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Organ transplantation is a lifesaving procedure,with 50,000 transplants happening every year in the United States. However,many patients harbor antibodies and B cells directed against allogeneic human leukocyte antigen (HLA) molecules,notably HLA-A2,greatly decreasing their likelihood of receiving a compatible organ. Moreover,antibody-mediated rejection is a significant contributor to chronic transplant rejection. Current strategies to desensitize patients non-specifically target circulating antibodies and B cells,resulting in poor efficacy and complications. Regulatory T cells (Tregs) are immune cells dedicated to suppressing specific immune responses by interacting with both innate and adaptive immune cells. Here,we genetically modified human Tregs with a chimeric anti-HLA antibody receptor (CHAR) consisting of an extracellular HLA-A2 protein fused to a CD28-CD3zeta intracellular signaling domain,driving Treg activation upon recognition of anti-HLA-A2 antibodies on the surface of alloreactive B cells. We find that HLA-A2 CHAR Tregs get activated specifically by anti-HLA-A2 antibody-producing cells. Of note,HLA-A2 CHAR activation does not negatively affect Treg stability,as measured by expression of the Treg lineage transcription factors FOXP3 and HELIOS. Interestingly,HLA-A2 CHAR Tregs are not cytotoxic towards anti-HLA-A2 antibody-producing cells,unlike HLA-A2 CHAR modified conventional CD4+ T cells. Importantly,HLA-A2 CHAR Tregs recognize and significantly suppress high affinity IgG antibody production by B cells from HLA-A2 sensitized patients. Altogether,our results provide proof-of-concept of a new strategy to specifically inhibit alloreactive B cells to desensitize transplant recipients. View Publication

ABSTRACTActivation of the cGAS‐STING pathway plays an important role in antitumor immunity through maturation of tumor‐infiltrating DCs. DCs engulf extracellular DNA released by dying cancer cells,supporting activation of the cGAS‐STING pathway and concomitant DC maturation. Extracellular DNA in the tumor microenvironment is primarily derived from cells undergoing uncontrolled necrosis or programmed inflammatory death,such as necroptosis,which can be induced when apoptosis pathways are inhibited. Here,we report that caspase inhibition primes activation of a RIPK1/3,MLKL,and STING signaling axis in DCs,resulting in maturation without the need for any further maturation stimuli such as LPS or TNF‐α. Notably,these signaling events do not induce DC death,indicating a nonnecroptotic role of the RIPK1‐RIPK3‐MLKL pathway and novel crosstalk with the STING pathway. Caspase inhibition in DC/cancer cell co‐cultures results in DC maturation,inducing TNF‐α secretion,which delivers the co‐signal to induce cancer cell necroptosis. In summary,we find a collaborative mechanism of the STING and necroptosis pathway in DC maturation,and that activation of the necroptosis pathway has opposite effects on cancer cells and DCs,proposing a possibility for new targets in cancer immunotherapy. In this study,we show that caspase‐8 inhibition induce maturation in DCs through a synergistic crosstalk between the STING signaling pathway and RIPK1‐RIPK3‐MLKL pathway,without induction of cell death in the DCs. The mature DCs secrete TNF‐α that induces cancer cell death in DC/cancer cell co‐cultures upon concomitant caspase‐8 inhibition. View Publication

IntroductionCCR2,a chemokine receptor critical for immune cell migration,inflammation,and HIV infection,is regulated by poorly understood mechanisms.MethodsThis study investigated the unusually long CCR2 3’-UTR’s role in post-transcriptional regulation.ResultsThe full-length 3′-UTR significantly inhibited reporter gene expression in primary CD4+ T cells and macrophages,likely mediated by RNA binding proteins (RBPs). HnRNPA0,was shown to bind directly to this region and influence CCR2 levels. When the RBP binding sites were mutagenized or the 3′- UTR removed using CRISPR-Cas9 and gRNAs,CCR2 mRNA and protein levels significantly increased. Cell fractionation experiments confirmed that these changes occurred in both the nucleus and cytoplasm. To directly test mRNA stability,we used a-amanitin and found that removing the 3′-UTR nearly doubled the half-life of CCR2 mRNA. Finally,pseudotyping studies revealed CCR2 functions as an HIV co-receptor at ~10% efficiency compared to CCR5.DiscussionThese results show that the CCR2 3′-UTR plays an important role in post-transcriptional regulation and may provide a novel approach to regulating CCR2 activity in inflammatory or infectious diseases. View Publication

