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BackgroundNeutrophils are granulocytes with essential antimicrobial effector functions and short lifespans. During infection or sterile inflammation,emergency granulopoiesis leads to release of immature neutrophils from the bone marrow,serving to boost circulating neutrophil counts. Steady state and emergency granulopoiesis are incompletely understood,partly due to a lack of genetically amenable models of neutrophil development.MethodsWe optimised a method for ex vivo production of human neutrophils from CD34+ haematopoietic progenitors. Using flow cytometry,we phenotypically compared cultured neutrophils with native neutrophils from donors experiencing emergency granulopoiesis,and steady state neutrophils from non-challenged donors. We carry out functional and proteomic characterisation of cultured neutrophils and establish genome editing of progenitors.ResultsWe obtain high yields of ex vivo cultured neutrophils,which phenotypically resemble immature neutrophils released into the circulation during emergency granulopoiesis. Cultured neutrophils have similar rates of ROS production and bacterial killing but altered degranulation,cytokine release and antifungal activity compared to mature neutrophils isolated from peripheral blood. These differences are likely due to incomplete synthesis of granule proteins,as demonstrated by proteomic analysis.ConclusionEx vivo cultured neutrophils are genetically tractable via genome editing of precursors and provide a powerful model system for investigating the properties and behaviour of immature neutrophils.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-024-05337-x. View Publication

BackgroundHematopoietic stem cell (HSC) regeneration underlies hematopoietic recovery from myelosuppression,which is a life-threatening side effect of cytotoxicity. HSC niche is profoundly disrupted after myelosuppressive injury,while if and how the niche is reshaped and regulates HSC regeneration are poorly understood.MethodsA mouse model of radiation injury-induced myelosuppression was built by exposing mice to a sublethal dose of ionizing radiation. The dynamic changes in the number,distribution and functionality of HSCs and megakaryocytes were determined by flow cytometry,immunofluorescence,colony assay and bone marrow transplantation,in combination with transcriptomic analysis. The communication between HSCs and megakaryocytes was determined using a coculture system and adoptive transfer. The signaling mechanism was investigated both in vivo and in vitro,and was consolidated using megakaryocyte-specific knockout mice and transgenic mice.ResultsMegakaryocytes become a predominant component of HSC niche and localize closer to HSCs after radiation injury. Meanwhile,transient insulin-like growth factor 1 (IGF1) hypersecretion is predominantly provoked in megakaryocytes after radiation injury,whereas HSCs regenerate paralleling megakaryocytic IGF1 hypersecretion. Mechanistically,HSCs are particularly susceptible to megakaryocytic IGF1 hypersecretion,and mTOR downstream of IGF1 signaling not only promotes activation including proliferation and mitochondrial oxidative metabolism of HSCs,but also inhibits ferritinophagy to restrict HSC ferroptosis. Consequently,the delicate coordination between proliferation,mitochondrial oxidative metabolism and ferroptosis ensures functional HSC expansion after radiation injury. Importantly,punctual IGF1 administration simultaneously promotes HSC regeneration and hematopoietic recovery after radiation injury,representing a superior therapeutic approach for myelosuppression.ConclusionsOur study identifies megakaryocytes as a last line of defense against myelosuppressive injury and megakaryocytic IGF1 as a novel niche signal safeguarding HSC regeneration.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12964-024-01651-5. View Publication

The antitumor efficacy of adoptively transferred T cells is limited by their poor persistence,in part due to exhaustion,but the underlying mechanisms and potential interventions remain underexplored. Here,we show that targeting histone demethylase LSD1 by chemical inhibitors reshapes the epigenome of in vitro activated and expanded CD8+ T cells,and potentiates their antitumor efficacy. Upon T cell receptor activation and IL-2 signaling,a timely and transient inhibition of LSD1 suffices to improve the memory phenotype of mouse CD8+ T cells,associated with a better ability to produce multiple cytokines,resist exhaustion,and persist in both antigen-dependent and -independent manners after adoptive transfer. Consequently,OT1 cells primed with LSD1 inhibitors demonstrate an enhanced antitumor effect in OVA-expressing solid tumor models implanted in female mice,both as a standalone treatment and in combination with PD-1 blockade. Moreover,priming with LSD1 inhibitors promotes polyfunctionality of human CD8+ T cells,and increases the persistence and antitumor efficacy of human CD19-CAR T cells in both leukemia and solid tumor models. Thus,pharmacological inhibition of LSD1 could be exploited to improve adoptive T cell therapy. Phenotypic changes in exhausted T cells are linked to chromatin remodeling. Here the authors show that pharmacological inhibition of the H3K4me1/2 demethylase LSD1 promotes the persistence and enhances the therapeutic activity of adoptively transferred T cells for cancer therapy. View Publication

