Cell Reports

The generation of an effective T cell response against an antigen depends on the recognition of the antigen by the T cell receptor (TCR), followed by T cell priming, initiating coordinated biophysical, biochemical and proliferative changes that drive differentiation into effector and memory clones. The immunological environments in which priming occurs, such as natural infection or vaccination, influences the quality and persistence of memory T cells, but the long-term impacts remain incompletely understood. Here, we investigate how the mode of priming shapes durable antigen-specific CD4+ T cell memory, utilising two cohorts 3-4 years after initial antigen encounter: individuals recovered from SARS-CoV-2 infection and infection-naive individuals who received a SARS-CoV-2 vaccination. Using ex vivo single-cell RNA sequencing, paired TCR sequencing and in vitro functional analyses, we characterise the transcriptional, clonal and functional profile of Spike-specific CD4+ T cells. Across both cohorts, CD4+ T cell responses against spike epitopes S166-180, S751-765 and S866-880, were immunodominant, with shared public TCR clonotypes indicating conserved antigen-recognition regardless of mode of priming. Despite this shared specificity, infection-primed individuals exhibited greater TCR repertoire diversity and lower CDR3{beta} sequence convergence. Transcriptionally, infection-primed cells exhibited a more cytotoxic and effector phenotype, while vaccine-primed cells preferentially adopted T follicular helper (Tfh)- and Th1-associated phenotypes. Infection-primed individuals also displayed enrichment of cell adhesion and integrin signalling pathways, with a greater proportion of spike-specific CD4+ T cells expressing 4{beta}1 integrin subunits, consistent with enhanced migratory and effector potential. Collectively, our findings demonstrate that the mode of antigen priming may influence the long-term CD4+ T cell memory states, influencing TCR repertoire diversity, functional differentiation and tissue-homing potential, years after the initial immune response.

The piriform cortex, the largest paleocortical domain and a central olfactory cortex, is classically described as a three-layered structure, with layer II subdivided into semilunar and superficial pyramidal neurons. However, its cellular composition, developmental logic, and evolutionary relationship with the six-layered neocortex remain incompletely defined. Here, we aimed to resolve piriform cortical cell types and laminar organization and compare their molecular programs across mammalian cortical regions and the reptilian cortex. We performed single-nucleus RNA sequencing of the microdissected piriform cortex and integrated neuronal lineage data with published datasets, with spatial validation via the Allen Mouse Brain Atlas. Interspecies analyses identified piriform-dominant glutamatergic populations marked by Unc13c/Lmo3/Rora and an Rorb/Reln/Ntng1-enriched sensory-recipient subtype localized to the superficial layer IIa, consistent with semilunar neurons. Spatial transcriptomic mapping revealed that piriform layer IIb contains neocortical upper-layer-like corticocortical neurons, while piriform layer III segregates into layer V-like (IIIa), layer VI-like (IIIb), and VIb/VII (subplate)-equivalent populations, indicating a neocortex-like laminar framework with an inverted positioning of sensory recipients and corticocortical compartments relative to the neocortex. GABAergic neurons largely conformed to canonical MGE- and CGE-derived lineages but included a piriform-specific Rgs9+Pde7b+ subtype consistent with an LGE-related origin, suggesting region-specific diversification of GABAergic neurons. Disease ontology enrichment linked hippocampus-dominant glutamatergic programs to Alzheimers-related genes and piriform-dominant (and pan-cortical) excitatory and inhibitory programs to autism spectrum disorder and intellectual disability, implicating coordinated E/I circuit specializations in area-selective vulnerability. These findings support a revised view of the paleocortex as a divergent specialization of a conserved, multilayered cortical program and provide molecular markers and a comparative framework for studying cortical evolution and region-specific disease susceptibility.

Type-I interferons (IFN-I) and IFN-stimulated genes (ISGs) are central to antiviral defense, while dysregulation can drive autoimmunity. IFNB1 expression is controlled by a highly ordered multiprotein complex composed of IRF3/7, NF{kappa}B, and ATF2/c-Jun (AP-1) that recruit coactivators and chromatin-remodeling proteins to expose the IFNB1 promoter for the RNA polymerase II (RNA Pol II) transcriptional machinery. Here, we identified the paraspeckle protein non-POU domain-containing octamer-binding protein (NONO) as a critical facilitator of innate immune activation. Loss of NONO enhanced replication of multiple orthoflaviviruses including West Nile virus due to impaired induction of IFN-I and ISGs. NONO did not affect upstream signaling but instead promoted chromatin accessibility and promoter access for RNA Pol II to drive expression of IFNB1, ISGs, and proinflammatory cytokines. These findings position NONO as a key regulator of antiviral gene expression and reveal chromatin-levels of control that determine effective antiviral immunity.