Atopic dermatitis (AD) is a chronic dermatological disorder characterized by intense pruritus and eczematous lesions. Repeated topical application of 2,4-dinitrofluorobenzene (DNFB) in NC/Nga mice produces AD-like clinical symptoms that closely resemble human AD. N-acetyl-L-alanine (L-NAA),a derivative of L-Alanine,has unknown biological and physiological effects on cutaneous tissue. In this study,we investigated whether L-NAA modifies AD-like symptoms elicited by ongoing DNFB exposure in NC/Nga mice. Topical administration of L-NAA markedly attenuated the development of AD-like cutaneous lesions triggered by DNFB. L-NAA treatment further suppressed DNFB-induced infiltration of eosinophils and mast cells and prevented the increase of serum IgE resulting from DNFB application. L-NAA treatment decreased DNFB-stimulated expression of IL-4,a Th2-associated cytokine,but increased IFN-γ expression,indicative of Th1 activity,within the skin lesions. In addition,L-NAA prevented the DNFB-driven upregulation of GATA3,a central regulator of Th2 lineage differentiation,in CD4＋ cells,with no effect on T-bet,the principal regulator of Th1 cells. These findings indicate that L-NAA can limit Th2 differentiation in the AD mouse model. Therefore,L-NAA may serve as a promising therapeutic and immunomodulatory compound against AD. View Publication

Toxocariasis,a neglected zoonotic disease caused by parasites of the Toxocara genus,represents a significant public health concern,with an estimated global seroprevalence of 19%. Despite the well-known respiratory symptoms associated with toxocariasis,the immune response in the lungs during toxocariasis is still poorly understood. This study analyzes both local lung and systemic immune response to T. canis infection and T. canis excretory-secretory antigens (TES) intranasal application in C57BL/6J mice. Lungs,blood,and spleens were collected at specific time points for histopathological analyses,flow cytometry,cytokine profiling,and gene expression studies. The systemic immune response was further assessed by cytokine measurements in splenocyte cultures and the detection of TES-specific antibodies. T. canis infection triggered severe pulmonary inflammation characterized by eosinophilia and mucus accumulation,with persistent inflammation lasting up to 28 days post-infection. Interestingly,this response was not solely driven by Th2-type interleukin production. Cytokine analysis of splenocyte cultures revealed elevated levels of IL-5 and IL-6,along with increased TES-specific IgE and IgG1 antibody concentrations. In contrast,TES application alone induced local eosinophil infiltration and upregulated genes associated with lung repair,though this response was less intense and shorter-lived compared to the infection. Our study is the first to present a comprehensive cytokine proteome analysis in mouse lungs during T. canis infection and stimulation by larval antigens,highlighting the key role of cytokines such as IL-5,IL-6,and IL-33. These findings provide new insights into the pathogenesis of toxocariasis and underscore the need for further research into potential therapeutic targets. View Publication

AbstractMucus is an important component of airway host defenses that acts by enabling the trapping and clearance of infectious materials such as bacteria and viruses. It can be difficult,however,to design experiments that independently determine the extent to which mucus contributes to innate barrier functions in the lung. Here,we provide detailed protocols to collect mucus from human airway epithelial cultures and evaluate how the properties of mucus impact mucociliary transport and protection from viral infection. We include recommended test parameters depending on the specific research question as it relates to respiratory infectious diseases. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Analysis of mucociliary transport and ciliary beat frequency in HAE cultures Basic Protocol 2: Collection of mucus from HAE cultures Basic Protocol 3: Transplantation of mucus to HAE cultures and infection with virus View Publication

