Therapeutically targeting pathogenic T cells in autoimmune diseases has been challenging. Although LAG-3, an inhibitory checkpoint receptor specifically expressed on activated T cells, is known to bind to major histocompatibility complex class II (MHC class II), we demonstrate that MHC class II interaction alone is insufficient for optimal LAG-3 function. Instead, LAG-3's spatial proximity to T cell receptor (TCR) but not CD4 co-receptor, facilitated by cognate peptide-MHC class II, is crucial in mediating CD4+ T cell suppression. Mechanistically, LAG-3 forms condensate with TCR signaling component CD3ε through its intracellular FSAL motif, disrupting CD3ε/lymphocyte-specific protein kinase (Lck) association. To exploit LAG-3's proximity to TCR and maximize LAG-3-dependent T cell suppression, we develop an Fc-attenuated LAG-3/TCR inhibitory bispecific antibody to bypass the requirement of cognate peptide-MHC class II. This approach allows for potent suppression of both CD4+ and CD8+ T cells and effectively alleviates autoimmune symptoms in mouse models. Our findings reveal an intricate and conditional checkpoint modulatory mechanism and highlight targeting of LAG-3/TCR cis-proximity for T cell-driven autoimmune diseases lacking effective and well-tolerated immunotherapies.

The vasculature and mesenchyme exhibit distinct organ-specific characteristics adapted to local physiological needs, shaped by microenvironmental and cell-cell interactions from early development. To recapitulate this entire process, we co-differentiated mesoderm and endoderm within the same spheroid to vascularize lung and intestinal organoids from induced pluripotent stem cells (iPSCs). Bone morphogenetic protein (BMP) signaling fine-tuned the endoderm-to-mesoderm ratio, a critical step in generating appropriate proportions of endothelial and epithelial progenitors with tissue specificity. Single-cell RNA sequencing (scRNA-seq) revealed organ-specific gene signatures of endothelium and mesenchyme and identified key ligands driving endothelial specification. The endothelium exhibited tissue-specific barrier function, enhanced organoid maturation, cellular diversity, and alveolar formation on the engineered lung scaffold. Upon transplantation into mice, the organoid vasculature integrated with the host circulation while preserving organ specificity, further promoting organoid maturation. Leveraging these vascularized organoids, we uncovered abnormal endothelial-epithelial crosstalk in patients with forkhead box F1 (FOXF1) mutations. Multilineage organoids provide an advanced platform to study intricate cell-to-cell communications in human organogenesis and disease.

Vascular dementia (VaD), the second-leading cause of dementia, is primarily a white matter ischemic disease with no direct therapies. Cell-cell interactions within lesion sites dictate disease progression or repair. To elucidate key intercellular pathways, we employ a VaD mouse model with focal ischemia replicating many elements of the complex pathophysiology of human VaD combined with transcriptomic and functional analyses. By integrating cell-type-specific mouse VaD transcriptomes and human VaD single-nucleus RNA sequencing (snRNA-seq) data plus a custom ligand-receptor database (4,053 human and 2,032 mouse pairs), conserved dysregulated intercellular pathways in both species are identified. We demonstrate that two intercellular signaling systems, Serpine2-Lrp1 and CD39-A3AR, are disrupted in VaD. Reduced Serpine2 expression enhances oligodendrocyte progenitor cell (OPC) differentiation, promoting repair, while an A3AR-specific agonist-currently in clinical trials for psoriasis-restores tissue integrity and behavioral function in the VaD model. This study reveals intercellular signaling targets and provides a foundation for developing innovative therapies for VaD.

Ataxia telangiectasia mutated (ATM), a large gene with 63 exons, plays a critical role in the DNA damage response, and its loss of function increases cancer risk and affects the prognosis of cancer patients. However, interpreting the functional impact of ATM variants remains challenging because most are variants of uncertain significance (VUSs). Here, we assessed the functions of all 27,513 possible single-nucleotide variants (SNVs) in ATM. By using prime editing, we experimentally evaluated the effects of 23,092 SNVs on cell fitness in the presence of olaparib, thereby identifying critical residues. Using cancer genetics data and UK Biobank data, we found that our results are useful for estimating both cancer risk and prognosis. We also developed a deep learning model, DeepATM, which predicted such functional effects of the remaining 4,421 SNVs with unprecedentedly high accuracy. This complete evaluation of ATM variants supports precision medicine and provides a framework for addressing VUSs in other genes.

