Biology subjects

Wang, Y.-H.

Publications and source records attributed to Wang, Y.-H..

At least 19 recordsLinked to original sources

bioRxiv2026

Subtyping active-site inhibitor binding mode to Abl kinase using super-resolution nanopore tweezers.

Accurate determination of kinase inhibitor binding modes could provide essential information for understanding resistance mechanisms and accelerating drug discovery. While conventional structural methods such as X-ray crystallography, cryo-EM and NMR provide high-resolution information but are low-throughput and capture largely static snapshots of dynamic protein-ligand interactions Here, we introduce a single-molecule nanopore tweezer platform that functionally subtypes ATP-competitive Abl kinase inhibitors by resolving distinct ionic current signatures of Abl-inhibitor complexes. This approach distinguishes Type I, Type IIA, and Type IIB inhibitors without structural determination. We further show how clinically relevant Abl variants (T315I and E255V) reshape inhibitor engagement and binding modes. By combining baseline probability features with wavelet-based time-frequency descriptors, ensemble machine-learning models achieved 97.5% classification accuracy across seven kinase inhibitor binding modes at sub-angstrom resolution and enabled deconvolution of mixed-inhibitor samples at nanomolar concentrations. These results establish nanopore tweezers as a label-free, super-resolution platform for profiling kinase conformational states and inhibitor binding modes, complementing structural approaches and supporting precision oncology.

biophysics↗

bioRxiv2026

A Quartet of Native Orai Channel Isoforms Orchestrates Graded NFAT Activation and Transcription

The Ca{superscript 2} release-activated Ca{superscript 2} (CRAC) channel mediates store-operated calcium entry (SOCE), a ubiquitous pathway essential for many cell types, including immune cells. Three Orai (Orai1/2/3) proteins constitute the plasma membrane pore-forming units of CRAC channels that are activated by the endoplasmic reticulum (ER) Ca2+-sensing STIM1/2 proteins when ER Ca2+ stores are depleted. Orai1/2/3 are differentially expressed across primary cells with discernible differences in their structures and biophysical properties. Further, Orai1 has two alternatively translated isoforms: long mammalian-specific Orai1 and the 63-residue shorter Orai1{beta}, which is evolutionarily older and conserved across vertebrates. Whether Orai1/1{beta}/2/3 produce unique cytosolic Ca{superscript 2} signatures that bias transcriptional responses through effectors like NFAT is unclear. Here, we used HEK293 cells engineered to express one native Orai isoform and show that all Orai isoforms couple to NFAT1/4 induction. The magnitude of NFAT1/4 induction for each Orai isoform matches that of SOCE, with the following profile: Orai1{beta}>Orai1>>Orai2>Orai3. Near-native re-expression of either Orai1 or Orai1{beta} in primary murine Orai1-/- CD4 T cells restored SOCE, NFAT activation, cytokine production and promoted near identical transcriptional responses enriched for immune activation pathways. An analysis of genetic and clinical data of human individuals showed that homozygous null mutations selectively abolishing Orai1 are not associated with disease resembling CRAC channelopathy. Primary T cells from individuals homozygous or heterozygous for an Orai1 null mutation showed enhanced, rather than impaired, SOCE and NFAT induction. Our data indicate that NFAT activation and transcriptional outputs are primarily driven by the graded strength of SOCE mediated by each isoform of the Orai quartet.

cell biology↗

bioRxiv2026

NPC1 deficiency engages a lysosome - genome - immune program linked to neurodegeneration and cellular aging signatures