An imbalance between suppressor and effector immune responses may preclude cure in chronic parasitic diseases. In the case of Trypanosoma cruzi infection,specialized regulatory Foxp3+ T (Treg) cells suppress protective type-1 effector responses. Herein,we investigated the kinetics and underlying mechanisms behind the regulation of protective parasite-specific CD8+ T cell immunity during acute T. cruzi infection. Using the DEREG mouse model,we found that Treg cells play a role during the initial stages after T. cruzi infection,restraining the magnitude of CD8+ T cell responses and parasite control. Early Treg cell depletion increased the frequencies of polyfunctional short-lived,effector T cell subsets,without affecting memory precursor cell formation or the expression of activation,exhaustion and functional markers. In addition,Treg cell depletion during early infection minimally affected the antigen-presenting cell response but it boosted CD4+ T cell responses before the development of anti-parasite effector CD8+ T cell immunity. Crucially,the absence of CD39 expression on Treg cells significantly bolstered effector parasite-specific CD8+ T cell responses,preventing increased parasite replication in T. cruzi infected mice adoptively transferred with Treg cells. Our work underscores the crucial role of Treg cells in regulating protective anti-parasite immunity and provides evidence that CD39 expression by Treg cells represents a key immunomodulatory mechanism in this infection model. Author summaryChagas disease,caused by Trypanosoma cruzi,can result in severe health complications. While the exact mechanisms underlying the disease’s pathogenesis remain incompletely understood,the host’s inflammatory immune response is believed to play a critical role. To shed light on disease mechanisms and potential treatments,we investigated the impact of regulatory T (Treg) cells on the development of effector immune responses against T. cruzi. Our findings reveal that Treg cells dampen parasite-specific CD8+ T cells,a crucial arm of the immune response in counteracting the parasite. Notably,this regulatory influence occurs primarily during the early stages of T. cruzi infection. Furthermore,we observed that while Treg cells have minimal effects on antigen-presenting cells,they modulate the magnitude and phenotype of conventional CD4+ T cells. Importantly,we identified CD39,a molecule involved in the purinergic pathway,as essential for the suppressive functions of Treg cells during T. cruzi infection. Our findings enhance the understanding of the regulatory response during the acute phase of T. cruzi infection and may have implications for the development of novel therapeutic strategies. View Publication

Only a minority of cancer patients benefit from immune checkpoint blockade therapy. Sophisticated cross-talk among different immune checkpoint pathways as well as interaction pattern of immune checkpoint molecules carried on circulating small extracellular vesicles (sEV) might contribute to the low response rate. Here we demonstrate that PD-1 and CD80 carried on immunocyte-derived sEVs (I-sEV) induce an adaptive redistribution of PD-L1 in tumour cells. The resulting decreased cell membrane PD-L1 expression and increased sEV PD-L1 secretion into the circulation contribute to systemic immunosuppression. PD-1/CD80+ I-sEVs also induce downregulation of adhesion- and antigen presentation-related molecules on tumour cells and impaired immune cell infiltration,thereby converting tumours to an immunologically cold phenotype. Moreover,synchronous analysis of multiple checkpoint molecules,including PD-1,CD80 and PD-L1,on circulating sEVs distinguishes clinical responders from those patients who poorly respond to anti-PD-1 treatment. Altogether,our study shows that sEVs carry multiple inhibitory immune checkpoints proteins,which form a potentially targetable adaptive loop to suppress antitumour immunity. Immune checkpoint inhibition is a successful form of immune therapy; however response rates vary widely among individual patients. Here authors show that circulating small extracellular vesicles might contribute to poor response to anti-PD-1 treatment by carrying PD-1 and CD80 which results in higher level of vesicular PD-L1 expression in the circulation at the expense of expression on tumour cell membranes,causing immunosuppression. View Publication

