In this article for the Highlights of 2024 series, we review the latest advances in the biology of the germinal center response. These discoveries provide key insights into germinal center function and dysregulation, uncovering new opportunities for the development of more effective vaccines.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis, are often associated with mutations in stress granule proteins. Aberrant stress granule condensate formation is associated with disease, making it a potential target for pharmacological intervention. Here, we identified lipoamide, a small molecule that specifically prevents cytoplasmic condensation of stress granule proteins. Thermal proteome profiling showed that lipoamide stabilizes intrinsically disordered domain-containing proteins, including SRSF1 and SFPQ, which are stress granule proteins necessary for lipoamide activity. SFPQ has redox-state-specific condensate dissolving behavior, which is modulated by the redox-active lipoamide dithiolane ring. In animals, lipoamide ameliorates aging-associated aggregation of a stress granule reporter protein, improves neuronal morphology and recovers motor defects caused by amyotrophic lateral sclerosis-associated FUS and TDP-43 mutants. Thus, lipoamide is a well-tolerated small-molecule modulator of stress granule condensation, and dissection of its molecular mechanism identified a cellular pathway for redox regulation of stress granule formation.

RNA binding proteins (RBP) of the ZFP36 family limit the differentiation and effector functions of CD4 and CD8 T cells, but little is known of their expression or function in regulatory T (Treg) cells. By using Treg cell-restricted deletion of Zfp36 family members we identify the role of Zfp36l1 and Zfp36l2 in Treg cells to maintain immune homeostasis. Mice with Treg cells deficient in these RBP display an inflammatory phenotype with an expansion in the numbers of type-2 conventional dendritic cells, T effector cells, T follicular helper and germinal center B cells and elevated serum cytokines and immunoglobulins. In the absence of Zfp36l1 and Zfp36l2, the pool of cycling CTLA-4 in naïve Treg cells is reduced, Treg cells are less sensitive to IL-2 and IL-7 but are more sensitive to IFNγ. In mice lacking both RBP in Treg cells, the deletion of a single allele of Ifng is sufficient to ameliorate the pathology. Our results indicate that ZFP36L1 and ZFP36L2 regulate the availability of IFNγ and are required for the maintenance of Treg cell stability. Thus, ZFP36L1 and ZFP36L2 regulate multiple pathways that enable Treg cells to enforce immune homeostasis.

Public dialogue is crucial for understanding societal views on human embryo research, and the complexity and sensitivity of this topic require special considerations of how such dialogues are facilitated. Here, we identify enablers of effective dialogue, which can improve the design and delivery of engagement exercises related to embryo research.

Porcine respiratory coronavirus (PRCV) is a naturally occurring pneumotropic coronavirus in the pig, providing a valuable large animal model to study acute respiratory disease. PRCV pathogenesis and the resulting immune response were investigated in pigs, the natural large animal host. We compared 2 strains, ISU-1 and 135, which induced differing levels of pathology in the respiratory tract to elucidate the mechanisms leading to mild or severe disease. The 135 strain induced greater pathology which was associated with higher viral load and stronger spike-specific antibody and T-cell responses. In contrast, the ISU-1 strain triggered mild pathology with a more balanced immune response and greater abundance of T regulatory cells. A higher frequency of putative T follicular helper cells was observed in animals infected with strain 135 at 11 days postinfection. Single-cell RNA-sequencing of bronchoalveolar lavage revealed differential gene expression in B and T cells between animals infected with 135 and ISU-1 at 1 day postinfection. These genes were associated with cell adhesion, migration, and immune regulation. Along with increased IL-6 and IL-12 production, these data indicate that heightened inflammatory responses to the 135 strain may contribute to pronounced pneumonia. Among bronchoalveolar lavage (BAL) immune cell populations, B cells and plasma cells exhibited the most gene expression divergence between pigs infected with different PRCV strains, highlighting their role in maintaining immune homeostasis in the respiratory tract. These findings indicate the potential of the PRCV model for studying coronavirus-induced respiratory disease and identifying mechanisms that determine infection outcomes.

