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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

AbstractLong single-stranded DNA (ssDNA) is a versatile molecular reagent with applications including RNA-guided genome engineering and DNA nanotechnology,yet its production is typically resource-intensive. We introduce a novel method utilizing an engineered Escherichia coli ‘helper’ strain and phagemid system that simplifies long ssDNA generation to a straightforward transformation and purification procedure. Our method obviates the need for helper plasmids and their associated contamination by integrating M13mp18 genes directly into the E. coli chromosome. We achieved ssDNA lengths ranging from 504 to 20 724 nt with titers up to 250 μg/l following alkaline lysis purification. The efficacy of our system was confirmed through its application in primary T-cell genome modifications and DNA origami folding. The reliability,scalability and ease of our approach promise to unlock new experimental applications requiring large quantities of long ssDNA. Graphical Abstract Graphical Abstract 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

A major limitation of chimeric antigen receptor (CAR) T cell therapies is the poor persistence of these cells in vivo1. The expression of memory-associated genes in CAR T cells is linked to their long-term persistence in patients and clinical efficacy2–6,suggesting that memory programs may underpin durable CAR T cell function. Here we show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR T cells. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes,promoted an exhaustion-like phenotype and impaired the antitumour activity of CAR T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR T cells that overexpressed FOXO1 retained their function,memory potential and metabolic fitness in settings of chronic stimulation,and exhibited enhanced persistence and tumour control in vivo. By contrast,overexpression of TCF1 (encoded by TCF7) did not enforce canonical memory programs or enhance the potency of CAR T cells. Notably,FOXO1 activity correlated with positive clinical outcomes of patients treated with CAR T cells or tumour-infiltrating lymphocytes,underscoring the clinical relevance of FOXO1 in cancer immunotherapy. Our results show that overexpressing FOXO1 can increase the antitumour activity of human CAR T cells,and highlight memory reprogramming as a broadly applicable approach for optimizing therapeutic T cell states. The transcription factor FOXO1 has a key role in human T cell memory,and manipulating FOXO1 expression could provide a way to enhance CAR T cell therapies by increasing CAR T cell persistence and antitumour activity. View Publication

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