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HexaBody-CD27 (GEN1053/BNT313) is an investigational novel agonistic CD27 antibody engineered to enhance T-cell costimulation and promote antitumor immunity. Through the introduction of a hexamerization-enhancing mutation in the IgG Fc domain, HexaBody-CD27 was designed to drive clustering and activation of CD27 via intermolecular Fc:Fc interactions between membrane-bound antibodies, independent of crosslinking by FcγR-bearing cells. HexaBody-CD27 carries an Fc-silencing mutation to prevent T-cell depletion through Fc-mediated effector functions. In vitro, HexaBody-CD27 induced CD27 receptor signaling independent of FcγR-mediated crosslinking in a reporter assay. It also enhanced T-cell proliferation, cytotoxic activity and proinflammatory cytokine secretion in primary human lymphocytes. In contrast to benchmark IgG1 CD27 antibodies, HexaBody-CD27 did not induce phagocytosis of T cells in vitro. HexaBody-CD27 promoted ex vivo tumor infiltrating lymphocyte (TIL) expansion in non-small cell lung cancer (NSCLC) specimens, in particular of CD8 + TILs. The combination of HexaBody-CD27 with an anti-PD-1 antibody enhanced T-cell proliferation, cytokine secretion, and cytotoxic activity in vitro compared to either compound alone. In conclusion, HexaBody-CD27 enhanced T-cell activation and effector functions in an FcγR-crosslinking-independent manner, without inducing T-cell depletion. The immune agonist activity of HexaBody-CD27 was potentiated in combination with PD-1 blockade.

Peroxisome proliferator-activated receptor gamma (PPARG) is a nuclear receptor family transcription factor (TF) critical for adipogenesis, lipid metabolism, insulin sensitivity, and inflammation. It has also been known to play essential roles in trophoblast development and placentation. Dysregulation of PPARG in trophoblast differentiation has been implicated in pregnancy complications, such as pre-eclampsia and gestational diabetes. However, the molecular mechanisms of PPARG-dependent target gene regulation and its interactions with other regulatory factors during human trophoblast differentiation remain unclear. Using human trophoblast stem cells (TSCs), mimicking placental cytotrophoblasts (CTs), and their differentiation into extravillous trophoblasts (EVTs) as our models, we reveal that PPARG has cell-type-specific targets in TSCs and EVTs. We also find that while PPARG is essential for both TSC self-renewal and EVT differentiation, only its role in EVT differentiation is ligand sensitive and requires ligand-binding domain (LBD)-mediated transcriptional activity, whereas its function in TSC self-renewal appears to be ligand insensitive. Combined analysis with chromosomal targets of previously defined key TFs in TSCs and EVTs shows that PPARG forms trophoblast cell-type-specific regulatory circuitries, leading to differential target gene regulation via transcriptional re-wiring during EVT differentiation. Additionally, the enhanced invasiveness of EVTs treated with a PPARG agonist suggests a potential connection between PPARG pathways and human placenta accreta.

