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Pancreatic ductal adenocarcinoma (PDAC) growth and metastasis are influenced by the tumor microenvironment (TME), which includes immune cells, endothelial cells, macrophages, and cancer-associated fibroblasts (CAFs). Since the novel integrin-targeted cytotoxin ProAgio can inhibit activated CAFs and endothelial cells, we investigated its combination with standard of care chemotherapies in genetically engineered mouse (GEM) and orthotopic murine models of PDAC. We established metastatic murine KPC-Ganji & Bassel-Luc (mKPC-GB-Luc) cell lines and validated them using bulk RNA sequencing. Two in vivo orthotopic mouse model were used to evaluate ProAgio with chemotherapy. mKPC-GB-Luc was used to evaluate the 5FU, oxaliplatin, and irinotecan (FOI combination), and KPC-ML1-Luc was used to evaluate the gemcitabine, nab-paclitaxel (GPTx combination). Immunohistochemistry was used to measure integrin β3, E-cadherin, and HIF-1α. Hypoxia was evaluated using pimonidazole. Stem cells, CAFs, and immune cell subtypes were quantified by flow cytometry. We evaluated the combination of ProAgio plus gemcitabine in a KPC genetically engineered mouse model (KPC GEM). Two sets of KPC GEM were developed. The first set was used for a survival study. The second set was terminated early and tumors were used for single-cell RNA seq and sequential multiplex immunofluorescence (COMET). Compared with the chemo regimen (GPTx or FOI), ProAgio, or sham, the combination of ProAgio plus chemo significantly modulated the TME, reduced tumor weight, reduced hypoxia, and eliminated metastasis to the lungs and liver. The combination of ProAgio and gemcitabine increased overall survival in KPC GEM mice compared with either treatment alone. Single-cell RNA sequencing, flow cytometry, immunofluorescence, immunohistochemistry, and COMET analyses demonstrated that ProAgio plus chemotherapy reprogrammed the PDAC-TME by changing activated CAFs toward a qCAFs (quiescent CAFs), macrophages from protumor to proinflammatory polarization, and activating natural killer (NK) cells, CD4⁺, and CD8⁺ T cells. Combination therapy inhibited PDAC stemness. ProAgio-treated CAFs in vitro exhibited reduced secretion of glycine and cysteine, vital metabolites that support stemness and tumor progression. These results confirm the novel mechanism of action of ProAgio, which includes reducing hypoxia and modulating TME. The current data provide evidence for potentiation of chemotherapy efficacy by ProAgio.

Synovial sarcoma (SS) is a rare and highly malignant soft tissue sarcoma. The efficacy of existing therapies is limited, and new strategies are urgently needed. Osteopontin (OPN) promotes disease progression by regulating tumor-immune cell interaction and immunosuppressive microenvironment, which is a potential therapeutic target. The aim of this study was to investigate the role of OPN in SS immune regulation and tumor progression. A stable cell line with OPN knockdown was constructed using lentiviral transduction. RNA-seq and bioinformatics analysis were used to predict the correlation between OPN and immunity. Flow cytometry and ELISA were used to detect the changes in the number and function of CD8 + T cells. EDU, CCK-8 and Transwell chamber experiments were used to detect the proliferation, invasion and migration ability of SS. The protein interaction between OPN and PD-L1 was detected by molecular docking and Co-IP. The expression of PD-L1 was detected by Western blot and flow cytometry. The expression levels of OPN, PD-L1 and CD8 were detected by immunohistochemistry. To predict the correlation of OPN, PD-L1 and CD8 in sarcoma tissues and their clinical prognostic significance. RNA-seq analysis revealed that OPN-associated differentially expressed genes were significantly enriched in immune response-related biological processes. Bioinformatics analysis demonstrated that OPN expression in sarcoma was associated with immune responses and T-cell activation, and was negatively correlated with immune cell infiltration. OPN induced CD8⁺ T-cell exhaustion, thereby promoting immune evasion and facilitating the proliferation, invasion, and migration of synovial sarcoma (SS) cells. Molecular docking and co-immunoprecipitation assays confirmed a direct protein-protein interaction between OPN and PD-L1, and knockdown of OPN resulted in a significant reduction in PD-L1 expression. In SS tissues, OPN was highly expressed, whereas PD-L1 and CD8 were expressed at low levels and exhibited a positive correlation. Furthermore, bioinformatics analysis showed that PD-L1 expression was positively correlated with CD8 expression in sarcoma, and low CD8 expression was associated with poor prognosis. We preliminarily propose that OPN promotes immune evasion in synovial sarcoma (SS) by regulating PD-L1 expression and subsequently inducing CD8⁺ T-cell exhaustion. This mechanism suggests that OPN may serve as a novel immunotherapeutic target and provides new insights into the development of immunotherapy strategies for SS.

