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T follicular helper (T) cells support B cell function by promoting memory B cell differentiation and sustaining long-lasting antibody responses, thereby enabling immunity that protects the host from subsequent infections. T cells are essential for orchestrating the antibody-mediated immunity achieved via vaccination, which has had a profound global impact in reducing the morbidity and mortality associated with infectious diseases. T cells provide help to B cells both within and outside germinal centres and deliver key signals that drive immunoglobulin class switching and affinity maturation. The formation and function of T cells is dynamic and adaptable, with significant cellular plasticity to ensure that robust antibody production accompanies most immune responses. However, when antibodies are directed against self or non-pathogenic antigens, they can contribute to the development of autoimmune diseases, allergic reactions and transplant rejection. Beyond supporting antibody responses, T cells have been implicated in cancer, diabetes and atherosclerosis. Given the multifaceted roles of T cells in health and disease, and the changes to their biology during normal ageing, the development of targeted strategies to modulate Tcell activity is an attractive approach to promote health across the lifespan.

PTEN (phosphatase and tensin homolog deleted on chromosome ten) is a tumor suppressor, the function of which is impaired in many diverse cancers. It has phosphoinositide lipid phosphatase activity by which it suppresses activation of the oncogenic PI3K signaling network but in vitro also displays activity against protein substrates and is able to auto-dephosphorylate its Thr366 residue. Here we generate germline knock-in mice expressing PTEN-Y138L, a mutant enzyme which selectively lacks protein phosphatase activity and retains lipid phosphatase activity. Homozygous Pten mice die in utero before E10.5. Primary MEFs and thymocytes with only a single Pten allele display normal low levels of AKT phosphorylation indicating effective regulation of PI3K signaling by endogenous PTEN-Y138L in vivo. Heterozygous Pten mice have reduced overall survival compared to wild-type littermates and develop tumors in multiple organs. Our data imply that in addition to its lipid phosphatase activity, the protein phosphatase activity of PTEN is also required for normal embryonic development and tumor suppression.

B cells generate plasma cells (PCs) and memory B cells (MBCs) to combat recurrent pathogens. B cell receptor (BCR) affinity dictates fate decisions in response to model antigens, but the factors regulating B cell fate during infection remain unknown. Here, we used respiratory and gastrointestinal infection models to study B cell selection across barrier tissues in mice. Memory selection was governed by tissue-specific cues: Selection in the lung was skewed toward MBCs, whereas the gut favored PC entry, even in response to the same pathogen. Divergence was linked to differential BCR isotype usage across barrier tissues rather than differential affinity maturation. In the gut, the commensal-induced TGF-β-rich milieu promoted class-switching to IgA, which skewed selection toward PCs, a process that was counteracted by the IgA cytosolic tail domain. Thus, mucosal B cell selection integrates tissue-specific cues by relying on BCR isotype usage, with implications for nasal and oral vaccine development.

Proteostasis, the maintenance of a healthy proteome, is a fundamental pillar of cellular and organismal health that declines with age. While the intracellular proteostasis network (PN) is well-characterised, proteostasis mechanisms acting in the extracellular space remain understudied. Yet, these mechanisms face unique challenges and are critical for ensuring functional systemic signalling, immune surveillance and structural integrity. In contrast to the cytosol, extracellular environments lack ATP-activated chaperones and a comprehensive ubiquitin-proteasome system and instead rely on specialised secreted chaperones, extracellular proteases and receptor-mediated clearance mechanisms. This review examines the emerging landscape of the extracellular proteostasis network (exPN) and its challenges with age. We discuss how age-related remodelling of the extracellular proteome, shifts in extracellular physicochemical properties and disrupted fluid dynamics collectively create a permissive environment for protein misfolding and aggregation. We evaluate current experimental models of extracellular protein damage and examine how exPN factors target specific stages of the aggregation process to cooperatively safeguard extracellular proteome integrity. Analysis of recent human proteomic data spanning the life course uncovers an unexpected upregulation of exPN components with age. We further explore the role of extracellular proteostasis in inflammageing, a defining hallmark of ageing. Finally, we highlight strategies that bolster extracellular proteostasis as a promising frontier for extending healthspan, limiting age-associated protein aggregation and restoring extracellular matrix homeostasis. By adopting an ageing-centred perspective, we move beyond the disease context to present a holistic overview of extracellular proteostasis in organismal health, thereby positioning the exPN as a critical yet under-exploited target for biomedical intervention.

