技术资料

AbstractIn this work,we have generated bispecific interleukin (IL)‐12 surrogate agonists based on camelid‐derived single‐domain antibodies (sdAbs) targeting the IL‐12 receptor (IL‐12R) subunits IL‐12Rβ1 and IL‐12Rβ2. Following immunization and antibody display‐based paratope isolation,respective sdAbs were combinatorially reformatted into a monovalent bispecific architecture by grafting resulting paratopes onto the hinge region of a heterodimeric Fc region. Functional characterization using NK‐92 cells enabled the identification of multiple different sdAb‐based bispecifics displaying divergent IL‐12R agonism capacities as analyzed by STAT4 phosphorylation. Further investigations by harnessing peripheral blood mononuclear cells (PBMCs) from healthy donors revealed attenuated pSTAT4 activation compared to recombinant human (rh) wild‐type IL‐12 regarding both natural killer (NK)‐cell and T‐cell activation but robust IL‐12R agonism on stimulated T cells. While several sdAb‐based IL‐12 mimetics were nearly inactive on NK cells as well as T cells obtained from PBMCs,they elicited significant STAT4 phosphorylation and interferon (IFN)‐γ release on stimulated T cells as well as an IL‐12‐like transcriptional signature. Furthermore,we demonstrate that the activity of receptor agonism of generated bispecific IL‐12 mimetics can also be biased towards stimulated T cells by changing the spatial orientation of the individual sdAbs within the molecular design architecture. Taken together,we present an alternative strategy to generate IL‐12‐like biologics with tailor‐made characteristics. View Publication

Current clinical strategies for the delivery of pulmonary therapeutics to the lung are primarily targeted to the upper portions of the airways,such as treatment with nebulized instillation and inhalation. However,targeted delivery to the lower regions of the lung is necessary for the treatment of parenchymal lung injury and disease. Here,we show the development of an mRNA therapeutic for the lower lung in mice using one-component Ionizable Amphiphilic Janus Dendrimers as a delivery vehicle. We deliver an anti-inflammatory cytokine mRNA,transforming growth factor-beta,to produce transient protein expression in the lower regions of the lung. This study highlights a method for precise,effective,and safe delivery of TGF-β mRNA to the lung in mice. 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. Targeted delivery to the lower regions of the lung is necessary for the treatment of parenchymal lung injury and disease but is challenging. Here,the authors develop an mRNA delivery platform to treat acute lung injury in mice and demonstrate that it can reach the lower regions of the lung. View Publication

Disease progression and drug resistance in patients with chronic lymphocytic leukaemia (CLL) depend on signals from the tumour microenvironment in lymphoid sites. GRK2 inhibits the egress of normal B cells from lymphoid tissues by inducing the downregulation of the S1P-receptor 1 (S1PR1). In this study we investigated the role of GRK2 in the context of CLL using in vitro and in vivo murine models,and also primary samples from CLL patients. We found that pharmacological inhibition of GRK2 enhanced the migration of leukemic cells from CLL patients towards S1P and impaired the S1P-induced downregulation of S1PR1. Likewise,CRISPR/Cas9-mediated GRK2 deletion in a murine leukemic cell line derived from the Eµ-TCL1 mouse model of CLL also increased migratory capacity toward S1P in vitro. Furthermore,when injected into mice,GRK2-deficient murine leukemic cells exhibited an altered in vivo localization,with a higher presence in the blood and spleen compared to the bone marrow. Within the spleen,these cells displayed reduced localization to the follicles compared to control murine leukemic cells. Deletion of GRK2 on murine leukemic cells did not affect their in vitro proliferation,but notably,conferred a growth disadvantage in vivo. These findings underscore GRK2 as a critical regulator of the localization of CLL cells in vivo and suggest its potential as a therapeutic target to disrupt survival niches in CLL.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-91536-5. View Publication

ABSTRACTCell therapy based on chimeric antigen receptor (CAR) T cells has represented a revolutionary new approach for treating tumors,especially hematological diseases. Complete remission rates (CRR) > 80%–97% and 50%–90% overall response rates (ORR) have been achieved with a treatment based on CAR‐T cells in patients with malignant B‐cell tumors that have relapsed or are refractory to previous treatments. Toxicity remains the major problem. Most patients treated with CAR‐T cells develop high‐grade cytokine release syndrome (CRS) and immune effector cell‐associated neurotoxicity syndrome (ICANS). However,the unprecedentedly high CRR and ORR have led to the approval of six CAR‐T cell therapeutics by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA),prompting researchers to improve existing products and develop new ones. By now,around 1000 clinical trials based on CAR‐T cells are registered at ClinicalTrials.gov: 82% are for hematological diseases,while the remaining 16% are for solid tumors. As a result of this increased research,an enormous amount of conflicting information has been accumulated in the literature,and each group follows its manufacturing protocols and performs specific in vitro testing. This review aimed to combine and compare clinical and preclinical information,highlighting the most used protocols to provide a comprehensive overview of the in vitro world of CAR‐T cells,from manufacturing to their characterization. The focus is on all steps of the CAR‐T cell manufacturing process,from the collection of patient or donor blood to the enrichment of T cells,their activation with anti‐CD3/CD28 beads,interleukin‐2 (IL‐2) or IL‐7 and IL‐15 (induction of a more functional memory phenotype),and their transfection (viral or non‐viral methods). Automation is crucial for ensuring a standardized final product. View Publication