The primary cause of alopecia areata (AA) is autoimmune-mediated hair follicle destruction, while breakdown of immune tolerance due to Treg cell homeostasis disruption critically contributes to this process. However, in AA, the factors leading to Treg cell impairment and the effective regulatory pathways remain unanswered. To assess the therapeutic effects of sCD83 on AA and elucidate the crucial role of sCD83-mediated Treg cell activation in remodeling the perifollicular microenvironment. Blood and scalp tissue were collected from AA patients and healthy controls to characterize sCD83 using ELISA, flow cytometry, and immunofluorescence. In graft-induced C3H/HeJ mouse model of AA, the therapeutic effect of sCD83 on early-onset AA was evaluated by H&E staining, immunofluorescence, and flow cytometry. In vitro, human hair follicle organ culture and primary outer root sheath keratinocyte (ORSK) culture were utilized to clarify the impact of sCD83 on the expression and activity of indoleamine 2,3-dioxygenase (IDO) in hair follicle. Co-culture of ORSKs and PBMCs, together with administration of an IDO inhibitor to AA mice, were performed to determine the necessity of IDO for Treg cell activation. Finally, GST-pulldown and co-IP assays were employed to identify potential sCD83 receptors on ORSKs. We showed here that AA patients exhibit sCD83 deficiency, which may be attributed to reduced sCD83 release from DCs, and that serum sCD83 levels are negatively correlated with disease severity. Supplementation with sCD83 in AA mouse reversed disease manifestations and promoted Treg cell proliferation. Mechanistically, IDO activity in ORSKs is essential for sCD83-mediated Treg cell activation, with TRX potentially serving as a sCD83 receptor to regulate IDO expression. Our study establish the functional role of sCD83 in AA, confirms the therapeutic potential of sCD83 supplementation, and provides some mechanistic insights.

While radiotherapy (RT) is effective for local tumor control, it rarely induces the regression of non-irradiated metastases, a phenomenon known as the abscopal effect. The mechanisms constraining this systemic immune response remain poorly understood. This study investigates galectin-9 (Gal-9), a ligand for the TIM-3 (T-cell immunoglobulin and mucin-domain containing-3) immune checkpoint, as a mediator of immune escape that limits RT systemic efficacy and evaluates combinatorial RT/Gal-9 blockade in preclinical models. METHODS: Multiplatform analysis (RNA sequencing, immunoblot, flow cytometry, ELISA, immunohistochemistry) characterized RT-induced Gal-9 regulation in human lung/colorectal cancer cell lines, murine tumors/serums, and paired patient tumors. Local and abscopal therapeutic efficacy was evaluated in homologous (CT26/CT26, LLC/LLC) and heterologous (CT26/4T1) two-tumor mouse models. Immune profiling of tumor microenvironment, tumor-draining lymph nodes (tdLNs), and splenic compartments was comprehensively assessed by flow cytometry. Mechanistic studies employed STING (Stimulator of Interferon Genes) inhibition (H151), CD8+ T-cell depletion (anti-CD8α), macrophage/monocyte depletion (PLX-3397), interferon-I (IFN-I) blockade (anti-IFNAR1), and lymphocyte egress inhibition (FTY720). RT upregulated Gal-9 predominantly within host myeloid compartments (dendritic cells, macrophages, monocytes, neutrophils) versus tumor cells, in both irradiated tumors and abscopal tumors. Mechanistically, RT-activated STING-IFN-I axis locally induced Gal-9+ myeloid cells that subsequently disseminated systemically. Clinically, elevated post-RT Gal-9 in patient biopsies correlated with poor therapeutic outcomes. Notably, combining Gal-9 blockade with RT elicited potent abscopal responses in homologous two-tumor mouse models. Furthermore, anti-Gal-9 markedly enhanced radio-immunotherapy efficacy in the poorly immunogenic Lewis lung carcinoma. Immunologically, Gal-9 blockade synergized with RT to activate the myeloid and T cell compartments, enhancing dendritic cell accumulation in tdLNs and boosting CD8+ T cell infiltration in abscopal tumors. Depletion of CD8+ T cells/monocytes or blocking lymphocyte egress from lymph nodes abrogated the abscopal efficacy, underscoring their essential roles. Our findings establish RT-induced Gal-9 as a novel dual myeloid/T-cell immune checkpoint restricting abscopal responses. Gal-9 blockade represents a promising strategy to potentiate radiotherapy against metastatic disease, defining a therapeutic paradigm distinct from conventional checkpoint inhibitors.