Innate antiviral factors are essential for effective defense against viral pathogens. However,the identity of major restriction mechanisms remains elusive. Current approaches to discover antiviral factors usually focus on the initial steps of viral replication and are limited to a single round of infection. Here,we engineered libraries of >1500 replication-competent HIV-1 constructs each expressing a single gRNAs to target >500 cellular genes for virus-driven discovery of antiviral factors. Passaging in CD4+ T cells robustly enriched HIV-1 encoding sgRNAs against GRN,CIITA,EHMT2,CEACAM3,CC2D1B and RHOA by >50-fold. Using an HIV-1 library lacking the accessory nef gene,we identified IFI16 as a Nef target. Functional analyses in cell lines and primary CD4+ T cells support that the HIV-driven CRISPR screen identified restriction factors targeting virus entry,transcription,release and infectivity. Our HIV-guided CRISPR technique enables sensitive discovery of physiologically relevant cellular defense factors throughout the entire viral replication cycle. Innate immune mechanisms are critical for antiviral defense. Here,the authors developed a CRISPR/Cas9-based HIV-driven approach to identify cellular factors compromising viral transcription,assembly,release or infectivity in human T cells. They identify targets of the Nef protein as antiviral factors. 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

Manufacturing chimeric antigen receptor (CAR) T cell therapies is complex,with limited understanding of how medium composition impacts T cell phenotypes. CRISPR-Cas9 ribonucleoproteins can precisely insert a CAR sequence while disrupting the endogenous T cell receptor alpha constant (TRAC) gene resulting in TRAC-CAR T cells with an enriched stem cell memory T cell population,a process that could be further optimized through modifications to the medium composition. In this study we generated anti-GD2 TRAC-CAR T cells using "metabolic priming" (MP),where the cells were activated in glucose/glutamine-low medium and then expanded in glucose/glutamine-high medium. T cell products were evaluated using spectral flow cytometry,metabolic assays,cytokine production,cytotoxicity assays in vitro,and potency against human GD2+ xenograft neuroblastoma models in vivo. Compared with standard TRAC-CAR T cells,MP TRAC-CAR T cells showed less glycolysis,higher CCR7/CD62L expression,more bound NAD(P)H activity,and reduced IFN-γ,IL-2,IP-10,IL-1β,IL-17,and TGF-β production at the end of manufacturing ex vivo,with increased central memory CAR T cells and better persistence observed in vivo. MP with medium during CAR T cell biomanufacturing can minimize glycolysis and enrich memory phenotypes ex vivo,which could lead to better responses against solid tumors in vivo. Graphical abstract Cappabianca and colleagues manufactured virus-free CAR T cells at scale with CRISPR-Cas9 and “metabolically primed” them by attenuating activation in low-glucose/glutamine medium with expansion in high-glucose/glutamine medium. Priming made CAR T cells with increased stem cell memory properties,including enriched central memory phenotypes in vivo while lysing solid tumors. 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

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

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

Midnolin is an essential gene with previously unknown effects in vivo. This paper shows that midnolin stimulates proteasome activity necessary for lymphopoiesis and B cell cancer growth in mice. In a genetic screen,we identified two viable missense alleles of the essential gene Midnolin (Midn) that were associated with reductions in peripheral B cells. Causation was confirmed in mice with targeted deletion of four of six MIDN protein isoforms. MIDN was expressed predominantly in lymphocytes where it augmented proteasome activity. We showed that purified MIDN directly stimulated 26S proteasome activity in vitro in a manner dependent on the ubiquitin-like domain and a C-terminal region. MIDN-deficient B cells displayed aberrant activation of the IRE-1/XBP-1 pathway of the unfolded protein response. Partial or complete MIDN deficiency strongly suppressed Eμ-Myc–driven B cell leukemia and the antiapoptotic effects of Eμ-BCL2 on B cells in vivo and induced death of Sp2/0 hybridoma cells in vitro,but only partially impaired normal lymphocyte development. Thus,MIDN is required for proteasome activity in support of normal lymphopoiesis and is essential for malignant B cell proliferation over a broad range of differentiation states. View Publication