技术资料

CD38,an ecto-enzyme involved in NAD + catabolism,is highly expressed in exhausted CD8 + T cells and has emerged as an attractive target to improve response to immune checkpoint blockade (ICB) by blunting T cell exhaustion. However,the precise role(s) and regulation of CD38 in exhausted T cells and the efficacy of CD38-directed therapeutic strategies in human cancer remain incompletely defined. Here,we show that CD38 + CD8 + T cells are induced by chronic TCR activation and type I interferon stimulation and confirm their association with ICB resistance in human melanoma. Disrupting CD38 restores cellular NAD + pools and improves T cell bioenergetics and effector functions. Targeting CD38 restores ICB sensitivity in a cohort of patient-derived organotypic tumor spheroids from explanted melanoma specimens. These results support further preclinical and clinical evaluation of CD38-directed therapies in melanoma and underscore the importance of NAD + as a vital metabolite to enhance those therapies. View Publication

Natural killer (NK) cell-based therapies represent a promising approach for acute myeloid leukemia (AML) relapse,yet their efficacy is hindered by immunosuppressive factors such as transforming growth factor beta (TGF-β) in the tumor microenvironment. This study investigated the effects of TGF-β on NK cell cytotoxicity and migration using 2D and 3D co-culture models that mimic the leukemic microenvironment. TGF-β production was evaluated in AML-derived leukemic cell lines and mesenchymal stromal cells (hTERT-MSCs) using ELISA. Bulk RNA sequencing (RNA-seq) was performed to analyze global gene expression changes in TGF-β-treated primary human NK cells. NK cell cytotoxicity and migration were assessed in 2D monolayer and 3D spheroid co-cultures containing hTERT-MSCs and leukemic cells using flow cytometry and confocal microscopy. Both leukemic cells and MSCs produced TGF-β,with increased levels observed in MSCs after co-culture with primary AML blasts. RNA sequencing revealed that TGF-β altered key gene pathways associated with NK cell cytotoxicity,adhesion,and migration,supporting its immunosuppressive role. In functional assays,TGF-β exposure significantly reduced NK cell-mediated cytotoxicity in a time-dependent manner and impaired NK cell infiltration into 3D spheroids,particularly in models incorporating MSCs. Additionally,MSCs themselves provided a protective environment for leukemic cells,further reducing NK cell effectiveness in 2D co-cultures. TGF-β suppresses both NK cell cytotoxicity and migration,limiting their ability to eliminate leukemic cells and infiltrate the bone marrow niche (BMN). These findings provide novel insights into TGF-β–mediated immune evasion mechanisms and provide important insights for the future design of NK-based immunotherapies and clinical trials. View Publication

Severe cutaneous adverse drug reactions (SCARs) are life-threatening diseases,which are associated with human leukocyte antigen ( HLA ) risk variants. However,the low positive predictive values of HLA variants suggest additional factors influence disease susceptibility. Using dapsone hypersensitivity syndrome (DHS) as a paradigm for SCARs,we show that the DHS patients harbor a sex-related global reduction in blood NK cells,contributing to the higher incidence of reactions in females. Single-cell RNA sequencing revealed a decrease in the immunoregulatory CD56 low XCL1/2 low NK cell subset and an expansion of CD56 high XCL1/2 high NK cell subsets with an effector phenotype in DHS patients compared to dapsone-tolerant individuals. Functionally,interleukin-15 superagonist-induced activation of NK cells exacerbated SCARs-like symptoms in a murine model. Mechanistically,TSC22 domain family member 3 (TSC22D3) deficiency enhanced NK cell effector function,shifting the immune response from CD4+ T cell to CD8+ T cell function. These results demonstrate that TSC22D3-regulated NK cells play a critical role in predisposing to drug hypersensitivity reactions,bridging innate and adaptive immune dysregulation in SCARs pathogenesis. Our study highlights the importance of NK cell heterogeneity and TSC22D3 in immune-mediated hypersensitivity disorders,offering potential therapeutic targets for SCARs and related conditions. Subject terms: Innate immunity,Innate immunity View Publication