Cryoablation (Cryo) is used for local tumor control in lung cancer patients who are not candidates for surgical resection, but its effects on distant tumor growth remain incompletely understood. The present study evaluated the effects of Cryo on distant Lewis lung tumors, explored whether Cryo-induced PD-1/PD-L1 axis upregulation may represent a treatment-limiting pathway, and assessed whether PD-1/PD-L1 inhibition in Cryo-treated mice was associated with remodeling of the immunosuppressive tumor microenvironment and reduced tumor proliferation. In the experiments, mice bearing bilateral Lewis tumors were assigned to Untreated, Cryo, In-PD-1, and Cryo + In-PD-1 groups. Immunofluorescence was used to assess PD-L1 and Ki-67 expression. Quantitative PCR, enzyme-linked immunosorbent assay, and flow cytometry were used to evaluate immunosuppressive cytokines and immune-cell populations, while western blotting was performed to analyze signaling-related proteins. Results showed that Cryo mildly inhibited distant tumor growth, increased tumor-infiltrating T-cell levels, and upregulated the PD-1/PD-L1 axis. In-PD-1 was associated with prolonged survival and further inhibition of distant tumor growth in Cryo-treated tumor-bearing mice. The Cryo + In-PD-1 group showed reduced regulatory T cells, M2-type tumor-associated macrophages, myeloid-derived suppressor cells, and the immunosuppressive cytokines interleukin-6 and VEGF, together with decreased Ki-67 expression and p-ERK/ERK ratio in tumor tissues. These findings suggest that Cryo-induced upregulation of the PD-1/PD-L1 axis may be associated with limited efficacy of Cryo monotherapy, and that PD-1/PD-L1 inhibition in Cryo-treated mice may contribute to broader remodeling of the immunosuppressive tumor microenvironment and improved tumor control, accompanied by a less proliferative tumor phenotype and decreased ERK pathway activation.

Resistance training has been shown to activate the protein synthesis pathway, leading to muscle growth in humans. However, this type of exercise has shown equivocal results in animal studies due to the difficulty of mimicking muscle overload in vivo. This study aimed to determine whether ladder-based exercise in mice induces canonical molecular, cellular, and functional adaptations to training. Mice performed a single exercise session or 6 weeks of training in the ladder climb. Acute responses included mTOR phosphorylation, puromycin incorporation, and mRNA levels of myogenic regulatory factors (MRF). Chronic adaptations were assessed by strength, fat-free mass, physical performance, and blood lactate levels to confirm the training load. Sarcomeric proteins were analyzed using Western blot, while histology measured muscle fiber diameter and satellite cell (SC) fusion. The SC amount was quantified by flow cytometry. After a single exercise bout, mTOR phosphorylation increased at one and 3 h, with puromycin incorporation and MRF mRNA levels elevated at 8 h. After 6 weeks of training, the mice showed increased skeletal muscle strength and fat-free mass, with no changes in physical performance. Muscle-specific adaptations included increases in sarcomeric proteins and fiber diameters. SC adaptations were associated with an increased pool and enhanced capacity to fuse with muscle fibers. Our results demonstrate that ladder-based resistance exercise in mice induces molecular, cellular, and functional responses that are directionally consistent with adaptations reported after human resistance training, supporting its value for investigating the molecular and cellular mechanisms underlying this training.

Combretastatin A4 (CA4) is a potent microtubule inhibitor, but its phenolic hydroxyl group is prone to oxidation into toxic quinone products, leading to poor metabolic stability and dose-limiting toxicity that restricts its clinical application. To overcome this limitation, we propose a novel dimer prodrug strategy that covalently links two CA4 molecules via an enzyme-cleavable linker, achieving temporary masking of the phenolic hydroxyl group. The resulting CA4-dimer1 and CA4-dimer2 exhibited potent in vitro antiproliferative activity. Of the two dimers evaluated, CA4-dimer1 demonstrated superior in vitro potency. Crucially, CA4-dimer1 achieved significant tumor suppression rates in patient-derived xenograft (PDX) liver cancer models without notable toxicity. CA4-dimer2 releases active CA4 through gradual hydrolysis but showed slightly lower in vivo release efficiency compared to CA4-dimer1. Flow cytometry and Western blot analysis revealed that this prodrug exerts antitumor activity through the same mechanism as the parent compound CA4. Collectively, the reported CA4-dimer prodrug strategy offers a highly promising approach for treating liver cancer and enhancing the therapeutic index of phenolic drugs, while also providing a potential strategy for structural optimization of drugs containing phenolic hydroxyl groups.

