参考文献

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

Heat shock protein 90 (Hsp90) is a molecular chaperone important for maintaining protein homeostasis (proteostasis) in the cell. Hsp90 inhibitors are being explored as cancer therapeutics because of their ability to disrupt proteostasis. Inhibiting Hsp90 increases surface density of the immunological receptor Major Histocompatibility Complex 1 (MHC1). Here we show that this increase occurs across multiple cancer cell lines and with both cytosol-specific and pan-Hsp90 inhibitors. We demonstrate that Hsp90 inhibition also alters surface expression of both IFNGR and PD-L1,two additional immunological receptors that play a significant role in anti-tumour or anti-immune activity in the tumour microenvironment. Hsp90 also negatively regulates IFN-γ activity in cancer cells,suggesting it has a unique role in mediating the immune system’s response to cancer. Our data suggests a strong link between Hsp90 activity and the pathways that govern anti-tumour immunity. This highlights the potential for the use of an Hsp90 inhibitor in combination with another currently available cancer treatment,immune checkpoint blockade therapy,which works to prevent immune evasion of cancer cells. Combination checkpoint inhibitor therapy and the use of an Hsp90 inhibitor may potentiate the therapeutic benefits of both treatments and improve prognosis for cancer patients. 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

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

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

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

BACKGROUND:Venous thromboembolism is a major health problem. After thrombus formation,its resolution is essential to re-establish blood flow,which is crucially mediated by infiltrating neutrophils and monocytes in concert with activated platelets and endothelial cells. Thus,we aimed to modulate leukocyte function during thrombus resolution post-thrombus formation by blocking P-selectin/CD62P-mediated cell interactions.METHODS:Thrombosis was induced by inferior vena cava stenosis through ligation in mice. After 1 day,a P-selectin-blocking antibody or isotype control was administered and thrombus composition and resolution were analyzed.RESULTS:Localizing neutrophils and macrophages in thrombotic lesions of wild-type mice revealed that these cells enter the thrombus and vessel wall from the caudal end. Neutrophils were predominantly present 1 day and monocytes/macrophages 3 days after vessel ligation. Blocking P-selectin reduced circulating platelet-neutrophil and platelet-Ly6Chigh monocyte aggregates near the thrombus,and diminished neutrophils and Ly6Chigh macrophages in the cranial thrombus part compared with isotype-treated controls. Depletion of neutrophils 1 day after thrombus initiation did not phenocopy P-selectin inhibition but led to larger thrombi compared with untreated controls. In vitro,P-selectin enhanced human leukocyte function as P-selectin-coated beads increased reactive oxygen species production by neutrophils and tissue factor expression of classical monocytes. Accordingly,P-selectin inhibition reduced oxidative burst in the thrombus and tissue factor expression in the adjacent vessel wall. Moreover,blocking P-selectin reduced thrombus density determined by scanning electron microscopy and increased urokinase-type plasminogen activator levels in the thrombus,which accelerated caudal fibrin degradation from day 3 to day 14. This accelerated thrombus resolution as thrombus volume declined more rapidly after blocking P-selectin.CONCLUSIONS:Inhibition of P-selectin-dependent activation of monocytes and neutrophils accelerates venous thrombosis resolution due to reduced infiltration and activation of innate immune cells at the site of thrombus formation,which prevents early thrombus stabilization and facilitates fibrinolysis. 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

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

Allogeneic CAR T–cell therapies are being developed for hematologic malignancies. The authors implement a Cas12a chRDNA platform to generate allogeneic immune-cloaked BCMA-specific CAR T cells with resistance to host response–mediated rejection for evaluation in multiple myeloma. AbstractAllogeneic chimeric antigen receptor (CAR) T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR T cells. Key considerations in the development of allogeneic CAR T cell therapies include prevention of graft-vs-host disease (GvHD) and suppression of allograft rejection. Here,we describe preclinical data supporting the ongoing first-in-human clinical study,the CaMMouflage trial (NCT05722418),evaluating CB-011 in patients with relapsed/refractory multiple myeloma. CB-011 is a hypoimmunogenic,allogeneic anti–B-cell maturation antigen (BCMA) CAR T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor–derived T cells using a Cas12a CRISPR hybrid RNA–DNA (chRDNA) genome-editing technology platform. To address allograft rejection,CAR T cells were engineered to prevent endogenous HLA class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. In addition,T-cell receptor (TCR) expression was disrupted at the TCR alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell cocultures derived from patients with multiple myeloma. In addition,CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer cell–mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M–HLA-E. Potent antitumor efficacy,when combined with an immune-cloaking armoring strategy to dampen allograft rejection,offers optimized therapeutic potential in multiple myeloma. See related Spotlight by Caimi and Melenhorst,p. 385 View Publication