技术资料

AbstractT cells in the human female genital tract (FGT) are key mediators of susceptibility to and protection from infection,including HIV and other sexually transmitted infections. There is a critical need for increased understanding of the distribution and activation of T cell populations in the FGT,but current sampling methods require a healthcare provider and are expensive,limiting the ability to study these populations longitudinally. To address these challenges,we have developed a method to sample immune cells from the FGT utilizing disposable menstrual discs which are noninvasive,self-applied,and low in cost. To demonstrate reproducibility,we sampled the cervicovaginal fluid of healthy,reproductive-aged individuals using menstrual discs across 3 sequential days. Cervicovaginal fluid was processed for cervicovaginal cells,and high-parameter flow cytometry was used to characterize immune populations. We identified large numbers of live,CD45+ leukocytes,as well as distinct populations of T cells and B cells. Within the T cell compartment,activation and suppression status of T cell subsets were consistent with previous studies of the FGT utilizing current approaches,including identification of both tissue-resident and migratory populations. In addition,the T cell population structure was highly conserved across days within individuals but divergent across individuals. Our approach to sample immune cells in the FGT with menstrual discs will decrease barriers to participation and empower longitudinal sampling in future research studies. View Publication

BackgroundDendritic cell (DC)-mediated antigen presentation is essential for the priming and activation of tumor-specific T cells. However,few drugs that specifically manipulate DC functions are available. The identification of drugs targeting DC holds great promise for cancer immunotherapy.MethodsWe observed that type 1 conventional DCs (cDC1s) initiated a distinct transcriptional program during antigen presentation. We used a network-based approach to screen for cDC1-targeting therapeutics. The antitumor potency and underlying mechanisms of the candidate drug were investigated in vitro and in vivo.ResultsSitagliptin,an oral gliptin widely used for type 2 diabetes,was identified as a drug that targets DCs. In mouse models,sitagliptin inhibited tumor growth by enhancing cDC1-mediated antigen presentation,leading to better T-cell activation. Mechanistically,inhibition of dipeptidyl peptidase 4 (DPP4) by sitagliptin prevented the truncation and degradation of chemokines/cytokines that are important for DC activation. Sitagliptin enhanced cancer immunotherapy by facilitating the priming of antigen-specific T cells by DCs. In humans,the use of sitagliptin correlated with a lower risk of tumor recurrence in patients with colorectal cancer undergoing curative surgery.ConclusionsOur findings indicate that sitagliptin-mediated DPP4 inhibition promotes antitumor immune response by augmenting cDC1 functions. These data suggest that sitagliptin can be repurposed as an antitumor drug targeting DC,which provides a potential strategy for cancer immunotherapy. View Publication

SUMMARYThe mechanistic target of rapamycin (mTOR) positively regulates multiple steps of the HIV-1 replication cycle. We previously reported that a 12-weeks supplementation of antiretroviral therapy (ART) with metformin,an indirect mTOR inhibitor used in type-2 diabetes treatment,reduced mTOR activation and HIV transcription in colon-infiltrating CD4+ T-cells,together with systemic inflammation in nondiabetic people with HIV-1 (PWH). Herein,we investigated the antiviral mechanisms of metformin. In a viral outgrowth assay performed with CD4+ T-cells from ART-treated PWH,and upon infection in vitro with replication-competent and VSV-G-pseudotyped HIV-1,metformin decreased virion release,but increased the frequency of productively infected CD4lowHIV-p24+ T-cells. These observations coincided with increased BST2/Tetherin (HIV release inhibitor) and Bcl-2 (pro-survival factor) expression,and improved recognition of productively infected T-cells by HIV-1 Envelope antibodies. Thus,metformin exerts pleiotropic effects on post-transcription/translation steps of the HIV-1 replication cycle and may be used to accelerate viral reservoir decay in ART-treated PWH. Graphical Abstract View Publication

Understanding the molecular and cellular processes involved in lung epithelial regeneration may fuel the development of therapeutic approaches for lung diseases. We combine mouse models allowing diphtheria toxin-mediated damage of specific epithelial cell types and parallel GFP-labeling of functionally dividing cells with single-cell transcriptomics to characterize the regeneration of the distal lung. We uncover cell types,including Krt13+ basal and Krt15+ club cells,detect an intermediate cell state between basal and goblet cells,reveal goblet cells as actively dividing progenitor cells,and provide evidence that adventitial fibroblasts act as supporting cells in epithelial regeneration. We also show that diphtheria toxin-expressing cells can persist in the lung,express specific inflammatory factors,and transcriptionally resemble a previously undescribed population in the lungs of COVID-19 patients. Our study provides a comprehensive single-cell atlas of the distal lung that characterizes early transcriptional and cellular responses to concise epithelial injury,encompassing proliferation,differentiation,and cell-to-cell interactions. This study uses single-cell transcriptomics to examine how lung cells respond to targeted damage. The authors employ genetically modified mouse models and cell sorting to enrich for rare,actively dividing cells,revealing cell types/states and alternative differentiation paths. View Publication