Cell surface glycans regulate key biological processes including immune signaling, cell communication, and pathogen recognition. Glycan-driven signaling is primarily mediated by glycan-binding proteins (lectins), whose functions depend on the identity and presentation of their glycoprotein ligands. However, identifying ligands for glycan-binding proteins remains challenging due to the structural complexity of carbohydrates and the importance of cellular context in determining binding specificity. Here, we describe a fluorescence-activated cell sorting (FACS)-based pooled CRISPR screening workflow for systematic identification of genetic factors that regulate lectin binding in living cells. The protocol covers lentiviral transduction of pooled sgRNA libraries and phenotypic selection of high- and low-lectin-binding populations by flow cytometry. Genomic DNA extraction, sequencing, and computational sgRNA enrichment analysis enable identification of genes influencing ligand biosynthesis and presentation. Subsequent analysis of these genetic factors can provide a comprehensive view of the structural determinants that govern lectin-glycan binding. The approach is compatible with CRISPR knockout, interference, and activation strategies, allowing broad interrogation of both loss- and gain-of-function effects. Key considerations for maintaining library coverage, optimizing sorting parameters, and performing robust statistical analysis are highlighted to maximize screening performance. Overall, this workflow offers a scalable framework for mapping glycan ligand landscapes in health and disease.

Cell replacement therapies aimed at restoring foveal vision require a robust source of red/green (long/medium wavelength, or L/M) cone photoreceptors with intrinsic properties conducive to functional integration into host retina. Recent evidence has shown that cones present within mature human retinal organoids (ROs) can generate light responses comparable to macaque foveal cones. However, only cone precursors from early developing ROs possess a capacity for cell-autonomous axonogenesis. Therefore, we sought to identify and enrich for a population of early L/M cone-competent precursors with intrinsically superior axon dynamics that would provide an ideal donor cell source for future foveal reconstruction efforts. We developed a dual L/M cone/rod reporter (L/M-CRR) line to unequivocally identify early L/M cone-competent precursors from human ROs. To do so, we used CRISPR/Cas9 to link a tdTomato transgene to the endogenous THRB2 promoter in the WA09 NRL+/eGFP rod reporter line. Differentiated ROs were characterized at early (day 50) and intermediate (day 100) developmental stages using a combination of live fluorescence imaging, immunocytochemistry, and flow cytometry, followed by fluorescence-activated cell sorting and bulk RNAseq analysis to delineate the unique molecular signature of early L/M cone-competent precursors. THRB2-driven tdTomato fluorescence faithfully demarcated L/M cone-competent precursors throughout RO development, although fluorescence declined in later ROs as L/M cones matured. Transcriptomic profiling revealed that day 50 sorted tdTomato+ cells were specifically enriched for genes associated with neural development, axon extension and guidance, and cell migration, which included a gene encoding the cell surface protein CD166/ALCAM. Magnetic-activated cell sorting using an anti-CD166 antibody resulted in specific enrichment of early, highly axonogenic L/M cone-competent precursors assessed by time-lapse imaging. The L/M-CRR line enables definitive identification and transcriptomic characterization of L/M cone-competent precursors throughout early to mid-stage RO development. Our investigation also revealed that CD166/ALCAM can be used to independently identify and enrich for a subset of early L/M cone-competent precursors that selectively display axon dynamicity conducive for retinal circuit integration. Our studies provide the first insights into early human L/M cone development and establish a method to isolate L/M cone-competent precursors with enhanced axon dynamics, which constitutes a compelling cell population for treating central vision loss caused by photoreceptor degeneration.

As a common and devastating complication of sepsis, sepsis-induced acute kidney injury (SAKI) confers a significant risk of mortality. This work focused on the utility of miR-126-5p as a biomarker for SAKI. A total of 95 patients with SAKI and 70 patients with sepsis alone were enrolled. Serum levels of miR-126-5p and Caspase-3 (CASP3) were measured using RT-qPCR. The regulatory interaction between miR-126-5p and CASP3 was validated using a dual-luciferase reporter assay. Cell proliferation, apoptosis, inflammatory cytokine levels, and oxidative stress markers were evaluated using the CCK-8 assay, flow cytometry, ELISA, and corresponding commercial kits, respectively. Serum miR-126-5p levels were downregulated in SAKI patients and showed significant negative correlations with kidney injury markers, including serum creatinine (Scr), cystatin C (Cys-C), neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1). Overexpression of miR-126-5p in lipopolysaccharide (LPS)-stimulated HK-2 cells was shown in vitro to facilitate cell proliferation, restrain apoptosis, and suppress the production of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Concurrently, it reduced oxidative stress markers, reactive oxygen species (ROS), and malondialdehyde (MDA), and enhanced superoxide dismutase (SOD) activity. Mechanistically, CASP3 was identified as a direct downstream target of miR-126-5p, and its expression was negatively regulated by miR-126-5p. Furthermore, overexpression of CASP3 significantly reversed the protective effects of miR-126-5p in vitro. MiR-126-5p showed diagnostic value for SAKI and exerted a protective effect in vitro through targeted regulation of CASP3 expression.