Uveal melanoma (UM) is the most common intraocular cancer in adults,with metastatic disease (mUM) occurring in approximately half of the patients. Tebentafusp,an immune-mobilizing monoclonal T cell receptor against cancer (ImmTAC),is a therapeutic shown to improve overall survival (OS) in HLA-A*02:01+ adult patients with mUM. Here we investigate the impact of tumor-associated macrophages (TAM) on ImmTAC activity. In vitro,M2 macrophages inhibit ImmTAC-mediated tumor-killing in a dose-dependent and contact-dependent manner. Accordingly,high baseline intratumoral TAM-to-T cell ratios correlate with shorter OS (HR = 2.09,95% CI,1.31–3.33,p = 0.002) in tebentafusp-treated mUM patients from a phase 2 trial. By contrast,IL-2 conditioning of T cells overcomes M2 macrophage-mediated suppression in vitro,while ImmTAC treatment leads to M2-to-M1 macrophage reprogramming both in vitro and in tebentafusp-treated mUM patients. Overall,we show that tebentafusp reshapes the tumor microenvironment to enhance anti-tumor T cell activity,whilst combining tebentafusp with IL-2 may enhance benefit in patients with high levels of TAM. ‘T cell engagers promote antitumor immunity,but how macrophage modulates this activity in tumor is still unclear. Here the authors show,using biopsies from patients with uveal melanoma and single cell analyses,that a T cell engager,tebentafusp,reprograms tumor-associated macrophages and ameliorates,in synergy with IL-2,immunosuppression to cancer. View Publication

AbstractThere is currently a lack of tools capable of perturbing genes in both a precise and a spatiotemporal fashion. The flexibility of CRISPR (clustered regularly interspaced short palindromic repeats),coupled with light’s unparalleled spatiotemporal resolution deliverable from a controllable source,makes optogenetic CRISPR a well-suited solution for precise spatiotemporal gene perturbations. Here,we present a new optogenetic CRISPR tool (Blue Light-inducible Universal VPR-Improved Production of RGRs,BLU-VIPR) that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of guide RNA (gRNA) production. We engineered BLU-VIPR around a new potent blue-light activated transcription factor (VPR-EL222) and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single messenger RNA transcript. This simplified spatiotemporal gene perturbation and allowed for several types of optogenetic CRISPR,including indels,CRISPRa,and base editing. BLU-VIPR also worked in vivo with cells previously intractable to optogenetic gene editing,achieving optogenetic gene editing in T lymphocytes in vivo. View Publication

Tetracyclines are essential bacterial protein synthesis inhibitors under continual development to combat antibiotic resistance yet suffer from unwanted side effects. Mitoribosomes - responsible for generating oxidative phosphorylation (OXPHOS) subunits - share structural similarities with bacterial machinery and may suffer from cross-reactivity. Since lymphocytes rely upon OXPHOS upregulation to establish immunity,we set out to assess the impact of ribosome-targeting antibiotics on human T cells. We find tigecycline,a third-generation tetracycline,to be the most cytotoxic compound tested. In vitro,5–10 μM tigecycline inhibits mitochondrial but not cytosolic translation,mitochondrial complex I,III and IV expression,and curtails the activation and expansion of unique T cell subsets. By cryo-EM,we find tigecycline to occupy three sites on T cell mitoribosomes. In addition to the conserved A-site found in bacteria,tigecycline also attaches to the peptidyl transferase center of the large subunit. Furthermore,a third,distinct binding site on the large subunit,aligns with helices analogous to those in bacteria,albeit lacking methylation in humans. The data provide a mechanism to explain part of the anti-inflammatory effects of these drugs and inform antibiotic design. Tetracyclines impair cellular function by targeting ribosomes. Here,the authors demonstrate that tigecycline impairs T cell function by selectively inhibiting mitochondrial protein synthesis and uncover the structural basis for mitoribosome inhibition and its role in immunosuppression. View Publication

Non-viral DNA donor templates are commonly used for targeted genomic integration via homologous recombination (HR),with efficiency improved by CRISPR/Cas9 technology. Circular single-stranded DNA (cssDNA) has been used as a genome engineering catalyst (GATALYST) for efficient and safe gene knock-in. Here,we introduce enGager,an enhanced GATALYST associated genome editor system that increases transgene integration efficiency by tethering cssDNA donors to nuclear-localized Cas9 fused with single-stranded DNA binding peptide motifs. This approach further improves targeted integration and expression of reporter genes at multiple genomic loci in various cell types,showing up to 6-fold higher efficiency compared to unfused Cas9,especially for large transgenes in primary cells. Notably,enGager enables efficient integration of a chimeric antigen receptor (CAR) transgene in 33% of primary human T cells,enhancing anti-tumor functionality. This ‘tripartite editor with ssDNA optimized genome engineering (TESOGENASE) offers a safer,more efficient alternative to viral vectors for therapeutic gene modification. Non-viral DNA donor templates are commonly used for targeted genomic integration via homologous recombination. Here the authors present the TESOGENASE system which enhances CRISPR-based gene integration by tethering circular single-stranded DNA to Cas9. 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

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

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

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