Mitochondrial dysfunction has long been known to underlie neurodegeneration, yet the contribution of mitochondrial turnover dynamics to functional aspects of defined synaptic circuits remains poorly understood. Here, we show that the mitochondrial proteome and turnover rates of hippocampal glutamatergic and GABAergic neurons is differentially remodelled by experience, with distal axon terminals of somatostatin-positive neurons exhibiting most dramatic changes, suggesting a form of metabolic plasticity at GABAergic synapses coupled to circuit activity. Conditional ablation of the mitochondrial transport proteins MIRO1 or TRAK1 (whose human mutations cause congenital epilepsy) stalled turnover at axon terminals driving loss of cristae, without affecting synapse or neuron integrity. The resulting reduction in synaptic GABA levels destabilized network oscillations and led to hyperexcitability, culminating in recurrent seizures and premature death. Post-weaning gene therapy efficiently reversed mitochondrial alterations and ameliorated the epileptic phenotype, underscoring the crucial role of mitochondrial turnover at central GABAergic synapses for balancing network excitability.

The long non-coding RNA Negative Regulator of Antiviral Response (NRAV) is known to suppress antiviral immunity by regulating interferon response, but its functional role in tumor immunology remains poorly understood. We examined the relevance of NRAV in melanoma and found that high NRAV expression was associated with poor survival, reduced inflammatory pathway activation, and resistance to immune checkpoint blockade. Bulk and single-cell transcriptomic profiling indicates that NRAV expression is selectively enriched in malignant cells suggesting a potential cancer cell-intrinsic function. To examine whether NRAV can regulate inflammatory responses in melanoma cells, we manipulated the levels of NRAV in the BRAF-mutant A375 melanoma model and characterized the expression of key interferon-stimulated genes (ISGs) following type-I and type-II interferon stimulation. Our findings reveal that the stable NRAV overexpression blunts the induction of key ISGs, whereas NRAV knockdown reciprocally amplifies their transcription. Subcellular fractionation revealed that NRAV is predominantly localized to the nuclear compartment of melanoma cells and the overexpression of NRAV altered regulatory histone marks on the target ISG promoters including MX1 and IFITM3. Collectively, these findings establish NRAV as a tumor-intrinsic epigenetic regulator of interferon signaling, highlighting its potential contribution to melanoma immune evasion.

Metabolic pathways are increasingly recognized as tractable targets in aging and disease. Building on prior work demonstrating that supplementation with low-molecular weight metabolites (amino acids, vitamins, and their intermediates) can extend lifespan in Caenorhabditis elegans, we focused on pantothenate (vitamin B5), which is dysregulated in sarcopenic muscle and in several neurodegenerative and metabolic disorders. Pantothenate is the obligate precursor of coenzyme A through a short, highly conserved biosynthetic pathway in which loss-of-function mutations can cause neurodegeneration with brain iron accumulation. In C. elegans, the longevity curtailing transcription factor DAF-16/FOXO has a conserved binding element in the promoter region of pnk-1, encoding the first enzyme (PNK-1) in the coenzyme A pathway, and pnk-1 is markedly upregulated in long-lived daf-2 (insulin/-like receptor) mutants, implicating coenzyme A metabolism in longevity. Here, we demonstrate that CoA levels naturally increase during early life and decrease towards older age in C. elegans. Dietary pantothenate supplementation increases coenzyme A levels with minimal effects on lifespan but systemic effects on lipid metabolism, mitochondrial dynamics, and muscle structure under basal conditions. Under DAF-16-associated stress conditions, including heat and oxidative stress, pnk-1 expression is upregulated and pantothenate supplementation robustly extends lifespan and improves mobility. Finally, we demonstrate dysregulation of daf-16 and pnk-1 expression in amyotrophic lateral sclerosis (ALS) models, in which pantothenate supplementation confers both lifespan extension and cholinergic neuroprotection.