Replication stress poses a major threat to genome integrity, yet how higher-order chromatin organization contributes to replication fork protection remains unclear. Here we show that replication stress induces the formation of transient chromatin loops that enclose de novo heterochromatin-enriched stalled replication forks. Stressed forks preferentially stall at convergent CTCF motifs, triggering stress-dependent CTCF enrichment that constrains loop extrusion and stabilizes these structures. Loop stabilization requires both CTCF anchoring and G9a-dependent heterochromatin (trimethylation of Lys9 of histone H3 (H3K9me3)) deposition on nascent DNA within the loop body. These loops function as protective scaffolds that shield stalled and reversed forks from degradation by multiple nucleases. By contrast, combined loss of stress-induced heterochromatin and CTCF enrichment destabilizes the loop scaffold, exposing multiple entry points for nucleolytic attack and resulting in extensive nascent-strand degradation through mechanisms distinct from classical fork-reversal-dependent pathways. This protective architecture is similarly critical in BRCA2-deficient cells, in which replication-stress-associated loops predominantly safeguard replication initiation zones, while nascent DNA outside these loops undergoes massive degradation and remains highly susceptible to mutations. Our study elucidates the fundamental role of replication-stress-induced three-dimensional genome reorganization in preserving replication fork stability, thereby mitigating mutagenesis and genomic instability.

Targeting tumour antigens is a major challenge in cancer-immunotherapy. We use active vaccination to induce antibodies targeting self-antigen Robo4, which is selectively expressed on tumour vascular endothelium, and supports vascular development. Our previous work showed that a conjugate of Robo4 with a foreign carrier protein induced autoantibodies specific to Robo4, which inhibited angiogenesis and tumour growth.

The development of complex tissues relies on the precise assignment of cell identity. At the molecular scale, this process depends on the deposition of epigenetic modifications-such as methylation-that are regulated by complex biochemical networks and occur at specific regions on the DNA and chromatin. Here we show that despite the complexity of epigenetic regulation, dynamical scaling and self-similarity of DNA methylation marks emerge in embryonic development. Drawing on single-cell multi-omics experiments, super-resolution microscopy and statistical physics, we demonstrate that these phenomena originate in dynamical feedback between DNA methylation and the formation of nanoscale dynamic chromatin aggregates. These nanoscale processes lead to genome-wide increase in DNA methylation marks following a power law and self-similar correlation functions. Using this framework, we identify methylation patterns that precede gene expression changes in embryonic symmetry breaking. Our work identifies linear sequencing measurements as a laboratory to study mesoscopic biophysical processes in vivo.

Direct interactions between the cell’s powerhouses and nuclear pores might channel energy straight into the nucleus, fuelling cell division and differentiation.

Aging is a complex multifactorial process that affects cellular function and tissue homeostasis over time. Despite substantial research, the molecular mechanisms driving cellular aging remain poorly understood. Many studies focused on changes in DNA methylation as an indicator of aging. In particular, methylation at polycomb CpG islands was shown to be predictive of phenotypic changes associated with aging. Since many age-related pathological processes are thought to originate from single cells, we asked whether polycomb CpG island methylation occurs preferentially in a subset of cells within a population. Using single-cell whole-genome methylation data across ages and tissues, we identify polycomb CpG methylation as a hallmark of cellular aging. This revealed that aging occurs at varying rates, with faster proliferating cells showing accelerated gain of methylation. Differential gene expression analysis identified changes in immune response, translation, tumorigenesis and neurodegeneration. These results challenge traditional models of homogeneous cellular aging and suggest that aging is a highly individualized process at the single-cell level, that may be driven by programmed changes in polycomb CpG island DNA methylation.

Poised enhancers (PEs), co-marked by H3K4me1 and Polycomb-associated H3K27me3, are common in primed human pluripotent stem cells (hPSCs) resembling post-implantation epiblast but scarce in naive hPSCs modeling pre-implantation epiblast. PEs form abundant chromosomal contacts with developmental genes, but the timing of their emergence, their relationship to enhancer poising, and their functional significance remain unclear. We devised high-resolution, PE-targeted Capture Hi-C to map PE contacts during the transition from naive to primed pluripotency. We find that enhancer poising emerges early in the transition, while the contacts show diverse dynamics. PROTAC-induced degradation of Polycomb repressive complex 2 early in the transition, but not inhibition of its H3K27 methyltransferase activity, weakens PE connectivity. Finally, PE contacts persist after developmental activation or ectopic CRISPRa targeting and can mediate long-range gene induction. Together, these findings reveal the temporal and mechanistic principles of PE connectivity and highlight a potential role of PE contacts in establishing developmental gene expression patterns.