The oncomutation lysine 27-to-methionine in histone H3 (H3K27M) is frequently identified in tumors of patients with diffuse midline glioma-H3K27 altered (DMG-H3K27a). H3K27M inhibits the deposition of the histone mark H3K27me3, which affects the maintenance of transcriptional programs and cell identity. Cells expressing H3K27M are also characterized by defects in genome integrity, but the mechanisms linking expression of the oncohistone to DNA damage remain mostly unknown. In this study, we demonstrate that expression of H3.1K27M in the model plant Arabidopsis thaliana interferes with post-replicative chromatin maturation mediated by the H3.1K27 methyltransferases ATXR5 and ATXR6. As a result, H3.1 variants on nascent chromatin remain unmethylated at K27 (H3.1K27me0), leading to ectopic activity of TONSOKU (TSK/TONSL), which induces DNA damage and genomic alterations. Elimination of TSK activity suppresses the genome stability defects associated with H3.1K27M expression, while inactivation of specific DNA repair pathways prevents survival of H3.1K27M-expressing plants. Overall, our results suggest that H3.1K27M disrupts the chromatin-based mechanisms regulating TSK activity, which causes genomic instability and may contribute to the etiology of DMG-H3K27a.

Extraembryonic tissues are essential for proper fetal development and exhibit great diversity across species. Despite its importance, human extraembryonic development has been relatively overlooked. Previously, we established an in vitro model to study human amniogenesis and extraembryonic mesoderm formation. In this article, I develop discussions on four topics inspired by this study: (1) Features of amniotic cell populations described to date. A recently reported early amniotic cell type is examined based on its signature genes to consider how this population should be incorporated into models of primate amniogenesis. (2) Molecular mechanisms underlying the effect of cell density in regulating non-neural ectoderm specification. Fate specification by positional cues in mouse is revisited and possible mechanisms are suggested by drawing insights from human epiblast models. (3) Potential applications of the three-dimensional culture we established. Primate amniotic ectoderm is postulated as a gastrulation-inducing signaling center, and our technique could be used to effectively model its interactions with epiblast. (4) Extraembryonic mesoderm development in human embryos. The obscure origin of primate extraembryonic mesoderm and implications from recent in vitro differentiation models using human pluripotent stem cells are explained. The key concepts explored here will stimulate further studies into both amnion and extraembryonic mesoderm during human and non-human primate development.

Insulin and other growth factors are key regulators of liver gene expression, including in metabolic diseases. Most of the phosphoinositide 3-kinase (PI3K) activity induced by insulin is considered to be dependent on PI3Kα. We used mice lacking p110α, the catalytic subunit of PI3Kα, to investigate its role in the regulation of liver gene expression in health and in metabolic dysfunction-associated steatotic liver disease (MASLD). The absence of hepatocyte PI3Kα reduced maximal insulin-induced PI3K activity and signaling, promoted glucose intolerance in lean mice and significantly regulated liver gene expression, including insulin-sensitive genes, in ad libitum feeding. Some of the defective regulation of gene expression in response to hepatocyte-restricted insulin receptor deletion was related to PI3Kα signaling. In addition, though PI3Kα deletion in hepatocytes promoted insulin resistance, it was protective against steatotic liver disease in diet-induced obesity. In the absence of hepatocyte PI3Kα, the effect of diet-induced obesity on liver gene expression was significantly altered, with changes in rhythmic gene expression in liver. Altogether, this study highlights the specific role of p110α in the control of liver gene expression in physiology and in the metabolic rewiring that occurs during MASLD.

Female human induced pluripotent stem cells frequently undergo X-chromosome inactivation (XCI) erosion, marked by X-inactive specific transcript (XIST) RNA loss and partial reactivation of the inactive X (Xi). This overlooked phenomenon limits our understanding of its impact on stem cell applications. Here, we show that XCI erosion is frequent and heterogeneous, leading to the reactivation of several X-linked genes. These are primarily located on the short arm of the X chromosome, particularly near escape genes and within H3K27me3-enriched domains, with reactivation linked to reduced promoter DNA methylation. Interestingly, escape genes further increase their expression from Xi upon XCI erosion, highlighting the critical role of XIST in their dosage regulation. Importantly, global (hydroxy)methylation levels and imprinted regions remain unaffected, and analysis of trilineage commitment and cardiomyocyte formation reveals that XCI erosion persists across differentiation. These findings underscore the need for greater awareness of the implications of XCI erosion for stem cell research and clinical applications.

The lungs are constantly exposed to the external environment and a myriad of antigenic challenges within the air. Chronic exposure to allergens and other airborne antigens can result in the formation of lymphocyte aggregates in the lung, which can harbor ectopic germinal centers (GCs). After allergen exposure, GCs that form in the lung are much smaller and less densely packed with B cells than lymph node GCs. Despite this, ectopic lung GCs support somatic hypermutation and affinity-based maturation as in lymph node GCs, and export memory B cells (MBCs) directly into the lung tissue. This demonstrates that the lung can locally diversify B cell responses and supports the generation of tissue MBC populations in situ.

