Biology subjects

Chou, T.-F.

Publications and source records attributed to Chou, T.-F..

At least 19 recordsLinked to original sources

bioRxiv2026

Unbiased and scalable reduction of diverse bacterial genomes

The genome is a complex, integrated system where the functions and regulatory interactions of its many components remain poorly understood. Genome minimization aims to reduce genomic complexity by removing non-essential elements to reveal the fundamental building blocks of cellular life. However, current minimization strategies are often slow and species-specific due to a reliance on prior information, and limited to producing single, isolated strains, which obscures the diverse ways a genome can adapt to large-scale DNA removal. Here we show the development and application of Stochastic Lineage-based Iterative Minimization (SLIM) a modular, high-throughput platform for unbiased genome reduction across phylogenetically diverse bacteria. We apply SLIM to generate a library of genome-reduced Escherichia coli lineages. We then interrogate the lineages, identifying both universal and lineage-specific transcriptional and translational reprogramming in response to deletions. We demonstrate that these expression dynamics drive environment-dependent fitness, allowing us to pinpoint a single gene deletion in one genome-reduced lineage as the driver of a measurable environmental growth defect. Beyond E. coli, we successfully deploy SLIM in phylogenetically distinct bacterial taxa to rapidly reduce the genomes of Shigella flexneri and Pseudomonas putida, distinct genus and order respectively from E. coli, without species-specific optimization. Our results establish a scalable, generalizable framework for navigating the vast landscape of minimized genomes, providing a powerful new tool for functional discovery and the rational design of synthetic genomic chassis.

synthetic biology↗

bioRxiv2026

FAM136A is an essential chaperone for mitochondrial membrane protein biogenesis

The metabolic and signaling function of mitochondria rely on a network of chaperones within the inner membrane space (IMS) that regulate the biogenesis of nascent mitochondrial proteins. Using a genome wide CRISPRi screen we found that in human cells FAM136A is required for biogenesis of all three voltage-dependent anion channel (VDAC) paralogs, an abundant and essential family of {beta}-barrel metabolite transporters in the outer mitochondrial membrane (OM). FAM136A is a ubiquitously expressed essential gene, that is conserved in metazoa and plants. Using a combination of experiments in human cells and in vitro reconstitution, we determined that FAM136A associates with unfolded VDACs in the IMS; solubilizes nascent VDAC through a direct interaction; and facilitates insertion of VDAC into the OM. FAM136A also binds and chaperones a subset of -helical subunits of the electron transport chain. We therefore conclude that FAM136A is an IMS-resident chaperone, necessary and sufficient to maintain nascent membrane proteins in a folding-competent state to mediate their integration into the bilayer.

molecular biology↗

bioRxiv2026

ENAH Binding to the Huntingtin Proline-Rich Domain Modulates HTT1a Assembly

The proline-rich domain (PRD) of huntingtin (HTT), located C-terminal to the polyglutamine tract within the exon-1 region, plays a critical role in modulating the aggregation and toxicity of mutant HTT1a. To identify PRD-interacting proteins, we performed mass spectrometry analysis of PRD peptide pulldowns from human neurons. ENAH, a member of the Ena/VASP family of actin regulatory proteins, emerged as the top enriched interactor from neuronal membrane fractions. In neurons, ENAH colocalized with HTT1a species, suggesting a biologically relevant interaction. Because the EVH1 domain of ENAH binds polyproline motifs with high affinity, we examined its interaction with HTT1a using recombinant proteins. EVH1 directly bound HTT1a and unexpectedly formed condensate-like assemblies capable of recruiting HTT1a monomers. Remarkably, EVH1 robustly suppressed HTT1a oligomerization and promoted remodeling of preformed HTT1a fibrils in vitro. These findings demonstrate that ENAH-PRD interactions modulate HTT1a aggregation dynamics and identify EVH1 assembly as a potential proteostatic mechanism regulating mutant HTT1a states. More broadly, this work establishes a framework for investigating how EVH1-containing proteins may influence HTT1a proteostasis and neurotoxicity in Huntington's disease.

neuroscience↗

bioRxiv2026

Tetramerization and RNA-guided filament assembly control Schlafen-Argonaute antiphage defense