Lysosomal dysfunction is a prominent feature of neurodegeneration and aging, yet how primary defects in lysosomal trafficking are converted into progressive cellular decline remains poorly understood. Niemann Pick disease type C (NPC), caused by impaired NPC1 dependent cholesterol export, provides a genetically defined model to address this question. Here, we show that NPC1 deficiency activates a lysosome, genome, immune axis linking cholesterol trafficking failure to neurodegeneration and hallmarks of cellular aging. In Npc1 mutant mice, NPC1 loss triggered DNA damage, neuroinflammation, microglial and astrocytic activation, Purkinje neuron degeneration, and motor dysfunction. Consistently, NPC patient-derived fibroblasts exhibited mitochondrial abnormalities and widespread DNA double-strand breaks. Genome-wide DNA break mapping and transcriptomic analyses revealed extensive genomic instability at regulatory regions, including enrichment of DNA breaks at transcription start sites and G quadruplex associated loci, accompanied by widespread transcriptional reprogramming, activation of innate immune pathways, disruption of fibroblast identity, and induction of cellular aging signatures. We further identify Fingolimod, an FDA approved sphingosine - 1 phosphate receptor modulator, as a potent modifier of this disease network. Fingolimod improved lysosomal cholesterol trafficking, increased LAMP1 abundance, attenuated STING associated inflammatory signaling, normalized mitochondrial function, reduced neuroinflammatory and neurodegenerative phenotypes in Npc1 mutant mice, and broadly shifted disease-associated transcriptional programs toward a healthier state. Extending these findings beyond NPC, Fingolimod improved age-associated phenotypes in C. elegans and prolonged lifespan in aged male mice. Together, these findings identify genome instability and chronic innate immune activation as major downstream consequences of lysosomal cholesterol trafficking failure and establish Fingolimod as a clinically actionable modulator of lysosomal dysfunction, neurodegeneration, and aging-related decline.

molecular biology↗

bioRxiv2026

Benchmarking AI Protein Structure Predictors Reveals a Persistent Bias in Multi-State Proteins

Protein structure predictors achieve high single-state accuracy, but it remains unclear whether they can recover functionally relevant conformational ensembles or account for the presence of ligands and/or binding partners. Here, we benchmark AlphaFold3, Boltz-2, Chai-1, and BioEmu on four canonical multi-state proteins (Pf-MATE, LAO, SecA, and {beta}2AR), quantifying state bias and sampling breadth against experimental reference structures. Models frequently default to a dominant state represented in the PDB; small-molecule ligands have weak or inconsistent effects, while large protein partners drive clear conformational switching between states. Multiple sequence alignment (MSA)-based approaches (AF-Cluster and random subsampling) recapitulate similar biases, indicating that this behavior is not unique to newer architectures. These results underscore current limitations for multi-state protein structure prediction and structure-guided ligand discovery. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/737860v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@278cc1org.highwire.dtl.DTLVardef@8a0b66org.highwire.dtl.DTLVardef@f28a21org.highwire.dtl.DTLVardef@14ab675_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

Dynamic partitioning shapes the in vivo organization of the E. coli RNA degradosome

The bacterial RNA degradosome is a central mediator of RNA turnover, yet how its components are organized and regulated in living cells remains unclear. Using live-cell single-molecule imaging, we quantified the spatial distribution and dynamics of the four major Escherichia coli degradosome components: RNase E, RhlB, PNPase, and enolase. These proteins occupied distinct membrane-associated and cytoplasmic pools whose relative abundance varied among proteins and changed with physiological state. In particular, PNPase underwent pronounced redistribution between membrane-associated and cytoplasmic states in response to altered RNA availability and growth conditions, whereas RNase E and RhlB remained largely membrane associated. To determine the functional consequences of this organization, we examined the degradation of lacZ reporter transcripts with different translation initiation strengths. PNPase and RhlB preferentially promoted degradation of weakly translated transcripts, whereas strongly translated transcripts were largely insensitive to their loss. Together, these results reveal that the bacterial RNA degradosome is not a static, uniformly assembled molecular machine. Instead, RNA decay is organized through dynamic intracellular partitioning of degradosome components, linking physiological state and translation status to transcript degradation.

microbiology↗

bioRxiv2026

Structural Insights into Native Intact Mycobacterium abscessus by Conventional and Ultrahigh-field solid-state NMR at 1.2 GHz

