参考文献

Plasmacytoid dendritic cells (pDCs) are multifunctional immune cells with roles in both the innate and adaptive immune system. Their hallmark function is production of large amounts of type I interferons in response to viral infections,but they are also capable of producing a range of other cytokines,antigen presentation,and cytotoxicity. Their potential as an immunotherapy for cancer and infectious disease is being explored,but broad application of these cells is challenged by low frequency in the blood and low viability during ex vivo culturing. We have previously developed an effective in vitro differentiation protocol for producing pDCs from CD34+ hematopoietic stem and progenitor cells (HSPC-pDCs),which provides an attainable and large source of pDCs. HSPC-pDCs present pDC characteristics and functions,and like naturally occurring pDCs they exhibit large phenotypic and functional heterogeneity. Here,we characterize different cell subsets from in vitro pDC differentiation and identify a distinct population,which is the major producer of IFNα in response to TLR9 stimulation and display a transcriptomic profile similar to what is seen for pDCs circulating in the blood. We also investigate the possibility of rerouting subset specification during HSPCs-to-pDC differentiation by controlling gene expression of key master transcription factors (TFs). We identify TFs associated with the pDC differentiation trajectory that are essential for the development of TLR9-responsive HSPC-pDCs,and we also identify TFs that increase their frequency. In conclusion,we phenotypically and functionally characterize different cell subsets and modulate their relative frequencies by manipulating TF expression during pDC differentiation. These findings provide a deeper understanding of in vitro-differentiated pDC cultures that may spur further developments in their use as an immunomodulatory cell therapy. View Publication

Microsporidia are single-celled intracellular parasites that cause opportunistic diseases in humans. Encephalitozoon intestinalis is a prevalent human-infecting species that invades the small intestine. Macrophages are potential reservoirs of infection,and dissemination to other organ systems is also observed. The macrophage response to infection and the developmental trajectory of the parasite are not well studied. Here we use single cell RNA sequencing to investigate transcriptional changes in both the parasite and the host during E. intestinalis infection of human macrophages in vitro. The parasite undergoes large transcriptional changes throughout the life cycle,providing a blueprint for parasite development. While a small population of infected macrophages mount a response,most remain transcriptionally unchanged,suggesting that the majority of parasites may avoid host detection. The stealthy microsporidian lifestyle likely allows these parasites to harness macrophages for replication. Together,our data provide insights into the host response in primary human macrophages and the E. intestinalis developmental program. Microsporidia such as Encephalitozoon intestinalis are single-celled intracellular parasites that cause opportunistic infections and disease in humans involving infection of macrophages. Here the authors infect human macrophages with E. intestinalis,in vitro and use single cell transcriptomics to assess the consequences of cellular infection compared to bystander effects on macrophages and provide insights into the E. intestinalis developmental program. View Publication

The excessive accumulation of neutrophils within the epidermis is a significant hallmark of cutaneous diseases; however,the mechanisms governing neutrophil transepidermal migration (NTEM) remain inadequately understood. In this study,we develop trichromatic-fluorescence-labeled chimeric mice by utilizing Cx3cr1GFP/+Lyz2RFP/+ mice as bone marrow donors and Krt14YFP/+ mice as recipients. This approach enables us to visualize the process of NTEM and the crosstalk between neutrophils and monocytes in a murine model of irritant contact dermatitis (ICD). Intravital imaging reveals a preferential transmigration of neutrophils through hair follicle (HF),where dermal neutrophils exhibit limited mobility and interact with dermal monocytes. Notably,18 h following hapten exposure,dermal neutrophils continuously migrate toward HF regions and form clusters within 3 h. Importantly,MMP-9 is identified as essential for the NTEM process; the depletion of dermal monocytes results in a significant reduction of MMP-9 expression in the skin and inhibits the NTEM process in ICD. Mechanistically,dermal monocytes are found to be a crucial source of the cytokines TNF-α and CXCL2,which promote the upregulation of MMP-9 in neutrophils. Therefore,our results highlight HF regions as crucial gateways for dermal monocyte-modulated NTEM and provide visual insights into the crosstalk between neutrophils and monocytes in inflammatory skin disorders. Intravital imaging reveals that dermal monocytes orchestrate neutrophil transepidermal migration through hair follicles in skin inflammation via TNF-α and CXCL2,driving enhanced MMP-9 expression in neutrophils for epidermal infiltration. View Publication

