技术资料

IntroductionBispecific antibodies (BsAbs) can simultaneously target two epitopes of different antigenic targets,bringing possibilities for diversity in antibody drug design and are promising tools for the treatment of cancers and other diseases. T-cell engaging bsAb is an important application of the bispecific antibody,which could promote T cell-mediated tumor cell killing by targeting tumor-associated antigen (TAA) and CD3 at the same time.MethodsThis study comprised antibodies purification,Elisa assay for antigen binding,cytotoxicity assays,T cell activation by flow cytometry in vitro and xenogenic tumor model in vivo.ResultsWe present a novel bsAb platform named PHE-Ig technique to promote cognate heavy chain (HC)-light chain (LC) pairing by replacing the CH1/CL regions of different monoclonal antibodies (mAbs) with the natural A and B chains of PHE1 fragment of Integrin β2 based on the knob-in-hole (KIH) technology. We had also verified that PHE-Ig technology can be effectively used as a platform to synthesize different desired bsAbs for T-cell immunotherapy. Especially,BCMA×CD3 PHE-Ig bsAbs exhibited robust anti-multiple myeloma (MM) activity in vitro and in vivo.DiscussionMoreover,PHE1 domain was further shortened with D14G and R41S mutations,named PHE-S,and the PHE-S-based BCMA×CD3 bsAbs also showed anti BCMA+ tumor effect in vitro and in vivo,bringing more possibilities for the development and optimization of different bsAbs. To sum up,PHE1-based IgG-like antibody platform for bsAb construction provides a novel strategy for enhanced T-cell immunotherapy. 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

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

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

SummarySensing of extracellular ATP (eATP) controls CD8+ T cell function. Their accumulation can occur through export by specialized molecules,such as the release channel Pannexin 1 (Panx1). Whether Panx1 controls CD8+ T cell immune responses in vivo,however,has not been previously addressed. Here,we report that T-cell-specific Panx1 is needed for CD8+ T cell responses to viral infections and cancer. We found that CD8-specific Panx1 promotes both effector and memory CD8+ T cell responses. Panx1 favors initial effector CD8+ T cell activation through extracellular ATP (eATP) export and subsequent P2RX4 activation,which helps promote full effector differentiation through extracellular lactate accumulation and its subsequent recycling. In contrast,Panx1 promotes memory CD8+ T cell survival primarily through ATP export and subsequent P2RX7 engagement,leading to improved mitochondrial metabolism. In summary,Panx1-mediated eATP export regulates effector and memory CD8+ T cells through distinct purinergic receptors and different metabolic and signaling pathways. Graphical abstract Highlights•The hemichannel Panx1 promotes in vivo effector and memory CD8+ T cell responses•Panx1 promotes effector CD8+ T cells via eATP and lactate extracellular accumulation•Panx1 promotes memory CD8+ T cell survival by eATP export and activation of AMPK Natural sciences; Biological sciences; Biochemistry; Immunology; Immune response; Immune system View Publication

Background & AimsType 2 innate lymphoid cells (ILC2s) and interleukin-13 (IL-13) promote the onset of spasmolytic polypeptide-expressing metaplasia (SPEM) cells. However,little is known about molecular effects of IL-13 in SPEM cells. We now sought to establish a reliable organoid model,Meta1 gastroids,to model SPEM cells in vitro. We evaluated cellular and molecular effects of ILC2s and IL-13 on maturation and proliferation of SPEM cells.MethodsWe performed single-cell RNA sequencing to characterize Meta1 gastroids,which were derived from stomachs of Mist1-Kras transgenic mice that displayed pyloric metaplasia. Cell sorting was used to isolate activated ILC2s from stomachs of IL-13-tdTomato reporter mice treated with L635. Three-dimensional co-culture was used to determine the effects of ILC2s on Meta1 gastroids. Mouse normal or metaplastic (Meta1) and human metaplastic gastroids were cultured with IL-13 to evaluate cell responses. Air-Liquid Interface culture was performed to test long-term culture effects of IL-13. In silico analysis determined possible STAT6-binding sites in gene promoter regions. STAT6 inhibition was performed to corroborate STAT6 role in SPEM cells maturation.ResultsMeta1 gastroids showed the characteristics of SPEM cell lineages in vitro even after several passages. We demonstrated that co-culture with ILC2s or IL-13 treatment can induce phosphorylation of STAT6 in Meta1 and normal gastroids and promote the maturation and proliferation of SPEM cell lineages. IL-13 up-regulated expression of mucin-related proteins in human metaplastic gastroids. Inhibition of STAT6 blocked SPEM-related gene expression in Meta1 gastroids and maturation of SPEM in both normal and Meta1 gastroids.ConclusionsIL-13 promotes the maturation and proliferation of SPEM cells consistent with gastric mucosal regeneration. Graphical Abstract View Publication