We present an ultrahigh-field magic-angle spinning (MAS) solid-state NMR (ssNMR) study to characterize intact nontuberculous mycobacteria (NTM) at the molecular level. Hydrated and dried whole-cell Mycobacterium abscessus samples were investigated by combining conventional high-field ssNMR at 750 MHz with ultrahigh-field ssNMR at 1.2 GHz and ultrafast MAS at 100 kHz. To improve sensitivity and enable multidimensional experiments, 13C/15N isotope labeling was performed after growth in synthetic cystic fibrosis medium (SCFM). We utilized 1D 13C and multidimensional 1H-13C and 13C-13C ssNMR experiments to characterize the chemical composition, dynamics, and structural organization of the M. abscessus cell envelope. The isotope-labeling efficiency was found to be non-uniform across different molecular classes, with high incorporation into polysaccharides and lower incorporation into lipid and peptide-associated signals. INEPT- and CP-based experiments selectively probed flexible and rigid fractions of the samples, revealing substantial differences in linewidth, dynamics, and sensitivity between hydrated and dried preparations. Conventional 750 MHz experiments provided high-resolution multidimensional spectra and enabled identification of distinct chemical environments associated with peptidoglycan, arabinogalactan, mycolic acids, lipids, and peptide-associated components. Ultrahigh-field ssNMR at 1.2 GHz combined with ultrafast MAS and 1H detection substantially improved spectral resolution and sensitivity in particular per mg of sample amount, allowing detection of weak and previously unresolved resonances, including polysaccharide and possible nucleic-acid-associated signals. Together, these results demonstrate that ultra-high-field and ultrafast-MAS ssNMR enables detailed characterization of intact NTM cell envelopes under near-native conditions and provides a framework for future molecular investigations of antimicrobial interactions.

microbiology↗

bioRxiv2026

A scalable approach to inoculate plant viral vectors into plant tissue using non-pathogenic, transgenic galls

Vascular plant pathogens transmitted by insects inflict devastating economic losses on crops worldwide. By obstructing and usurping the natural flow of nutrients, plant infection by these pathogens drastically reduces yields, vigor and productivity. Treating fruit-bearing trees against persistent vascular pathogens poses a unique challenge, as systemic delivery of therapeutics must navigate the compartmentalized architecture of the trees vascular system under changing environmental conditions without disrupting fruit production or long-term tree health. Plant viruses have gained traction as a novel approach to deliver therapies to crop plants, including fruit trees, but delivery of viral vectors to orchards at scale remains a significant challenge. We tested whether transgenic galls can be used to systemically infect plants with a plant virus infectious clone. We combined the plant growth regulator gene cassette from Agrobacterium tumefaciens strain C58 with the wild-type strain of citrus yellow vein associated virus-1 (CY1) on a single plasmid within the T-DNA for plant cell transformation. Using EHA105, a disarmed strain of A. tumefaciens, we inoculated stems with these gall-forming plasmids and initiated systemic CY1 infection in citrus and Arabidopsis thaliana over three independent experiments. We provide proof-of-concept that transgenic galls, referred to as symbionts, can launch the systemic infection of CY1 in economically important and model plants. Symbiont delivery of therapeutic viral vectors is theoretically scalable from inoculation of mother trees within the nursery to millions of trees in the field and may be a valuable tool for the commercial delivery of therapeutic plant viral vectors.

plant biology↗

bioRxiv2026

Dynamic RNA Polymerase compartments organize the transcription of gene clusters

Spatial organization of transcription machinery is emerging as a key regulator of gene expression, yet how RNA polymerases are organized at gene clusters remains unclear. Here, we show that RNA polymerases II and polymerase III form distinct nuclear foci at the 5S ribosomal DNA (rDNA)-Spliced leader 1 (SL1) cluster in C. elegans. Within this cluster, polymerase II binds to the SL1 gene, while polymerase III associates with 5S rDNA. Both polymerase foci display dynamic but distinct behaviors within the nucleus. The assembly of these polymerase foci is regulated across the cell cycle. ATTF-6, an AT-hook transcription factor, is essential for polymerases II foci formation but dispensable for polymerases III foci. While Pol III foci are largely resistant to temperature changes, Pol II foci are temperature-sensitive, and their dissolution correlates with reduced SL1 expression. Together, these results reveal a spatial and temporal regulation of two RNA polymerases that organize gene cluster transcription.