The antitumor effects of Lasiokaurin (LAS), a natural compound derived from traditional Chinese medicine extracts, remain poorly understood in hepatocellular carcinoma (HCC). Therefore, this study investigates the anticancer effects of LAS in liver cancer. The experiment employed Wound healing assay, colony formation assays, Transwell assays, flow cytometry, Western blot analysis, and reverse transcription quantitative polymerase chain reaction (RT-qPCR) to evaluate the relationship between LAS and apoptosis, migration, proliferation, and cell cycle arrest. RNA-sequencing, RT-qPCR, and Western blot techniques were used to detect markers associated with the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway. The anti-tumor efficacy was assessed using a subcutaneous tumor-bearing mouse model. LAS promotes apoptosis and cell cycle arrest, significantly inhibiting tumor growth. These findings indicate that LAS mediates apoptosis through the JAK2/STAT3 pathway, suggesting its potential as a novel anticancer agent.

Recurrent spontaneous abortion (RSA) is commonly attributed to immune dysregulation at the maternal-fetal interface. The contribution of the spleen, as a peripheral immune organ, remains unclear. This study investigated spleen-associated immune alterations in a murine model of RSA. An RSA model was established by mating CBA/J females with DBA/2 males. Splenic and decidual immune-cell distributions were assessed by histology, flow cytometry, and cytokine analysis. Splenectomy and macrophage depletion were performed as experimental interventions. Decidual transcriptomic changes following splenectomy were analyzed by bulk RNA sequencing and transcriptomic enrichment analyses. RSA mice exhibited splenomegaly, altered splenic histological features, and changes in splenic immune-cell composition, including increased numbers of Ly6C+ CX3CR1lo macrophages. Decidual immune alterations mirrored several changes observed in the spleen. Splenectomy was associated with increased embryo viability, reduced histological alterations, and changes in decidual immune-cell distributions. Similar effects on embryo viability and decidual immune-cell distributions were observed following effective macrophage depletion. Transcriptomic analyses of decidual tissue following splenectomy identified transcriptional differences involving mitochondrial-associated, Wnt-related, and protein synthesis-associated pathways. These findings identify an association between spleen-associated immune alterations and RSA and suggest that splenectomy is accompanied by changes in decidual immune-cell distributions, tissue-level transcriptional patterns, and embryo viability.

SMARCA4-deficient non-small cell lung cancer (SD-NSCLC) is associated with poor prognosis and lacks a standard treatment strategy. This multicenter retrospective study evaluated immunotherapy-based regimens and characterized the immune features of advanced SD-NSCLC using next-generation sequencing, flow cytometry, multiplex immunofluorescence, single-cell RNA and TCR sequencing, and a CRISPR/Cas9-mediated SMARCA4 knockout model. A total of 162 patients with advanced SD-NSCLC were enrolled from four Chinese cancer centers. Genomic analysis identified TP53, KEAP1, STK11, and KRAS as the primary co-mutated genes. Compared to single-agent chemotherapy, immunotherapy combined with chemotherapy and antiangiogenesis therapy demonstrated significantly longer median progression-free survival (mPFS) (12.10 vs. 2.77 months, p < 0.001). Furthermore, responders to this combination also exhibited elevated CD8+ T cell infiltration (p < 0.01). In vivo, the three-drug combination regimen significantly inhibited the growth of SMARCA4-knock-out mouse tumor cells in mice, increased infiltration of CD8+ T cells, and reduced the presence of regulatory T cells (Tregs) in the tumor microenvironment. In this mouse model, single-cell RNA-seq revealed a significant decrease in CTLA4+ Tregs with active intercellular communication following anti-PD-1 therapy. Ex vivo experiments demonstrated that treatment with anti-PD-1 markedly reduced the proportion of Tregs and concurrently increased the proportions of IFN-&#x3b3; and TNF-&#x3b1; producing cells. Lastly, single-cell TCR-seq confirmed the expansion of specific TCR clones, particularly within CCR7+ and LEF1+ naive T cells. Collectively, these findings suggest that immunotherapy-based combination regimens may provide substantial clinical benefit for patients with SD-NSCLC by enhancing CD8+ T cell activity, suppressing CTLA4+ Tregs, and expanding tumor-reactive TCR clones.