Striatal cholinergic interneurons (CINs) exhibit a transient pause in tonic firing in response to salient stimuli, a hallmark of reinforcement learning that becomes synchronized with learning. Although thalamostriatal and dopaminergic inputs have been implicated in this pause, the underlying circuit mechanisms remain unclear. Here, we combine optogenetics, electrophysiology, and genetic approaches to examine inhibitory interactions within the CIN network. Synchronized activation of CINs in striatal slices elicited robust feedback inhibition in CINs, suppressing firing and generating pause-like responses. This inhibition was mediated by GABAA receptors and required beta2-containing nicotinic acetylcholine receptors (beta2-nAChR), and could be recruited by thalamostriatal activation. Dopamine is not required for this circuit but modulates it via D2 receptors. Surprisingly, cell-type-specific silencing and striatal beta2-nAChR deletion excluded local GABAergic sources, whereas retrograde beta2-nAChR deletion abolished inhibition, revealing an extrastriatal pathway. These findings identify a long-range inhibitory mechanism linking synchronized cholinergic activity to pause generation in striatal circuits.

The ability to adapt to changing environmental conditions is essential for cellular survival. A central feature of the eukaryotic stress response is the inhibition of bulk mRNA nuclear export, while stress-induced transcripts are specifically exported. However, the molecular mechanisms that simultaneously inhibit bulk mRNA export while mediating selective export of specific transcripts remain poorly understood. Here, we performed comparative phosphoproteomic analyses of S. cerevisiae under different stress conditions. We identified a heat shock-induced increase in phosphorylation within the N-terminal domain of the mRNA export adaptor Yra1. Preventing this phosphorylation significantly reduces nuclear accumulation of poly(A)+ RNA during heat stress and concomitantly enhances the export of heat-induced transcripts. Mechanistically, Yra1 phosphorylation appears to weaken its interaction with the export receptor Mex67, thereby contributing to nuclear accumulation of bulk poly(A)+ RNA under heat stress. Together, our findings establish Yra1 phosphorylation as a previously unrecognized regulatory mechanism that promotes nuclear mRNA accumulation during heat stress and contributes to selective mRNA export.

Aging is associated with a progressive decline in cognitive function, including the ability to adapt behavior based on its consequences. While classical conditioning in Drosophila melanogaster has provided key insights into reinforcement learning, how aging impacts operant learning and adjusting behavior based on action outcomes remains unclear. Here, we used a closed-loop optogenetic paradigm to test how aging affects operant learning and the role of dopaminergic neurons (DANs; PPL1 and PAM) in this process. Activation of distinct DAN subsets revealed that both young and aged flies retain PPL1-dependent avoidance learning, indicating preserved action-outcome learning with age. However, learning in aged flies depended on prolonged reinforcement: unlike young flies, they failed to learn under shorter optogenetic stimulation durations, suggesting an aging-associated reduced dopaminergic reinforcement rather than a loss of learning capacity. Moreover, reducing mitochondrial antioxidant capacity via SOD2 knockdown in PPL1 neurons phenocopied the aging-related deficit, implicating oxidative stress in impaired reinforcement signaling. In contrast, broad PAM neuron activation drove robust learning across ages and stimulation regimes. Nevertheless, functional dissection of PAM subpopulations revealed subtype-specific aging-associated vulnerability within dopaminergic circuits. In line with the behavioral data, PPL1 but not PAM neurons exhibited age-dependent reductions in cell size. Together, our findings suggest that aging selectively reduces dopaminergic reinforcement in a DAN subtype-dependent manner while preserving the capacity for operant learning. Increasing reinforcement length rescues this deficit, indicating that altered dopaminergic signaling, rather than impaired learning capacity, is a key driver of age-related cognitive decline.

