significantly downregulated mRNA in resistant cells. Techniques including immunofluorescence,immunohistochemistry,and Western blotting were employed to determine PDK4’s localization and expression in resistant cells and its regulatory role in phosphorylation of Histone deacetylase 8 (HDAC8). Furthermore,we engineered advanced exosome nanoparticle complexes,aCD20@ExoCTX/siPDK4,through cellular,genetic,and chemical engineering methods. These nanoparticles underwent characterization via Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM),and their cellular uptake was assessed through flow cytometry. We evaluated the nanoparticles’ effects on apoptosis in DLBCL-resistant cells and immune cells using CCK-8 assays and flow cytometry. Additionally,their capacity to counteract resistance and exert anti-tumor effects was tested in a resistant DLBCL mouse model.ResultsWe found that PDK4 initiates HDAC8 activation by phosphorylating the Ser-39 site,suppressing CD20 protein expression through deacetylation. The aCD20@ExoCTX/siPDK4 nanoparticles served as effective intracellular delivery mechanisms for gene therapy and monoclonal antibodies,simultaneously inducing apoptosis in resistant DLBCL cells and triggering immunogenic cell death in tumor cells. This dual action effectively reversed the immunosuppressive tumor microenvironment,showcasing a synergistic therapeutic effect in a subcutaneous mouse tumor resistance model.ConclusionsThis study demonstrates that PDK4 contributes to rituximab resistance in DLBCL by modulating CD20 expression via HDAC8 phosphorylation. The designed exosome nanoparticles effectively overcome this resistance by targeting the PDK4/HDAC8/CD20 pathway,representing a promising approach for drug delivery and treating patients with Rituximab-resistant DLBCL.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12943-024-02057-0. View Publication

Synthetic Notch (synNotch) receptors are genetically encoded,modular synthetic receptors that enable mammalian cells to detect environmental signals and respond by activating user-prescribed transcriptional programs. Although some materials have been modified to present synNotch ligands with coarse spatial control,applications in tissue engineering generally require extracellular matrix (ECM)-derived scaffolds and/or finer spatial positioning of multiple ligands. Thus,we develop here a suite of materials that activate synNotch receptors for generalizable engineering of material-to-cell signaling. We genetically and chemically fuse functional synNotch ligands to ECM proteins and ECM-derived materials. We also generate tissues with microscale precision over four distinct reporter phenotypes by culturing cells with two orthogonal synNotch programs on surfaces microcontact-printed with two synNotch ligands. Finally,we showcase applications in tissue engineering by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined micropatterns. These technologies provide avenues for spatially controlling cellular phenotypes in mammalian tissues. Synthetic Notch (synNotch) receptors are genetically encoded,modular synthetic receptors that enable mammalian cells to detect environmental signals and respond by activating user-prescribed transcriptional programs. Here the authors apply synNotch receptors to spatially control differentiation of endothelial and skeletal muscle cells in a multicellular construct on assorted biomaterials. View Publication

Immune evasion is one of the critical hallmarks of malignant tumors,especially non-small cell lung cancer (NSCLC). Emerging findings have illustrated the roles of N6-methyladenosine (m6A) on NSCLC immune evasion. Here,this study investigated the function and underlying mechanism of m6A reader YTH domain family protein 3 (YTHDF3) on NSCLC immune evasion. YTHDF3 was found to be highly expressed in NSCLC tissue and act as an independent prognostic factor for overall survival. Functionally,up-regulation of YTHDF3 impaired the CD8+ T antitumor activity to deteriorate NSCLC immune evasion,while YTHDF3 silencing recovered the CD8+ T antitumor activity to inhibit immune evasion. Besides,YTHDF3 up-regulation reduced the apoptosis of NSCLC cells. Mechanistically,PD-L1 acted as the downstream target for YTHDF3,and YTHDF3 could upregulate the transcription stability of PD-L1 mRNA. Overall,YTHDF3 targeted PD-L1 to promote NSCLC immune evasion partially through escaping effector cell cytotoxicity CD8+ T mediated killing and antitumor immunity. In summary,this study provides an essential insight for m6A modification on CD8+ T cell-mediated antitumor immunity in NSCLC,which might inspire an innovation for lung cancer tumor immunotherapy. View Publication

