Biology subjects

Salerno, F.

Publications and source records attributed to Salerno, F..

13 recordsLinked to original sources

bioRxiv2025

Oligosaccharyltransferase (OST) complex inhibition effectively treats rodent and human prions

Prion diseases are invariably fatal neurodegenerative diseases that occur when the prion protein misfolds into a pathogenic form. There are currently no clinical treatments or cures for prion disease. Current challenges in the development of prion therapeutics include prion strain specificity, which can cause the emergence of drug-resistant prions, and lack of efficacy in treating human prions despite promising results in rodent models. Here we identify a novel therapeutic target for prion disease: the oligosaccharyltransferase (OST) complex. The OST complex is responsible for transferring the mature glycan to the acceptor polypeptide during Nglycosylation. We found that inhibiting OST effectively treats rodent prions in various dividing and non-dividing cell types. Importantly, we also demonstrate efficacy in treating human sCJD prions in non-dividing cerebral organoids. Inhibition of OST results in a 50% reduction in cell surface expression of the prion protein, PrPC. In addition, lysates of cells treated with the OST inhibitor NGI-1 were unable to amplify PrPSc seeds in Protein Misfolding Cyclic Amplification (PMCA) reactions. In summary, our results identify OST as a novel therapeutic target that regulates both the abundance of cell surface PrPC as well as its ability to convert into multiple strains of PrPSc, including human prions, in various in vitro systems. Author SummaryPrion diseases, such as Creutzfeldt-Jakob disease, are fatal brain disorders caused when a normal protein (PrPC) misfolds into a harmful form that spreads through the brain. There are no effective treatments, and drug development has been hampered by "strain" differences in prions that can lead to resistance and by therapies that work in rodents but not in humans. This study identifies a new treatment target: the oligosaccharyltransferase (OST) complex, a cellular machine that adds sugar groups to proteins. Blocking OST with a small molecule (NGI1) limited prion growth in multiple types of rodent cells, including non-dividing cells, and-- critically--also worked against human sporadic CJD prions in laboratory-grown human brain organoids. OST inhibition cut the amount of normal prion protein on cell surfaces in half, reducing the raw material available to convert into the disease-causing form. In addition, extracts from NGI-1-treated cells could no longer drive prion formation in a sensitive lab amplification test. These results suggest that targeting OST may offer a new strategy that works across different prion strains, including human ones.

cell biology↗

bioRxiv2025

Requirements for development of T-helper1 and T-follicular helper cells from a common precursor

Activated CD4 T cells become either T-helper (Th) or T-follicular helper (Tfh) cells that support cellular or humoral immunity. We have investigated how polyclonal, vaccine induced T cells in mice bifurcate into Th1- and Tfh trajectories. We show that Th1 and Tfh cells originate from the same, highly proliferative precursor clones that co-express Th1- and Tfh-related transcription factors and chemokine receptors, including T-bet, BCL6, CXCR3 and CXCR5. Generation of the common Th1/Tfh precursor pool from antigen-specific CD4 T cells relies on CD28 costimulation but occurs independently of type-1 conventional dendritic cells (cDC1s) or B cells. Differentiation of Th1/Tfh precursors into the Th1 lineage relies on CD40 costimulation and cDC1s, while differentiation into the Tfh lineage relies on ICOS costimulation and B cells. Thus, activated CD4 T cells give rise to a bipotent Th1/Tfh precursor and differentiation into Th1 or Tfh cells depends on interactions with antigen-presenting cDC1s or B cells.

immunology↗

bioRxiv2025

RNA helicase DDX1 regulates germinal centre selection and affinity maturation by promoting tRNA ligase activity

Clonal expansion of antigen-specific B-cells defines effective germinal centre responses and is key for the generation of high-affinity antibodies. While positive selection in germinal centres has been associated with anabolic metabolism and cell growth, the downstream drivers of B-cell proliferation are not well understood. Here we report that the RNA helicase DDX1 is required for germinal centre maturation and accrual of dark-zone cellularity. Upon interaction with T-follicular helper cells, DDX1-deficient B-cells upregulate c-MYC but do not clonally expand. We show that positive selection is coupled with an increase in mRNA translation, that is dependent on DDX1. DDX1 endows B-cells with the protein biosynthetic capability that is required for rapid cell proliferation. It does so by modulating the activity of the tRNA ligase complex and tRNA splicing. Our data reveal that mRNA translation efficiency is a key determinant of B-cell fitness during germinal centre responses.