The hypoxic tumor microenvironment, particularly hypoxia-conditioned cancer-associated fibroblasts (CAFs), drives breast cancer (BC) progression and therapy resistance. However, the molecular mechanisms linking hypoxic CAFs to BC plasticity and chemoresistance remain elusive. Primary CAFs were isolated from high-grade BC tissues (Grade III) and characterized (α-SMA⁺/CD34⁻/pan-CK⁻), with normal fibroblasts (NFs) from reduction mammoplasty as controls. Hypoxic CAF-derived exosomal circSTAT3 stability was validated using RNase R resistance and actinomycin D assays. Exosomes were characterized via transmission electron microscopy (TEM), dynamic light scattering (DLS), and marker profiling (CD63⁺/TSG101⁺/Alix⁺, calnexin⁻). Functional effects of hypoxic CAF exosomes on TNBC cells (MDA-MB-231, SUM159) were assessed through proliferation/migration assays, stemness/epithelial-mesenchymal transition (EMT) marker analysis, and siRNA-mediated circSTAT3 knockdown. Mechanistic studies employed luciferase assays and RNA immunoprecipitation (RIP). Chemoresistance was evaluated by cisplatin half-maximal inhibitory concentration (IC₅₀). In vivo tumor growth and stemness enrichment were analyzed in xenografts. Clinical validation used BC tissues (n = 60) and plasma exosomes from BC patients (n = 40) versus healthy controls (n = 25). Hypoxic CAF-derived exosomes efficiently transferred circSTAT3 to TNBC cells, promoting proliferation, migration, EMT, and stemness marker expression. SiRNA-mediated circSTAT3 knockdown reversed these effects. Mechanistically, circSTAT3 acted as a competitive endogenous RNA (ceRNA), sponging miR-671-5p to derepress NOTCH1. Hypoxic CAF exosomes increased cisplatin IC₅₀ in TNBC cells, while circSTAT3 depletion restored chemosensitivity. In vivo, hypoxic CAF exosomes accelerated tumor growth, enriched CD44⁺/NOTCH1⁺ populations, and elevated circulating exosomal circSTAT3. Clinically, circSTAT3 was significantly upregulated in advanced BC tissues (p < 0.01) and patient plasma exosomes (p < 0.01), correlating with lymph node metastasis. This study identifies a hypoxia-driven feedforward loop wherein CAF-derived exosomal circSTAT3 promotes TNBC stemness and chemoresistance via miR-671-5p/NOTCH1 signaling. CircSTAT3 redefines stromal-tumor crosstalk as a circRNA-driven process and serves as both a circulating non-invasive biomarker and a promising therapeutic target to disrupt stromal-mediated resistance in aggressive TNBC. The online version contains supplementary material available at 10.1186/s12967-025-06794-8.

Background: Human cytomegalovirus (CMV) is a major pathogen that poses significant risks to immunocompromised individuals and neonates. The tegument protein pp71, encoded by the UL82 gene, plays a pivotal role in initiating viral lytic replication and evading host immune responses. Despite its clinical relevance, standardized monoclonal antibodies (mAbs) for pp71 remain limited, prompting the need to expand the available repertoire of antibodies targeting this critical protein. Methods: In this study, we constructed a diverse human single-chain variable fragment (scFv) library using RNA derived from the B cells of four healthy donors. The library was expressed in Saccharomyces cerevisiae , and iterative rounds of magnetic-activated cell sorting (MACS) were performed against recombinant pp71. Clonal enrichment was monitored using flow cytometry. Results: Among the isolated clones, one designated ID2 exhibited high sensitivity and specificity for pp71, as demonstrated by flow cytometry, immunofluorescence, an enzyme-linked immunosorbent assay (ELISA), and biolayer interferometry (BLI). Conclusions: Collectively, these findings establish a novel pp71-specific mAb and underscore the utility of yeast surface display combined with MACS for expanding the antibody toolkit available for CMV research and diagnostics.

Respiratory syncytial virus (RSV) infection in the upper respiratory tract promotes disease progression and transmission, with excessive inflammation contributing to severe lower respiratory tract involvement. This study investigates the immunomodulatory effects of Lactobacillus rhamnosus D3189 on viral kinetics and innate immune responses in well-differentiated nasal epithelial cells (WD-NECs). WD-NECs from healthy adult donors (N = 8) were cultured in vitro , treated with L. rhamnosus D3189, and then infected with RSV (strain RS4) 24 hours later. Viral replication and shedding were assessed via RT-qPCR and plaque assays. Cytotoxicity and epithelial integrity were evaluated using LDH release and transepithelial electrical resistance (TEER). Inflammatory and antiviral responses were investigated using multiplex immunoassays, AlphaLISA, and ELISA. RSV infection induced robust viral replication and shedding, disrupted epithelial barrier integrity, and triggered the release of pro-inflammatory cytokines and type I/III interferons. L. rhamnosus D3189 alone did not induce cytotoxicity or inflammation. While it had no effect on viral replication, TEER, LDH release, or IFN-λ1/3 levels, D3189 significantly enhanced IFN-β production, reduced viral shedding, and attenuated RSV-induced cytokine and chemokine responses. L. rhamnosus D3189 modulates the epithelial immune response to RSV, reducing inflammation and viral shedding without compromising epithelial integrity. These findings support its potential as a novel strategy to limit RSV-associated infection and transmission.