BackgroundTriple negative breast cancer (TNBC) is an aggressive breast cancer subtype characterized with poor prognosis and high metastatic potential. Although traditional chemotherapy,radiation,and surgical resection remain the standard treatment options for TNBC,bispecific antibody-based immunotherapy is emerging as new strategy in TNBC treatment. Here,we found that the receptor tyrosine kinase-like Orphan Receptor 1 (ROR1) was highly expressed in TNBC but minimally expressed in normal tissue. A bispecific ROR1-targeted CD3 T cell engager (TCE) was designed in IgG-based format with extended half-life.MethodThe expression of ROR1 in TNBC was detected by RT-qPCR and immunohistology analysis. The killing of ROR1/CD3 antibody on TNBC cells was determined by the in vitro cytotoxicity assay and in vivo PBMC reconstituted mouse model. The activation of ROR1/CD3 on T cells was analyzed by the flow cytometry and ELISA assay. Pharmacokinetics study of ROR1/CD3 was performed in mouse.ResultsThe ROR1/CD3 TCE triggered T cell activation and proliferation,which showed potent and specific killing to TNBC cells in ROR1-depedent manner. In vivo mouse model indicated that ROR1/CD3 TCE redirected the cytotoxic activity of T cells to lyse TNBC cells and induced significant tumor regression. Additionally,the ROR1/CD3 bispecific antibody exhibited an extended half-life in mouse,which may enable intermittent administration in clinic.ConclusionsCollectively,these results demonstrated that ROR1/CD3 TCE has a promising efficacy profile in preclinical studies,which suggested it as a possible option for the treatment of ROR1-expressing TNBC.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13058-025-02005-w. View Publication

ABSTRACTBackgroundIdiopathic pulmonary fibrosis (IPF) is a lethal disease with an unknown etiology and complex pathophysiology that are not fully understood. The disease involves intricate cellular interplay,particularly among various immune cells. Currently,there is no treatment capable of reversing the fibrotic process or aiding lung regeneration. Hepatocyte growth factor (HGF) has demonstrated antifibrotic properties,whereas the adoptive transfer of modified T cells is a well‐established treatment for various malignancies. We aimed to understand the dynamics of T cells in the progression of lung fibrosis and to study the therapeutic benefit of adoptive T cell transfer in a bleomycin‐injured mouse lung (BLM) model.MethodsT cells were isolated from the spleen of naïve mice and transfected in vitro with mouse HGF plasmid and were administered intratracheally to the mice lungs 7 days post‐bleomycin injury to the lung. Lung tissue and bronchoalveolar lavage were collected and analyzed using flow cytometry,histology,qRT‐PCR,ELISA,and hydroxyproline assay.ResultsOur findings demonstrate the successful T cell therapy of bleomycin‐induced lung injury through the adoptive transfer of HGF‐transfected T cells in mice. This treatment resulted in decreased collagen deposition and a balancing of immune cell exhaustion and cytokine homeostasis compared with untreated controls. In vitro testing showed enhanced apoptosis in myofibroblasts induced by HGF‐overexpressing T cells.ConclusionsTaken together,our data highlight the great potential of adoptive T cell transfer as an emerging therapy to counteract lung fibrosis. This study explores the potential of T cells as a therapeutic strategy against idiopathic pulmonary fibrosis (IPF),a progressive lung disease for which there is currently no treatment to reverse fibrosis or restore normal lung function. To investigate an innovative approach using adoptive T cell transfer,T cells isolated from healthy mice were genetically modified to carry a plasmid containing hepatocyte growth factor (HGF). The modified cells were delivered directly into the airways of mice with bleomycin‐induced lung fibrosis. The results showed a significant reduction in fibrotic scarring,improved immune regulation,and increased apoptosis of pathogenic myofibroblasts. These results highlight the potential of HGF‐engineered T cells as a promising therapeutic approach to combat IPF. View Publication