Mitochondria react to infection with sub-lethal signals in the apoptosis pathway. Mitochondrial signals can be inflammatory but mechanisms are only partially understood. We show that activation of the caspase-activated DNase (CAD) mediates mitochondrial pro-inflammatory functions and substantially contributes to host defense against viral infection. In cells lacking CAD,the pro-inflammatory activity of sub-lethal signals was reduced. Experimental activation of CAD caused transient DNA-damage and a pronounced DNA damage response,involving major kinase signaling pathways,NF-κB and cGAS/STING,driving the production of interferon,cytokines/chemokines and attracting neutrophils. The transcriptional response to CAD-activation was reminiscent of the reaction to microbial infection. CAD-deficient cells had a diminished response to viral infection. Influenza virus infected CAD-deficient mice displayed reduced inflammation in lung tissue,higher viral titers and increased weight loss. Thus,CAD links the mitochondrial apoptosis system and cell death caspases to host defense. CAD-driven DNA damage is a physiological element of the inflammatory response to infection. View Publication

Human regulatory T cells (Treg) suppress other immune cells. Their dysfunction contributes to the pathophysiology of autoimmune diseases,including type 1 diabetes (T1D). Infusion of Tregs is being clinically evaluated as a novel way to prevent or treat T1D. Genetic modification of Tregs,most notably through the introduction of a chimeric antigen receptor (CAR) targeting Tregs to pancreatic islets,may improve their efficacy. We evaluated CAR targeting of human Tregs to monocytes,a human β cell line and human islet β cells in vitro. Targeting of HLA-A2-CAR (A2-CAR) bulk Tregs to HLA-A2+ cells resulted in dichotomous cytotoxic killing of human monocytes and islet β cells. In exploring subsets and mechanisms that may explain this pattern,we found that CD39 expression segregated CAR Treg cytotoxicity. CAR Tregs from individuals with more CD39low/- Tregs and from individuals with genetic polymorphism associated with lower CD39 expression (rs10748643) had more cytotoxicity. Isolated CD39− CAR Tregs had elevated granzyme B expression and cytotoxicity compared to the CD39+ CAR Treg subset. Genetic overexpression of CD39 in CD39low CAR Tregs reduced their cytotoxicity. Importantly,β cells upregulated protein surface expression of PD-L1 and PD-L2 in response to A2-CAR Tregs. Blockade of PD-L1/PD-L2 increased β cell death in A2-CAR Treg co-cultures suggesting that the PD-1/PD-L1 pathway is important in protecting islet β cells in the setting of CAR immunotherapy. In summary,introduction of CAR can enhance biological differences in subsets of Tregs. CD39+ Tregs represent a safer choice for CAR Treg therapies targeting tissues for tolerance induction. View Publication

How the microbiome regulates responses of systemic innate immune cells is unclear. In the present study,our purpose was to document a novel mechanism by which the microbiome mediates crosstalk with the systemic innate immune system. We have identified a family of microbiome Bacteroidota-derived lipopeptides—the serine-glycine (S/G) lipids,which are TLR2 ligands,access the systemic circulation,and regulate proinflammatory responses of splenic monocytes. To document the role of these lipids in regulating systemic immunity,we used oral gavage with an antibiotic to decrease the production of these lipids and administered exogenously purified lipids to increase the systemic level of these lipids. We found that decreasing systemic S/G lipids by decreasing microbiome Bacteroidota significantly enhanced splenic monocyte proinflammatory responses. Replenishing systemic levels of S/G lipids via exogenous administration returned splenic monocyte responses to control levels. Transcriptomic analysis demonstrated that S/G lipids regulate monocyte proinflammatory responses at the level of gene expression of a small set of upstream inhibitors of TLR and NF-κB pathways that include Trem2 and Irf4. Consistent with enhancement in proinflammatory cytokine responses,decreasing S/G lipids lowered gene expression of specific pathway inhibitors. Replenishing S/G lipids normalized gene expression of these inhibitors. In conclusion,our results suggest that microbiome-derived S/G lipids normally establish a level of buffered signaling activation necessary for well-regulated innate immune responses in systemic monocytes. By regulating gene expression of inflammatory pathway inhibitors such as Trem2,S/G lipids merit broader investigation into the potential dysfunction of other innate immune cells,such as microglia,in diseases such as Alzheimer’s disease. View Publication