Coronaviruses and influenza A viruses are major respiratory pathogens with pandemic potential. Using pigs as a translational large-animal model, we compare the virulence, pathogenesis, and immune responses to porcine respiratory coronavirus (PRCV) and pandemic H1N1 2009 influenza virus (pH1N1). Here we show that PRCV induces higher viral load and prolonged viral shedding, stronger systemic and mucosal T cell activation, expansion of memory B cells, and distinct nasal microbiome changes. In contrast, pH1N1 results in rapid neutralising antibody production, robust Tfh and germinal centre B cell responses, and broader early nasal microbial diversity. Transcriptional responses to PRCV and pH1N1 infection start with the activation of shared interferon-stimulated genes but later diverge as pathways involving stromal-immune interactions and vascular integrity shapes lung pathology and subsequent immune responses. These findings demonstrate fundamental differences in coronavirus and influenza virus-host interactions and establish the pig as a powerful comparative model for studying respiratory virus pathogenesis and immunity.

The germinal center (GC) is the engine room of the humoral immune response, driving the evolution of B cells into memory and long-lived plasma cells that can protect against (re)infection. Understanding GC biology remains a key research focus for promoting lifelong health. In this annual update, we highlight several influential studies on GC biology published in 2025. Space constraints prevent us from discussing in depth all published work.

Noncanonical conjugation of ATG8 proteins, including LC3, to single membranes implicates the autophagy machinery in cell functions unrelated to metabolic stress. One such pathway is LC3-associated phagocytosis (LAP), which aids in phagosome maturation and subsequent signaling upon cargo uptake mediated by certain innate immunity-associated receptors. Here, we show that a specific isoform of RAB5 GTPases, the molecular switches controlling early endosome traffic, is necessary for LAP. We demonstrate that RAB5c regulates phagosome recruitment and function of complexes required for phosphatidylinositol 3-phosphate [PI(3)P] and reactive oxygen species (ROS) generation by macrophages. RAB5c facilitates phagosome translocation of the V-ATPase transmembrane core, which is needed for ATG16L1 binding and consequent LC3 conjugation. RAB5c depletion impaired macrophage elimination of the fungal pathogen and disruption of the V-ATPase-ATG16L1 axis increased susceptibility in vivo. Thus, early endosome-to-phagosome trafficking can be selectively engaged to promote pathogen elimination by directing phagosomal maturation toward LAP.

As lipidomics approaches its 25th anniversary, we explore how lipid research has matured over the years while highlighting emerging innovations that are expanding our ability to study these diverse, life-critical biomolecules. In particular, we showcase the community-driven, open-access databases, software, and educational resources made freely available through the ELIXIR Core Data Resource LIPID MAPS for the benefit of both established and new researchers.

The P-Rex family proteins P-Rex1 and P-Rex2 are Dbl-type guanine-nucleotide exchange factors (GEFs) that activate Rac small GTPases upon synergistic stimulation by PIP and Gβγ, acting as coincidence detectors for PI3K and GPCR signalling. P-Rex Rac-GEFs control physiological responses ranging from inflammation, innate and adaptive immunity to GPCR trafficking, glucose homeostasis, and the function of the vascular endothelium, nervous system, and adipose tissue. P-Rex2 also increases PI3K-signalling through its catalysis-independent inhibition of the tumour suppressor PTEN. Deregulated levels of P-Rex1 are linked to fibrotic diseases, asthma, and autism spectrum disorders, and both P-Rex1 and P-Rex2 are deregulated in metabolic diseases. Upregulation of P-Rex1 and P-Rex2 as well as activating P-Rex2 mutations also occur in many types of cancer, including breast, prostate, lung, liver and colorectal cancer, as well as in melanoma and glioma. and contribute to tumour growth or metastasis depending on the P-Rex protein and cancer type. Deregulation of P-Rex1 in cancer typically promotes tumour growth or metastasis, whereas upregulation or mutation of P-Rex2 in cancer is mostly associated with tumour growth. Recently, structural data have increased our understanding of P-Rex regulation, the first P-Rex inhibitors have been developed, and GEF-activity independent functions of P-Rex proteins in GPCR trafficking, neutrophil-responses, innate immunity, and glucose homeostasis have been described. This review summarises the P-Rex literature from the discovery of the P-Rex protein family in 2002 to the present, with a focus on recent advances.