Two deeply conserved protein families, Argonaute (Ago) and Schlafen (SLFN), play defense roles in diverse prokaryotes and eukaryotes, including humans. Here, we identify a monophyletic group of proteins broadly distributed across bacteria and archaea that fuse a SLFN domain with an Ago core and a GHKL-family ATPase. Using SLFN-pAgo from Runella zeae as a model, we show that these proteins protect bacterial cells against bacteriophages by employing the Ago core as a guide-dependent sensor and the SLFN domain as a nuclease effector to induce abortive infection via tRNAs cleavage. Structural and biochemical analyses reveal that ATP binding by the GHKL domain drives tetramerization of SLFN-pAgo, reconstituting the canonical SLFN nuclease architecture found in mammalian proteins. Prior to target detection, non-canonical guide RNA binding induces the formation of long helical filaments, locking the SLFN domains in an inactive configuration. Guide-dependent recognition of complementary target DNA triggers massive structural rearrangements leading to filament disassembly and the induction of SLFN nuclease activity. Together, our findings uncover a new antiphage system that employs reversible guide- and ATP-mediated oligomerization to strictly regulate cooperation between an Ago sensor and a SLFN RNAse effector.

molecular biology↗

bioRxiv2026

TXNDC15 modulated quality control at the endoplasmic reticulum shapes ciliogenesis

At the endoplasmic reticulum (ER), membrane protein quality control is tightly regulated to ensure excess subunits are recognized and degraded to protect cellular homeostasis. Using genome wide CRISPR screens, we identified a factor of unknown function, thioredoxin domain containing protein 15 (TXNDC15), and showed that it regulates membrane protein stability by tuning the activity of the E3-ubiquitin ligase, MARCHF6. TXNDC15 modulates MARCHF6 in two opposing ways: first, it enhances the binding, ubiquitination, and degradation of membrane protein subunits with soluble cytosolic domains; and second, it prevents the inappropriate recruitment and ubiquitination of subunits with globular lumenal domains. Patient mutations to TXNDC15 that cause the ciliopathy Meckel-Gruber syndrome, disrupted its binding to MARCHF6, allowing degradation of critical ciliary proteins as they transit through the ER leading to defects in ciliogenesis. The regulatory function of TXNDC15 therefore exemplifies how protein quality control maintains the integrity of the proteome to prevent disease.

molecular biology↗

bioRxiv2025

Proteome-wide identification of metamorphic protein candidates using mass spectrometry

How protein folds remodel at low temperature remains poorly mapped at the proteome scale. We combine brief, temperature-controlled proteolysis with LC-MS/MS to chart cold-dependent conformational changes in Escherichia coli lysate. Methodological validation with the metamorphic protein Sa1V90T--whose /{beta}-plait and 3 states interchange between 30 {degrees}C and 5 {degrees}C--showed that a high-protease, short-digestion regime detects temperature-dependent exposure at four peptides, whereas the temperature-insensitive variant Sa1V90TV52D is largely unchanged. Applied to lysate, the workflow identifies >750 proteolytic sites across >250 abundant proteins (>10% of the identified proteome) whose local susceptibility shifts as temperature decreases from 35 {degrees}C to 5 {degrees}C, including five Sa1V90T peptides recapitulating purified-protein behavior. A site-level scoring model yields 338 destabilized peptides (177 proteins) and 426 stabilized peptides (138 proteins). Cold stabilization prominently marks translation machinery (>15 ribosomal proteins), while cooling also exposes regions in chaperones and cell-division factors. Sequence analysis links temperature sensitivity to charge-enriched composition, consistent with thermodynamic expectations. These data provide a proteome-wide view of cold-sensitive folding, implicate translation and cytokinetic systems in low-temperature remodeling, and illuminate a new strategy for identifying metamorphic proteins on an unprecedented scale.

biophysics↗

bioRxiv2025

Parkinsons disease modeling in regenerative spiny mice (Acomys dimidiatus) captures key disease-relevant behavioral, histological, and molecular signatures