AbstractThe effect of targeted therapeutics on anticancer immune responses is poorly understood. The BRAF inhibitor dabrafenib has been reported to activate the integrated stress response (ISR) kinase GCN2,and the therapeutic effect has been partially attributed to GCN2 activation. Because ISR signaling is a key component of myeloid-derived suppressor cell (MDSC) development and function,we measured the effect of dabrafenib on MDSC differentiation and suppressive activity. Our data showed that dabrafenib attenuated MDSC ability to suppress T-cell activity,which was associated with a GCN2-dependent block of the transition from monocytic progenitor to polymorphonuclear (PMN)-MDSCs and proliferative arrest resulting in PMN-MDSC loss. Transcriptional profiling revealed that dabrafenib-driven GCN2 activation altered metabolic features in MDSCs enhancing oxidative respiration,and attenuated transcriptional programs required for PMN development. Moreover,we observed a broad downregulation of transcriptional networks associated with PMN developmental pathways,and increased activity of transcriptional regulons driven by Atf5,Mafg,and Zbtb7a. This transcriptional program alteration underlies the basis for PMN-MDSC developmental arrest,skewing immature MDSC development toward monocytic lineage cells. In vivo,we observed a pronounced reduction in PMN-MDSCs in dabrafenib-treated tumor-bearing mice suggesting that dabrafenib impacts MDSC populations systemically and locally,in the tumor immune infiltrate. Thus,our data reveal transcriptional networks that govern MDSC developmental programs,and the impact of GCN2 stress signaling on the innate immune landscape in tumors,providing novel insight into potentially beneficial off-target effects of dabrafenib.Significance:An important,but poorly understood,aspect of targeted therapeutics for cancer is the effect on antitumor immune responses. This article shows that off-target effects of dabrafenib activating the kinase GCN2 impact MDSC development and function reducing PMN-MDSCs in vitro and in vivo. This has important implications for our understanding of how this BRAF inhibitor impacts tumor growth and provides novel therapeutic target and combination possibilities. View Publication

ObjectivesIn SLE,deregulation of haematopoiesis is characterised by inflammatory priming and myeloid skewing of haematopoietic stem and progenitor cells (HSPCs). We sought to investigate the role of extramedullary haematopoiesis (EMH) as a key player for tissue injury in systemic autoimmune disorders.MethodsTranscriptomic analysis of bone marrow (BM)-derived HSPCs from patients with SLE and NZBW/F1 lupus-prone mice was performed in combination with DNA methylation profile. Trained immunity (TI) was induced through β-glucan administration to the NZBW/F1 lupus-prone model. Disease activity was assessed through lupus nephritis (LN) histological grading. Colony-forming unit assay and adoptive cell transfer were used to assess HSPCs functionalities.ResultsTranscriptomic analysis shows that splenic HSPCs carry a higher inflammatory potential compared with their BM counterparts. Further induction of TI,through β-glucan administration,exacerbates splenic EMH,accentuates myeloid skewing and worsens LN. Methylomic analysis of BM-derived HSPCs demonstrates myeloid skewing which is in part driven by epigenetic tinkering. Importantly,transcriptomic analysis of human SLE BM-derived HSPCs demonstrates similar findings to those observed in diseased mice.ConclusionsThese data support a key role of granulocytes derived from primed HSPCs both at medullary and extramedullary sites in the pathogenesis of LN. EMH and TI contribute to SLE by sustaining the systemic inflammatory response and increasing the risk for flare. View Publication