Sepsis-associated muscle wasting (SAMW) causes long-term functional decline, even after recovery. Emerging evidence indicates that adenosine 5'-monophosphate (AMP) confers organ-protective effects in response to physiological stress or injury, potentially through the activation of AMP-activated protein kinase (AMPK) signalling pathways. In this study, we investigated the effects of AMP on SAMW to evaluate its efficacy as a therapeutic agent to alleviate SAMW. In vivo, a mouse model of cecum ligation and puncture sepsis was established using male C57BL/6 mice, which received intraperitoneal AMP (0.5&#xa0;mg/g) or saline as a control. In vitro, C2C12 myoblasts and RAW264.7 macrophages were cultured under standard conditions and treated with AMP. Forelimb grip strength, blood and muscle sampling, western blotting, AMP assays, RNA sequencing, ELISA, flow cytometry, real-time PCR, immunohistochemistry, histology, and computed tomography imaging were performed to assess molecular, cellular, and physiological responses. In addition, plasma samples from patients with sepsis were analysed to explore translational relevance. AMP suppressed sepsis-induced inflammatory cytokine production and improved muscle strength by attenuating mammalian target of rapamycin complex 1 activation and modulating AMPK signaling, thereby contributing to the preservation of muscle mass and a reduction in systemic inflammation. In vitro, AMP suppressed LPS- induced IL-1&#x3b2; production in RAW264.7 macrophages and attenuated LPS- or IL-1&#x3b2;-induced myotube atrophy in C2C12 cells and shifted the cells towards a fast-twitch phenotype. Evaluation of clinical samples revealed elevated inflammatory cytokines in patients with sepsis exhibiting muscle wasting. This study demonstrates that AMP effectively mitigates SAMW by activating AMPK and suppressing IL-1&#x3b2;-mediated molecular pathways. These findings highlight the potential of AMP as a novel therapeutic agent for preserving skeletal muscle functionality and morphology in sepsis.

The clinical significance of PNH Type II white blood cells (WBCs) remains unclear. We assessed the relative percentage (rel%) of Type II neutrophils in 355 patients with a PNH clone &#x2265;&#x2009;1% on neutrophils enrolled by 33 flow cytometry laboratories in the 5-year French nation-wide multicenter prospective observational study. We first analyzed 127 of 133 patients with a major PNH clone (&#x2265;&#x2009;50%) and evaluable Type II neutrophil rel%. Using hemolysis data available for 107 patients, a threshold of 3% Type II neutrophils rel% distinguished two groups: 22 "high Type II" and 105 "low Type II" patients. Hemolysis was less frequent in the "high Type II" group. Thrombosis was more frequent at diagnosis and during cumulative long-term follow-up in this group. High neutrophil rel%&#x2009;&#x2265;&#x2009;3% was independently associated with thrombotic events. Among 222 patients with smaller PNH clones (1%-50%), 207 displayed evaluable Type II neutrophil rel%, of which 87 were classified as "high Type II" and 120 as "low Type II". Although no thrombosis was present at diagnosis, two thrombotic events occurred during follow-up in the "high Type II" group, whereas none were observed in the "low Type II" group. Long-term follow-up showed no major changes in Type II neutrophil rel%, irrespective of variations in total clone size. These findings suggest that, regardless of the total PNH clone size, PNH Type II neutrophil rel% is a stable parameter and may represent a reliable predictor of thrombosis, potentially supporting earlier consideration of anticomplement therapy initiation.

Streptococcus agalactiae infection remains a major constraint on the productivity of Nile tilapia (Oreochromis niloticus), a species of considerable economic importance in global aquaculture. Although selective breeding programs have successfully produced tilapia strains with enhanced resistance to this pathogen, the B cell-mediated immune mechanisms underlying this resistance remain unclear. In this study, we systematically investigated differences in IgM+ B cell mediated immune responses between S. agalactiae-resistant and susceptible Nile tilapia. Transcriptomic analysis of splenic leukocytes revealed that genes associated with immunoglobulin production and B cell differentiation were markedly upregulated in resistant tilapia following infection. Consistent with these findings, flow cytometry further demonstrated a more pronounced expansion of splenic IgM+ B cells in resistant tilapia, which was primarily driven by significantly increased proliferative activity. Importantly, this expansion coincided with elevated activation induced apoptosis, suggesting an accelerated B cell turnover that could facilitate the selection and maintenance of functionally competent cells during the immune response. Functionally, IgM+ B cells from resistant tilapia exhibited sustained enhancement of antigen processing capacity following infection. Meanwhile, macrophage-mediated phagocytosis of S. agalactiae increased progressively and became particularly evident at later stages of infection. Moreover, IgM+ B cells in resistant tilapia maintained relatively stable intracellular ROS levels, whereas susceptible tilapia exhibited persistent oxidative stress. Collectively, these findings indicate that resistance to S. agalactiae infection in Nile tilapia is closely associated with coordinated functional remodeling of IgM+ B cells, characterized by efficient proliferation, functional maturation, and stringent homeostatic regulation. This refined B cell-mediated immunity likely contributes to the effective pathogen clearance and provides an immunological basis for the evaluation and selective breeding of disease-resistant tilapia strains.