Streptococcus pneumoniae (Sp),a leading cause of community-acquired pneumonia,can spread from the lung into the bloodstream to cause septicemia and meningitis,with a concomitant three-fold increase in mortality. Limitations in vaccine efficacy and a rise in antimicrobial resistance have spurred searches for host-directed therapies that target pathogenic immune processes. Polymorphonuclear leukocytes (PMNs) are essential for infection control but can also promote tissue damage and pathogen spread. The major Sp virulence factor,pneumolysin (PLY),triggers acute inflammation by stimulating the 12-lipoxygenase (12-LOX) eicosanoid synthesis pathway in epithelial cells. This pathway is required for systemic spread in a mouse pneumonia model and produces a number of bioactive lipids,including hepoxilin A3 (HXA3),a hydroxy epoxide PMN chemoattractant that has been hypothesized to facilitate breach of mucosal barriers. To understand how 12-LOX-dependent inflammation promotes dissemination during Sp lung infection and dissemination,we utilized bronchial stem cell-derived air-liquid interface (ALI) cultures that lack this enzyme to show that HXA3 methyl ester (HXA3-ME) is sufficient to promote basolateral-to-apical PMN transmigration,monolayer disruption,and concomitant Sp barrier breach. In contrast,PMN transmigration in response to the non-eicosanoid chemoattractant fMLP did not lead to epithelial disruption or bacterial translocation. Correspondingly,HXA3-ME but not fMLP increased release of neutrophil elastase (NE) from Sp-infected PMNs. Pharmacologic blockade of NE secretion or activity diminished epithelial barrier disruption and bacteremia after pulmonary challenge of mice. Thus,HXA3 promotes barrier disrupting PMN transmigration and NE release,pathological events that can be targeted to curtail systemic disease following pneumococcal pneumonia. View Publication

TIGIT is an alternative checkpoint receptor (CR) whose inhibition promotes Graft-versus-Leukemia effects of NK cells. Given the significant immune-permissiveness of NK cells circulating in acute myeloid leukemia (AML) patients,we asked whether adoptive transfer of activated NK cells would benefit from additional TIGIT-blockade. Hence,we characterized cytokine-induced memory-like (CIML)-NK cells and NK cell lines for the expression of inhibitory CRs. In addition,we analyzed the transcription of CR ligands in AML patients (CCLE and Beat AML 2.0 cohort) in silico and evaluated the efficacy of CR blockade using in vitro cytotoxicity assays,CD69,CD107a and IFN-γ expression. Alternative but not classical CRs were abundantly expressed on healthy donor NK cells and even further upregulated on CIML-NK cells. In line with our finding that CD155,one important TIGIT-ligand,is reliably expressed on AMLs,we show improved killing of CD155+-AML blasts by NK-92 but interestingly not CIML-NK cells in the presence of TIGIT-blockade. Additionally,our in silico data (n = 671) show that poor prognosis AML patients rather displayed a CD86low CD112/CD155high phenotype,whereas patients with a better outcome rather exhibited a CD86high CD112/CD155low phenotype. Collectively,our data evidence that the complex CR ligand expression profile on AML blasts may be one explanation for the intrinsic NK cell exhaustion observed in AML patients which might be overcome with adoptive NK-92 transfer in combination with TIGIT-blockade.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00262-024-03766-7. View Publication

AbstractObjectiveUnique metabolic requirements accompany the development and functional fates of immune cells. How cellular metabolism is important in natural killer (NK) cells and their memory‐like differentiation in bacterial infections remains elusive.MethodsHere,we utilise our established NK cell memory assay to investigate the metabolic requirement for memory‐like NK cell formation and function in response to the Gram‐negative intracellular bacteria Burkholderia pseudomallei (BP),the causative agent of melioidosis.ResultsWe demonstrate that CD160+ memory‐like NK cells upon BP stimulation upregulate glucose and amino acid transporters in a cohort of recovered melioidosis patients which is maintained at least 3‐month post‐hospital admission. Using an in vitro assay,human BP‐specific CD160+ memory‐like NK cells show metabolic priming including increased expression of glucose and amino acid transporters with elevated glucose uptake,increased mTOR activation and mitochondrial membrane potential upon BP re‐stimulation. Antigen‐specific and cytokine‐induced IFN‐γ production of this memory‐like NK cell subset are highly dependent on oxidative phosphorylation (OXPHOS) with some dependency on glycolysis,whereas the formation of CD160+ memory‐like NK cells in vitro is dependent on fatty acid oxidation and OXPHOS and further increased by metformin.ConclusionThis study reveals the link between metabolism and cellular function of memory‐like NK cells,which can be exploited for vaccine design and for monitoring protection against Gram‐negative bacterial infection. This study reveals the link between metabolism and cellular function of memory‐like NK cells in melioidosis. We demonstrate that CD160+ memory‐like NK cells upon Burkholderia pseudomallei (BP) stimulation upregulate glucose and amino acid transporters in a cohort of recovered melioidosis patients. Using an in vitro assay,human BP‐specific CD160+ memory‐like NK cells show metabolic priming including increased expression of glucose and amino acid transporters with elevated glucose uptake,increased mTOR activation and mitochondrial membrane potential upon BP re‐stimulation. View Publication