cell biology↗

bioRxiv2025

Intracellular glutamine fluctuates with nitrogen availability and regulates Mycobacterium smegmatis biofilm formation

Biofilm formation allows pathogenic nontuberculous mycobacteria (NTM) to adhere to household plumbing systems and has been observed in vivo during human infection. Glucose drives NTM aggregation in vitro, and ammonium inhibits it, but the regulatory systems controlling this early step in biofilm formation are not understood. Here, we show that a variety of carbon and nitrogen sources have similar impacts on aggregation in the model NTM Mycobacterium smegmatis as glucose and ammonium, suggesting that the response to these nutrients is general and likely sensed through downstream, integrated signals. Next, we performed a transposon screen in M. smegmatis to uncover these putative regulatory nodes. Our screen revealed that mutating specific genes in the purine and pyrimidine biosynthesis pathways caused an aggregation defect, but supplementing with adenosine and guanosine had no impact on aggregation either in a purF mutant or WT. Realizing that the only genes we hit in purine or pyrimidine biosynthesis were those that utilized glutamine as a nitrogen donor, we pivoted to the hypothesis that intracellular glutamine could be a nitrogen-responsive node affecting aggregation. We tested this hypothesis in defined M63 medium using targeted mass spectrometry. Indeed, intracellular glutamine increased with nitrogen availability and correlated with planktonic growth. Furthermore, a garA mutant, which has an artificially expanded glutamine pool in growth phase, grew solely as planktonic cells even without nitrogen supplementation. Altogether these results establish that intracellular glutamine controls M. smegmatis aggregation, and they introduce flux-dependent sensors as key components of the NTM biofilm regulatory system. ImportanceA subset of nontuberculous mycobacteria (NTM), including Mycobacterium abscessus, are opportunistic pathogens that can cause severe pulmonary infections. M. abscessus is present in showerhead biofilms, one of the reservoirs from which it can infect susceptible individuals. Moreover, M. abscessus can exist as biofilms during pulmonary infections, and biofilm formation in vitro renders M. abscessus more tolerant to antibiotics. The ability to inhibit NTM biofilm formation could therefore help us better prevent and treat NTM infections. However, the regulatory systems controlling NTM biofilm formation, which could include targets for anti-biofilm therapeutics, are poorly understood. The significance of this work is that it reveals intracellular glutamine as an important node controlling initiation of biofilm formation in the model NTM Mycobacterium smegmatis. Building on this foundation, future studies will investigate how NTM biofilms can be dispersed by altering glutamine levels and will describe how NTM translates intracellular glutamine to alteration of surface adhesins.

microbiology↗

bioRxiv2025

Practical considerations for accurate estimation of diffusion parameters from single-particle tracking in living cells

Advances in fluorescence microscopy have enabled high-resolution tracking of individual biomolecules in living cells. However, accurate estimation of diffusion parameters from single-particle trajectories remains challenging due to static and dynamic localization errors inherent in these measurements. While previous studies have characterized how such errors affect mean-squared displacement (MSD) analysis, practical guidelines for minimizing them during data acquisition and correcting them during analysis are still lacking. Here, we combine theoretical modeling and simulations to evaluate how exposure time and sampling rate influence the accuracy of MSD-based inference under fractional Brownian motion (FBM), a canonical model of anomalous diffusion. We demonstrate that decoupling exposure and sampling times enables escape from the error-prone regime, thus improving inference accuracy, and that incorporating an offset in nonlinear MSD fitting substantially improves the estimation of the anomalous diffusion exponent. We validate this framework using trajectories of cytoplasmic particles in Escherichia coli, recovering consistent diffusion parameters across multiple data sets. Our findings offer practical strategies to improve both experimental design and data analysis in single-particle tracking of live or synthetic systems.