Wnt4 signaling promotes somatic cell development in the female embryo, but its role in germline differentiation during meiosis remains poorly characterized. To explore Wnt4 functions in female embryonic gonads, we isolated germ cells from Wnt4 knock-out mice to investigate histone modifications and DNA methylation distribution patterns. The lack of the Wnt4 signaling pathway deregulates germ cell cycle markers, such as cyclins, alters the cell cycle by impairing meiosis progression, maintains the germ cells in the G1-GO and S phases, and supporting DNMT3A and DNMT1 enzyme expression at meiosis entry. Conversely, in the nucleus of the Wnt4 knock-out female germ cells, an increase of H3K27me3 pattern persists at the entry of meiosis, leading to altered methylation at the Sycp3 promoters combined with an acetylation of Stra8 promoter at E14.5. This changed pattern might be explained by the overexpression of Creb-binding protein (CBP) in the mutant female germ cells, leading to deregulation of histone marks on meiosis genes. Our findings reveal that the Wnt4 signal is necessary for inducing meiosis by inhibiting germ cell proliferation via the regulation of histone modification. Wnt4 signaling plays a crucial role in regulating the delicate balance between DNA methylation and acetylation in female germ cells. This fascinating interaction highlights the complexities of cellular processes that contribute to reproductive health and development.

TrAEL-seq is a robust method for profiling DNA replication genome-wide that works in unsynchronized cells and does not require drugs or nucleotide analogues. Here, we provide an updated method for TrAEL-seq that improves sample quality and includes multiplexing of up to six samples which dramatically improves throughput, and we validate TrAEL-seq in multiple mammalian cell lines. The updated protocol is straightforward and robust yet provides excellent resolution comparable to OK-seq in mammalian cell samples. High resolution replication profiles can be obtained across large panels of samples and in dynamic systems, for example during the progressive onset of oncogene induced senescence. In addition to mapping zones where replication initiates and terminates, TrAEL-seq is sensitive to replication fork speed, revealing effects of both transcription and proximity to replication Initiation Zones on fork progression. Although forks move more slowly through transcribed regions, this does not have a significant impact on the broader dynamics of replication fork progression, and instead replication forks accelerate across the first ∼1 Mb of travel irrespective of local transcriptional activity. We propose that this is a consequence of fewer replication forks being active later in S-phase when these distal regions replicate and there being less competition for replication factors.

New biomarkers are needed for better stratification and personalized treatment of Systemic Lupus Erythematosus (SLE). Phosphoinositide 3-kinase δ (PI3Kδ) has been implicated in SLE pathogenesis. Here, we investigated whether a subset of SLE patients has increased PI3Kδ activity after T cell activation.

We developed a 50-plex imaging protocol using the MACSima platform to characterize the microarchitecture of germinal centers in secondary lymphoid tissues. This workflow combines tissue processing, automated cyclic imaging, image preprocessing, and a new analysis method that detects morphological changes in irregularly shaped stromal cells.

Vaccines stimulate protective humoral immunity by coordinating lymphocyte activity in the germinal center (GC) response. However, the degree of protection varies across the population, with older individuals often showing lower titer antibody responses to vaccines. Previous work has correlated increased exposure to Th1 cytokines, like IL-2, with poor antibody responses in aging. Whether this is a causal relationship is unknown. Here we used Il2; Rosa26 mice, which cannot shut down IL-2 production once initiated, to test the hypothesis that sustained IL-2 exposure affects the GC response and antibody production post vaccination. Prolonged IL-2 production impeded T follicular helper (Tfh) cell differentiation, with reduced numbers of GC Tfh, pre-Tfh and T follicular regulatory cells consistently observed in the draining lymph node of Il2; Rosa26 mice at day 10 post immunization, relative to control mice. Numbers of Tfh cells remained suppressed at day 21 post immunization and corresponded with reduced GC B cell formation, including NP-specific IgG1 class-switched GC B cells. As a core output of the GC response, B220IRF4CD138 antibody-secreting cells (ASCs) were significantly reduced in the bone marrow of Il2; Rosa26 mice, 10 days post NP-KLH immunization compared to control mice. However, by day 21, antigen-specific humoral immunity was restored, with similar levels of B220IRF4CD138 and NP-specific ASCs in both experimental and control groups. In summary, while sustained IL-2 production delayed Tfh differentiation and early GC formation, it did not affect overall antibody titers after vaccination.