IntroductionThe early transcription unit 3 (E3) of human adenoviruses (HAdVs) encodes several immunoevasins,including the E3/49K protein,which is unique for species D of HAdVs. It is expressed as surface transmembrane protein and shed. E3/49K of HAdV-D64 binds to the protein tyrosine phosphatase surface receptor CD45,thereby modulating activation of T and NK cells.MethodsConsidering that E3/49K represents the most polymorphic viral protein among species D HAdVs,we demonstrate here that all tested E3/49K orthologs bind to the immunologically important regulator CD45. Thus,this feature is conserved regardless of the pathological associations of the respective HAdV types.ResultsIt appeared that modulation of CD45 is a unique property restricted to HAdVs of species D. Moreover,E3/49K treatment inhibited B cell receptor (BCR) signaling and impaired BCR signal phenotypes. The latter were highly comparable to B cells having defects in the expression of CD45,suggesting E3/49K as a potential tool to investigate CD45 specific functions.ConclusionWe identified B cells as new direct target of E3/49K-mediated immune modulation,representing a novel viral immunosubversive mechanism. View Publication

CD19-targeted chimeric antigen receptor (CAR) T cell therapies have driven a paradigm shift in the treatment of relapsed/refractory B-cell malignancies. However,>50% of CD19-CAR-T-treated patients experience progressive disease mainly due to antigen escape and low persistence. Clinical prognosis is heavily influenced by CAR-T cell function and systemic cytokine toxicities. Furthermore,it remains a challenge to efficiently,cost-effectively,and consistently manufacture clinically relevant numbers of virally engineered CAR-T cells. Using a highly efficient piggyBac transposon-based vector,Quantum pBac™ (qPB),we developed a virus-free cell-engineering system for development and production of multiplex CAR-T therapies. Here,we demonstrate in vitro and in vivo that consistent,robust and functional CD20/CD19 dual-targeted CAR-T stem cell memory (CAR-TSCM) cells can be efficiently produced for clinical application using qPB™. In particular,we showed that qPB™-manufactured CAR-T cells from cancer patients expanded efficiently,rapidly eradicated tumors,and can be safely controlled via an iCasp9 suicide gene-inducing drug. Therefore,the simplicity of manufacturing multiplex CAR-T cells using the qPB™ system has the potential to improve efficacy and broaden the accessibility of CAR-T therapies. View Publication

Small extracellular vesicles (sEVs) are important mediators of intercellular communication between tumor cells and their surrounding environment. Furthermore,the mechanisms by which miRNAs carried in tumor sEVs regulate macrophage polarization remain largely unknown. To concentrate sEVs,we used the traditional ultracentrifugation method. Western blot,NanoSight,and transmission electron microscopy were used to identify sEVs. To determine the function of sEVs-miR-487a,we conducted in vivo and in vitro investigations. The intercellular communication mechanism between osteosarcoma cells and M2 macrophages,mediated by sEVs carrying miR-487a,was validated using luciferase reporter assays,transwell assays,and Western blot analysis. In vitro,sEVs enriched in miR-487a and delivered miR-487a to macrophages,promoting macrophage polarization toward an M2-like type,which promotes proliferation,migration,invasion,and epithelial-mesenchymal transition (EMT) of osteosarcoma cells. In vivo,sEVs enriched in miR-487a facilitate lung metastasis of osteosarcoma. Moreover,plasma miR-487a in sEVs was shown to be a potential biomarker applicable for osteosarcoma diagnosis. In summary,miR-487a derived from osteosarcoma cells can be transferred to macrophages via sEVs,then promote macrophage polarization towards an M2-like type by targeting Notch2 and activating the GATA3 pathway. In a feedback loop,the activation of macrophages accelerates epithelial-mesenchymal transition (EMT),which in turn promotes the migration,invasion,and lung metastasis of osteosarcoma cells. This reciprocal interaction between activated macrophages and osteosarcoma cells contributes to the progression of the disease. Our data demonstrate a new mechanism that osteosarcoma tumor cells derived exosomal-miR-487a which is involved in osteosarcoma development by regulating macrophage polarization in tumor microenvironment (TME).Supplementary InformationThe online version contains supplementary material available at 10.1186/s12935-024-03488-x. View Publication