To explore the relevance of non-malignant B cells (NMBCs) in chronic lymphocytic leukaemia (CLL),201 blood samples from 79 previously untreated CLL patients receiving chemoimmunotherapy (CIT) in a phase III clinical trial were subjected to mass cytometry analysis using a bespoke panel of 24 antibodies. One memory (CD27+CD38−) and two naïve (CD27−CD38+) CD19+ clusters with a non-malignant phenotype (NMP; CD5−CD43−ROR1−CD20hiCD79bhiCD81+) were identified by FlowSOM and UMAP algorithms. NMP clusters were most prevalent in healthy controls,almost undetectable in untreated and relapsed CLL,and variable following CIT,where they correlated with haematopoietic recovery (naïve NMP clusters) and polyclonal antibody production (memory NMP cluster). Larger NMP cluster size at the early post-treatment timepoint was associated with a significantly longer progression free survival (PFS; HR 0.40 [95% CI: 0.22–0.75]; P = 0.003). Similar findings were obtained when NMBCs were prospectively defined by Boolean gating using the non-redundant features of the NMP clusters. NMBCs measured in this way negatively correlated with MRD and provided complementary prognostic information in patients with persistent MRD. Our findings raise the possibility that re-emergence of circulating NMBCs after treatment reflects clearance of CLL from secondary lymphoid organs,thereby complementing MRD as a biomarker of bone marrow clearance.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-16558-5. View Publication

Cysteine-rich angiogenic inducer 61 (CYR61) promotes prostate cancer (PCa) cell growth,but its role in disease progression remains unclear. Given its insulin-like growth factor (IGF)-binding domain and the known involvement of insulin-like growth factor-1 (IGF1) in PCa,we investigated the molecular interplay between CYR61 and IGF1. CYR61 was silenced using small interfering RNA (siRNA) in prostate carcinoma 3 (PC3),lymph node carcinoma of the prostate (LNCaP),and androgen receptor (AR)-positive 22Rv1 cells,followed by assessments of their proliferation,viability,colony formation,migration,and signaling pathway activation. CYR61 knockdown significantly reduced cell growth,viability,prostasphere formation,and migration across all three cell lines. Mechanistically,CYR61 silencing inhibited PI3K/AKT signaling but had no effect on MAPK activation. In addition,treatment with recombinant IGF1 induced CYR61 expression in a time-dependent manner,and the inhibition of PI3K/AKT signaling suppressed both CYR61 expression and cell proliferation. These findings suggest that IGF1 promotes PCa progression through CYR61 and that CYR61 may serve as a potential therapeutic target for limiting tumor growth and metastasis. View Publication

AbstractSeveral gene editing tools have entered the clinic,representing varied options for eliminating or correcting mutations. Although gene editing by homologous recombination (HR) can potentially accomplish any type of gene edit (insertions,deletions,and replacements),as the outcome is defined by a recombinant repair template,gene editing enzymes that support efficient HR are rare. ARCUS nucleases,engineered from the homing endonuclease I-CreI,have programmable sequence specificity and support precise,high-frequency transgene insertion. In this study,we demonstrate that the 3′ overhangs that ARCUS nucleases generate when cutting DNA are key to triggering high rates of HR. We show that a single editor can be used to accomplish the full range of currently understood DNA editing approaches,allowing all combinations of single base changes,introducing small,specific deletions,small and large insertions,and the ability to replace large segments of genomic DNA with efficiencies ranging from 60% to 90% in lymphocytes. ARCUS also supports precise,efficient insertion (30%–40%) in noncycling hepatocytes via nonclassical HR pathways. Collectively,this work characterizes a flexible and efficient gene insertion system for potential therapeutic use. Graphical Abstract Graphical Abstract View Publication