Long-lasting immunological memory is a core feature of the adaptive immune system that allows an organism to have a potent recall response to foreign agents that have been previously encountered. Persistent humoral immunity is afforded by long-lived memory B cells and plasma cells, which can mature in germinal centers (GCs) in secondary lymphoid organs. The development of new GC-derived immunity diminishes with age, thereby impairing our immune system's response to both natural infections and vaccinations. This review will describe the current knowledge of how aging affects the cells and microenvironment of the GC. A greater understanding of how the GC changes with age, and how to circumvent these changes, will be critical for tailoring vaccines for older people. This area of research is critical given the twenty-first century will witness a doubling of the aging population and an increased frequency of pandemics.

Metastasis is the spread of cancer cells from primary tumours to distant organs and is the cause of 90% of cancer deaths globally. Metastasizing cancer cells are uniquely vulnerable to immune attack, as they are initially deprived of the immunosuppressive microenvironment found within established tumours. There is interest in therapeutically exploiting this immune vulnerability to prevent recurrence in patients with early cancer at risk of metastasis. Here we show that inhibitors of cyclooxygenase 1 (COX-1), including aspirin, enhance immunity to cancer metastasis by releasing T cells from suppression by platelet-derived thromboxane A (TXA). TXA acts on T cells to trigger an immunosuppressive pathway that is dependent on the guanine exchange factor ARHGEF1, suppressing T cell receptor-driven kinase signalling, proliferation and effector functions. T cell-specific conditional deletion of Arhgef1 in mice increases T cell activation at the metastatic site, provoking immune-mediated rejection of lung and liver metastases. Consequently, restricting the availability of TXA using aspirin, selective COX-1 inhibitors or platelet-specific deletion of COX-1 reduces the rate of metastasis in a manner that is dependent on T cell-intrinsic expression of ARHGEF1 and signalling by TXA in vivo. These findings reveal a novel immunosuppressive pathway that limits T cell immunity to cancer metastasis, providing mechanistic insights into the anti-metastatic activity of aspirin and paving the way for more effective anti-metastatic immunotherapies.

Cell mechanical properties determine many physiological functions, such as cell fate specification, migration, or circulation through vasculature. Identifying factors that govern the mechanical properties is therefore a subject of great interest. Here, we present a mechanomics approach for establishing links between single-cell mechanical phenotype changes and the genes involved in driving them. We combine mechanical characterization of cells across a variety of mouse and human systems with machine learning-based discriminative network analysis of associated transcriptomic profiles to infer a conserved network module of five genes with putative roles in cell mechanics regulation. We validate in silico that the identified gene markers are universal, trustworthy, and specific to the mechanical phenotype across the studied mouse and human systems, and demonstrate experimentally that a selected target, , changes the mechanical phenotype of cells accordingly when silenced or overexpressed. Our data-driven approach paves the way toward engineering cell mechanical properties on demand to explore their impact on physiological and pathological cell functions.

Vaccines are less immunogenic in older adults, partly due to immunosenescence. Having previously shown that morning influenza vaccination may be more immunogenic in older adults (mean age 71), we assessed if this could be replicated in a younger cohort (mean age 57) and with a T-cell independent vaccine. This study examined whether diurnal timing of a single dose of Pneumovax® (PPV-23) and seasonal influenza vaccine influenced antibody responses in 140 healthy adults over the age of 50. Pneumococcal serotype-specific (PnPS) antibodies and Haemagglutination Inhibition Assays (HAI) were used to characterize antibody responses at Baseline, 1, 4, and 52 weeks post-vaccination. Protective thresholds were set at 0.35 μg/mL for two-thirds of PnPS tested (WHO) and a titre of ≥40 HAI for H1N1, H3N2, and B/Victoria strains. Both AM and PM cohorts showed increased Pn-specific antibodies to one PPV-23 dose at weeks 1, 4, and 52; however, time of day did not significantly influence antibody responses. Baseline immunity for pneumococcus was high (57.1 % AM, 50.0 % PM had WHO), and immunity was maintained with at least 7/12 serotypes elevated at 52 weeks. Time of day did not alter short- or long-term influenza antibody responses. H1N1 had the highest baseline immunity (67.6 % AM, 48.6 % PM had ≥40 HAI) and the most increased responses at week 4 post-vaccination (92.8 % AM, 94.1 % PM) that were maintained at 52 weeks post-vaccination (91.7 % AM, 89.3 % PM). The poorest serotype immunity was for the B/Victoria strain at all time points. Although time of day did not influence vaccine immunogenicity in AM and PM cohorts, sustained cohort-wide antibody responses were demonstrated in an older population. Identifying 18 % of the total cohort exhibited suboptimal responses to pneumococcal or influenza vaccines underscores the imperative for enhancing vaccine efficacy within this age group to reduce morbidity and mortality.