The prognosis of patients with metastatic/relapsed/refractory Ewing sarcoma (ES) is dismal. Natural killer (NK) cells are highly cytotoxic to ES but limited by resistance within the ES tumor microenvironment (TME). Here we sought to overcome ES resistance to NK cells by a combinatorial immunotherapy approach simultaneously enabling NK tumor-specific-targeting via chimeric antigen receptor (CAR) against a novel ES target interleukin-1 receptor accessory protein (IL1RAP), circumventing transforming growth factor beta (TGF&#x3b2;)-mediated NK immunosuppression by TGF&#x3b2;1-imprinting, increasing NK cell antibody-dependent cellular cytotoxicity (ADCC) via an anti-GD2 antibody dinutuximab, and improving NK cell persistence and ADCC by an IL-15 agonist, NKTR-255. Peripheral blood mononuclear cells were expanded into NK and TGF&#x3b2;1-imprinted-NK (imNK) cells using antigen-presenting feeder cells co-expressing IL-21 and 4-1&#x2009;BBL. Anti-IL1RAP-CAR messenger RNA was electroporated into NK or imNK cells. In vitro cytotoxicity assays were performed to investigate the efficacy of anti-IL1RAP-CAR-NK/imNK cell alone or combined with NKTR-255 and/or dinutuximab against ES cells. Xenograft mouse models of ES were used to investigate the antitumor efficacy of the combinatorial CAR-NK/imNK cell therapy against ES in vivo. Single-cell RNA sequencing and mass cytometry analyses of cells from xenograft tumors were performed to identify mechanisms of response/resistance to this combinatorial immunotherapy. We found that anti-IL1RAP-CAR-NK cells significantly and specifically enhanced NK cytotoxicity in vitro and decreased tumor growth and lung metastasis in vivo against IL1RAP+ES. TGF&#x3b2;1-imprinting significantly enhanced in vitro cytotoxicity and tumor infiltration of CAR-NK cells, leading to significantly reduced tumor growth and improved animal survival in the ES orthotopic mouse model. Compared with single agent or double combinations, the triple combination of imprinted-CAR-NK (CAR-imNK) cells and NKTR-255 with dinutuximab had superior antitumor efficacy against IL1RAP+GD2+ ES. Mechanistic studies on single cells from the xenograft tumors revealed increased apoptosis of ES cells, upregulated expression of ligands to NK inhibitory receptors on ES cells, and enhanced mouse macrophage migration in the ES TME in response to the CAR-imNK+NKTR-255+dinutuximab therapy. Our preclinical data demonstrate that combinatorial innate immunotherapy leveraging tumor-targeting TGF&#x3b2;1-imprinted IL1RAP-CAR-NK cells combined with an IL-15 agonist and an anti-GD2 antibody is a promising novel therapeutic strategy for targeting metastatic/relapsed/refractory ES.

Intervertebral disc degeneration (IDD) serves as a predominant pathogenic driver of spinal degenerative diseases and low back pain. Long non-coding RNAs (lncRNAs) have emerged as important regulators involved in the pathogenesis of IDD. Despite this, the specific contributions of LINC00324, a particular lncRNA, to the advancement of IDD are not yet well understood. The study seeks to elucidate the functional role and explore the potential molecular mechanisms of LINC00324 in IDD. RT-qPCR was employed to analyze LINC00324, miR-143-3p, and TRAIL levels in intervertebral disc (IVD) tissues from IDD patients and control subjects, followed by correlation analysis. Bioinformatics tools, dual-luciferase reporter assays, and RNA pull-down experiments were conducted to validate the regulatory interactions within LINC00324/miR-143-3p/TRAIL in nucleus pulposus cells (NPCs). In vitro, NPCs were stimulated with lipopolysaccharide (LPS) to construct an IDD cell model. RT-qPCR, western blot, CCK-8 assay, and flow cytometry were utilized to evaluate the effects on the levels of LINC00324/miR-143-3p/TRAIL/DR4/DR5, as well as the proliferation and apoptosis of NPCs. Additionally, extracellular matrix (ECM)-related proteins and inflammatory factors were detected by western blot and ELISA, respectively. In vivo, a rat IDD model was established through fibroannular puncture. Radiological analysis (X-ray), HE staining, TUNEL staining, and western blot were used to assess disc structural changes, NPC apoptosis, and the levels of miR-143-3p/TRAIL/DR4/DR5 and apoptosis-related proteins. Clinical evidence showed that LINC00324 and TRAIL levels were increased, and miR-143-3p was decreased in IDD samples. miR-143-3p had the negative correlation coefficient with TRAIL and LINC00324. Furthermore, LINC00324 acted as a sponge for miR-143-3p, and miR-143-3p specifically attenuated the downstream of TRAIL level. In vitro, the LPS-induced IDD model showed decreased NPC viability, increased apoptosis, disrupted ECM metabolism (downregulated Collagen II and Aggrecan, upregulated MMP13), elevated inflammatory factors, and activated TRAIL/DR4/DR5 axis. Silencing LINC00324 or overexpressing miR-143-3p alleviated these pathological changes. Furthermore, miR-143-3p partially counteracted the promoting influences of LINC00324 overexpression on NPC apoptosis, ECM degradation, inflammatory response, and TRAIL/DR4/DR5 axis activation in the IDD model. In vivo, LINC00324 overexpression exacerbated IDD in rats (decreased disc height index, disrupted NPC tissue structure, enhanced NPC apoptosis, aggravated ECM degradation, inhibited miR-143-3p expression, and activated TRAIL/DR4/DR5 axis), while LINC00324 knockdown attenuated these degenerative changes. In conclusion, LINC00324 promotes IDD progression by inducing NPC apoptosis, disrupting ECM metabolism, and enhancing inflammatory response via sponging miR-143-3p to activate the TRAIL/DR4/DR5 axis. Not applicable.