biophysics↗

bioRxiv2025

Granulocyte Derived Resistin Inhibits Monocyte Maturation and Induces Immune Suppression in CMML

Chronic myelomonocytic leukaemia (CMML) is a haematological malignancy characterised by overlapping myeloid dysplasia and proliferation with persisting monocytosis. While monocytes are the cardinal malignant cell type in CMML, as a stem cell neoplasm the disease clone comprises most lineages and differentiation stages, including granulocytes. To investigate the pathogenic contribution of granulocytes in CMML maintenance and progression, we performed phenotypic, transcriptomic and functional characterization of CMML granulocytes. Compared with healthy age-matched controls, CMML granulocytes exhibit defective maturation with reduced granularity and phagocytic capacity. Transcriptome analysis revealed activation of pathways linked to proliferation, Myc activity and inflammation. Notably, RETN, which encodes the inflammatory mediator resistin, was upregulated approximately 100-fold in CMML granulocytes; but not differentially expressed in CMML PBMNCs, sorted monocytes, or stem and progenitor cells compared to their healthy counterparts. Accordingly, resistin protein levels were 10-fold higher in plasma from CMML patients and higher plasma resistin levels correlate with poor overall survival and AML-free survival. Remarkably, exposure of healthy monocytes to exogenous recombinant resistin inhibited monocyte maturation and macrophage differentiation. Transcriptome analysis of resistin treated monocytes revealed that resistin induces gene signatures related to immune suppression and myeloid-derived suppressor cell phenotype. We found SEMA4A to be a downstream target of resistin and overexpressed in CMML monocytes. Consistent with known roles for SEMA4A, CMML patients displayed higher percentage of Tregs and elevated Th2/Th1 ratio compared with healthy controls and percentage of Tregs corresponding with associated resistin levels. Furthermore, we demonstrated that resistin directly skews the Th2/Th1 ratio via binding to monocytes. In conclusion, we showed that immature granulocytes in CMML produce high levels of resistin, which contributes to defective monocyte maturation and immune suppression.

cancer biology↗

bioRxiv2025

Harnessing landscape genomics to evaluate genomic vulnerability and future climate resilience in an East Asia perennial

In this era of rapid climate change, understanding the adaptive potential of organisms is imperative for buffering biodiversity loss. Genomic forecasting provides invaluable insights into population vulnerability and adaptive potential under diverse climatic conditions, thereby facilitating management interventions, bolstering population resilience, and shaping germplasm conservation strategies tailored to specific species. Here we integrated population genomics and landscape genomics approaches, leveraging single-nucleotide polymorphisms obtained through whole-genome resequencing of 43 Rheum palmatum complex populations, to pinpoint adaptive variation and its significance in the context of future climates, delineate seed zones, and establish guidelines for ex situ germplasm conservation to capture the majority of existing adaptive diversity. Our analysis unveiled that the species complex comprised two distinct genetic clusters, exhibiting differential climate adaptation and genomic vulnerabilities across its distribution range. We also determined that the species range could be subdivided into three distinct seed zones, with varying sample requirements per seed zone corresponding to differed conservation efforts. Overall, our findings provide a genome-wide perspective on climate adaptation and valuable insights into germplasm conservation strategies aimed at enhancing population resilience in future climates, serving as a blueprint for restoration plans of other vulnerable species.

evolutionary biology↗

bioRxiv2025

Loss of DHX36/G4R1, a G4 resolvase, drives genome instability and regulates innate immune gene expression in cancer cells