Parkinsons disease (PD) is a multifactorial neurodegenerative disorder that has been modeled extensively in animals, primarily rodents, but also in non-human primates and non-mammalian organisms. However, no single animal model fully recapitulates the hallmarks of PD pathology. Here, we extend this work by modeling PD for the first time in the spiny mouse (Acomys dimidiatus), a mammal notable for its robust regeneration of multiple tissues. We show that the nigrostriatal pathway of A. dimidiatus is vulnerable to both acute 6-hydroxydopamine (6-OHDA) toxicity and chronic -synuclein (Syn) preformed fibril-induced aggregation. Mouse Syn PFFs produced widespread pS129-positive Syn inclusions across multiple brain regions, mirroring a key pathological hallmark of PD. Compared to C57BL/6J mice, A. dimidiatus exhibited more pronounced behavioral impairments, greater nigrostriatal degeneration, and higher pS129-Syn inclusion burden within substantia nigra pars compacta (SNpc) neurons. To probe the molecular underpinnings behind the vulnerability, we performed single-cell spatial proteomics, which revealed extensive proteomic alterations in dopaminergic neurons associated with Syn aggregation. Multiple proteins were dysregulated in A. dimidiatus, including those involved in proteasomal function, mitochondrial pathways, and oxidative stress regulation, which are processes commonly implicated in PD. Notably, proteomic analysis identified heightened astrocytic activation in the SNpc, which we validated histologically, suggesting a distinct glial response compared to mice. Together, these findings expand our understanding of PD-relevant pathophysiology across species and establish A. dimidiatus as a model for studying mechanisms of neurodegeneration.

neuroscience↗

bioRxiv2025

Determining Multivalent Interactomes of Intrinsically Disordered Proteins in Phase-Separated Condensates in Live Cells

Many intrinsically disordered protein regions (IDRs) can form liquid-liquid phase separation (LLPS) condensates via multivalent interactions, but how the interaction behaviors contribute to specific cellular functions remains unclear due to the unknown composition of the condensates. Here, we report phase-separation-induced interactome detection (PhaseID), a new method that determines the protein components within the LLPS condensates driven by any given IDR in live human cells. Using PhaseID, we demonstrated that transactivation IDRs each have unique multivalent interactomes, thereby executing transactivation through distinct pathways. In-depth analyses of PhaseID-determined IDR interactomes revealed that LLPS tunes the interaction selectivity of IDRs, enabling them to play new roles in specific cellular processes, and different LLPS condensates recruit proteins with distinct physicochemical properties encoded in their sequences.

molecular biology↗

bioRxiv2025

ATM promotes reversed fork processing during DNA interstrand cross-link repair

During replication-coupled DNA interstrand cross-link (ICL) repair, fork reversal is thought to enable the Fanconi anemia (FA) pathway to resolve the ICL through nucleolytic incisions. Subsequent fork restoration then allows nascent DNA strand extension past the lesion. Although these fork remodeling events are crucial for ICL repair, their regulation remains poorly understood. Here, we use cell-free Xenopus egg extracts to investigate fork dynamics during ICL repair by the FA pathway. We find that the ataxia telangiectasia-mutated (ATM) kinase is activated concomitantly with fork reversal and promotes resection of the reversed fork intermediate. This resection depends on the coordinated activities of the EXO1 and DNA2 nucleases. Our data indicate that EXO1 initiates 5 to 3 resection of nascent lagging strands in the regressed arm, while DNA2 performs 5 to 3 resection of recessed lagging strands. We further show that the inhibition of protein phosphatase 2A (PP2A) during ICL repair results in ATM hyperactivation, reversed fork over-resection, and formation of aberrant end-joining products, indicating that PP2A counteracts ATM signaling to constrain reversed fork resection. Taken together, this work implicates reversed forks as substrates for ATM activation and reveals a phospho-regulatory circuit that governs reversed fork processing during ICL repair.

molecular biology↗

bioRxiv2025

stk32a links sleep homeostasis to suppression of sensory and motor systems

Sleep is regulated by a homeostatic process and associated with an increased arousal threshold, but the genetic and neuronal mechanisms that implement these essential features of sleep remain poorly understood. To address these fundamental questions, we performed a zebrafish genetic screen informed by human genome-wide association studies. We found that mutation of serine/threonine kinase 32a (stk32a) results in increased sleep and impaired sleep homeostasis in both zebrafish and mice, and that stk32a acts downstream of neurotensin signaling and the serotonergic raphe in zebrafish. stk32a mutation reduces phosphorylation of neurofilament proteins, which are co-expressed with stk32a in neurons that regulate motor activity and in lateral line hair cells that detect environmental stimuli, and ablating these cells phenocopies stk32a mutation. Neurotensin signaling inhibits specific sensory and motor populations, and blocks stimulus-evoked responses of neurons that relay sensory information from hair cells to the brain. Our work thus shows that stk32a is an evolutionarily conserved sleep regulator that links neuropeptidergic and neuromodulatory systems to homeostatic sleep drive and changes in arousal threshold, which are implemented through suppression of specific sensory and motor systems.