immunology↗

bioRxiv2024

A proteomic map of B cell activation and its shaping by mTORC1, MYC and iron

Using high resolution quantitative mass spectrometry, we have explored how immune activation and the metabolic checkpoint kinase mTORC1 (mammalian target of rapamycin complex 1) regulate the proteome of B lymphocytes. B cell activation via the B cell receptor, CD40 and the IL4 receptor induced considerable re-modelling of the B cell protein landscape, with a 5-fold increase in total cellular protein mass within 24 hours of activation. Analysis of copy numbers per cell of >7,500 proteins revealed increases in the metabolic machinery that supports B cell activation and nutrient and amino acid transporters that fuel B cell biosynthetic capacity. Comparison of T cell-dependent versus T cell-independent B cell stimulation along with an analysis of the synergistic effects of stimuli, identified shared and unique impacts of stimuli and highlighted the drivers of B cell proteome remodelling. In addition, we reveal that mTORC1 controls activation-induced B cell growth and inhibiting mTORC1 impairs the expression of amino acid transporters that fuel B cell protein production. We also show that mTORC1 activity regulates the expression of the transcription factor MYC and the transferrin receptor CD71. Blocking MYC activity phenocopied mTORC1 inhibition in many ways including impaired CD71 expression, while limiting iron availability during B cell activation impaired B cell growth and protein synthesis. This work provides a detailed map of naive and immune activated B cell proteomes and a greater understanding of the cellular machinery that directs B cell phenotypes. This work also provides new insights into the role of mTORC1, MYC and iron in regulating activation-induced proteome remodelling and protein production in B cells.

immunology↗

bioRxiv2024

RNA-binding proteins control the G2-M checkpoint of the germinal centre B cell

How germinal centre (GC) B cells undergo rapid cell division while maintaining genome stability is poorly understood. Here, we show that the RNA-binding proteins ZFP36L1 and ZFP36L2 act downstream of antigen-sensing and protect GC B cells from replication stress by controlling a cell cycle-related RNA post-transcriptional regulon. ZFP36L1 and ZFP36L2 safeguard faithful completion of mitosis by restraining the expression of CDK1 and cyclin B1, whilst controlling their activity through regulation of a p21-mediated negative feedback loop. In the absence of ZFP36L1 and ZFP36L2, GC B cells arrest in G2-M and die by apoptosis, resulting in curtailed GC responses. This is associated with stalling of the DNA replication fork at active replication initiation zones, which causes replication stress and increased activity of the ATR/CHK1 DNA damage response. Our findings reveal that gene regulation by RNA-binding proteins is essential for a functional G2-M checkpoint to operate in GC B cells.

immunology↗

bioRxiv2023

Zfp36l1 establishes the high affinity CD8 T cell response by directly linking TCR affinity to cytokine sensing

How individual T cells compete for and respond to IL2 at the molecular level, and, as a consequence, how this shapes population dynamics and the selection of high affinity clones is still poorly understood. Here we describe how the RNA binding protein ZFP36L1, acts as a sensor of TCR affinity to promote clonal expansion of high affinity CD8 T cells. As part of an incoherent feed forward loop ZFP36L1 has a non-redundant role in suppressing negative regulators of cytokine signalling and mediating a selection mechanism based on competition for IL2. We suggest that ZFP36L1 acts as a sensor of antigen affinity and establishes dominance of high affinity T cells by installing a hierarchical response to IL2.

immunology↗

bioRxiv2022

An integrated proteome and transcriptome of B cell maturation defines poised activation states of transitional and mature B cells

