技术资料

Gene silencing without gene editing holds great potential for the development of safe therapeutic applications. Here,we describe a novel strategy to concomitantly repress multiple genes using zinc finger proteins fused to Krüppel-Associated Box repression domains (ZF-Rs). This was achieved via the optimization of a lentiviral system tailored for the delivery of ZF-Rs in hematopoietic cells. We showed that an optimal design of the lentiviral backbone is crucial to multiplex up to three ZF-Rs or two ZF-Rs and a chimeric antigen receptor. ZF-R expression had no impact on the integrity and functionality of transduced cells. Furthermore,gene repression in ZF-R-expressing T cells was highly efficient in vitro and in vivo during the entire monitoring period (up to 10 weeks),and it was accompanied by epigenetic remodeling events. Finally,we described an approach to improve ZF-R specificity to illustrate the path toward the generation of ZF-Rs with a safe clinical profile. In conclusion,we successfully developed an epigenetic-based cell engineering approach for concomitant modulation of multiple gene expressions that bypass the risks associated with DNA editing. Graphical abstract David Fenard and colleagues developed a lentiviral backbone for the multiplexing of up to three ZF-R sequences,allowing an efficient,stable,and specific epigenetic control of multiple genes in T cells or Tregs after a single lentiviral transduction event. View Publication

IntroductionB-cell activation triggers the release of endoplasmic reticulum calcium stores through the store-operated calcium entry (SOCE) pathway resulting in calcium influx by calcium release-activated calcium (CRAC) channels on the plasma membrane. B-cell-specific murine knockouts of SOCE do not impact humoral immunity suggesting that alternative channels may be important.MethodsWe identified a member of the calcium-permeable transient receptor potential (TRP) ion channel family,TRPV5,as a candidate channel expressed in B cells by a quantitative polymerase chain reaction (qPCR) screen. To further investigate the role of TRPV5 in B-cell responses,we generated a murine TRPV5 knockout (KO) by CRISPR–Cas9. ResultsWe found TRPV5 polarized to B-cell receptor (BCR) clusters upon stimulation in a PI3K–RhoA-dependent manner. TRPV5 KO mice have normal B-cell development and mature B-cell numbers. Surprisingly,calcium influx upon BCR stimulation in primary TRPV5 KO B cells was not impaired; however,differential expression of other calcium-regulating proteins,such as ORAI1,may contribute to a compensatory mechanism for calcium signaling in these cells. We demonstrate that TRPV5 KO B cells have impaired spreading and contraction in response to membrane-bound antigen. Consistent with this,TRPV5 KO B cells have reduced BCR signaling measured through phospho-tyrosine residues. Lastly,we also found that TRPV5 is important for early T-dependent antigen specific responses post-immunization. DiscussionThus,our findings identify a role for TRPV5 in BCR signaling and B-cell activation. View Publication

The immune system has a critical role in orchestrating tissue healing. As a result,regenerative strategies that control immune components have proved effective1,2. This is particularly relevant when immune dysregulation that results from conditions such as diabetes or advanced age impairs tissue healing following injury2,3. Nociceptive sensory neurons have a crucial role as immunoregulators and exert both protective and harmful effects depending on the context4–12. However,how neuro–immune interactions affect tissue repair and regeneration following acute injury is unclear. Here we show that ablation of the NaV1.8 nociceptor impairs skin wound repair and muscle regeneration after acute tissue injury. Nociceptor endings grow into injured skin and muscle tissues and signal to immune cells through the neuropeptide calcitonin gene-related peptide (CGRP) during the healing process. CGRP acts via receptor activity-modifying protein 1 (RAMP1) on neutrophils,monocytes and macrophages to inhibit recruitment,accelerate death,enhance efferocytosis and polarize macrophages towards a pro-repair phenotype. The effects of CGRP on neutrophils and macrophages are mediated via thrombospondin-1 release and its subsequent autocrine and/or paracrine effects. In mice without nociceptors and diabetic mice with peripheral neuropathies,delivery of an engineered version of CGRP accelerated wound healing and promoted muscle regeneration. Harnessing neuro–immune interactions has potential to treat non-healing tissues in which dysregulated neuro–immune interactions impair tissue healing. Experiments in mouse models show that NaV1.8+ nociceptors innervate sites of injury and provide wound repair signals to immune cells by releasing calcitonin gene-related peptide (CGRP). 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

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

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

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

IntroductionLoss of proteasome function,proteinopathy,and proteotoxicity may cause neurodegeneration across the human lifespan in several forms of brain injury and disease. Drugs that activate brain proteasomes in vivo could thus have a broad therapeutic impact in neurology.MethodsUsing pigs,a clinically relevant large animal with a functionally compartmental gyrencephalic cerebral cortex,we evaluated the localization and biochemical activity of brain proteasomes and tested the ability of small molecules to activate brain proteasomes.ResultsBy Western blotting,proteasome protein subunit PSMB5 and PSMA3 levels were similar in different pig brain regions. Immunohistochemistry for PSMB5 showed localization in the cytoplasm (diffuse and particulate) and nucleus (cytoplasm < nucleus). Some PSMB5 immunoreactivity was colocalized with mitochondrial (voltage-gated anion channel and cyclophilin D) and cell death (Aven) proteins in the neuronal soma and neuropil in the neocortex of pig and human brains. In the nucleus,PSMB5 immunoreactivity was diffuse,particulate,and clustered,including perinucleolar decorations. By fluorogenic assay,proteasome chymotrypsin-like activities (CTL) in crude tissue soluble fractions were generally similar within eight different pig brain regions. Proteasome CTL activity in the hippocampus was correlated with activity in nasal mucosa biopsies. In pilot analyses of subcellular fractions of pig cerebral cortex,proteasome CTL activity was highest in the cytosol and then ~50% lower in nuclear fractions; ~15–20% of total CTL activity was in pure mitochondrial fractions. With in-gel activity assay,26S-singly and -doubly capped proteasomes were the dominant forms in the pig cerebral cortex. With a novel in situ histochemical activity assay,MG132-inhibitable proteasome CTL activity was localized to the neuropil,as a mosaic,and to cell bodies,nuclei,and centrosome-like perinuclear satellites. In piglets treated intravenously with pyrazolone derivative and chlorpromazine over 24 h,brain proteasome CTL activity was modestly increased.DiscussionThis study shows that the proteasome in the pig brain has relative regional uniformity,prominent nuclear and perinuclear presence with catalytic activity,a mitochondrial association with activity,26S-single cap dominance,and indications from small molecule systemic administration of pyrazolone derivative and chlorpromazine that brain proteasome function appears safely activable. 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