Histologic variant (HV) subtypes of bladder cancer are clinically aggressive tumors that are more resistant to standard therapy compared to conventional urothelial carcinoma (UC). Little is known about the transcriptional programs that account for their biological differences. Here we show using single cell analysis that HVs harbor a tumor cell state characterized by expression of MUC16 (CA125),MUC4,and KRT24 . This cell state is enriched in metastases,predicted to be highly resistant to chemotherapy,and linked with poor survival. We also find enriched expression of TM4SF1,a transmembrane protein,in HV tumor cells. Chimeric antigen receptor (CAR) T cells engineered against TM4SF1 protein demonstrated in vitro and in vivo activity against bladder cancer cell lines in a TM4SF1 expression-dependent manner,highlighting its potential as a therapeutic target. Subject terms: Bladder cancer,Tumour biomarkers,Targeted therapies View Publication

Targeted therapy interventions using tyrosine kinase inhibitors (TKIs) provide encouraging treatment responses in patients with ALK -rearranged lung adenocarcinomas,yet resistance occurs almost inevitably. In addition to tumor cell-intrinsic resistance mechanisms,accumulating evidence suggests that cancer-associated fibroblasts (CAFs) within the tumor microenvironment contribute to therapy resistance. This study aimed to investigate CAF-driven molecular networks that shape the therapeutic susceptibility of ALK -driven lung adenocarcinoma cells. Three-dimensional (3D) spheroid co-cultures comprising ALK -rearranged lung adenocarcinoma cells and CAFs were utilized to model the tumor microenvironment. Single-cell RNA sequencing was performed to uncover transcriptional differences between TKI-treated homotypic and heterotypic spheroids. Functional assays assessed the effects of CAF-conditioned medium and CAF-secreted factors on tumor cell survival,proliferation,lipid metabolism,and downstream AKT signaling. The therapeutic potential of targeting metabolic vulnerabilities was evaluated using pharmacological inhibition of lipid metabolism and by ferroptosis induction. CAFs significantly diminished the apoptotic response of lung tumor cells to ALK inhibitors while simultaneously enhancing their proliferative capacity. Single-cell RNA sequencing identified lipogenesis-associated genes as a key transcriptional difference between TKI-treated homotypic and heterotypic lung tumor spheroids. CAF-conditioned medium and the CAF-secreted factors HGF and NRG1 activated AKT signaling in 3D-cultured ALK-rearranged lung tumor cells,leading to increased de novo lipogenesis and suppression of lipid peroxidation. These metabolic adaptations were critical for promoting tumor cell survival and fostering therapy resistance. Notably,both dual inhibition of ALK and the lipid-regulatory factor SREBP-1,as well as co-treatment with ferroptosis inducers such as erastin or RSL3,effectively disrupted the CAF-driven metabolic-supportive niche and restored sensitivity of resistant lung tumor spheroids to ALK inhibition. This study highlights a critical role for CAFs in mediating resistance to ALK-TKIs by reprogramming lipid metabolism in ALK-rearranged lung cancer cells. It suggests that targeting these metabolic vulnerabilities,particularly through inhibition of lipid metabolism or induction of ferroptosis,could provide a novel therapeutic approach to overcome resistance and improve patient outcomes. The online version contains supplementary material available at 10.1186/s40170-025-00400-7. View Publication

Cystic Fibrosis (CF) is a common genetic disease in the United States,resulting from mutations in the Cystic Fibrosis transmembrane conductance regulator (cftr) gene. CFTR modulators,particularly Elexacaftor/Tezacaftor/Ivacaftor (ETI),have significantly improved clinical outcomes for patients with CF. However,many CFTR mutations are not eligible for CFTR modulator therapy due to their rarity. In this study,we report that a patient carrying rare complex CFTR mutations,c.1680-877G>T and c.3067_3072delATAGTG,showed positive clinical outcomes after ETI treatment. We demonstrate that ETI was able to increase the expression of CFTR harboring c.3067_3072delATAGTG in a heterologous system. Importantly,patient-derived nasal epithelial cells in an air–liquid interface (ALI) culture showed improved CFTR function following ETI treatment. These findings supported the initiation of ETI with the patient. Retrospective studies have suggested that the patient has shown small but steady improvement over the past two years in several clinical metrics,including lung function,body mass index (BMI),and sweat chloride levels. Our studies suggest that ETI could be beneficial for patients carrying c.3067_3072delATAGTG. View Publication