Recognition of exogenous RNA by Toll-like receptors (TLRs) is central to pathogen defense. Using two distinct binding pockets, TLR7 and TLR8 recognize RNA degradation products generated by endolysosomal nucleases. RNA modifications present in endogenous RNA prevent TLR activation; notably, pseudouridine-containing RNA lacks immunostimulatory activity. Indeed, this property has been critical to the successful implementation of mRNA technology for medical purposes. However, the molecular mechanism for this immune evasion has remained elusive. Here, we report that RNase T2 and PLD exonucleases do not adequately process pseudouridine-containing RNA to generate TLR-agonistic ligands. As a second safety mechanism, TLR8 neglects pseudouridine as a ligand for its first binding pocket and TLR7 neglects pseudouridine-containing RNA as a ligand for its second pocket. Interestingly, the medically used N1-methylpseudouridine also evades RNase T2, PLD3, and PLD4 processing but is able to directly activate TLR8. Taken together, our findings provide a molecular basis for self-avoidance by RNA-sensing TLRs.

The development of a multicellular organism is a highly intricate process tightly regulated by numerous genes and pathways in both spatial and temporal manners. Here, we present Flysta3D-v2, a comprehensive multi-omics atlas of the model organism Drosophila spanning its developmental lifespan from embryo to pupa. Our datasets encompass 3D single-cell spatial transcriptomic, single-cell transcriptomic, and single-cell chromatin accessibility information. Through the integration of multimodal data, we generated developmentally continuous in silico 3D models of the entire organism. We further constructed tissue development trajectories that uncover the detailed profiles of cell-type differentiation. With a focus on the midgut, we identified transcription factors involved in midgut cell-type regulation and validated exex as a key regulator of copper cell development. This extensive atlas provides a rich resource and serves as a systematic platform for studying Drosophila development with integrated single-cell data at ultra-high spatiotemporal resolution.

India has been underrepresented in genomic surveys. We generated whole-genome sequences from 2,762 individuals in India, capturing the genetic diversity across most geographic regions, linguistic groups, and historically underrepresented communities. We find most Indians harbor ancestry primarily from three ancestral groups: South Asian hunter-gatherers, Eurasian Steppe pastoralists, and Neolithic farmers related to Iranian and Central Asian cultures. The extensive homozygosity and identity-by-descent sharing among individuals reflects strong founder events due to a recent shift toward endogamy. We uncover that most of the genetic variation in Indians stems from a single major migration out of Africa that occurred around 50,000 years ago, followed by 1%-2% gene flow from Neanderthals and Denisovans. Notably, Indians exhibit the largest variation and possess the highest amount of population-specific Neanderthal ancestry segments among worldwide groups. Finally, we discuss how this complex evolutionary history has shaped the functional and disease variation on the subcontinent.

Virtual cells are an emerging frontier at the intersection of artificial intelligence and biology. A key goal of these cell state models is predicting cellular responses to perturbations. The Virtual Cell Challenge is being established to catalyze progress toward this goal. This recurring and open benchmark competition from the Arc Institute will provide an evaluation framework, purpose-built datasets, and a venue for accelerating model development.

The reproductive cycle in females influences their social and sexual behaviors. In this issue of Cell, Wang et al. find that the calcium channel Cav3.2 in the brain's prefrontal cortex functions as a hormone-regulated electrophysiological switch. It connects hormonal state to brain activity, enabling the processing of signals from potential male partners and facilitating sexual behavior during the reproductive period.

The SARS-CoV-2 polymerase NiRAN domain initiates RNA capping. Previous results showed that both GTP and GDP can be utilized by NiRAN to yield GpppA together with RNAylated nsp9 (RNA-nsp9); however, the G-pocket substrate selection and the working mechanism of NiRAN remain unclear. Small et al. questioned the binding of the non-hydrolyzable GTP analog GMPPNP in the G-pocket of the RTC:RNA-nsp9:GMPPNP structure (PDB: 8GWE) and proposed that the GTP-mediated RNA-capping mechanism remains unresolved. Here, we show the optimized density derived from the original data to support the modeling of GMPPNP, and we reveal why the alternative data processing method failed to obtain density results. We provide additional biochemical and structural evidence by using GTP, GDP, GMPPNP, and GDP⋅BeF3- as probes to clarify the GTP-mediated RNA-capping mechanism and reconcile the two currently known models by using GTP and GDP as substrates. This Matters Arising Response addresses the Small et al. (2025) Matters Arising paper, published concurrently in Cell.