BackgroundDifferences in male vs. female immune responses are well-documented and have significant clinical implications. While the immunomodulatory effects of sex hormones are well established,the contributions of sex chromosome complement (XX vs. XY) and gut microbiome diversity on immune sexual dimorphisms have only recently become appreciated. Here we investigate the individual and collaborative influences of sex chromosome complements and gut microbiota on humoral immune activation.MethodsMale and female Four Core Genotype (FCG) mice were immunized with heat-killed Streptococcus pneumoniae (HKSP). Humoral immune responses were assessed,and X-linked immune-related gene expression was evaluated to explain the identified XX-dependent phenotype. The functional role of Kdm6a,an X-linked epigenetic regulatory gene of interest,was evaluated ex vivo using mitogen stimulation of B cells. Additional influences of the gut microbiome on sex chromosome-dependent B cell activation was also evaluated by antibiotically depleting gut microbiota prior to HKSP immunization. Reconstitution of the depleted microbiome with short-chain fatty acid (SCFA)-producing bacteria tested the impact of SCFAs on XX-dependent immune activation.ResultsXX mice exhibited higher HKSP-specific IgM-secreting B cells and plasma cell frequencies than XY mice,regardless of gonadal sex. Although Kdm6a was identified as an X-linked gene overexpressed in XX B cells,inhibition of its enzymatic activity did not affect mitogen-induced plasma cell differentiation or antibody production in a sex chromosome-dependent manner ex vivo. Enhanced humoral responses in XX vs. XY immunized FCG mice were eliminated after microbiome depletion,indicating that the microbiome contributes to the identified XX-dependent immune enhancement. Reconstituting microbiota-depleted mice with select SCFA-producing bacteria enhanced fecal SCFA concentrations and increased humoral responses in XX,but not XY,FCG mice. However,exposure to the SCFA propionate alone did not enhance mitogenic B cell stimulation in ex vivo studies.ConclusionsFCG mice have been used to assess sex hormone and sex chromosome complement influences on various sexually dimorphic traits. The current study indicates that the gut microbiome impacts humoral responses in an XX-dependent manner,suggesting that the collaborative influence of gut bacteria and other sex-specific factors should be considered when interpreting data aimed at delineating the mechanisms that promote sexual dimorphism.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13293-024-00597-0. Highlights Humoral immune responses against HKSP immunization are influenced by the possession of an XX vs. XY sex chromosome complement. While gonadal sex differentially influenced the number of antigen-specific IgM-secreting cells,the overall percentage of CD138 + plasma cells generated in response to HKSP immunization was not influenced by gonadal sex.Kdm6a is overexpressed in XX vs. XY B cells and splenocytes of HKSP-immunized mice and is demonstrated to be biallelically expressed in a subset of B cells.Ex vivo inhibition of KDM6a enzymatic activity promotes plasma cell differentiation in response to mitogenic stimulation. However,this effect was not sex chromosome-dependent. KDM6a inhibition did not impact total IgM concentrations in culture supernatants following mitogenic stimulation.XX-dependent immune enhancement is microbiome-dependent. Reconstitution of the antibiotic-depleted gut microbiome with select SCFA-producing bacteria rescued the XX-dependent immune phenotype observed in XX,but not XY,FCG mice. Supplementary InformationThe online version contains supplementary material available at 10.1186/s13293-024-00597-0. Plain language summaryMale and female immune systems differ in their ability to respond to infectious challenge. While males tend to be more susceptible to infection and produce lower amounts of antibodies in response to vaccination,females are more prone to develop autoimmune and inflammatory diseases. Key contributors to these differences include sex hormones,sex chromosome complement (XX in females vs. XY in males),and distinct gut microbial communities capable of regulating immune activation. While each factor has been studied individually,this research underscores the potential for these factors to collaboratively impact immune activation. Here,possession of an XX vs. XY sex chromosome complement was demonstrated to enhance antibody responses to heat-killed Streptococcus pneumoniae vaccination. While attempting to determine the underlying cause of this immune enhancement,the gut microbiome was identified to play a critical role. In the absence of an intact gut microbiome,XX immune activation was reduced to levels similar to those seen in XY sex chromosome complement-possessing mice. Replacement of the depleted gut microbiomes with select SCFA-producing bacterial species enhanced SCFA levels in antibiotic-treated mice and rescued the XX-dependent immune enhancement,suggesting a SCFA-mediated contribution. Further studies are needed to determine exactly how these select bacteria impact immune activation in a sex chromosome complement-dependent manner. Our findings highlight the need to consider the collaborative effects of individual sex-specific factors when attempting to understand immune sex biases,as a better understanding of these interactions will likely pave the way for improving therapeutics and vaccines tailored to both sexes.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13293-024-00597-0. View Publication

More than 80% of patients with myasthenia gravis (MG) are positive for anti-acetylcholine receptor (AChR) antibodies. Regulatory T cells (Tregs) suppress overproduction of these antibodies,and patients with AChR antibody-positive MG (AChR MG) exhibit impaired Treg function and reduced Treg numbers. The gut microbiota and their metabolites play a crucial role in maintaining Treg differentiation and function. However,whether impaired Tregs correlate with gut microbiota activity in patients with AChR MG remains unknown. Here,we demonstrate that butyric acid-producing gut bacteria and serum butyric acid level are reduced in patients with AChR MG. Butyrate supplementation effectively enhanced Treg differentiation and their suppressive function of AChR MG. Mechanistically,butyrate activates autophagy of Treg cells by inhibiting the mammalian target of rapamycin. Activation of autophagy increased oxidative phosphorylation and surface expression of cytotoxic T-lymphocyte-associated protein 4 on Treg cells,thereby promoting Treg differentiation and their suppressive function in AChR MG. This observed effect of butyrate was blocked using chloroquine,an autophagy inhibitor,suggesting the vital role of butyrate-activated autophagy in Tregs of patients with AChR MG. We propose that gut bacteria derived butyrate has potential therapeutic efficacy against AChR MG by restoring impaired Tregs.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-024-01588-9. View Publication