ABSTRACTImmune checkpoint inhibitors (ICIs) have provided new hope for melanoma patients,however,not all patients benefit. Furthermore,ICI‐related therapies cause significant immune‐related adverse events that adversely affect patient outcomes. Therefore,there is a pressing need for reliable biomarkers to identify patients most likely to benefit from these treatments. In this study,we employed an extracellular vesicles (EVs) protein expression array to explore the longitudinal membrane protein profiles of plasma‐derived EVs from 32 melanoma patients receiving anti‐PD‐1 and anti‐angiogenesis therapy at baseline and early treatment. We found that the dynamic changes in PD‐L2 on the EV membrane were associated with treatment response and patient survival. The dynamic change of EV PD‐L2 as an indication of treatment efficacy was validated in an independent cohort of melanoma patients treated with anti‐PD‐1 monotherapy. Plasma‐derived PD‐L2+ EVs from patients with mucosal melanoma significantly reduced the frequency of granzyme B+ CD8 T cells within the peripheral blood mononuclear cells (PBMCs) of healthy individuals. The inhibitory effect of PD‐L2+ EVs on CD8 T cells was further validated using human melanoma cell lines and the B16‐F10 mouse model. Although intratumoural injection of PD‐L2+ EVs could promote melanoma growth in vivo,tumours with PD‐L2+ EVs showed a higher response to anti‐PD‐1 than those without PD‐L2+ EVs. Collectively,our study demonstrates that PD‐L2+ EVs inhibit CD8 T cell activation and promote melanoma growth,and changes in PD‐L2 on circulating EVs during early treatment could serve as a biomarker for ICI‐based therapy. View Publication

AbstractThe landscape of cancer treatment has been transformed by immune checkpoint inhibitors; however,the failure to benefit a large number of patients with cancer has underlined the need to identify promising targets for more effective interventions. In this study,we leverage 23andMe,Inc.’s large-scale human germline genetic and health database to uncover the previously unknown role of UL16-binding protein 6 (ULBP6),a high-affinity NK group 2D (NKG2D) ligand,in cancer and its promise as an immuno-oncology therapeutic target. We confirm ULBP6 expression in human tumors and demonstrate that soluble ULBP6 shed from tumors circumvents NKG2D activation provided by membrane-anchored NKG2D ligands to inhibit immune cell activation and tumor cell killing. Based on these findings,we developed 23ME-01473,a humanized Fc effector–enhanced antibody that binds to ULBP6 and its closely related family members,ULBP2 and ULBP5. 23ME-01473 effectively blocks soluble ULBP6-mediated immunosuppression to restore the NKG2D axis on NK and T cells to elicit tumor growth control. Moreover,the Fc effector–enhanced design of 23ME-01473 increases its binding affinity to fragment crystallizable gamma receptor IIIa,which,together with 23ME-01473’s binding to membrane-anchored ULBP6/2/5 on cancer cells,allows for augmented antibody-dependent cellular cytotoxicity induction,providing a second activation node for NK cells. Our studies demonstrate the therapeutic potential of an Fc effector–enhanced anti-ULBP6/2/5 antibody to reinvigorate NK cell and T-cell activation and cytotoxicity for the treatment of cancer.Significance:This study emphasizes the utility of population-based genome-wide assessments for discovering naturally occurring genetic variants associated with lifetime risks for cancer or immune diseases as novel drug targets. We identify ULBP6 as a potential keystone member of the NKG2D pathway,which is important for antitumor immunity. Targeting ULBP6 may hold therapeutic promise for patients with cancer. View Publication