Exercise has well-established health benefits, yet its molecular underpinnings remain incompletely understood. We conducted an integrated multi-omics analysis to compare the effects of acute vs. long-term exercise in healthy males. Acute exercise induced transient responses, whereas repeated exercise triggered adaptive changes, notably reducing cellular senescence and inflammation and enhancing betaine metabolism. Exercise-driven betaine enrichment, partly mediated by renal biosynthesis, exerts geroprotective effects and rescues age-related health decline in mice. Betaine binds to and inhibits TANK-binding kinase 1 (TBK1), retarding the kinetics of aging. These findings systematically elucidate the molecular benefits of exercise and position betaine as an exercise mimetic for healthy aging.

Eukaryotic life evolved over a billion years ago when ancient cells engulfed and integrated prokaryotes to become modern mitochondria and chloroplasts. Sacoglossan "solar-powered" sea slugs possess the ability to acquire organelles within a single lifetime by selectively retaining consumed chloroplasts that remain photosynthetically active for nearly a year. The mechanism for this "animal photosynthesis" remains unknown. Here, we discovered that foreign chloroplasts are housed within novel, host-derived organelles we term "kleptosomes." Kleptosomes use ATP-sensitive ion channels to maintain a luminal environment that supports chloroplast photosynthesis and longevity. Upon slug starvation, kleptosomes digest stored chloroplasts for additional nutrients, thereby serving as a food source. We leveraged this discovery to find that organellar retention and digestion of photosynthetic cargo has convergently evolved in other photosynthetic animals, including corals and anemones. Thus, our study reveals mechanisms underlying the long-term acquisition and evolutionary incorporation of intracellular symbionts into organelles that support complex cellular function.

The Nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain initiates mRNA capping in coronaviruses through a GDP-polyribonucleotidyltransferase reaction, with RNA covalently linked to nsp9. GDP is the preferred substrate for this reaction, but the NiRAN domain can also utilize GTP to produce an authentic 5' RNA cap structure, though the GTP-mediated mechanism is unclear. Yan and colleagues claimed to have delineated the reaction mechanism from the analysis of a cryoelectron microscopy (cryo-EM) structure of a trapped catalytic intermediate of the SARS-CoV-2 NiRAN domain with a β-γ-non-hydrolyzable GTP analog (GMPPNP) and RNA-nsp9 (PDB: 8GWE). We show that the cryo-EM data used to derive PDB: 8GWE do not support the presence of GMPPNP in the NiRAN active site, and the resulting atomic model is incompatible with fundamental chemical principles. We conclude that Yan and colleagues' conclusions are not experimentally supported and the mechanism for GTP-mediated RNA capping by the SARS-CoV-2 NiRAN domain remains unresolved. This Matters Arising paper is in response to Yan et al. (2022), published in Cell. See also the response by Huang et al. (2025), published in this issue.

This study reports the first-in-human application of iPSC-derived CD19/BCMA dual-targeting chimeric antigen receptor-natural killer (CAR-NK) cells (QN-139b) in a patient with severe, diffuse cutaneous systemic sclerosis. The allogeneic product was genetically edited for reduced alloreactivity and improved in vivo performance, with no structural chromosomal abnormalities detected. The treatment led to significant B cell depletion with minimal toxicity, similar to CAR T cell therapy. The patient showed marked clinical improvements during the 6-month follow-up, including reduced autoantibodies and reversed fibrosis, which are resistant to conventional treatments. Single-cell analysis of peripheral blood revealed that the treatment shifted B cells toward more naive phenotypes and eliminated pathogenic B cells. Proteomic studies demonstrated suppression of inflammation and fibrosis, enhanced tissue regeneration, and improved angiogenesis. Pathological evaluation confirmed the elimination of infiltrated lymphocytes from affected skin along with restored skin and microvascular structure. These findings suggest QN-139b is a promising immune-modulatory treatment for severe autoimmune diseases.

Sleep is known to promote tissue growth and regulate metabolism, partly by enhancing growth hormone (GH) release, but the underlying circuit mechanism is unknown. We demonstrate how GH release, which is enhanced during both rapid eye movement (REM) and non-REM (NREM) sleep, is regulated by sleep-wake-dependent activity of distinct hypothalamic neurons expressing GH-releasing hormone (GHRH) and somatostatin (SST). SST neurons in the arcuate nucleus suppress GH release by inhibiting nearby GHRH neurons that stimulate GH release, whereas periventricular SST neurons inhibit GH release by projecting to the median eminence. GH release is associated with strong surges of both GHRH and SST activity during REM sleep but moderately increased GHRH and decreased SST activity during NREM sleep. Furthermore, we identified a negative feedback pathway in which GH enhances the excitability of locus coeruleus neurons and increases wakefulness. These results elucidate a circuit mechanism underlying bidirectional interactions between sleep and hormone regulation.

