Biology subjects

Dong, T.

Publications and source records attributed to Dong, T..

At least 55 records · Page 3Linked to original sources

bioRxiv2021

IFITM3 regulates virus-induced inflammatory cytokine production by titrating Nogo-B orchestration of TLR responses

Interferon induced transmembrane protein 3 (IFITM3) is an important viral restriction factor in viral pathogenesis that also exhibits poorly understood immune regulatory functions. Here, using human and mouse models, we demonstrate that IFITM3 regulates MyD88-dependent TLR-mediated cytokine production following dendritic cell exposure to cytomegalovirus (CMV), and this process limits viral pathogenesis in vivo. IFITM3 also restricted pro-inflammatory (IL-6) cytokine production in response to influenza. IFITM3 bound to and promoted ubiquitination and proteasomal degradation of the reticulon 4 isoform Nogo-B. We reveal that Nogo-B mediates TLR-dependent pro-inflammatory cytokine production and promotes viral pathogenesis in vivo, and this process involved alteration of TLR dynamics. The anti-inflammatory function of IFITM3 was intrinsically linked to its ability to regulate Nogo-B. Thus, we uncover Nogo-B as an unappreciated driver of viral pathogenesis and highlight a novel immune regulatory pathway where IFITM3 fine-tunes TLR responsiveness of myeloid cells to viral stimulation.

immunology↗

bioRxiv2021

Breaching the cell-envelope barriers of gram-positive and fungal microbes by a type VI secretion system in Acidovorax citrulli

The type VI secretion system (T6SS) is a double-tubular toxin-injection nanomachine widely found in gram-negative human and plant pathogens. The current model depicts that the T6SS spear-like Hcp tube is powered by the contraction of an outer sheath to drill through the envelope of a neighboring cell, achieving cytosol to cytosol delivery. However, gram-positive bacteria seem to be impenetrable to such T6SS action. Here we report that a plant pathogen Acidovorax citrulli (AC) deploys a highly potent T6SS to kill a range of bacteria including Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Mycobacterium smegmatis as well as fungal species including Candida albicans and Pichia pastoris. Using bioinformatic and biochemical assays, we identified a group of T6SS effectors and characterized one effector RhsB that is critical for interspecies interaction. We report that RhsB contains a conserved YD-repeat domain and a C-terminal nuclease domain. Toxicity of RhsB was neutralized by its downstream immunity proteins through direct interaction. RhsB was cleaved at the C-terminal end and a catalytic mutation within the internal aspartic protease abolished such cleavage. Collectively, the T6SS of AC displays potent activities to penetrate the cell envelope barriers of gram-positive and fungal species, highlighting the greatly expanded capabilities of T6SS in modulating microbiome compositions in complex environments.

microbiology↗

bioRxiv2021

Synthetic Antigen Presenting cells reveal the diversity and functional specialization of extracellular vesicles composing the fourth signal of T cell immunological synapses.

The T cell Immunological Synapse (IS) is a pivotal hub for the regulation of adaptive immunity by endowing the exchange of information between cells engaged in physical contacts. Beyond the integration of antigen (signal one), co-stimulation (signal two), and cytokines (signal three), the IS facilitates the delivery of T-cell effector assemblies including supramolecular attack particles (SMAPs) and extracellular vesicles (EVs). How these particulate outputs differ among T -cell subsets and how subcellular compartments and signals exchanged at the synapse contribute to their composition is not fully understood. Here we harnessed bead-supported lipid bilayers (BSLBs) as a tailorable and versatile technology for the study of synaptic particle biogenesis and composition in different T-cell subsets, including CART. These synthetic antigen-presenting cells (APCs) facilitated the characterisation of trans-synaptic vesicles (tSV) as a heterogeneous population of EVs comprising among others PM-derived synaptic ectosomes and CD63+ exosomes. We harnessed BSLB to unveil the factors influencing the vesicular release of CD40L, as a model effector, identifying CD40 trans presentation, T-cell activation, ESCRT upregulation/recruitment, antigen density/potency, co-repression by PD-1 ligands, and its processing by ADAM10 as major determinants. Further, BSLB made possible the comparison of microRNA (miR) species associated with tSV and steadily released EVs. Altogether, our data provide evidence for a higher specialisation of tSV which are enriched not only in effector immune receptors but also in miR and RNA-binding proteins. Considering the molecular uniqueness and functional complexity of the tSV output, which is also accompanied by SMAPs, we propose their classification as signal four. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/445691v3_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@100f3aorg.highwire.dtl.DTLVardef@57adfforg.highwire.dtl.DTLVardef@6052fcorg.highwire.dtl.DTLVardef@1e85c21_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIBead Supported Lipid Bilayers (BSLB) reconstituting antigen-presenting cells support synapse assembly by T cells and the release of effector particles. C_LIO_LIBSLB facilitate the dissection of the cellular machineries and synapse composition shaping the released tSV. C_LIO_LItSV and their steadily released counterparts have a different composition. TSV show a higher enrichment of effectors including immune receptors, miR, RNA- and other nucleic acid-binding proteins, than EVs. C_LI

immunology↗

bioRxiv2021

Abiotic factors modulate interspecies competition mediated by the type VI secretion system effectors in Vibrio cholerae