G-quadruplexes (G4s) are four-stranded alternative secondary structures formed by guanine-rich nucleic acids and are prevalent across the human genome. G4s are enzymatically resolved using specialized helicases. Previous in vitro studies showed that DEAH-box Helicase 36 (DHX36/G4R1/RHAU), has the highest specificity and affinity for G4 structures. Here, by mapping genome-wide DNA double-strand breaks (DSBs), we demonstrate that knockout (KO) of DHX36 helicase increases DSB enrichment at G4 sites and that the presence of the G4 motif is a significant mediator of genome instability at regulatory regions. The loss of DHX36 corresponds with the significant upregulation of NF-{kappa}B transcriptional programs, culminating in the production and secretion of proinflammatory cytokines. Loss of DHX36 expression results in an increase in the innate immune signaling stimulator of interferon response cGAMP interactor 1 (STING1) expression and activation of genes involved in immune response pathways. Importantly, higher levels of DHX36 mRNA expression in human B-cell acute lymphoblastic leukemia correlate with improved overall survival relative to lower expression of DHX36, highlighting its critical role in preserving genome integrity at a cellular level and in the context of cancer.

molecular biology↗

bioRxiv2024

Mechanistic Insights into Dengue Virus Inhibition by a Clinical Trial Compound NITD-688

Dengue, caused by the dengue virus (DENV), presents a significant public health challenge with limited effective treatments. NITD-688 is a potent pan-serotype DENV inhibitor currently in Phase II clinical trials. However, its mechanism of action is not fully understood. Here, we present the molecular details of how NITD-688 inhibits DENV. NITD-688 binds directly to the nonstructural protein 4B (NS4B) with nanomolar affinities across all four DENV serotypes and specifically disrupts the interaction between NS4B and nonstructural protein 3 (NS3) without significantly changing the interactions between NS4B and other viral or host proteins. NS4B mutations that confer resistance to NITD-688 reduce both NITD-688 binding to NS4B and disruption of the NS4B/NS3 interaction. Specifically, NITD-688 blocks the interaction of NS3 with a cytosolic loop within NS4B. This inhibits the formation of new NS4B/NS3 complexes and disrupts pre-existing complexes in vitro and DENV-infected cells, ultimately inhibiting viral replication. Consistent with this mechanism, NITD-688 retains greater potency in cellular assays with delayed treatment compared to JNJ-1802, another NS4B inhibitor that has been studied in Phase II clinical trials. Together, these findings provide critical insights into the mechanism of action of NITD-688, facilitating the development of novel flavivirus NS4B inhibitors and informing future clinical interventions against DENV.

microbiology↗

bioRxiv2024

Single-molecule imaging reveals the role of membrane-binding motif and C-terminal domain of RNase E in its localization and diffusion in Escherichia coli

In Escherichia coli, RNase E, a central enzyme in RNA processing and mRNA degradation, contains a catalytic N-terminal domain (NTD), a membrane-targeting sequence (MTS), and a C-terminal domain (CTD). We investigated how MTS and CTD influence RNase E localization, diffusion, and function. Super-resolution microscopy revealed that [~]93% of RNase E localizes to the inner membrane and exhibits slow diffusion similar to polysomes. Comparing the native amphipathic MTS with a transmembrane motif showed that the MTS confers slower diffusion and stronger membrane binding. The CTD further slows diffusion by increasing mass but unexpectedly weakens membrane association. RNase E mutants with partial cytoplasmic localization displayed enhanced co-transcriptional degradation of lacZ mRNA. These findings indicate that variations in the MTS and the presence of the CTD shape the spatiotemporal organization of RNA processing in bacterial cells, providing mechanistic insight into how RNase E domain architecture influences its cellular function.

microbiology↗

bioRxiv2024

Re-defining how mRNA degradation is coordinated with transcription and translation in bacteria