The epithelial gut barrier and gut microbiota significantly contribute to human health by controlling molecule absorption, a regulated transport that dictates bioavailability. Effective public health strategies, like dietary reference values, require a complete understanding of nutrient absorption. However, the lack of internationally harmonized nutritional recommendations indicates that gut barrier mechanisms are not fully unraveled. The conventional in vitro model Caco-2/HT29-MTX cultured on cell culture inserts, established for drug development, is limited in representing complex human gut physiology. The new bioelectronic e-transmembrane platform leverages technological and biological advances to generate more meaningful in vitro predictions. The soft electroactive Poly (3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) scaffold enables direct cell-electrode coupling for more sensitive barrier impedance measurements, especially required for testing commonly low physiological nutrient concentrations. Promoted epithelial-fibroblast interactions result in modulated protein signal transduction and expression of genes regulating gut barrier integrity. Overall, the e-transmembrane gut barrier more closely mimicked physiological effects for humans as demonstrated using the dietary compound butyrate.

T follicular helper (T) cells are essential for germinal center (GC) formation and long-lived humoral immunity. Here, we used an fate mapping (Il21-fm) strategy to remove T cells from established GCs to disentangle their function from that of other T cells in a temporal manner. We confirm their role in driving proliferation and positive selection of GC B cells but show that GCs can survive the transient absence of T cells. After ablation of T cells, both the GC response and affinity maturation recover via ingress of new T cells. Despite recovery of the B cell response, T cell numbers are never fully restored. This feeds through into the T cell memory response, resulting in diminished recall GC responses. This work shows an unappreciated resilience of primary GC responses to perturbations in T cells and demonstrates critical memory T cell generation during this phase.

Dynamic regulation of protein tyrosine phosphorylation (pTyr) by kinases and phosphatases enables cells to sense and respond to environmental changes. The widely used chemical pervanadate induces the accumulation of pTyr in mammalian cell lines. This effect is primarily attributed to its inhibition of protein tyrosine phosphatases (PTPs), leading to the assertion that PTPs are master gatekeepers of intracellular pTyr homeostasis. Here, we used several approaches to reveal that pervanadate disrupted cellular redox homeostasis and directly activated tyrosine kinases of the SRC family through the oxidation of specific cysteine residues. Mass spectrometry and biophysical approaches showed that pervanadate-induced oxidation of cysteine-188 and cysteine-280 activated SRC by disrupting autoinhibitory intramolecular interactions between the catalytic domain and the SH2/SH3 domains and by impairing SH2 domain binding to phosphopeptides, including the regulatory carboxyl-terminal tail phosphotyrosine-530. Redox-sensitive cysteine residues were essential for SRC to promote the overgrowth of mouse fibroblasts. Our findings call for a reevaluation of pervanadate-based experiments and demonstrate that SRC cysteines control its oncogenic properties.

Infection triggers one of the most dramatic systemic responses in the body, and the coordinated activation and function of immune cells requires a dynamic regulation of transcriptomes and proteomes. This is achieved by RNA-binding proteins, which, together with RNA, form ribonucleoproteins. These proteins expand the information content of the genome and determine the lifespan, localization and function of RNA. Moreover, they control when, where and how much protein is produced. They can also mediate cell-autonomous immunity to foreign RNA and to misfolded self-RNAs and ensure the fidelity of the transcriptome by acting as RNA modifiers and chaperones to prevent RNA misfolding. These activities are integrated with gene expression programmes that are induced by the pathogen-sensing mechanisms of immune cells, which together activate, and later resolve, immune responses. Here, we review the activities of RNA-binding proteins in immune cells and discuss how perturbations of their function can result in immunodeficiency, autoimmunity and chronic inflammation.