Vibrio cholerae, the etiological pathogen of cholera, relies on its type VI secretion system (T6SS) as an effective weapon to survive in highly competitive communities. The anti-bacterial and anti-eukaryotic functions of T6SS depend on its secreted effectors that target multiple essential cellular processes. However, the mechanisms that account for effector diversity and different effectiveness during interspecies competition remain elusive. Here, we report that environmental cations and temperature play a key role in dictating effector-mediated competition of Vibrio cholerae. We found that V. cholerae could employ its cell-wall-targeting effector TseH to outcompete the otherwise resistant Escherichia coli and the V. cholerae immunity deletion mutant{Delta} tsiH when Ca2+ and Mg2+ were supplemented. The E. coli {Delta}phoQ mutant was more sensitive to TseH-mediated killing during competition, suggesting the metal-sensing PhoPQ two-component system is protective to E. coli from TseH activity. Using transcriptome analysis, we found multiple stress response systems, including acid stress response, oxidative stress response, and osmotic stress response, were activated in E. coli expressing TseH in comparison with E. coli expressing the inactive mutant TseHH64A. The membrane-targeting lipase effector TseL also exhibited reduced killing against E. coli when divalent cations were removed. In addition, competition analysis of E. coli with V. cholerae single-effector active strains reveals a temperature-dependent susceptibility of E. coli to effectors, VasX, VgrG3, and TseL. These findings suggest that abiotic factors, that V. cholerae frequently encounters in natural habitats, play a crucial role in dictating the competitive fitness conferred by the type VI secretion system in complex multispecies communities.

microbiology↗

bioRxiv2021

Engineered type six secretion systems deliver active exogenous effectors and Cre recombinase

Genetic editing has revolutionized biotechnology but delivery of endonuclease genes as DNA can lead to aberrant integration or overexpression, leading to off-target effects. Here we develop a mechanism to deliver Cre recombinase as a protein by engineering the bacterial type six secretion system (T6SS). Using multiple T6SS fusion proteins, Aeromonas dhakensis or attenuated Vibrio cholerae donor strains, and a gain-of-function cassette for detecting Cre recombination, we demonstrate successful delivery of active Cre directly into recipient cells. Most efficient transfer was achieved using a truncated version of PAAR2 from V. cholerae, resulting in a relatively small (118 amino acid) delivery tag. We further demonstrate the versatility of this system by delivering an exogenous effector, TseC, enabling V. cholerae to kill Pseudomonas aeruginosa. This implicates that P. aeruginosa is naturally resistant to all native effectors of V. cholerae and that the TseC chaperone protein is not required for its activity. Moreover, it demonstrates that the engineered system can improve T6SS efficacy against specific pathogens, proposing future application in microbiome manipulation or as a next-generation antimicrobial. Inexpensive and easy to produce, this protein delivery system has many potential applications ranging from studying T6SS effectors to genetic editing. ImportanceDelivery of protein-based drugs, antigens, and gene-editing agents has broad applications. The type VI protein secretion system (T6SS) can target both bacteria and eukaryotic cells and deliver proteins of diverse size and function. Here we harness the T6SS to successfully deliver Cre recombinase to genetically edit bacteria without requiring the introduction of exogenous DNA into the recipient cells. This demonstrates a promising advantage over current genetic editing tools that require transformation or conjugation of DNA. The engineered secretion tag can also deliver a heterologous antimicrobial toxin that kills an otherwise unsusceptible pathogen, Pseudomonas aeruginosa. These results demonstrate the potential of T6SS-mediated delivery in areas including genome editing, killing drug-resistant pathogens, and studying toxin functions.

