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Chimeric Antigen receptor T cell (CAR-T) treatments for solid cancers have been compromised by limited expansion and survival in the tumor microenvironment following interaction with antigen-expressing target cells. Using B7H3 as a model antigen with broad clinical applicability, we evaluate the relationship between the antibody/antigen affinity of three clinical candidate binders and the three following characteristics: cellular avidity, duration of sustained cytotoxicity in tumoroid re-stimulation assays, and in vivo anti-tumoral responses. Next, BEHAV3D video microscopy is used to assess CAR-T cell interaction with tumor cells at single cell resolution. These data are consistent with a threshold avidity of CAR-T / tumor cell interaction and target cell B7H3 expression level, where enhanced functionality is characterized by longer cumulative CD8+ CAR-T / tumor target interaction times, CAR-T cell expansion and sustained tumor control. Lower checkpoint receptor expression does not correlate with enhanced anti-tumor function. These results provide further insights into design of anti-B7H3 CAR-T cells for antigen-dim cell targeting, and avoidance of antigen-dim tumor relapse. CAR-T cells have been found to be less effective as treatment for solid tumours. Here the authors, utilising B7H3 as an antigen, consider how changes in B7H3 binders lead to functional changes of CAR-T cells and differences in tumour outcomes in humanised mouse tumour models.

AbstractMacrophages engulf apoptotic bodies and cellular debris as part of homeostasis, but they can also phagocytose live cells, such as aged red blood cells. Pharmacologic reprogramming with the SMAC mimetic LCL161 in combination with T-cell-derived cytokines can induce macrophages to phagocytose live cancer cells in mouse models. Here we extend these findings to encompass a wide range of monovalent and bivalent SMAC mimetic compounds, demonstrating that live cell phagocytosis is a class effect of these agents. We demonstrate robust phagocytosis of live pancreatic and breast cancer cells by primary human macrophages across a range of healthy donors. Unlike mouse macrophages, where a combination of SMAC mimetics with lymphotoxin enhanced phagocytosis, human macrophages were more efficiently polarized to phagocytose live cells by the combination of SMAC mimetics and IFNg. We profiled phagocytic macrophages by transcriptional and proteomic methodologies, uncovering a positive feedback loop of autocrine TNFa production. Graphical Abstract Graphical Abstract

IntroductionElevated glucose levels during pregnancy disrupt placental structure, signaling, and cellular interactions, impairing its immune response. In mothers with gestational diabetes mellitus (GDM), Streptococcus agalactiae (Group B Streptococcus, GBS) is the second leading cause of bacterial infections. GDM is also linked to altered chemokine profiles in maternal serum and placenta tissue. However, the impact of hyperglycemia on placental immune responses to bacterial infections remains poorly understood. This work aimed to evaluate cytokine and chemokine production, as well as chemotactic responses, in the placenta following GBS infection under hyperglycemic conditions.MethodsHuman villous explants from term, normoevolutive pregnancies were cultured with 5, 10 or 50 mM glucose, and subsequently infected or not with GBS. Bacterial growth and adherence to villous tissue, syncytial disruption, cytokine and chemokine mRNA expression and secretion, leukocyte chemotaxis using intervillous blood mononuclear cells (IVMC), and TLR-2 expression at both mRNA and protein levels, were evaluated.ResultsUnder high glucose conditions, GBS showed increased proliferation and invasiveness, while villous explants presented evidence of syncytial barrier degradation. Also, placental TNF-α, MCP-1, and MIP-1β were induced by GBS infection. However, the dual challenge of high glucose and infection reduced the above inflammatory markers’ gene and protein synthesis. GBS infection enhanced IVMC migration compared to uninfected groups, but the combination of GBS and hyperglycemia led to a reduced migration of IVMC, particularly monocytes and NK cells. TLR-2 placental expression was also downregulated by this dual challenge.ConclusionAt the placental level, hyperglycemia attenuates the immune response against GBS infection, promoting syncytial disruption, bacterial growth, and tissue colonization. The combined stimulus of hyperglycemia and GBS resulted in reduced placental expression of TLR-2, TNF-α, MCP-1, and MIP-1β, thereby impairing the chemotaxis of IVMC, monocytes, and NK cells. This dysregulated immune response may compromise bacterial clearance and placental integrity, favoring pathogen persistence. Our findings suggest a potential mechanism by which hyperglycemia increases susceptibility to GBS-associated complications, offering novel insight into the interplay between metabolic and infectious stressors at the maternal-fetal interface.