Brain capillary pericytes are essential components of the neurovascular unit, yet the extent of their molecular heterogeneity within intact vascular networks remains poorly understood. Here, we combined spatial imaging with reanalysis of independent single-cell transcriptomic datasets to investigate the molecular organization of adult mouse brain pericytes. We identified spatial organization of pericyte molecular heterogeneity associated with anatomical region and position within the vascular network, including recurrent differences in ACE2, CASQ2, Igf2, and OPN expression. Moreover, pericyte molecular phenotypes varied with aging, acute ischemia, and circadian phase. Notably, light-dark phase emerged as a major axis of transcriptional variation, with pericytes exhibiting distinct circadian phase-associated molecular states. Together, these data demonstrate that adult brain pericytes exhibit spatially organized and dynamically regulated molecular heterogeneity associated with vascular and physiological context.

Black-footed ferrets (Mustela nigripes) are highly susceptible to sylvatic plague caused by Yersinia pestis, but the genetic basis of this vulnerability remains poorly understood. Here, comparative immunogenomic analyses across Carnivora species identified a conserved class of immunoglobulin lambda variable (IGLV) genes with unusually long antigen-binding sites (CDRL1) that are common among Caniformia species but absent in Feliformia species. First discovered in the domestic ferret (Mustela putorius furo), these genes encode tyrosine-rich and anionic motifs resembling the chemokine receptor CCR5 and contain experimentally validated sulfotyrosines previously associated with pathogen-interacting interfaces. Evolutionary analyses revealed distinct selective pressures across Caniformia lineages and showed strong purifying selection acting on long-CDRL1 IGLV genes in mustelids and bears. Antibody repertoire sequencing demonstrated that these genes are actively utilized in expressed repertoires and that their usage correlates with evolutionary conservation. Functional analyses of monoclonal antibodies derived from the long-CDRL1 IGLV gene identified an antibody that significantly reduced intracellular Y. pestis survival in macrophages and revealed a positive correlation between anti-plague activity and sulfotyrosine signal. Notably, all analyzed black-footed ferrets carried a frameshifting deletion in the long-CDRL1 IGLV gene resulting in loss of its expression in antibody repertoires. Together, these findings uncover a germline-encoded immunoglobulin feature conserved across dog-like carnivores and suggest a potential link between antibody germline variation and immune responses to plague.

Cognitive flexibility (CF) declines during aging and is further impaired in neurodegenerative diseases such as Huntingtons disease (HD), yet the molecular mechanisms underlying these deficits remain poorly understood. Thalamostriatal (T-S) synapses are critical for CF, and we previously identified Heat Shock Factor 1 (HSF1) as a regulator of T-S density in HD. However, how HSF1 regulates T-S synapses and whether it modulates cognitive flexibility (CF) remained unclear. Here, we combined HSF1 ChIP-seq, transcriptomics, synapto-proteomics, targeted genetic manipulations and behavioral analyses to study how HSF1 regulates T-S synapses and CF. We found HSF1 directly controls a transcriptional program governing postsynaptic architecture and actin cytoskeletal dynamics, which are disrupted in aging and HD. Loss of HSF1 drives selective destabilization of actin patches at T-S shaft synapses and impaired CF decline. Our results underscore a novel function for HSF1 in the regulation of striatal neural circuits with essential implications in the neurobiology of cognitive flexibility.

CXCL4 (PF4) is a chemokine stored in platelets that has pleiotropic effects across biological settings. These effects include driving of inflammation and fibrosis as well as reversal of the effects of ageing. We have recently demonstrated that CXCL4 function is driven, independently of known chemokine receptors, through binding to glycosaminoglycan (GAG) side chains on proteoglycans within the cell surface glycocalyx. In this study, we have used intravital imaging and radioactive tracer studies, in combination with an exogenous inhibitor and a GAG-binding CXCL4 mutant, to demonstrate that CXCL4 can enter the brain parenchyma of mice by binding to proteoglycans within the cell surface of the endothelial glycocalyx of the blood-brain barrier (BBB). Furthermore, we have also demonstrated that CXCL4 directly promotes neurogenesis in vitro, which is mediated by its ability to oligomerise and bind to GAGs. These findings provide a molecular mechanism for CXCL4 uptake and function within the brain. Furthermore, these data have important implications for understanding CXCL4 during health and disease that may enable development of CXCL4-related therapeutics for inflammatory diseases and ageing.