molecular biology↗

bioRxiv2021

The impact of viral mutations on recognition by SARS-CoV-2 specific T-cells

We identify amino acid variants within dominant SARS-CoV-2 T-cell epitopes by interrogating global sequence data. Several variants within nucleocapsid and ORF3a epitopes have arisen independently in multiple lineages and result in loss of recognition by epitope-specific T-cells assessed by IFN-{gamma} and cytotoxic killing assays. These data demonstrate the potential for T-cell evasion and highlight the need for ongoing surveillance for variants capable of escaping T-cell as well as humoral immunity.

immunology↗

bioRxiv2021

Benchtop Carbon Fiber Microelectrode Array Fabrication Toolkit

BackgroundConventional neural probes are primarily fabricated in a cleanroom, requiring the use of multiple expensive and highly specialized tools. New methodWe propose a cleanroom "light" fabrication process of carbon fiber neural electrode arrays that can be learned quickly by an inexperienced cleanroom user. This carbon fiber electrode array fabrication process requires just one cleanroom tool, a parylene-c deposition machine, that can be learned quickly or outsourced to a commercial processing facility at marginal cost. Our fabrication process also includes hand-populating printed circuit boards, insulation, and tip optimization. ResultsThe three different tip optimizations explored here (Nd:YAG laser, blowtorch, and UV laser) result in a range of tip geometries and 1kHz impedances, with blowtorched fibers resulting in the lowest impedance. While previous experiments have proven laser and blowtorch electrode efficacy, this paper also shows UV laser cut fibers can record neural signals in vivo. Comparison with existing methodsExisting carbon fiber arrays either do not have individuated electrodes in favor of bundles or require cleanroom fabricated guides for population and insulation. The proposed arrays use only tools that can be used at a benchtop for fiber population. ConclusionsThis carbon fiber electrode array fabrication process allows for quick customization of bulk array fabrication at a reduced price compared to commercially available probes.

bioengineering↗

bioRxiv2021

Antibody evasion by the Brazilian P.1 strain of SARS-CoV-2

Terminating the SARS-CoV-2 pandemic relies upon pan-global vaccination. Current vaccines elicit neutralizing antibody responses to the virus spike derived from early isolates. However, new strains have emerged with multiple mutations: P.1 from Brazil, B.1.351 from South Africa and B.1.1.7 from the UK (12, 10 and 9 changes in the spike respectively). All have mutations in the ACE2 binding site with P.1 and B.1.351 having a virtually identical triplet: E484K, K417N/T and N501Y, which we show confer similar increased affinity for ACE2. We show that, surprisingly, P.1 is significantly less resistant to naturally acquired or vaccine induced antibody responses than B.1.351 suggesting that changes outside the RBD impact neutralisation. Monoclonal antibody 222 neutralises all three variants despite interacting with two of the ACE2 binding site mutations, we explain this through structural analysis and use the 222 light chain to largely restore neutralization potency to a major class of public antibodies.

microbiology↗

bioRxiv2021

Characterization of humoral and SARS-CoV-2 specific T cell responses in people living with HIV

There is an urgent need to understand the nature of immune responses generated against SARS-CoV-2, to better inform risk-mitigation strategies for people living with HIV (PLWH). Although not all PLWH are considered immunosuppressed, residual cellular immune deficiency and ongoing inflammation could influence COVID-19 disease severity, the evolution and durability of protective memory responses. Here, we performed an integrated analysis, characterizing the nature, breadth and magnitude of SARS-CoV-2-specific immune responses in PLWH, controlled on ART, and HIV negative subjects. Both groups were in the convalescent phase of predominately mild COVID-19 disease. The majority of PLWH mounted SARS-CoV-2 Spike- and Nucleoprotein-specific antibodies with neutralizing activity and SARS-CoV-2-specific T cell responses, as measured by ELISpot, at levels comparable to HIV negative subjects. T cell responses against Spike, Membrane and Nucleocapsid were the most prominent, with SARS-CoV-2-specific CD4 T cells outnumbering CD8 T cells. Notably, the overall magnitude of SARS-CoV-2-specific T cell responses related to the size of the naive CD4 T cell pool and the CD4:CD8 ratio in PLWH, in whom disparate antibody and T cell responses were observed. Both humoral and cellular responses to SARS-CoV-2 were detected at 5-7 months post-infection, providing evidence of medium-term durability of responses irrespective of HIV serostatus. Incomplete immune reconstitution on ART and a low CD4:CD8 ratio could, however, hamper the development of immunity to SARS-CoV-2 and serve as a useful tool for risk stratification of PLWH. These findings have implications for the individual management and potential effectiveness of vaccination against SARS-CoV-2 in PLWH. One Sentence SummaryAdaptive immune responses to SARS-CoV-2 in the setting of HIV infection

immunology↗

bioRxiv2020

Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent COVID-19 patients.