Biphasic in vitro oocyte maturation (IVM) can be offered as a patient-friendly alternative to conventional ovarian stimulation in in vitro fertilization (IVF) patients predicted to be hyper-responsive to ovarian stimulation. However, cumulative live birth rates after IVM per cycle are lower than after conventional ovarian stimulation for IVF. In different animal species, supplementation of IVM media with oocyte-secreted factors (OSFs) improves oocyte developmental competence through the expression of pro-ovulatory genes in cumulus cells. Whether the addition of OSFs in human biphasic IVM culture impacts the transcriptome of oocytes and cumulus cells retrieved from small antral follicles in minimally stimulated non-hCG-triggered IVM cycles remains to be elucidated. To answer this, human cumulus oocyte complexes (COCs) that were fully surrounded by cumulus cells or partially denuded at the time of retrieval were cultured in a biphasic IVM system either without or with the addition of pro-cumulin, a GDF9: BMP15 heterodimer. Oocytes and their accompanying cumulus cells were collected separately, and single-cell RNA-seq libraries were generated. The transcriptomic profile of cumulus cells revealed that pro-cumulin upregulated the expression of genes involved in cumulus cell expansion and proliferation while downregulating steroidogenesis, luteinization and apoptosis pathways. Moreover, pro-cumulin modulated the immature oocyte transcriptome during the prematuration step, including regulating translation, apoptosis and mitochondria remodeling pathways in the growing germinal vesicle (GV) oocytes. The addition of pro-cumulin also restored the transcriptomic profile of matured metaphase II (MII) oocytes that were partially denuded at collection. These results suggest that cumulus cell and oocyte transcriptome regulation by pro-cumulin may increase the number of developmentally competent oocytes after biphasic IVM treatment. Future studies should assess the effects of pro-cumulin addition in human biphasic IVM at the proteomic level and the embryological outcomes, particularly its potential to enhance outcomes of oocytes that are partially denuded at COC collection.

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The placental DNA methylation landscape is unique, with widespread partially methylated domains (PMDs). The placental "methylome" is conserved across mammals, a shared feature of many cancers, and extensively studied for links with pregnancy complications. Human trophoblast stem cells (hTSCs) offer exciting potential for functional studies to better understand this epigenetic feature; however, whether the hTSC epigenome recapitulates primary trophoblast remains unclear. We find that hTSCs exhibit an atypical methylome compared with trophectoderm and 1 trimester cytotrophoblast. Regardless of cell origin, oxygen levels, or culture conditions, hTSCs show localized DNA methylation within transcribed gene bodies and a complete loss of PMDs. Unlike early human trophoblasts, hTSCs display a notable absence of DNMT3L expression, which is necessary for PMD establishment in mouse trophoblasts. Remarkably, we demonstrate that ectopic expression of DNMT3L in hTSCs restores placental PMDs, supporting a conserved role for DNMT3L in de novo methylation in trophoblast development in human embryogenesis.

During the latter stages of their development, mammalian oocytes under dramatic chromatin reconfiguration, transitioning from a non-surrounded nucleolus (NSN) to a surrounded nucleolus (SN) stage, and concomitant transcriptional silencing. Although the NSN-SN transition is known to be essential for developmental competence of the oocyte, less is known about the accompanying molecular changes. Here we examine the changes in the transcriptome and DNA methylation during the NSN to SN transition in mouse oocytes.