The liver is a metabolic hub with a high protein turnover that renders it uniquely susceptible to proteotoxic stress. Perturbation of proteostasis, either by proteasomal inhibitors or in chronic liver diseases, could adversely impact liver physiology. Here, we show that proteasomal inhibition unexpectedly suppresses basal type I interferon (IFN-I) signaling in the murine liver. Proteasomal inhibition by bortezomib selectively downregulates a subset of interferon-stimulated genes (ISGs), among which USP18 and ISG15 emerge as critical determinants of hepatocellular survival. We identify USP18 as a central cytoprotective factor that prevents proteotoxic apoptosis independently of its deubiquitinase activity, but strictly requires its scaffolding function mediated by isoleucine-60 and interaction with STAT2. Mechanistically, proteotoxic stress disrupts IRF9 nuclear translocation, attenuating USP18 transcription, and drives USP18 and other ISGs into insoluble aggregates with kinetics distinct from canonical IFN-I-induced insolubility. Strikingly, IFN-I priming preserves ISG solubility, restores USP18 abundance, and confers resistance to proteotoxic cell death. Together, these findings uncover an unanticipated link between proteostasis and innate immune signaling, and establish the USP18-STAT2 axis to enhance hepatic resilience under proteotoxic stress.

Caenorhabditis elegans, like other animals, relies on attractive odours for foraging and on aversive odours for avoidance of pathogens. An odour produced by pathogenic bacterium Pseudomonoas aeruginosa induces immune response as well as avoidance in C. elegans via AWB olfactory neurons. We asked whether AWB neurons provide broader immunity to a wide range of pathogens. We activated AWB neurons using three chemically distinct bacterial odours and found that activation indeed induces protective immunity. Conversely, silencing of AWB neurons early during infection enhances susceptibility to infections. Mechanistic investigation revealed that the activation of AWB using odours upregulates detoxification pathways and other protective pathways in non-neuronal tissues. Specifically, odour exposure activates UDP-glucuronosyltransferase UGT-18 in the intestine that protects worms from the phenazine toxin of Pseudomonas aeruginosa and promotes broader immunity to Gram-negative bacteria (P. aeruginosa and Salmonella enterica), Gram-positive bacteria, (Enterococcus faecalis and Staphylococcus aureus), and yeast (Cryptococcus neoformans). Altogether, our findings highlights microbial odours as non-canonical molecular patterns for activating immune responses.

RTTN (rotatin) is a centrosomal protein mutated in severe malformations of cortical development, yet how its dysfunction disrupts human corticogenesis has remained unclear. Here, we show that RTTN has an unrecognized function at the core of the translation machinery. Using human telencephalic and hippocampal organoids carrying distinct RTTN alleles, together with single-cell and bulk transcriptomics, polysome profiling, and tRNA pseudouridine sequencing, we find that RTTN is enriched in cycling first-trimester neural progenitors and physically associates with ribosome-biogenesis and RNA-processing factors. RTTN mutations impair rRNA biogenesis and polysome assembly, reduce cytoplasmic ribosome density and nascent protein synthesis, and remodel the tRNA pseudouridylation landscape through both a PUS7L-dependent variable-arm signature and a broader RTTN-specific defect. These translational deficits are accompanied by prolonged mitosis, reduced entry into S-phase, and impaired interkinetic nuclear migration in mutant progenitors. Our findings redefine RTTN as a regulator of ribosome homeostasis and mRNA translation and implicate defective translational capacity as a driver of RTTN-associated microcephaly. Graphical AbstractRTTN sustains ribosome and tRNA homeostasis in human neural progenitors; its mutation disrupts mRNA translation, stalling progenitor proliferation and interkinetic nuclear migration, and driving cortical malformation and growth failure. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/744412v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@193ebb1org.highwire.dtl.DTLVardef@56c7d0org.highwire.dtl.DTLVardef@1586876org.highwire.dtl.DTLVardef@1322e21_HPS_FORMAT_FIGEXP M_FIG C_FIG