Immune checkpoint blockade (ICB) therapies for solid tumors often fail due to resistance, necessitating new strategies. While efforts target IFNγ signaling or antigen presentation, other immune evasion mechanisms are unclear. Here, we identify Monoacylglycerol O-Acyltransferase 1 (Mogat1) as a critical modulator of tumor immune evasion using an in vivo transcriptomic screen in progressing tumors. We find that tumors exploit Mogat1 to sequester fatty acids into triglycerides, a metabolic adaptation that fuels growth and fosters an immunosuppressive microenvironment, enabling immune escape. Genetic inhibition of Mogat1 suppresses tumor growth by promoting T-cell infiltration and enhancing their tumor-killing ability. Importantly, Mogat1 loss sensitizes tumors to PD-1 blockade, overcoming resistance and suggesting reduced reliance on conventional antigen presentation. Our findings reveal a lipid metabolism-centered immune evasion mechanism and highlight Mogat1 as a potential target to improve cancer immunotherapy. Despite initial clinical success of immune checkpoint blockade, unfortunately not all patients respond to these therapies due to immune evasion mechanisms. Here, the authors discover that Monoacylglycerol OAcyltransferase 1 (Mogat1) regulation of lipid homeostasis modulates tumor immune evasion in breast cancer.

Oncogenic alterations in fibroblast growth factor receptor (FGFR)-family proteins occur across cancers, including pediatric gliomas. Our genomic analysis of 11,635 gliomas across ages finds that 5.3% of all gliomas harbor FGFR alterations, with an incidence of almost 9% in pediatric gliomas. Alterations in FGFR proteins are differentially enriched by age, tumor grade, and histology, with FGFR1 alterations associated with glioneuronal histologies. Leveraging isogenic systems, we confirm FGFR1 alterations to induce downstream Mitogen Activated Protein Kinase (MAPK) and mTOR signaling pathways, drive gliomagenesis, activate neuronal transcriptional programs and exhibit sensitivity to MAPK pathway and pan-FGFR inhibitors. Finally, we perform a retrospective analysis of clinical responses in children diagnosed with FGFR-altered gliomas and find that treatment with currently available inhibitors is largely associated with stability of disease. This study provides key insights into the biology of FGFR1-altered gliomas, therapeutic strategies to target them and associated challenges that still need to be overcome. A subset of pediatric gliomas harbour alterations in fibroblast growth factor receptor (FGFR)-family proteins. Here, the authors characterise the genomic landscape of 11,635 gliomas across ages and use isogenic model systems to explore the underlying biology of FGFR1-altered gliomas and potential therapeutic vulnerabilities.

AbstractBackgroundThe hyporesponsiveness of tumor-infiltrating exhausted CD8+ T cells to tumor cells contributes to immune escape of renal cell carcinoma (RCC), representing a major challenge in current immunotherapy. However, the underlying molecular mechanism of CD8+ T-cell exhaustion in the tumor microenvironment remains largely unknown.MethodsWe first examined myocardial infarction associated transcript (MIAT) expression in RCC cell lines and clinical specimens, and analyzed its correlation with CD8+ T-cell exhaustion markers. To investigate the immunoregulatory role of MIAT, we evaluated its effects on CD8+ T-cell function using T-cell co-culture systems and humanized-peripheral blood mononuclear cells RCC patient-derived xenograft models. To determine the direct effects of MIAT on tumor cells, we assessed RCC cell malignant phenotypes following MIAT knockdown both in vitro and in immunodeficient nude mouse orthotopic xenograft models. Mechanistically, we employed RNA fluorescence in situ hybridization, chromatin isolation by RNA purification followed by mass spectrometry, RNA immunoprecipitation, and chromatin immunoprecipitation assays to identify the molecular interactions between MIAT, transcription factors, and target genes.ResultsMIAT was highly expressed in RCC cells and positively correlated with CD8+ T-cell exhaustion status. MIAT knockdown significantly enhanced CD8+ T-cell function with increased perforin and interferon-γ production, while reducing the expression of exhaustion markers programmed cell death protein 1 and T-cell immunoreceptor with Ig and ITIM domains. Mechanistically, MIAT was predominantly localized in the nucleus and formed a trimeric complex with transcription factor ETS proto-oncogene 1 (ETS1) and janus kinase 3 (JAK3) promoter, thereby upregulating JAK3 expression and activating the JAK3/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Importantly, ectopic expression of JAK3 largely abolished both the tumor-suppressive effects and enhanced T-cell function induced by MIAT depletion.ConclusionsOur study demonstrates that the MIAT/JAK3/STAT3 pathway plays a critical role in malignant progression and immune escape of RCC through regulating CD8+ T-cell exhaustion, suggesting its potential as a therapeutic target for RCC immunotherapy.