In eukaryotic cells, transcription, translation, and mRNA degradation occur in distinct subcellular regions. How these mRNA processes are organized in bacteria, without employing membrane-bound compartments, remains unclear. Here, we present generalizable principles underlying coordination between these processes in bacteria. In Escherichia coli, we found that co-transcriptional degradation is rare for mRNAs except for those encoding inner membrane proteins, due to membrane localization of the main ribonuclease, RNase E. We further found, by varying ribosome binding sequences, that translation affects mRNA stability not because ribosomes protect mRNA from degradation, but because low translation leads to premature transcription termination in the absence of transcription-translation coupling. Extending our analyses to Bacillus subtilis and Caulobacter crescentus, we established subcellular localization of RNase E (or its homolog) and premature transcription termination in the absence of transcription-translation coupling as key determinants that explain differences in transcriptional and translational coupling to mRNA degradation across genes and species.

microbiology↗

bioRxiv2024

Evaluation of Peregrinus maidis transformer-2 as a target for CRISPR-based control

The corn planthopper, Peregrinus maidis, is an economically important pest of corn and sorghum. Here we report the initial steps towards developing a CRISPR-based control method, precision guided sterile insect technique (pgSIT), for this hemipteran pest. Specifically, we evaluated the potential of transformer-2 (tra-2) as a target for sterilizing insects. First, we identified tra-2 transcripts within our P. maidis transcriptome database and performed RNA interference (RNAi) to confirm functional conservation. RNAi-mediated knockdown of Pmtra-2 in nymphs transformed females into pseudomales with deformed ovipositors resembling male claspers. While males showed no overt difference in appearance, they were indeed sterile. Importantly, the results were similar to those observed in another planthopper, Nilaparvata lugens. We also used CRISPR/Cas9 genome editing to assess the impact of tra-2 knockout in injectees. CRISPR-mediated knockout of Pmtra-2 had lethal effects on embryos, and hence not many injectees reached adulthood. However, mosaic knockout of Pmtra-2 did impact female and male fertility, which supports the use of tra-2 as a target for pgSIT in this hemipteran species.

genetics↗

bioRxiv2024

Regulatory T cells and IFN-γ-producing Th1 cells play a critical role in the pathogenesis of Sjögren's Syndrome

ObjectivesSjogrens Disease (SjD) is an autoimmune disorder characterized by progressive dysfunction, inflammation and destruction of salivary and lacrimal glands, and by extraglandular manifestations. Its etiology and pathophysiology remain incompletely understood, though a role for autoreactive B cells has been considered key. Here, we investigated the role of effector and regulatory T cells in the pathogenesis of SjD. MethodsHistological analysis, RNA-sequencing and flow cytometry were conducted on glands, lungs, eyes and lymphoid tissues of mice with regulatory T cell-specific deletion of stromal interaction proteins (STIM) 1 and 2 (Stim1/2Foxp3), which play key roles in calcium signaling and T cell function. The pathogenicity of T cells from Stim1/2Foxp3mice was investigated through adoptively transfer into lymphopenic host mice. Additionally, single-cell transcriptomic analysis was performed on peripheral blood mononuclear cells (PBMCs) of patients with SjD and control subjects. ResultsStim1/2Foxp3 mice develop a severe SjD-like disorder including salivary gland (SG) and lacrimal gland (LG) inflammation and dysfunction, autoantibodies and extraglandular symptoms. SG inflammation in Stim1/2Foxp3 mice is characterized by T and B cell infiltration, and transcriptionally by a Th1 immune response that correlates strongly with the dysregulation observed in patients with SjD. Adoptive transfer of effector T cells from Stim1/2Foxp3 mice demonstrates that the SjD-like disease is driven by interferon (IFN)-{gamma} producing autoreactive CD4+ T cells independently of B cells and autoantiboodies. scRNA-seq analysis identifies increased Th1 responses and attenuated memory Treg function in PBMCs of patients with SjD. ConclusionsWe report a more accurate mouse model of SjD while providing evidence for a critical role of Treg cells and IFN-{gamma} producing Th1 cells in the pathogenesis of SjD, which may be effective targets for therapy.

immunology↗