Accumulating data have shown that targeting breast cancer stem cells (CSCs) is an auspicious way for anticancer therapies. This study demonstrated that the antirheumatic drug auranofin is a potent CSC inhibitor with anti-CSC action on breast cancer. This research focused on investigating the effect of auranofin on breast cancer and CSCs and its cellular mechanism. Mammosphere formation,colony formation,levels of CD44 high /CD24 low,and aldehyde dehydrogenase 1 expression in the cells were evaluated after auranofin treatment. The anti-CSC properties of auranofin were further examined by gel shift assay and cytokine detection. Auranofin suppressed cell growth,colony formation,migration,and mammosphere formation and triggered apoptosis in breast cancer. Auranofin decreased the CD44 high /CD24 low - and aldehyde dehydrogenase-expressed subpopulations,as well as the Stat3-DNA interaction and phosphorylated Stat3 level. Auranofin also decreased the extracellular levels of interleukin-8 (IL-8) in the mammosphere media. Auranofin suppressed the Stat3/IL-8 signal and killed CSCs; therefore,it may be a potential target for CSCs. View Publication

Anti-interleukin (IL)-4Rα monoclonal antibodies (mAb) improve lung function and decrease the number of exacerbations in patients with COPD type (T)2 inflammation. However,the involvement of early innate immune responses underlying these treatment effects is not well known. We sought to understand the effect and mechanisms of IL-4Rα mAb treatment on bronchial epithelial cells (BECs) from COPD patients under T2 inflammatory conditions with and without rhinoviral infection. Primary BECs from healthy and COPD patients were grown at an air–liquid interface and stimulated with IL-4 or IL-13 cytokines in the presence of IL-4Rα mAb. Cells were infected with human rhinovirus 1B and collected 24 h after infection. Antiviral mediators ( i.e.,interferons (IFNs) and pattern recognition receptors (PRRs)),as well as chemokine and alarmin expression,were measured by reverse transcriptase quantitative PCR and ELISA. Treatment with IL-4Rα mAb (100 nM) inhibited the eotaxin-3 (CCL26) gene after IL-4/IL-13 induction (p<0.05) in COPD BECs. However,no significant changes in rhinovirus-induced IFN-β,PRRs or thymic stromal lymphopoietin gene responses were observed with IL-4/IL-13 stimulation and IL-4Rα mAb treatment. A significant increase in mucin 5AC gene expression was observed with both IL-4 and IL-13 stimulation,but it was not reduced with IL-4Rα treatment in BECs. Inhibition of IL-4Rα reduced CCL26 levels without affecting antiviral immune responses in BECs from COPD patients. Inhibition of IL-4Rα reduced IL-4/IL-13 signalling without broadly suppressing the immune system,which might suggest that inhibition of the IL-4Rα pathways may prevent COPD exacerbations through reduction of eosinophil chemotaxis. View Publication

This study investigates the impact of minimal residual disease (MRD) on relapse in patients with acute myeloid leukemia (AML),focusing on its interaction with immune cells function. A total of 49 AML patients were enrolled in this prospective study and categorized into four groups: MRD − positive with relapse,MRD − positive without relapse,MRD − negative with relapse,and MRD − negative without relapse. Peripheral blood T lymphocyte subpopulations were analyzed using ten-color flow cytometry. CD4 + T cells were co-cultured with leukemia cell lines to assess the impact of CD4 + T cells on leukemia cell proliferation,apoptosis,and cytokine release. In MRD − positive patients,relapsed individuals exhibited significantly higher levels of CD4 + T cells,regulatory T (Treg) cells,and CD4 + CD45RA + naïve T cells compared to non-relapsed patients ( P < 0.0001,P = 0.0016,and P = 0.0066,respectively). Conversely,in MRD − negative patients,relapsed individuals showed a significantly lower percentage of Treg cells ( P = 0.0068). Furthermore,we observed that CD4 + T cells were associated with enhanced leukemia cell proliferation and reduced apoptosis,along with markedly increased IL-10 expression. The available data raise the possibility that CD4 + T cell-derived IL-10 participates in immune microenvironment regulation,a process that may have implications for MRD maintenance and disease recurrence in AML. View Publication