Bovine trypanosomosis caused by Trypanosoma vivax is a health problem of economic importance in South America. In Ecuador, the presence of T. vivax was first reported in 2018; however, the isolates found in Ecuador are still being studied, mainly on issues related to virulence, pathogenicity, and immune response. To this end, this study aimed to evaluate the cellular and humoral adaptive immune response in vivo in experimentally infected cattle with T. vivax. The study lasted 42 days (with samples collected twice weekly) and was conducted in two cattle experimentally infected with an isolate of T. vivax circulating in Ecuador (TvET1) and two uninfected cattle as controls. Parasitemia was determined by the Brener method and relative gene expression (RGE) of six cytokines was evaluated by RT-qPCR to determine the Th1 response (IFN-γ, TNF-α, IL-1β, IL-12) and the Th2 response (IL-4 and IL-10). Additionally, the total IgG and the IgG1 (Th2) and IgG2 (Th1) subclasses levels were measured using an in-house iELISA. During the study, the animals exhibited four parasitemia peaks concomitant with the cytokines IFN-γ and IL-10. These cytokines, like TNF-α, showed a significant RGE increase (p < 0.05) in infected animals. The presence of total IgG, IgG1 and IgG2 was significant (p < 0.05) in infected animals, and presented a solid monotonic relationship over time. The predominant immunoglobulin subclass was IgG1, and we found that this response was similar to the total IgG. The present study allowed us to highlight the Th response of cattle to T. vivax infection, which is polarized into both a Th1 and a Th2 response. This information contributes to understanding the host-pathogen interaction with strains circulating in Ecuador. The thoroughness of our study can provide the needed knowledge to develop new diagnostic tests and even possible alternatives for vaccine development.

Well-differentiated airway epithelial cultures are commonly used to study airway stem cell lineages, ion and fluid transport, respiratory virus infection and replication, and disease mechanisms in vitro. This culture model involves the isolation and expansion of airway stem cells followed by their differentiation at an air–liquid interface (ALI), a process that has been previously documented in humans and mice. Domestic ferrets (Mustela putorius furo) have gained considerable importance in respiratory disease research due to their notable susceptibility to these conditions and their anatomical similarities to humans. Here, we present a comprehensive description of the isolation and culture of stem/progenitor cells from the ferret airway, along with a protocol for their differentiation at the ALI. Our findings have demonstrated that this ferret culture system not only supports the differentiation of the predominant airway epithelial cell types but also facilitates the generation of rare airway epithelial subpopulations, including pulmonary ionocytes, tuft cells, and pulmonary neuroendocrine cells. Additionally, we provide a detailed procedure for measuring transepithelial ion transport relevant to airway diseases, particularly cystic fibrosis. The ability to isolate and culture ferret airway stem cells, combined with ALI differentiation and functional assessment of transepithelial ion transport, offers a powerful platform for evaluating genetic and pharmacologic interventions related to cystic fibrosis. Key features • A protocol for isolating ferret airway basal cells and generating air–liquid interface (ALI) cultures for electrophysiologic research.• Detailed procedures for propagating ferret airway basal cells and culturing in vitro well-differentiated airway epithelium.• A protocol for measuring ion transport, conductance, and immunofluorescence to identify airway cell types.

SummaryHemolytic uremic syndrome (HUS), the main cause of acute renal failure in early childhood, is associated with infections by Escherichia coli strains producing Shiga toxin 2 (Stx2). The microangiopathic injuries caused by the toxin occur mainly in the renal microvasculature when the glycolipid receptor globotriaosylceramide (Gb3Cer) is targeted. Before entering the kidney, Stx2 binds to circulating cells through Gb3Cer and Toll-like receptor 4 (TLR4) and is subsequently delivered in extracellular vesicles to target cells. Here, we have found a specific inhibitor of the Stx2/TLR4 interaction, the preclinical polymyxin B derivative NAB815. The compound impairs the formation of Stx2-containing extracellular vesicles produced by leukocytes and platelets and also reduces their toxic effects in cellular (Vero cells) and animal models (CD-1 mice). NAB815 would represent a useful tool in preventing HUS and is effective at sub-bactericidal concentrations, thus overcoming the concern that antibiotics are harmful to patients infected with Stx2-producing E. coli. Graphical abstract Highlights•In children, life-threatening HUS is caused by bacteria producing Shiga toxins•There are no specific treatments for HUS, and antibiotics are not recommended•We have found an antibiotic impairing toxin actions at sub-bactericidal concentration•Administration of this drug in patients may represent a useful tool in preventing HUS Pharmacology; Natural sciences; Biological sciences; Microbiology; Medical Microbiology