B cell-derived IgG in the dorsal root ganglia (DRG) drives neuropathic pain after peripheral nerve injury (PNI), but the site of B cell organization is unclear. Here, PNI induced leukocyte clusters in the DRG meninges, enveloped by lymphatic endothelium and apposed to high endothelial venules. These clusters resemble tertiary lymphoid structures (TLSs) with germinal center-like features, including germinal center B cells and plasma cells, and follicular dendritic and follicular helper T cells. Single-cell RNA sequencing revealed enrichment of germinal center B cells in the DRG meninges after PNI. Germinal center B cells regulate TLS organization: TLSs were absent after deletion of Ezh2 from germinal center-experienced B cells. Intrathecal CD20 monoclonal antibody to locally deplete B cells also disrupted TLS organization. Conversely, intrathecal B cell transfer to B cell-deficient (muMT) mice was sufficient for TLS organization after PNI. Allodynia did not develop when TLS organization was disordered. Similar TLSs formed in pig DRG after tail docking and in human donors with chronic pain, where B cell receptor clonotype analysis confirmed functional maturity. Together, these data establish that germinal center B cells are required for TLS organization, and that disrupting this process abolishes the development of neuropathic pain after PNI.

Macrophages play an important role in coordinating the antiviral response and post-transcriptional regulation of mRNA is an important element of the inflammatory gene expression required for defense against viral pathogens. N6-methyladenosine (m6A) deposition on mRNA by METTL3 constitutes one such post-transcriptional event which facilitates a cascade of downstream regulation via RNA decay and translation. We discovered that in THP1-derived and peripheral blood macrophages, m6A depletion with the METTL3 inhibitor STM2457 leads to enhanced proliferation of the human coronavirus OC43. Using TimeLapse-seq to comprehensively measure changes in abundance, RNA decay and transcription, we find that STM2457 downregulates the interferon response far upstream by reducing expression of both the type I interferon receptor and STAT1. We conclude that macrophages depend on m6A to support expression of interferon sensing machinery and in m6As absence, fail to mount as strong of a type I interferon response.

The Plasmodium falciparum circumsporozoite protein (PfCSP) is the major surface antigen on Pf sporozoites. WHO-recommended vaccines RTS,S/AS01E and R21/Matrix-M target the PfCSP major repeat region and C-terminal domain (ctCSP). Although multiple studies associated protection with antibody responses to ctCSP, only a few ctCSP-specific monoclonal antibodies (mAbs) have been characterized. Here, crystal structures of 11 Fab-ctCSP complexes reveal how mAbs against the conserved {beta}-epitope region achieve diverse modes of strain-transcending recognition, in contrast to mAbs to the hypervariable -epitope. Consistent with previous studies, ctCSP on sporozoites could be unmasked by mAbs that bind CSP repeats, with unmasking dependent on the mAb fine-specificity and binding mode. In vitro, ctCSP mAbs promoted stronger Fc-receptor signaling, cellular cytotoxicity, and phagocytosis than repeat region mAbs, while mAb combinations targeting distinct PfCSP epitopes modulated Fc-signaling and cellular cytotoxicity. This study provides a rationale for optimization of PfCSP-based immunogens to enhance Fc-mediated contributions to malaria vaccine efficacy.

Excitation/inhibition (E:I) imbalance is a convergent mechanism in neurodevelopmental disorders (NDDs), yet whether NDD risk genes disrupt excitatory and inhibitory neurons through shared or distinct molecular programs remains poorly understood. Using human pluripotent stem cell-derived cortical projection and medial ganglionic eminence-like inhibitory neurons, we show that loss-of-function mutations in the chromatin reader ZMYND11 produce cell-type-selective vulnerability in cortical excitatory neurons. ZMYND11-deficient excitatory neurons exhibit hyperexcitability accompanied by de-repression of BMP signaling, dysregulation of glutamate receptor expression, and a shift toward non-brain splicing isoforms, whereas these molecular signatures are largely absent in ZMYND11-deficient inhibitory neurons. This cell-type selectivity is associated with differential upregulation of RBFOX family splicing regulators. Structure-function analysis reveals that MYND domain is required for normal progenitor dynamics and neuronal excitability. Together, these findings indicate that E:I imbalance in ZMYND11-associated NDD arises primarily from cell-type-selective vulnerability of excitatory cortical projection neurons and identify MYND domain as a critical determinant of neuronal function. HighlightsO_LIZMYND11-deficient excitatory neurons (ExNs) show hyperexcitability. C_LIO_LIAltered BMP signaling & glutamate receptor levels are implicated in mutant ExNs. C_LIO_LIInhibitory neurons show attenuated response to ZMYND11 loss. C_LIO_LIThe MYND domain in ZMYND11 is a critical regulator of neuronal excitability. C_LI