Tumor progression is regulated by cancer-associated fibroblasts (CAFs), pro-inflammatory cytokines (e.g., TNF&#x3b1;, IL-1&#x3b2;, IFN&#x3b3;) and acquired immunity (such as inhibitory immune checkpoints: PD-L1, PD-L2). Here, we determined the interactions between these elements by analyzing the impact of pro-inflammatory cytokines on the proportions of PD-L1&#x2009;+&#x2009;PD-L2-expressing CAFs; the cell-autonomous regulation of pro-tumorigenic characteristics and functions of CAFs by PD-L1/PD-L2; and the control of PD-L1/PD-L2 activities in CAFs by their N-glycosylation. CAFs were obtained from breast and lung cancer patients (BC-CAFs, Lung-CAFs). PD-L1/PD-L2 surface and whole-cell expression were determined by flow cytometry and Western blot, respectively. CAFs were transduced to express WT-PD-L1, WT-PD-L2, WT-PD-L1&#x2009;+&#x2009;WT-PD-L2, PD-L1/PD-L2 N-glycosylation mutants or vector controls, followed by determination of the pro-tumorigenic factors osteopontin (qRT-PCR) and CXCL8 (IL-8)/CCL2 (MCP-1)(ELISA). The effects of factors produced by CAFs expressing WT-PD-L1/WT-PD-L2/their N-glycosylation mutants on tumor cell migration were analyzed by wound-healing assays. TNF&#x3b1;&#x2009;+&#x2009;IL-1&#x3b2; potently increased the expression of CXCL8, whereas IFN&#x3b3; stimulation up-regulated the proportions of BC-CAFs and Lung-CAFs expressing PD-L1&#x2009;+&#x2009;PD-L2 at cell surface. Combined TNF&#x3b1;&#x2009;+&#x2009;IL-1&#x3b2;&#x2009;+&#x2009;IFN&#x3b3; stimulation has prominently increased intracellular pools of N-glycosylated PD-L1 and PD-L2, in BC-CAFs and Lung-CAFs. WT-PD-L1 induced in cell-autonomous manners pro-tumorigenic characteristics and functions in BC-CAFs: elevated the expression of osteopontin, CXCL8 and CCL2 and induced the production of factors that promoted tumor cell migration. WT-PD-L2 also acted in a cell-autonomous manner in BC-CAFs, generally down-regulating these phenotypes and activities. When co-expressed in BC-CAFs, the cancer-supporting effects of WT-PD-L1 dominated the anti-tumorigenic impacts of WT-PD-L2. The cell-autonomous functions of PD-L1 and PD-L2 were regulated, in context- and function-dependent manners, by their N-glycosylation sites, primarily N35/N192 in PD-L1 and N37/N163 in PD-L2. Chronic inflammation can up-regulate the proportions of PD-L1&#x2009;+&#x2009;PD-L2 expressing CAFs. PD-L1 and PD-L2 act in cell-autonomous manners to control the pro-tumorigenic characteristics and functions of CAFs, with dominance of the tumor-promoting effects of PD-L1 over the tumor-inhibiting roles of PD-L2. The cell-intrinsic functions of PD-L1 and PD-L2 in CAFs depended on their N-glycosylation. These observations emphasize the need to consider the expression of PD-L1 and PD-L2 in stromal cells when designing treatments with immune checkpoint blockades in patients.