During B cell maturation, transitional and mature B cells acquire cell-intrinsic features that determine their ability to exit quiescence and mount effective immune responses. We used label-free mass spectrometry to quantify the proteome of B cell subsets from the mouse spleen and map the differential expression of environmental sensing, transcription- and translation initiation-factors that define cellular identity and function. By comparing the full-length transcriptome and proteome within the same sample, we identified mRNAs linked to B cell activation and antibody secretion that are expressed without detectable protein. These "poised" mRNAs might enable rapid protein production through increased translation or protein stability. In addition, through interrogation of our proteomic dataset, we found that the translational repressor PDCD4 restrains the response of marginal zone B cells to a T-independent antigen. Our molecular characterization of B cell maturation is a valuable resource to further explore the mechanisms underpinning the specialised functions of B cell subsets.

immunology↗

bioRxiv2021

Time-dependent regulation of cytokine production by RNA binding proteins defines T cell effector function

Potent T cell responses against infections and malignancies depend on the release of effector molecules, such as pro-inflammatory cytokines. Because effector molecules can be toxic, their production is tightly regulated through post-transcriptional events at 3 Untranslated Regions (3UTRs). RNA binding proteins (RBPs) were shown to be key regulators herein. With an RNA aptamer-based capture assay from human T cells, we identified >130 RBPs interacting with the IFNG, TNF and IL2 3UTRs in human T cells. T cell activation altered RBP-RNA interactions, revealing that RBP-target mRNA interactions rapidly respond to stimulation. Furthermore, we uncovered the intricate and time-dependent regulation of cytokine production by RBPs: whereas HuR supports early cytokine production, ZFP36L1, ATXN2L and ZC3HAV1 dampen and shorten the production duration, each at different time points. Strikingly, even though ZFP36L1 deletion did not phenotypically rescue T cell dysfunction in tumors, the increased production of cytokines and cytotoxic molecules resulted in superior anti-tumoral T cell responses in vivo. Our findings thus show that identifying RBP-RNA interactions reveals key modulators of T cell responses in health and disease.

immunology↗

bioRxiv2021

The RNA m6A binding protein YTHDF2 promotes the B cell to plasma cell transition.

To identify roles of RNA binding proteins (RBPs) in the differentiation of B cells to antibody-secreting plasma cells we performed a CRISPR/Cas9 knockout screen of 1213 mouse RBPs for their ability to affect proliferation and/or survival, and the emergence of differentiated CD138+ cells in vitro. We identified RBPs that promoted the appearance of CD138+ cells including CSDE1 and STRAP, as well as RBPs that inhibited CD138+ cell appearance such as EIF3 subunits EIF3K and EIF3L. Furthermore, we identified RBPs that share the property of recruiting the CCR4-NOT complex to their target transcripts have the potential to mediate opposing outcomes on B cell differentiation. One such RBP, the m6A binding protein YTHDF2 promotes the appearance of CD138+ cells in vitro. In chimeric mouse models YTHDF2-deficient B cells formed germinal centers in a cell-autonomous manner, however plasma cells failed to accumulate.

immunology↗

bioRxiv2021

The timing of differentiation and potency of CD8 effector function is set by RNA binding proteins

CD8+ T cell differentiation into effector cells is initiated early after antigen encounter by signals from the T cell antigen receptor and costimulatory molecules. The molecular mechanisms that determine the timing and rate of differentiation however are not defined. Here we show that the RNA binding proteins (RBP) ZFP36 and ZFP36L1 limit the rate of differentiation of activated naive CD8+ T cells and the potency of the resulting cytotoxic lymphocytes. The RBP act in an early and short temporal window to enforce dependency on costimulation via CD28 for full T cell activation and effector differentiation by directly binding mRNA of NF-B, IRF8 and NOTCH1 transcription factors and cytokines, including IL2. Their absence in T cells, or the adoptive transfer of a small numbers of CD8+ T cells lacking the RBP, promotes resilience to influenza A virus infection without immunopathology. These findings highlight ZFP36 and ZFP36L1 as nodes for the integration of the early T cell activation signals determining the speed and quality of the CD8 response.