neuroscience↗

bioRxiv2025

Subcellular proteomics of Paramecium tetraurelia reveals mosaic localization of glycolysis and gluconeogenesis

Ciliates are unicellular heterotrophic eukaryotes, most of which consume other microbes as prey. They exhibit nuclear dimorphism which requires reconstruction of a transcriptionally active macronucleus from the germline micronucleus after sexual recombination. This complex genomic structure has prevented the development of highly tractable genetic models leaving much of ciliate cell biology unexplored. To complicate matters further, some ciliates tend to accumulate many gene duplicates either singly or via whole genome duplications. Thus, extensive insight into the cell biology of ciliates requires the use of high-throughput tools like subcellular proteomics. Here, we use a subcellular proteomics workflow to classify over 9,000 proteins to 16 subcellular compartments in Paramecium tetraurelia. From these data, we identify a small but robust subcellular cluster containing canonical mitochondrial outer membrane proteins as well as some ER proteins, putatively at membrane contact sites. Within this cluster, we identified the important glycolytic enzyme phosphofructokinase, which contained a transmembrane domain. Further investigation revealed that several latter-acting glycolytic enzymes were localized to the mitochondrial cluster. The location of phosphoenol pyruvate carboxykinase and pyruvate carboxylase in the mitochondria but pyruvate kinase in the cytosol suggests that ciliates prefer gluconeogenesis over glycolysis. The localization of these enzymes was confirmed in a preliminary subcellular proteome of Tetrahymena thermophila. In sum, our findings suggest that mitochondrial localization of glycolytic/gluconeogenic enzymes is widespread across ciliates and that several may preferentially undergo gluconeogenesis over glycolysis using amino acids as a primary carbon source in both catabolic and anabolic metabolism. HighlightsSubcellular proteomics of Paramecium tetraurelia revealed that glycolytic and gluconeogenic enzymes are mosaically distributed between the cytosol, mitochondrial matrix, and mitochondrial outer membrane. A distinct mitochondrial outer membrane compartment was identified with 105 classified proteins, including core mitochondrial biogenesis proteins and a putative Tom70-like protein. Phosphofructokinase, a key glycolytic enzyme, was found embedded in the mitochondrial outer membrane. Localization of biochemical pathways suggest ciliates favor gluconeogenesis over glycolysis. In total, over 9000 Paramecium proteins were identified using subcellular proteomics and classified into 16 different cellular compartments.

cell biology↗

bioRxiv2025

VDAC isoforms play distinct roles in modulating metabolism and maintaining mitochondrial function

The Voltage Dependent Anion Channel (VDAC) is the most ubiquitous protein in the mitochondrial outer membrane. This channel facilitates the flux of water-soluble metabolites and ions like calcium across the mitochondrial outer membrane. Beyond this canonical role, VDAC has been implicated, through interactions with protein partners, in several cellular processes such as apoptosis, calcium signaling, and lipid metabolism. There are three VDAC isoforms in mammalian cells, VDAC 1, 2, and 3, with varying tissue-specific expression profiles. From a biophysical standpoint, all three isoforms can conduct metabolites and ions with similar efficiency. However, isoform knockouts (KOs) in mice lead to distinct phenotypes, which may be due to differences in VDAC isoform interactions with partner proteins. To understand the functional role of each VDAC isoform within a single cell type, we created functional KOs of each isoform in HeLa cells and performed a comparative study of their metabolic activity and proteomics. We found that each isoform KO alters the proteome differently, with VDAC3 KO dramatically downregulating key members of the electron transport chain (ETC) while shifting the mitochondria into a glutamine-dependent state. Importantly, this unexpected dependence of mitochondrial function on the VDAC3 isoform is not compensated by the more ubiquitously expressed VDAC1 and VDAC2 isoforms. In contrast, VDAC2 KO did not affect respiration but upregulated ETC components and decreased key enzymes in the glutamine metabolic pathway. VDAC1 KO specifically reduced glycolytic activity linked to decreased hexokinase localization to mitochondria. These results reveal non-redundant roles of VDAC isoforms in cancer cell metabolic adaptability.

cell biology↗

bioRxiv2025

Molecularly-guided spatial proteomics captures single-cell identity and heterogeneity of the nervous system