Biased agonism of G protein-coupled receptors (GPCRs) offers potential for safer medications. Current efforts have explored the balance between G proteins and β-arrestin; however, other transducers like GPCR kinases (GRKs) remain understudied. GRK2 is essential for β2 adrenergic receptor (β2AR)-mediated glucose uptake, but β2AR agonists are considered poor clinical candidates for glycemic management due to Gs/cyclic AMP (cAMP)-induced cardiac side effects and β-arrestin-dependent desensitization. Using ligand-based virtual screening and chemical evolution, we developed pathway-selective agonists of β2AR that prefer GRK coupling. These compounds perform well in preclinical models of hyperglycemia and obesity and demonstrate a lower potential for cardiac and muscular side effects compared with standard β2-receptor agonists and incretin mimetics, respectively. Furthermore, the lead candidate showed favorable pharmacokinetics and was well tolerated in a placebo-controlled clinical trial. GRK-biased β2AR partial agonists are thus promising oral alternatives to injectable incretin mimetics used in the treatment of type 2 diabetes and obesity.

Next-generation sequencing is pivotal for diagnosing inborn errors of immunity (IEI) but predominantly yields variants of uncertain significance (VUS), creating clinical ambiguity. Activated PI3Kδ syndrome (APDS) is caused by gain-of-function (GOF) variants in PIK3CD or PIK3R1, which encode the PI3Kδ heterodimer. We performed massively parallel base editing of PIK3CD/PIK3R1 in human T cells and mapped thousands of variants to a clinically important readout (phospho-AKT/S6), nominating >100 VUS and unannotated variants for functional classification and validating 27 hits. Leniolisib, an FDA-approved PI3Kδ inhibitor, rescued aberrant signaling and dysfunction in GOF-harboring T cells and revealed partially drug-resistant PIK3R1 hotspots that responded to novel combination therapies of leniolisib with mTORC1/2 inhibition. We confirmed these findings in T cells from APDS patients spanning the functional spectrum discovered in the screen. Integrating our screens with population-level genomic studies revealed that APDS may be more prevalent than previously estimated. This work exemplifies a broadly applicable framework for removing ambiguity from sequencing in IEI.

Glutamatergic neuronal activity promotes proliferation of both oligodendrocyte precursor cells (OPCs) and gliomas, including diffuse midline glioma (DMG). However, the role of neuromodulatory brainstem neurons projecting to midline structures where DMGs arise remains unexplored. Here, we demonstrate that midbrain cholinergic neuronal activity modulates OPC and DMG proliferation in a circuit-dependent manner. Optogenetic stimulation of the cholinergic pedunculopontine nucleus (PPN) promotes glioma growth in pons, while stimulation of the laterodorsal tegmentum nucleus (LDT) drives proliferation in thalamus. DMG-bearing mice exhibit higher acetylcholine release and increased cholinergic neuronal activity over the disease course. In co-culture, cholinergic neurons enhance DMG proliferation, and acetylcholine directly acts on DMG cells. Single-cell RNA sequencing revealed high CHRM1 and CHRM3 expression in primary DMG samples. Pharmacological or genetic blockade of M1/M3 receptors abolished cholinergic activity-driven DMG proliferation. Taken together, these findings demonstrate that midbrain cholinergic long-range projections promote activity-dependent DMG growth, mirroring a parallel proliferative effect on healthy OPCs.

Developmental gene expression is regulated by the dynamic interplay of histone H3 lysine 4 (H3K4) and histone H3 lysine 27 (H3K27) methylation, yet the physiological roles of these epigenetic modifications remain incompletely understood. Here, we show that mice depleted for all forms of H3K4 methylation, using a dominant histone H3-lysine-4-to-methionine (H3K4M) mutation, succumb to a severe loss of all major blood cell types. H3K4M-expressing hematopoietic stem cells (HSCs) and committed progenitors are present at normal numbers, indicating that H3K4 methylation is dispensable for HSC maintenance and commitment but essential for progenitor cell maturation. Mechanistically, we reveal that H3K4 methylation opposes the deposition of repressive H3K27 methylation at differentiation-associated genes enriched for a bivalent (i.e., H3K4/H3K27-methylated) chromatin state in HSCs and progenitors. Indeed, by concomitantly suppressing H3K27 methylation in H3K4-methylation-depleted mice, we rescue the acute lethality, hematopoietic failure, and gene dysregulation. Our results provide functional evidence for the interaction between two crucial chromatin marks in mammalian tissue homeostasis.