Promoters of developmental genes in embryonic stem cells (ESCs) are marked by histone H3 lysine 4 trimethylation (H3K4me3) and H3K27me3 in an asymmetric nucleosomal conformation, with each sister histone H3 carrying only one of the two marks. These bivalent domains are thought to poise genes for timely activation upon differentiation. Here, we show that asymmetric bivalent nucleosomes recruit repressive H3K27me3 binders but fail to enrich activating H3K4me3 binders, thereby promoting a poised state. Strikingly, the bivalent mark combination further promotes recruitment of specific chromatin proteins that are not recruited by each mark individually, including the lysine acetyltransferase (KAT) complex KAT6B. Knockout of KAT6B blocks neuronal differentiation, demonstrating that KAT6B is critical for proper bivalent gene expression during ESC differentiation. These findings reveal how readout of the bivalent histone marks directly promotes a poised state at developmental genes while highlighting how nucleosomal asymmetry is critical for histone mark readout and function.

Animal genomes are packaged into chromatin, a highly dynamic macromolecular structure of DNA and histone proteins organised into nucleosomes. This accommodates packaging of lengthy genomic sequences within the physical confines of the nucleus while also enabling precise regulation of access to genetic information. However, histones existed before chromatin and have lesser-known functions beyond genome regulation. Most notably, histones are potent antimicrobial agents, and the release of chromatin to the extracellular space is a defence mechanism nearly as ancient and widespread as chromatin itself. Histone sequences have changed very little throughout evolution, suggesting the possibility that some of their 'non-canonical' functions are at play in parallel or in concert with their genome regulatory functions. In this Review, we take an evolutionary perspective of histone, nuclear chromatin and extracellular chromatin biology and describe the known extranuclear and extracellular functions of histones. We detail molecular mechanisms of chromatin release and extracellular chromatin sensing, and we discuss their roles in physiology and disease. Finally, we present evidence and give a perspective on the potential of extracellular histones to act as bioactive, cell modulatory factors.

Regulatory T (Treg) cells are essential for the maintenance of immunological tolerance, yet the molecular components required for their maintenance and effector functions remain incompletely defined. Inactivation of VPS34 in Treg cells led to an early, lethal phenotype, with massive effector T cell activation and inflammation, like mice lacking Treg cells completely. However, VPS34-deficient Treg cells developed normally, populated the peripheral lymphoid organs and effectively supressed conventional T cells . Our data suggest that VPS34 is required for the maintaining normal numbers of mature Treg. Functionally, we observed that lack of VPS34 activity impairs cargo processing upon transendocytosis, that defective autophagy may contribute to, but is not sufficient to explain this lethal phenotype, and that loss of VPS34 activity induces a state of heightened metabolic activity that may interfere with metabolic networks required for maintenance or suppressive functions of Treg cells.

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Metastatic melanoma remains a major clinical challenge. Large-scale genomic sequencing of melanoma has identified bona fide activating mutations in RAC1, which are associated with resistance to BRAF-targeting therapies. Targeting the RAC1-GTPase pathway, including the upstream activator PREX2 and the downstream effector PI3Kβ, could be a potential strategy for overcoming therapeutic resistance, limiting melanoma recurrence, and suppressing metastatic progression. Here, we used genetically engineered mouse models and patient-derived BRAFV600E-driven melanoma cell lines to dissect the role of PREX2 in melanomagenesis and response to therapy. While PREX2 was dispensable for the initiation and progression of melanoma, its loss conferred sensitivity to clinically relevant therapeutics targeting the MAPK pathway. Importantly, genetic and pharmacological targeting of PI3Kβ phenocopied PREX2 deficiency, sensitizing model systems to therapy. These data reveal a druggable PREX2/RAC1/PI3Kβ signaling axis in BRAF-mutant melanoma that could be exploited clinically.

Replication fork collision with a DNA nick can generate a one-ended break, fostering genomic instability. The opposing fork's collision with the nick could form a second DNA end, enabling conservative repair by homologous recombination (HR). To study mechanisms of nickase-induced HR, we developed the Flp recombinase "step arrest" nickase in mammalian cells. A Flp-nick induces two-ended, BRCA2/RAD51-dependent short tract gene conversion (STGC), BRCA2/RAD51-independent long tract gene conversion, and discoordinated two-ended invasions. HR pathways induced by a replication-independent break and the Flp-nickase differ in their dependence on BRCA1, MRE11, and CtIP. To determine the origin of the second DNA end during Flp-nickase-induced STGC, we blocked the opposing fork using a Tus/Ter replication fork barrier (RFB). Flp-nickase-induced STGC remained robust and two ended. Thus, a single replication fork's collision with a Flp-nick triggers two-ended HR, possibly reflecting replicative bypass of lagging strand nicks. This response may limit genomic instability during replication of nicked DNA.