Single-cell spatial proteomics (scSP) holds substantial potential for profiling healthy and diseased tissues. The emerging method of molecularly-guided unbiased scSP has mostly been applied to peripheral somatic tissues. Here, we optimize and apply scSP to the healthy and diseased mammalian brain, using molecularly-guided laser capture microdissection and unbiased mass spectrometry. We systematically evaluate the effects of tissue fixation, marker staining, and sample input size on proteome coverage and quantitative accuracy. We benchmark this workflow by profiling region-specific neuronal proteomes and describing the response of non-neuronal cells to acute brain injury. Across these applications, we integrate complementary transcriptomic resources to evaluate cross-modality trends and refine neuronal proteomic results by filtering out protein signals likely arising from non-neuronal cells, an essential consideration in heterogeneous tissues such as the brain. Finally, we leverage this approach to resolve proteomic differences between dopaminergic neuron subpopulations with differential vulnerability to Parkinsons disease and to uncover disease-specific disruptions in -synuclein-aggregate-bearing single dopaminergic neurons. Together, these data demonstrate the utility of scSP in neuroscience research for understanding fundamental biology and the molecular drivers of neurological conditions.

neuroscience↗

bioRxiv2025

Reducing Cofilin dosage makes embryos resilient to heat stress

In addition to regulating actin dynamics, Cofilin also senses and responds to physiological stress and can determine cell survival outcomes. Yet, the full picture of Cofilin's role in stress response is lacking. Here, we used imaging and RNA-seq methods to show that exposing early Drosophila melanogaster embryos to either acute or chronic heat stress (32{degrees}C) induces a Cofilin-mediated Actin Stress Response (ASR) and leads to upregulation of genes associated with both heat shock and Endoplasmic Reticulum (ER) unfolded protein responses. Reducing cofilin gene dosage (cofilin+/-) in heat-stressed embryos modulates all observed stress responses and partially rescues embryo survival. Unexpectedly we find that heat shock- and ER- stress response modulation arises because non-stressed cofilin+/- embryos already show upregulation of heat shock- and ER- stress response genes, prior to heat exposure. Our data support a model whereby cofilin heterozygosity activates specific stress responses associated with reduced protein homeostasis, thus priming embryos to be more resilient when they encounter subsequent heat stress. We conclude that Cofilin dosage serves as a determinant of stress outcomes, impacting both the actin cytoskeleton and inducible stress response pathways. In the embryo, we identify Cofilin as a novel link between inducible stress response and thermotolerance.

developmental biology↗

bioRxiv2024

The gut microbiome promotes mitochondrial respiration in the brain of a Parkinson's disease mouse model

The pathophysiology of Parkinsons disease (PD) involves gene-environment interactions that impair various cellular processes such as autophagy, lysosomal function, or mitochondrial dysfunction. Specifically, mitochondria-associated gene mutations increase PD risk, mitochondrial respiration is altered in the PD brain, and mitochondrial-damaging toxins cause PD-like motor and gastrointestinal symptoms in animal models. The gut microbiome is altered in PD patients and represents an environmental risk, however a relationship between mitochondrial function and the microbiome in PD has not been previously established. Herein, we report that striatal mitochondria are functionally overactive in -synuclein-overexpressing (ASO) mice, a model of PD, and that microbiome depletion restores respiration and mitochondria-associated gene expression patterns to wild-type levels. ASO mice harboring a complex microbiome produce increased reactive oxygen species in the striatum whereas germ-free counterparts express elevated levels of antioxidant proteins that may buffer against oxidative damage. Indeed, antioxidant treatment improves motor performance in ASO mice and, remarkably, blocking oxidant scavenging in germ-free mice induces -synuclein-dependent motor deficits. Thus, the gut microbiome increases mitochondrial respiration and oxidative stress in the brain, which enhances motor symptoms in a mouse model of PD.

physiology↗

bioRxiv2024

Optimizing protein expression in the One-Pot PURE system: insights into reaction composition and translation efficiency