SUMMARYRegulatory T cells (Tregs) are promising cellular therapies to induce immune tolerance in organ transplantation and autoimmune disease. The success of chimeric antigen receptor (CAR) T-cell therapy for cancer has sparked interest in using CARs to generate antigen-specific Tregs. Here,we compared CAR with endogenous T cell receptor (TCR)/CD28 activation in human Tregs. Strikingly,CAR Tregs displayed increased cytotoxicity and diminished suppression of antigen-presenting cells and effector T (Teff) cells compared with TCR/CD28 activated Tregs. RNA sequencing revealed that CAR Tregs activate Teff cell gene programs. Indeed,CAR Tregs secreted high levels of inflammatory cytokines,with a subset of FOXP3+ CAR Tregs uniquely acquiring CD40L surface expression and producing IFNγ. Interestingly,decreasing CAR antigen affinity reduced Teff cell gene expression and inflammatory cytokine production by CAR Tregs. Our findings showcase the impact of engineered receptor activation on Treg biology and support tailoring CAR constructs to Tregs for maximal therapeutic efficacy. Graphical Abstract View Publication

SUMMARY Tumor-associated macrophages (TAMs) shape tumor immunity and therapeutic efficacy. However,it is poorly understood whether and how post-translational modifications (PTMs) intrinsically affect the phenotype and function of TAMs. Here,we reveal that peptidylarginine deiminase 4 (PAD4) exhibits the highest expression among common PTM enzymes in TAMs and negatively correlates with the clinical response to immune checkpoint blockade. Genetic and pharmacological inhibition of PAD4 in macrophages prevents tumor progression in tumor-bearing mouse models,accompanied by an increase in macrophage major histocompatibility complex (MHC) class II expression and T cell effector function. Mechanistically,PAD4 citrullinates STAT1 at arginine 121,thereby promoting the interaction between STAT1 and protein inhibitor of activated STAT1 (PIAS1),and the loss of PAD4 abolishes this interaction,ablating the inhibitory role of PIAS1 in the expression of MHC class II machinery in macrophages and enhancing T cell activation. Thus,the PAD4-STAT1-PIAS1 axis is an immune restriction mechanism in macrophages and may serve as a cancer immunotherapy target. Graphical Abstract In brief Pitter et al. demonstrate that the PAD4-mediated citrullination of STAT1 in macrophages enforces the STAT1-PIAS1 interaction restraining STAT1 transcriptional activity and MHC class II machinery expression and,consequently,limits T cell-mediated anti-tumor immunity. View Publication

Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3′ ends of CRISPR–Cas guide RNAs1. To identify cellular determinants of prime editing,we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens,a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2,PE3,PE4 and PE5),edit types (substitutions,insertions and deletions),endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard,unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3′ ends of RNA polymerase III transcripts2. We found that La functionally interacts with the 3′ ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results,we developed a prime editor protein (PE7) fused to the RNA-binding,N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs),as well as with synthetic pegRNAs optimized for La binding. Together,our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein. Genome-scale genetic screens identify the small RNA-binding protein La as a strong mediator of prime editing. View Publication

AbstractPolysialic acid (polySia) is a linear polymer of α2,8-linked sialic acid residues that is of fundamental biological interest due to its pivotal roles in the regulation of the nervous,immune,and reproductive systems in healthy human adults. PolySia is also dysregulated in several chronic diseases,including cancers and mental health disorders. However,the mechanisms underpinning polySia biology in health and disease remain largely unknown. The polySia-specific hydrolase,endoneuraminidase NF (EndoN),and the catalytically inactive polySia lectin EndoNDM,have been extensively used for studying polySia. However,EndoN is heat stable and remains associated with cells after washing. When studying polySia in systems with multiple polysialylated species,the residual EndoN that cannot be removed confounds data interpretation. We developed a strategy for site-specific immobilization of EndoN on streptavidin-coated magnetic beads. We showed that immobilizing EndoN allows for effective removal of the enzyme from samples,while retaining hydrolase activity. We used the same strategy to immobilize the polySia lectin EndoNDM,which enabled the enrichment of polysialylated proteins from complex mixtures such as serum for their identification via mass spectrometry. We used this methodology to identify a novel polysialylated protein,QSOX2,which is secreted from the breast cancer cell line MCF-7. This method of site-specific immobilization can be utilized for other enzymes and lectins to yield insight into glycobiology. View Publication