Severely impaired mucociliary airway function is the primary pathomechanism in Cystic Fibrosis (CF) lung disease. Despite significant advances in CF therapy,there is still a critical need for alternative,individualized treatment options,especially for patients with untreatable CFTR mutations. Although intestinal organoids and primary airway cells are widely used as preclinical models of CF,both systems exhibit limitations with regard to the proper modelling of mucociliary clearance or the availability of sufficient cell quantities. Patient-specific human induced pluripotent stem cells (hiPSCs) are a promising alternative due to their unlimited expansion potential and capacity to differentiate into airway epithelia. However,cellular inhomogeneities in iPSC-derived airway cultures complicated conventional assays that determine CFTR function such as Ussing chamber measurements,and a comprehensive demonstration of CF pathophysiology in hiPSC-derived airway models has been largely lacking. This study provides comprehensive data demonstrating very similar gene expression,(ultra)structure and CFTR function in CF iPSC-derived airway (iALI) and primary airway (pALI) cultures. Addressing current limitations,we have implemented a sensitive,straightforward,and automatable ciliary beat frequency (CBF) assay,which is largely unaffected by inhomogeneities and directly reflects disturbed mucus viscosity and mucociliary transport in CF lung disease. Electron microscopy images confirmed the disease phenotype showing a highly dense and dehydrated mucus layer on top of CF iALI cultures. Furthermore,established CFTR modulator drugs partially rescued the disease phenotype in CF iALI cultures,which validated the utility of iALI cultures as a scalable,patient-specific platform for CF research and personalized drug development.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04737-0. View Publication

Neurons,as long-lived non-dividing cells with complex morphology,depend on a highly elaborate secretory trafficking system which enables a constant turnover of proteins and membranes. Previously,it was shown that simplified,Golgi-related structures called Golgi satellites (GS) are present in the dendrites of primary hippocampal neurons. These organelles are distinct from the somatic Golgi complex and are involved in de novo glycosylation and local forward trafficking of membrane proteins. However,the question of whether GS are also targeted to the axons of principal neurons remained unanswered. In this study,we investigated the subcellular distribution of GS in adult hippocampal neurons. Our findings showed that GS are present all along the axon,extending to the distal tips of the growth cone. Similar to dendritic GS,the axonal organelles are labeled by the same GS markers and are capable of mature glycosylation. Live imaging experiments revealed the presence of both mobile and immobile GS in the axon,and that the switch between active transport and stalling of GS was modulated by neuronal firing. We found that GS frequently pause at en passant synapses and remain stationary for longer time periods at activated pre-synaptic boutons. This behavior is dependent on the actin cytoskeleton and the actin-based motor protein myosin VI. Overall,our study demonstrates that neuronal activity can dynamically regulate the positioning of GS in the axon,shedding light on the intricate mechanisms underlying organelle trafficking in neurons.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-025-05896-2. View Publication

T-cell acute lymphoblastic leukaemia (T-ALL) is a haematological malignancy commonly driven by NOTCH1 activating mutations. A concomitant feature associated with NOTCH1 mutations is heightened oxidative metabolism enabling the exponential proliferation of T-ALL blasts. As such,targeting mitochondrial metabolism in T-ALL is an attractive therapeutic avenue. Related to this,canagliflozin (cana),is an FDA-approved sodium glucose co-transporter 2 inhibitor with known off-target effects on complex I and glutamate dehydrogenase,but its potential anti-leukaemic effects remain unexplored. Here,we show that cana possesses potent anti-leukaemic effects underpinned by proliferative defects,cell cycle disruption and apoptosis. These anti-leukaemic effects driven by cana,are attributed to a perturbed tricarboxylic acid (TCA) cycle and mitochondrial metabolism,and elevated mitochondrial ROS. Proteomic analysis revealed that cana treatment resulted in a compensatory increase in the expression of ATF4 targets,including upregulation of serine biosynthesis pathway and one-carbon metabolism enzymes. As such,restriction of serine and glycine synergized with cana treatment,further enhancing its anti-leukaemic effects. Collectively,our study reveals a cana-driven metabolic vulnerability that can be further exploited via dietary manipulation to treat T-ALL. Highlights•Canagliflozin impairs human T-ALL proliferation,and impacts viability,extending to a broad range of genetic subtypes.•Cana reprogrammes the proteome in T-ALL leading to compensatory increases in serine,glycine and one carbon metabolism.•Restriction of serine/glycine synergistically complemented canagliflozin treatment by heightening T-ALL apoptosis. View Publication