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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

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

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

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

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

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

BackgroundAnti-CD19 chimeric antigen receptor T (CAR-T) cell therapy has proven effective for treating relapsed or refractory acute B cell leukemia. However,challenges such as cytokine release syndrome,T cell dysfunction,and exhaustion persist. Enhancing CAR-T cell efficacy through changing CAR internalization and recycling is a promising approach. The transmembrane domain is the easiest motif to optimize for modulating CAR internalization and recycling without introducing additional domains,and its impact on CAR internalization and recycling has not yet been thoroughly explored. In this study,we aim to enhance CAR-T cell function by focusing on the solely transmembrane domain design.MethodsUtilizing plasmid construction and lentivirus generation,we get two different transmembrane CAR-T cells [19CAR-T(1a) and 19CAR-T(8α)]. Through co-culture with tumor cells,we evaluate CAR dynamic change,activation levels,exhaustion markers,mitochondrial function,and differentiation in both CAR-T cells. Furthermore,immunofluorescence microscopy analysis is performed to reveal the localization of internalized CAR molecules. RNA sequencing is used to detect the transcriptome of activated CAR-T cells. Finally,a mouse study is utilized to verify the anti-tumor efficacy of 19CAR-T(1a) cells in vivo.ResultsOur findings demonstrate that 19CAR-T(1a) has lower surface CAR expression,faster internalization,and a higher recycling rate compared to 19CAR-T(8α). Internalized 19CAR(1a) co-localizes more with early and recycling endosomes,and less with lysosomes than 19CAR(8α). These features result in lower activation levels,less cytokine release,and reduced exhaustion markers in 19CAR-T(1a). Furthermore,CAR-T cells with CD1a transmembrane domain also exhibit a superior anti-tumor ability and reduced exhaustion in vivo.ConclusionOverall,we demonstrate that the transmembrane domain plays a critical role in CAR-T cell function. An optimized transmembrane domain can alleviate cytokine release syndrome and reduce CAR-T cell exhaustion,providing a direction for CAR design to enhance CAR-T cell function. View Publication

CD4+ T cells have been well-regarded as “helper” cells in activating the cytotoxicity of CD8+ T cells for effective tumor eradication,while few studies have focused on whether CD8+ T cells regulate CD4+ T cells. Our previous studies provided evidence for an interaction between CD4+ and CD8+ T cells after cryo-thermal therapy,but the mechanism remains unclear,especially pertaining to how CD8+ T cells promote the Th1 differentiation of CD4+ T cells. This study revealed that activated CD4+ and CD8+ T cells are critical for CTT-induced antitumor immunity,and the interaction between activated T cells is enhanced. The reciprocal regulation of activated CD8+ and CD4+ T cells was through LFA-1/ICAM-1 interactions,in which CD8+ T cells facilitate Notch1-dependent CD4+ Th1-dominant differentiation and promote IL-2 secretion of CD4+ T cells. Meanwhile,IL-2 derived from CD4+ T cells enhances the cytotoxicity of CD8+ T cells and establishes a positive feedback loop via increasing the expression of LFA-1 and ICAM-1 on T cells. Clinical analyses further validated that LFA-1/ICAM interactions between CD4+ and CD8+ T cells are correlated with clinical outcomes. Our study extends the functions of the LFA-1/ICAM-1 adhesion pathway,indicating its novel role in the interaction of CD4+ and CD8+ T cells. View Publication

Hermansky-Pudlak Syndrome (HPS) type 1 (HPS-1) is an autosomal recessive disorder characterized by oculocutaneous albinism,platelet dysfunction,and pulmonary fibrosis (HPS-PF),the leading cause of mortality in these patients. HPS-PF manifests earlier than idiopathic pulmonary fibrosis,typically between 30 and 40 years of age. The etiology and drivers of HPS-PF progression remain poorly understood,and no FDA-approved therapies exist. Neutrophil extracellular traps (NETs) and neutrophil-derived mediators have emerged as key players in fibrosis,promoting lung injury,inflammation,and fibroblast activation. This study evaluates the role of neutrophil activation in age-related changes in patients with HPS-1,focusing on differences in inflammatory markers,neutrophil granules,and NETosis capacity. We observed significantly elevated levels of NETs,neutrophil granule proteins (NE,NGAL,LF),and inflammatory cytokines (IL-8,IL-6) in patients with HPS-1 older than 40 years compared to younger patients and healthy controls. Additionally,fibrosis-related markers (MMP-7 and MMP-8) were significantly higher in older patients. Elevated levels of anandamide (AEA),a circulating marker of HPS-PF,were positively associated with neutrophil granule markers in older patients,suggesting its association with fibrosis. Neutrophils from older patients also demonstrated increased NETosis capacity. These findings suggest that age-related neutrophil activation may contribute to an inflammatory environment that promotes fibrosis progression in HPS-1.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13023-025-03758-5. View Publication

PurposeNuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial cytoprotective protein that shields cells from electrophilic and oxidative stress. Mice lacking Nrf2 exhibit heightened susceptibility to myelosuppression due to impaired hematopoietic reconstitution. In this study,we examined the altered sensitivity to ionizing radiation (IR) and 7,12-dimethylbenz(a)anthracene (DMBA) in Nrf2−/− mice separately.Materials and MethodsIrradiate Nrf2−/− or wild-type mice with a dose of 4 Gy to observe changes in body weight,survival rate,and blood routine at 12 months. DMBA was used to treat Nrf2−/− and wild-type mice,and the body weight and survival rate of the mice were measured. The changes of heme oxygenase-1(HO1) and NAD(P)H: quinone oxidoreductase 1(NQO1) in mice treated with IR or DMBA were detected by RT-qPCR and western blotting.ResultsOur results indicate that Nrf2 deficiency leads to more severe blood and immune system injury in mice exposed to IR or DMBA. Additionally,long-term monitoring revealed that Nrf2 deletion resulted in more severe myelosuppression,leukemia-like symptoms,and higher cancer rates. At the mRNA and protein levels,there was no significant increase in HO1 and NQO1 levels in the Nrf2−/− mice treated with IR or DMBA. These adverse effects might be attributed to the inhibited protein levels of HO1 and NQO1 and significant DNA damage in hematopoietic stem and progenitor cells (HSPCs).ConclusionsWe demonstrate that the genetic deficiency of Nrf2 in mice leads to reduced antioxidant capacity and suppression of hematopoietic and immune system function,resulting in increased sensitivity to IR or DMBA. Graphical Abstract View Publication

Chimeric Antigen Receptor (CAR) T cell therapy is a pivotal treatment for hematological malignancies. However,CAR T cell products exhibit batch-to-batch variability in cell number,quality,and in vivo efficacy due to donor-to-donor heterogeneity,and pre/post-manufacturing processes,and the manufacturing of such products necessitates careful testing,both post-manufacturing and pre-infusion. Here,we introduce the Cell Trajectory Modulation (CTM) assay,a microfluidic,label-free approach for the rapid evaluation of the functional attributes of CAR T cells based on biophysical features (i.e.,size,deformability). CTM assay correlates with phenotypic metrics,including CD4:CD8 ratio,memory subtypes,and cytotoxic activity. Validated across multiple donors and culture platforms,the CTM assay requires fewer than 10,000 cells and delivers results within 10 minutes. Compared to labeled flow cytometry processing,the CTM assay offers real-time data to guide adaptive manufacturing workflows. Thus,the CTM assay offers an improvement over existing phenotypic assessments,marking a step forward in advancing CAR T cell therapy manufacturing. CAR T cell manufacturing faces significant challenges that impact cell quality and in vivo efficacy. This necessitates reliable cellular characterization methods. Here the authors present a real-time,label-free,microfluidic method that profiles cellular biophysical properties and correlates them to activation state and CAR T potency,facilitating the rapid phenotypic cell assessment during production. View Publication

The lack of curative therapies for acute myeloid leukaemia (AML) remains an ongoing challenge despite recent advances in the understanding of the molecular basis of the disease. Here we identify the WNK1-OXSR1/STK39 pathway as a previously uncharacterised dependency in AML. We show that genetic depletion and pharmacological inhibition of WNK1 or its downstream phosphorylation targets OXSR1 and STK39 strongly reduce cell proliferation and induce apoptosis in leukaemia cells in vitro and in vivo. Furthermore,we show that the WNK1-OXSR1/STK39 pathway controls mTORC1 signalling via regulating amino acid uptake through a mechanism involving the phosphorylation of amino acid transporters,such as SLC38A2. Our findings underscore an important role of the WNK1-OXSR1/STK39 pathway in regulating amino acid uptake and driving AML progression. With-No-lysine (K) kinase 1 (WNK1) is an atypical serine-threonine kinase that has been implicated in ion transport. Here,the authors show that WNK1 regulates amino acid transport and mTORC1 activity,and that the axis is a vulnerability for acute myeloid leukemia View Publication