Highlights•Convalescent plasma is an alternative COVID-19 therapy when no other is available.•We evaluated the long-term immune response in treated and untreated individuals.•A higher seroconversion rate early post infusion is observed compared to untreated.•However, long-term humoral and cellular immune responses are similar between groups. During the SARS-CoV-2 pandemic, the use of convalescent plasma (CP) in high-risk patients was proposed and widely implemented in several countries as a potential COVID-19 therapy. Nonetheless, CP therapy’s impact on immune response is nowadays poorly understood, including the correlation between IgG levels, neutralization capacity, and cellular immune response against SARS-CoV-2. Here we evaluated, in a cohort of patients with COVID-19 requiring hospitalization and having received or not CP, as well as in CP donors (recovered from mild disease), the humoral and cellular immune response assessed by titers of SARS-CoV-2 IgG, neutralizing antibodies, and IFN-γ+/IL-2+ ELISpot during the first month (early) and up to nine months (long-term) after symptom onset. Results showed higher seropositivity and seroconversion rates between 7–12 days after plasma infusion in CP-recipients. However, similar IgG and neutralizing immune response kinetics between CP-recipients and non-recipients was observed during the first and until the ninth month of analysis. A positive correlation between IgG and neutralizing levels was detected. Compared to outpatient donors, hospitalized individuals showed a higher response at 3 and 6 months after symptoms onset. A sustained SARS-CoV-2-specific CD4+ and CD8+ T cell response was observed in outpatients and hospitalized patients, regardless of the CP treatment. We concluded that the CP infusion did not affect the long-term SARS-CoV-2 specific humoral and cellular immune responses. Nonetheless, CP may provide a therapeutic window by promoting a higher humoral response during the acute phase of COVID-19. Graphical abstractImage, graphical abstract

The lymphocyte-specific transcription factor interferon regulatory factor 4 (IRF4) is a key player in immune evasion in cancers, with the complex mechanism(s) being barely understood. In this study, we have focused on the role of IRF4 in regulating T cell functions through its transcriptional regulation of programmed death 1 (PD1) and its ligand PD1 ligand 1 (PD-L1), which were identified as IRF4 transcriptional targets in multi-omics analysis. We have shown that IRF4 transcriptionally regulates both PD1 and PD-L1, promoting immune suppression in the context of Epstein–Barr virus (EBV) infection. Co-culturing EBV+ JiJoye lymphoma cells with CD4+ T cells or with peripheral blood mononuclear cells (PBMCs) downregulates CD4+ T cell functions, but the depletion of IRF4 in EBV+ JiJoye lymphoma cells reduces PD1 and PD-L1 expression, and partially restores CD4+ T cell functions. Moreover, CD4+ T cell depletion from PBMCs enhances EBV transformation, and EBV has a greater efficiency in transforming PBMCs from HIV patients with impaired CD4+ T cell functions. These findings support the role of IRF4 in immune evasion by upregulating PD1/PD-L1 during EBV transformation, and that functional CD4+ T cells are essential for limiting EBV transformation.

BackgroundGadolinium (Gd) deposition in the brain was observed in patients with history of gadolinium-based contrast agent (GBCA) administration. However, the exact mechanism behind this deposition remains unclear, especially given that an intact blood-brain barrier (BBB) is considered impermeable to GBCA. In this study, we propose that immune cells might play a role in facilitating GBCA entry into the brain despite an intact BBB.MethodsGadoterate meglumine, gadoteridol, gadobutrol and gadodiamide were investigated as GBCAs. Immune cells from human donor buffy coats were isolated, incubated with the GBCA and used in the experiments. Gd associated with the immune cells were measured using single-cell inductively coupled mass spectrometry (SC-ICP-MS). Flow cytometry analysis was performed to characterise the adhesion molecule expression profile on the immune cells and binding assay was employed to check the binding of Gd treated immune cells with endothelial ligands in static conditions. An in vitro model of the human BBB that prevents free diffusion of GBCA across was further used to observe immune cell behaviour at the BBB under physiological flow, in vitro.ResultsOur findings confirm that various immune cells, including CD4+ T cells, CD8+ T cells, monocytes, NK cells and B cells are capable of taking up the different GBCAs. Furthermore, we demonstrate that GBCA loading does not impair immune cell interaction with the endothelial ligands required for successful extravasation across the BBB under static conditions. Most importantly, we show that T cells and monocytes, loaded with the different contrast agents, extravasated across an in vitro BBB model under physiological flow conditions in a comparable manner to non GBCA loaded cells.ConclusionsTaken together, our in vitro observations show that immune cells can transport GBCA across the BBB and could lead to permanent deposition of Gd in the brain.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12987-025-00674-5.