immunology↗

bioRxiv2018

Costimulation through TLR2 drives polyfunctional CD8+ T cell responses

Optimal T cell activation requires antigen recognition through the T cell receptor (TCR), engagement of costimulatory molecules, and cytokines. T cells can also directly recognize danger signals through the expression of toll-like receptors (TLRs). Whether TLR ligands have the capacity to provide costimulatory signals and enhance antigen-driven T cell activation is not well understood. Here, we show that TLR2 and TLR7 ligands potently lower the antigen threshold for cytokine production in T cells. To investigate how TLR triggering supports cytokine production, we adapted the protocol for flow cytometry-based fluorescence in situ hybridization (Flow-FISH) to mouse T cells. The simultaneous detection of cytokine mRNA and protein with single-cell resolution revealed that TLR triggering primarily drives de novo mRNA transcription. Ifng mRNA stabilization only occurs when the TCR is engaged. TLR2, but not TLR7-mediated costimulation, can enhance mRNA stability at low antigen levels. Importantly, TLR2 costimulation increases the percentage of polyfunctional T cells, a hallmark of potent T cell responses. In conclusion, TLR-mediated costimulation effectively potentiates T cell effector function to suboptimal antigen levels.

immunology↗

bioRxiv2018

Critical role of post-transcriptional regulation for IFN-γ in tumor-infiltrating T cells

Protective T cell responses against tumors require the production of Interferon gamma (IFN-{gamma}). However, tumor-infiltrating T cells (TILs) gradually lose their capacity to produce IFN-{gamma} and therefore fail to clear malignant cells. Dissecting the underlying mechanisms that block cytokine production is thus key for improving T cell products. Here we show that although TILs express substantial levels of Ifng mRNA, post-transcriptional mechanisms impede the production of IFN-{gamma} protein due to loss of mRNA stability. CD28 triggering, but not PD1 blocking antibodies, effectively restores the stability of Ifng mRNA. Intriguingly, TILs devoid of AU-rich elements within the 3untranslated region maintain stabilized Ifng mRNA and produce more IFN-{gamma} protein than wild-type TILs. This sustained IFN-{gamma} production translates into effective suppression of tumor outgrowth, which is almost exclusively mediated by direct effects on the tumor cells. We therefore conclude that post-transcriptional mechanisms could be modulated to potentiate effective T cell therapies in cancer.

immunology↗

bioRxiv2018

Computational assessment of blood flow heterogeneity in dialysis patients’ cardiac ventricles

Dialysis prolongs life but augments cardiovascular mortality. Imaging data suggests that dialysis increases myocardial blood flow (BF) heterogeneity, but its causes remain poorly understood. A biophysical model of human coronary vasculature was used to explain the imaging observations, and highlight causes of coronary BF heterogeneity.\n\nPost-dialysis CT images from patients under control, pharmacological stress (adenosine), therapy (cooled dialysate), and adenosine and cooled dialysate conditions were obtained. The data presented disparate phenotypes. To dissect vascular mechanisms, a 3D human coronary vasculature model was implemented. Simulations were performed to investigate the effects of altered aortic pressure and blood vessel diameters on myocardial BF heterogeneity which was quantified using relative dispersion, fractal dimension, and transmural BF distribution.\n\nImaging showed that stress and therapy potentially increased mean and total BF, while reducing heterogeneity. BF histograms of one patient showed multi-modality. Using the model, it was found that total coronary BF increased as coronary perfusion pressure (CPP) was increased. BF heterogeneity was differentially affected by large or small vessel blocking. BF heterogeneity was found to be inversely related to small blood vessel diameters. Simulation of large artery stenosis indicates that BF became heterogeneous (increase relative dispersion) and gave multi-modal histograms. The total transmural BF as well as transmural BF heterogeneity reduced due to large artery stenosis, generating large patches of very low O_SCPCAPBFC_SCPCAP regions downstream. Blocking of arteries at various orders showed that blocking larger arteries results in multi-modal BF histograms and large patches of low BF, whereas smaller artery blocking results in augmented relative dispersion and fractal dimension. Transmural heterogeneity was also affected. Finally, the effects of augmented aortic pressure in the presence of blood vessel blocking shows differential effects on BF heterogeneity as well as transmural BF.\n\nImproved aortic blood pressure may lead to improved BF. Stress and therapy may be effective if they dilate small vessels. A potential cause for the observed complex BF distributions (multi-modal BF histograms) may indicate existing large vessel stenosis.\n\nThe intuitive BF heterogeneity methods used can be readily used in clinical studies. Further development of the model and methods will permit personalised assessment of patient BF status.

biophysics↗