Background and aimsMortality rates for hepatocellular carcinoma (HCC) remain high,while multimodal treatment approaches offer new perspectives. Here,we investigated the association of extracellular nicotinamide adenine dinucleotide (eNAD+) on ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (CD203a,ENPP1 or PC-1) on Th17 cells in relation to the likelihood of HCC recurrence following liver resection.MethodThe study compared heparinized blood plasma samples from 95 patients who underwent liver resection,including 25 patients with HCC and 24 control patients without liver disease. Plasma eNAD+ concentrations were determined using a heat-based dichotomous pH extraction method,followed by enzymatic cycling and a colorimetric assay for quantification. Fibrosis was graded histologically using the Desmet score (F0–F4). Surface expression analysis was performed using flow cytometry.ResultsWith increasing grades of liver fibrosis predominant in HCC patients,a significant reduction in plasma eNAD+ concentrations was measured (p < 0.05). Further,a significant correlation was found between HCC patients and CD203a expression on CD4+,CCR4+ as well as CCR6+ T cells (p < 0.05). Patients who exhibited high proportions of CD203a expressing Th17 cells (CD4+,CCR6+ CCR4+) post surgery were found to be at a sixfold increased risk (HR 6.38,95% Cl 1.51–27.00) of HCC recurrence and had a median recurrence-free survival of 233 days (p < 0.05),compared to patients with low CD203a expressing Th17 cells (CD4+ CCR6+ CCR4+). Similarly,patients who had a high proportion of CD203a expressing Th17 cells (CD4+ CCR6+) following surgery had a fivefold increased risk (HR 5.56,95% Cl 1.58–19.59) of HCC recurrence and a median recurrence-free survival of 334 days (p < 0.05) compared to those with low CD203a expressing Th17 cells (CCR6+).ConclusionThe data indicates that eNAD+ levels are decreased in patients with liver fibrosis or cirrhosis. Strikingly,patients with high CD203a expression on Th17 cells had a significantly increased likelihood of recurrence,highlighting its potential as a valuable prognostic marker and a possible therapeutic target.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00432-025-06155-4. View Publication

BackgroundMyeloid-derived suppressor cells (MDSCs) in tumor microenvironment reduce the efficacy of immunotherapy. PKN2 plays a role in colon cancer,but its function in esophageal cancer (EC) remains unclear. This study investigated PKN2 expression in MDSCs derived from EC tissues and determined whether PKN2 regulates immunosuppressive activity of MDSCs by mediating fatty acid oxidation (FAO).Materials and methodsPKN2 expression was determined in GEO database,EC patients,and 4-NQO-induced EC mice,as well as in different types of immune cells. The effect of PKN2 on the function of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) was investigated by co-culture of PMN-MDSCs and CD4+/CD8+ T cells. The co-culture of patient-derived organoids and autologous immune cells was performed to observe the effect of PKN2 on the immunosuppressive function of PMN-MDSCs.ResultsPKN2 is highly expressed in EC tumor tissues compared to normal tissues,especially in tumor-infiltrated PMN-MDSCs. Overexpressing PKN2 in PMN-MDSCs contributes to the immunosuppressive activity of PMN-MDSCs in vitro. PKN2-overexpressing PMN-MDSCs inhibited the killing ability of cytotoxic T lymphocytes and promoted EC organoid growth. PKN2 promotes FAO in PMN-MDSCs via CPT1B (a key enzyme of FAO). Mechanistically,PKN2 promotes CPT1B transcription by upregulating STAT3 phosphorylation.ConclusionsPKN2 expression was increased in PMN-MDSCs derived from human and mouse EC tissues. PKN2 plays a role in enhancing the immunosuppressive activity of PMN-MDSCs by facilitating STAT3 phosphorylation and CPT1B transcription,which in turn leads to increased CPT1B-mediated FAO in PMN-MDSCs. Targeted inhibition of PKN2 is expected to improve immunotherapeutic efficacy in EC patients.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10020-025-01132-6. View Publication

This protocol offers an ex vivo method for screening host-targeting antivirals (HTAs) using human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs). Unlike virus-targeting antivirals (VTAs),HTAs provide advantages in overcoming drug resistance and offering broad-spectrum protection,especially against rapidly mutating or newly emerging viruses. By focusing on PBMCs or pDCs,known for their high production of humoral factors such as Type I interferons (IFNs),the protocol enables the screening of antivirals that modulate immune responses against viruses. Targeting host pathways,especially innate immunity,allows for species-independent antiviral activity,reducing the likelihood of viral escape mutations. Additionally,the protocol's versatility makes it a powerful tool for testing potential antivirals against various viral pathogens,including emerging viruses,positioning it as an essential resource in both pandemic preparedness and broad-spectrum antiviral research. This approach differentiates itself from existing protocols by focusing on host immune modulation through pDCs,offering a novel avenue for HTA discovery. Key features • Optimized protocol for screening HTAs against dengue virus (DENV),chikungunya virus (CHIKV),and Zika virus (ZIKV). • This protocol is ideal for screening soluble or intravenous-formulated compounds for evaluating their efficacy in experimental settings. • This protocol builds upon the method developed by Tsuji et al. [1] and extends its application to PBMCs and testing against DENV,CHIKV,and ZIKV. View Publication