Regulatory T (Treg)-based cell therapy holds promise for autoimmune and inflammatory diseases,yet challenges remain regarding the functional stability and persistence of transferred Tregs. Here we engineer Tregs to express a partial agonist form of IL-2 (IL-2pa) to enhance persistence while avoiding toxicity from excessive signaling. Mouse Tregs expressing wild-type IL-2 (Tregs-IL2wt) have only a transient growth advantage,limited by toxicity from likely excessive signaling. By contrast,mouse Tregs-IL2pa exhibit sustained expansion,long-term survival in immunocompetent mice for over a year,and bystander expansion of endogenous Tregs. Tregs-IL2pa maintain a stable activated phenotype,Treg-specific demethylation,and a diverse TCR repertoire. In vivo,prophylactic transfer of Tregs-IL2pa ameliorates multi-organ autoimmunity in a Treg depletion-induced mouse autoimmune model. Lastly,compared with control Treg,human Tregs-IL2pa show enhanced survival in the IL-2-depleted environment of immune-deficient mice and improved control of xenogeneic graft-versus-host disease. Our results thus show that IL-2pa self-sufficiency enhances the stability,durability and efficacy of Treg therapies in preclinical settings. Subject terms: Cell delivery,Regulatory T cells,Autoimmune diseases,Interleukins View Publication

Enhanced glycolysis plays a pivotal role in fueling the aberrant proliferation,survival and therapy resistance of acute myeloid leukemia (AML) cells. Here,we aimed to elucidate the extent of glycolysis dependence in AML by focusing on the role of lactate dehydrogenase A (LDHA),a key glycolytic enzyme converting pyruvate to lactate coupled with the recycling of NAD + . We compared the glycolytic activity of primary AML patient samples to protein levels of metabolic enzymes involved in central carbon metabolism including glycolysis,glutaminolysis and the tricarboxylic acid cycle. To evaluate the therapeutic potential of targeting glycolysis in AML,we treated AML primary patient samples and cell lines with pharmacological inhibitors of LDHA and monitored cell viability. Glycolytic activity and mitochondrial oxygen consumption were analyzed in AML patient samples and cell lines post-LDHA inhibition. Perturbations in global metabolite levels and redox balance upon LDHA inhibition in AML cells were determined by mass spectrometry,and ROS levels were measured by flow cytometry. Among metabolic enzymes,we found that LDHA protein levels had the strongest positive correlation with glycolysis in AML patient cells. Blocking LDHA activity resulted in a strong growth inhibition and cell death induction in AML cell lines and primary patient samples,while healthy hematopoietic stem and progenitor cells remained unaffected. Investigation of the underlying mechanisms showed that LDHA inhibition reduces glycolytic activity,lowers levels of glycolytic intermediates,decreases the cellular NAD + pool,boosts OXPHOS activity and increases ROS levels. This increase in ROS levels was however not linked to the observed AML cell death. Instead,we found that LDHA is essential to maintain a correct NAD + /NADH ratio in AML cells. Continuous intracellular NAD + supplementation via overexpression of water-forming NADH oxidase from Lactobacillus brevis in AML cells effectively increased viable cell counts and prevented cell death upon LDHA inhibition. Collectively,our results demonstrate that AML cells critically depend on LDHA to maintain an adequate NAD + /NADH balance in support of their abnormal glycolytic activity and biosynthetic demands,which cannot be compensated for by other cellular NAD + recycling systems. These findings also highlight LDHA inhibition as a promising metabolic strategy to eradicate leukemic cells. The online version contains supplementary material available at 10.1186/s40170-025-00392-4. View Publication

Hematologic adverse events are common dose-limiting toxicities in drug development. Classical animal models for preclinical safety assessment of immunotherapies are often limited due to insufficient cross-reactivity with non-human homologous proteins,immune system differences,and ethical considerations. Therefore,we evaluate a human bone marrow (BM) microphysiological system (MPS) for its ability to predict expected hematopoietic liabilities of immunotherapeutics. The BM-MPS consists of a closed microfluidic circuit containing a ceramic scaffold covered with human mesenchymal stromal cells and populated with human BM-derived CD34+ cells in chemically defined growth factor-enriched media. The model supports on-chip differentiation of erythroid,myeloid and NK cells from CD34+ cells over 31 days. The hematopoietic lineage balance and output is responsive to pro-inflammatory factors and cytokines. Treatment with a transferrin receptor-targeting IgG1 antibody results in inhibition of on-chip erythropoiesis. The immunocompetence of the chip is established by the addition of peripheral blood T cells in a fully autologous setup. Treatment with T cell bispecific antibodies induces T cell activation and target cell killing consistent with expected on-target off-tumor toxicities. In conclusion,this study provides a proof-of-concept that this BM-MPS is applicable for in vitro hematopoietic safety profiling of immunotherapeutics. Subject terms: Biologics,Haematopoiesis,Lab-on-a-chip,Drug safety View Publication