The One-Pot PURE (Protein synthesis Using Recombinant Elements) system simplifies the preparation of traditional PURE systems by co-culturing and purifying 36 essential proteins for gene expression in a single step, thereby improving accessibility and affordability for widespread laboratory adoption and customization. However, replicating this protocol to match the productivity of traditional PURE systems can take considerable time and effort due to uncharacterized variability in the systems biochemical composition. In this work, we observed unstable PURE protein expression in E. coli strains M15/pREP4 and BL21(DE3) and addressed this using glucose-mediated catabolite repression to minimize burdensome background expression. We also identified differences in optimal protein induction timing between these two strains, leading to growth incompatibility in co-culture, and observed proteolysis of PURE proteins expressed in M15/pREP4. We showed that consolidating all expression vectors into a protease-deficient BL21(DE3) strain could minimize proteolysis. This single-strain system also led to more uniform cell growth at the time of protein induction, improving the stoichiometry of critical translation initiation factors in the PURE reaction for efficient protein production. In addition to optimizing One-Pot PURE protein composition, we found that variations in commercial energy solution formulations could compensate for suboptimal PURE protein stoichiometry. Moreover, our study revealed significant differences in the expression capacity of commercially available E. coli tRNAs, suggesting the potential of optimizing tRNA codons to improve protein translation. Taken together, this work highlights the complex biochemical interplay influencing protein expression capacity in the One-Pot PURE system and presents strategies to improve its robustness and productivity.

synthetic biology↗

bioRxiv2024

The Fanconi anemia pathway repairs colibactin-induced DNA interstrand cross-links

Colibactin is a secondary metabolite produced by bacteria present in the human gut and is implicated in the progression of colorectal cancer and inflammatory bowel disease. This genotoxin alkylates deoxyadenosines on opposite strands of host cell DNA to produce DNA interstrand cross-links (ICLs) that block DNA replication. While cells have evolved multiple mechanisms to resolve ("unhook") ICLs encountered by the replication machinery, little is known about which of these pathways promote resistance to colibactin-induced ICLs. Here, we use Xenopus egg extracts to investigate replication-coupled repair of plasmids engineered to contain site-specific colibactin-ICLs. We show that replication fork stalling at a colibactin-ICL leads to replisome disassembly and activation of the Fanconi anemia ICL repair pathway, which unhooks the colibactin-ICL through nucleolytic incisions. These incisions generate a DNA double-strand break intermediate in one sister chromatid, which can be repaired by homologous recombination, and a monoadduct ("ICL remnant") in the other. Our data indicate that translesion synthesis past the colibactin-ICL remnant depends on Pol{eta} and a Pol{kappa}-REV1-Pol{zeta} polymerase complex. Although translesion synthesis past colibactin-induced DNA damage is frequently error-free, it can introduce T>N point mutations that partially recapitulate the mutation signature associated with colibactin exposure in vivo. Taken together, our work provides a biochemical framework for understanding how cells tolerate a naturally-occurring and clinically-relevant ICL.

molecular biology↗

bioRxiv2024

Internal regulation between constitutively expressed T cell coinhibitory receptors BTLA and CD5 and tolerance in recent thymic emigrants

Several coinhibitory receptors are upregulated upon activation, whereas a small number of coinhibitory receptors are expressed constitutively by naive T cells. The relationship between constitutively expressed coinhibitors is unknown. We found an inverse relationship between two constitutively expressed coinhibitors, CD5 and BTLA; BTLA expression was low in the thymus and high in the periphery, corresponding respectively with high and low CD5 expression. Germline or induced deletion of Btla in somatic cells demonstrated a causal relationship between BTLA expression and CD5 levels in T cells of central and peripheral lymphoid tissues. The effect of BTLA on CD5 expression on thymic and peripheral CD4 T cells was due to BTLA signaling, rather than signaling by its ligand, the herpes virus mediator (HVEM). Regulation was maintained in mice with a non-signaling HVEM mutant but was lost in Tnfrsf14-/- (Hvem-/-) mice. Increased CD5 levels have been positively associated with increased recognition of self-peptide MHC complexes. Thus, control of CD5 expression by BTLA signals early in T cell ontogeny suggested that BTLA might be important for establishing self-tolerance in newly generated T cells. Consistent with this concept, we found that BTLA, as well as the inducible coinhibitor PD-1, were needed post thymic selection in recent thymic emigrants (RTE) to establish self-tolerance. RTE lacking BTLA caused a multiorgan autoimmune disease whose development required CD4 T cells and MHC class II. Together, our findings identify a negative regulatory pathway allowing constitutively expressed coinhibitory receptors to calibrate their expression in thymic T cell differentiation. Expression of constitutive and induced coinhibitory receptors is needed to establish tolerance in the periphery for RTE.

immunology↗