Resistance to chemotherapy remains a major clinical challenge in triple-negative breast cancer (TNBC), an intrinsic subtype with limited available therapeutic options. The expression of moesin (MSN) is upregulated in TNBC patients, but little is known about the role of MSN in breast carcinogenesis. We investigated the MSN-dependent autocrine loop between extracellular interleukin 6 (IL-6) and NF-κB, along with a signaling cascade involving GTPase-mediated STAT3 phosphorylation. Various in vitro and in vivo assays were used to evaluate tumor initiation, growth, and stemness properties in TNBC models. High MSN expression was correlated with shorter overall and disease-free survival in TNBC patients. In vivo, MSN promotes tumor initiation and growth. Mechanistically, MSN-mediated IL-6/NF-κB autoregulatory feedback enhances IL-6 transcription. IL-6 binding to LPAR1 activated MSN phosphorylation, which then sequentially phosphorylated the CDC42-PAK4 complex, triggering nuclear translocation of the pSTAT3-MSN complex. This led to pSTAT3-mediated activation of cancer stemness genes (IGFN1, EML1, and SRGN), contributing to Adriamycin resistance. Notably, combination treatment with the FDA-approved STAT3 inhibitor Atovaquone and Adriamycin restored drug sensitivity. Our findings uncover the critical role of MSN in regulating STAT3-mediated cancer stemness via the IL-6/NF-κB signaling axis. These results provide a strong rationale for repositioning STAT3 inhibitors such as Atovaquone as a therapeutic strategy in Adriamycin-resistant TNBC patients exhibiting pSTAT3-MSN complex upregulation. The online version contains supplementary material available at 10.1186/s13058-025-02072-z.

RNA 5-methylcytosine (m 5 C), a prevalent epitranscriptomic modification that critically regulates gene expression and cellular homeostasis. While its roles in solid tumors have been increasingly recognized, the functional landscape of m 5 C in acute myeloid leukemia (AML) remains unexplored. Here, we identified NSUN2, the principal RNA m 5 C methyltransferase, as a key regulator of AML progression. NSUN2 was aberrantly upregulated in AML patient samples and correlated with poor prognosis. Functional studies demonstrated that NSUN2 promoted leukemic cell proliferation, enhanced tumor growth in xenograft models, and conferred resistance to ferroptosis—a regulated cell death process driven by lipid peroxidation. Mechanistically, NSUN2 catalyzed m⁵C deposition on the 3’UTR of FSP1 (ferroptosis suppressor protein 1) mRNA, facilitating its recognition and stabilization by the m 5 C reader protein YBX1. This NSUN2-YBX1-FSP1 axis protected AML cells from ferroptotic stress by suppressing lipid peroxidation and oxidative damage. Depletion of NSUN2 or FSP1 induced mitochondrial remodeling, which primed cells for ferroptosis. Reconstitution of wild-type NSUN2 or FSP1 rescued ferroptosis resistance, whereas catalytically inactive NSUN2 (C271A/C321A) or non-functional FSP1 mutants (G2A/E156A) failed to reverse this phenotype. Pharmacological inhibition of NSUN2 with MY-1B or targeting FSP1 with iFSP1 exhibited potent anti-leukemic effects, synergizing robustly with ferroptosis inducers, standard chemotherapy, and the BCL-2 inhibitor venetoclax. Our study unveils NSUN2 and FSP1 as prognostic biomarkers and therapeutic targets in AML. We highlight a novel epitranscriptomic mechanism linking RNA methylation to ferroptosis evasion, providing a dual-strategy approach to overcome AML treatment resistance. The online version contains supplementary material available at 10.1186/s12943-025-02394-8.

Borisyuk et al. identify a signaling regulatory network of peroxisome proliferation, uncovering PKC as a positive regulator of peroxisome–ER interaction. During neuronal differentiation, activation of PKC contributes to an increase in peroxisome formation.