Regulatory T cells (Tregs) are crucial immune cells for tissue repair and regeneration. However,their potential as a cell-based regenerative therapy is not yet fully understood. Here,we show that local delivery of exogenous Tregs into injured mouse bone,muscle,and skin greatly enhances tissue healing. Mechanistically,exogenous Tregs rapidly adopt an injury-specific phenotype in response to the damaged tissue microenvironment,upregulating genes involved in immunomodulation and tissue healing. We demonstrate that exogenous Tregs exert their regenerative effect by directly and indirectly modulating monocytes/macrophages (Mo/MΦ) in injured tissues,promoting their switch to an anti-inflammatory and pro-healing state via factors such as interleukin (IL)-10. Validating the key role of IL-10 in exogenous Treg-mediated repair and regeneration,the pro-healing capacity of these cells is lost when Il10 is knocked out. Additionally,exogenous Tregs reduce neutrophil and cytotoxic T cell accumulation and IFN-γ production in damaged tissues,further dampening the pro-inflammatory Mo/MΦ phenotype. Highlighting the potential of this approach,we demonstrate that allogeneic and human Tregs also promote tissue healing. Together,this study establishes exogenous Tregs as a possible universal cell-based therapy for regenerative medicine and provides key mechanistic insights that could be harnessed to develop immune cell-based therapies to enhance tissue healing. Regulatory T cells (Tregs) are known for suppressing inflammatory processes,but their full capacity for tissue regeneration is yet to be harnessed. Here,the authors demonstrate the efficiency of Tregs in facilitating tissue healing in mouse models of bone,muscle,and skin injury,with monocytes/macrophages and interleukin-10 playing a key mechanistic role in the process. View Publication

Immunoglobulin G (IgG) is the main isotype of antibody in human blood. IgG consists of four subclasses (IgG1 to IgG4),encoded by separate constant region genes within the Ig heavy chain locus (IGH). Here,we report a genome-wide association study on blood IgG subclass levels. Across 4334 adults and 4571 individuals under 18 years,we discover ten new and identify four known variants at five loci influencing IgG subclass levels. These variants also affect the risk of asthma,autoimmune diseases,and blood traits. Seven variants map to the IGH locus,three to the Fcγ receptor (FCGR) locus,and two to the human leukocyte antigen (HLA) region,affecting the levels of all IgG subclasses. The most significant associations are observed between the G1m (f),G2m(n) and G3m(b*) allotypes,and IgG1,IgG2 and IgG3,respectively. Additionally,we describe selective associations with IgG4 at 16p11.2 (ITGAX) and 17q21.1 (IKZF3,ZPBP2,GSDMB,ORMDL3). Interestingly,the latter coincides with a highly pleiotropic signal where the allele associated with lower IgG4 levels protects against childhood asthma but predisposes to inflammatory bowel disease. Our results provide insight into the regulation of antibody-mediated immunity that can potentially be useful in the development of antibody based therapeutics. Immunoglobulin G (IgG) is the main isotype of antibody in human blood. Here the authors describe 14 genetic variants that affect IgG levels in blood. The data provide new insight into the regulation of humoral immunity that could be useful in the development of antibody-based therapeutics. View Publication

Mature erythrocytes are known to lack major histocompatibility complex (MHC) proteins. However,the presence of MHC molecules on erythrocytes has been occasionally reported,though without a defined function. In this study,we designed erythrocyte conjugated solely with a fusion protein consisting of an antigenic peptide linked to MHC class I (MHC-I) protein,termed MHC-I‒Ery. The modified erythrocyte,decorated with the peptide derived from human papillomavirus (HPV) 16 oncoprotein E6/E7,effectively activated antigen-specific CD8+ T cells in peripheral blood mononuclear cells (PBMCs) from HPV16+ cervical cancer patients. Additionally,MHC-I‒Ery monotherapy was shown to inhibit antigen-positive tumor growth in mice. This treatment immediately activated CD8+ T cells and reduced suppressive myeloid cells in the spleen,leading to systemic anti-tumor activity. Safety and tolerability evaluations of MHC-I‒Ery in non-human primates further supported its clinical potential. Our results first demonstrated that erythrocytes equipped solely with antigen peptide‒MHC-I complexes can robustly stimulate the immune system,suggesting a novel and promising approach for advancing cancer immunotherapy. View Publication