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

SummaryEnergy metabolism in the context of erythropoiesis and related diseases remains largely unexplored. Here,we developed a primary cell model by differentiating hematopoietic stem progenitor cells toward the erythroid lineage and suppressing the mitochondrial oxidative phosphorylation (OXPHOS) pathway. OXPHOS suppression led to differentiation failure of erythroid progenitors and defects in ribosome biogenesis. Ran GTPase-activating protein 1 (RanGAP1) was identified as a target of mitochondrial OXPHOS for ribosomal defects during erythropoiesis. Overexpression of RanGAP1 largely alleviated erythroid defects resulting from OXPHOS suppression. Coenzyme Q10,an activator of OXPHOS,largely rescued erythroid defects and increased RanGAP1 expression. Patients with Diamond-Blackfan anemia (DBA) exhibited OXPHOS suppression and a concomitant suppression of ribosome biogenesis. RNA-seq analysis implied that the substantial mutation (approximately 10%) in OXPHOS genes accounts for OXPHOS suppression in these patients. Conclusively,OXPHOS disruption and the associated disruptive mitochondrial energy metabolism are linked to the pathogenesis of DBA. Graphical abstract Highlights•Disruptive energy metabolism associates with the pathology of DBA•Suppression of OXPHOS leads to differentiation failure of erythroid progenitors•Energy metabolism disruption decreases overall ribosome levels in erythropoiesis•RanGAP1 is a target of OXPHOS pathway for ribosome biogenesis during erythropoiesis Cellular physiology; Cell biology; Developmental biology View Publication

AbstractBackgroundAcute graft‐versus‐host disease (aGVHD) arises from the imbalance of host T cells. Galectin‐9 negatively regulates CD4 effector T cell (Th1 and Th17) function by binding to Tim‐3. However,the relationship between Galectin‐9/Tim‐3 and CD4+ T subsets in patients with aGVHD after Haplo‐HSCT (haploidentical peripheral blood hematopoietic stem cell transplantation) has not been fully elucidated. Here,we investigated the role of Galectin‐9 and CD4+T subsets in aGVHD after haplo‐HSCT.MethodsForty‐two patients underwent Haplo‐HSCT (26 without aGVHD and 16 with aGVHD),and 20 healthy controls were included. The concentrations of Galectin‐9,interferon‐gamma (IFN‐γ),interleukin (IL)‐4,transforming growth factor (TGF)‐β,and IL‐17 in the serum and culture supernatant were measured using enzyme‐linked immunosorbent assay or cytometric bead array. The expression levels of Galectin‐9,PI3K,p‐PI3K,and p‐mTOR protein were detected by western blot analysis. Flow cytometry was used to analyze the proportions of CD4+ T cell subsets. Bioinformatics analysis was performed.ResultsIn patients with aGVHD,regulatory T (Treg) cells and Galectin‐9 decreased,and the Th1,Th17,and Treg cells were significantly imbalanced. Moreover,Treg and Galectin‐9 were rapidly reconstituted in the early stage of patients without aGVHD after Haplo‐HSCT,but Th17 cells were reconstituted slowly. Furthermore,Tim‐3 upregulation on Th17 and Th1 cells was associated with excessive activation of the PI3K/AKT pathway in patients with aGVHD. Specifically,in vitro treatment with Galectin‐9 reduced IFN‐γ and IL‐17 production while augmenting TGF‐β secretion. Bioinformatics analysis suggested the potential involvement of the PI3K/AKT/mTOR pathway in aGVHD. Mechanistically,exogenous Galectin‐9 was found to mitigate aGVHD by restoring the Treg/Teffs (effector T cells) balance and suppressing PI3K.ConclusionGalectin‐9 may ameliorate aGVHD after haplo‐HSCT by modulating Treg/Teffs balance and regulating the PI3K/AKT/mTOR pathway. Targeting Galectin‐9 may hold potential value for the treatment of aGVHD. In patients with acute graft‐versus‐host disease (aGVHD),the expression of Tim‐3 is significantly increased. Galectin‐9 binding to Tim‐3 may inhibit the activation of the PI3K/AKT pathway and enhance the function of Treg cells. On the other hand,transforming growth factor (TGF)‐β promotes the differentiation of Treg cells through autocrine secretion,while TGF‐β induces the expression of Galectin‐9 in a paracrine manner. The increased Treg cells can inhibit the activation of Th1 and Th17 cells by secreting TGF‐β,thus alleviating aGVHD and inducing immune tolerance View Publication