COVID-19 is an ongoing global crisis in which the development of effective vaccines and therapeutics will depend critically on understanding the natural immunity to the virus, including the role of SARS-CoV-2-specific T cells. We have conducted a study of 42 patients following recovery from COVID-19, including 28 mild and 14 severe cases, comparing their T cell responses to those of 16 control donors. We assessed the immune memory of T cell responses using IFN{gamma} based assays with overlapping peptides spanning SARS-CoV-2 apart from ORF1. We found the breadth, magnitude and frequency of memory T cell responses from COVID-19 were significantly higher in severe compared to mild COVID-19 cases, and this effect was most marked in response to spike, membrane, and ORF3a proteins. Total and spike-specific T cell responses correlated with the anti-Spike, anti-Receptor Binding Domain (RBD) as well as anti-Nucleoprotein (NP) endpoint antibody titre (p<0.001, <0.001 and =0.002). We identified 39 separate peptides containing CD4+ and/or CD8+ epitopes, which strikingly included six immunodominant epitope clusters targeted by T cells in many donors, including 3 clusters in spike (recognised by 29%, 24%, 18% donors), two in the membrane protein (M, 32%, 47%) and one in the nucleoprotein (Np, 35%). CD8+ responses were further defined for their HLA restriction, including B*4001-restricted T cells showing central memory and effector memory phenotype. In mild cases, higher frequencies of multi-cytokine producing M- and NP-specific CD8+ T cells than spike-specific CD8+ T cells were observed. They furthermore showed a higher ratio of SARS-CoV-2-specific CD8+ to CD4+ T cell responses. Immunodominant epitope clusters and peptides containing T cell epitopes identified in this study will provide critical tools to study the role of virus-specific T cells in control and resolution of SARS-CoV-2 infections. The identification of T cell specificity and functionality associated with milder disease, highlights the potential importance of including non-spike proteins within future COVID-19 vaccine design.

immunology↗

bioRxiv2020

Role of YAP1 gene in proliferation and osteogenic differentiation of human periodontal ligament stem cells induced by TNF-α

BackgroundPeriodontitis is a chronic inflammatory disease that occurs in periodontal tissues and can cause tooth loosening and loss in severe cases. As the main effector of downstream of Hippo signaling pathway, Yes-related protein 1 (YAP1) plays an important role in cell proliferation and differentiation. However, the role of YAP1 in periodontitis has not been reported. MethodsCell activity was detected by CCk-8. YAP1 was overexpressed by cell transfection, and then RT-qPCR and western blot were used to detect the expression of YAP1. The cell proliferation was determined by clone formation assay, and the expression of proliferation-related proteins was determined by western blot. The cell differentiation was detected by Elisa kit of ALP and alizarin red staining. Finally, western blot was used to detect the expression of differentiation-related protein and Hippo signaling pathway-related proteins. ResultsWith the increase of concentration induced by TNF-, the cell survival rate of human periodontal ligament stem cells (HPDLSC) decreased significantly. After the overexpression of YAP1, cell proliferation and proliferation-related protein expression increased. Overexpression of YAP1 can improve the differentiation and the formation of osteoblasts of HPDLSCs induced by TNF-. The expression of Hippo signaling pathway-related proteins transcriptional coactivators with PDZ binding domains (TAZ), TEA domain family member (TRED) increased and proliferation-related protein P27 decreased. ConclusionTNF- can inhibit proliferation and osteogenic differentiation of HPDLSCs, which can be ameliorated by the YAP1 gene through the Hippo signaling pathway. Our paper suggested that YAP1 may be a potential therapeutic target for periodontitis.

cell biology↗

bioRxiv2019

Low basal expression and slow induction of IFITM3 puts immune cells at risk of influenza A infection

The interferon-induced transmembrane protein, IFITM3, has been shown to restrict influenza virus infection in murine and in vitro settings for ten years, but no explanation has been found to explain why this virus infection is so highly contagious and infects most individuals it comes in contact with. We confirm that the expression level of IFITM3 plays a role in determining the level of viral infection through manipulation of IFITM3 levels with interferon (IFN) stimulation and overexpression systems. Low basal expression may put some immune cells, including lymphocytes and lung-resident macrophages, at risk of influenza virus infection. Investigating the induction of IFITM3 by IFN, we find a strong preference for Type I IFN in IFITM3 induction in both cell lines and primary human cells. While myeloid cells can increase expression following stimulation by Type I IFN, lymphocytes show minimal induction of IFITM3 following IFN stimulation, suggesting that they are always at risk of viral infection. Surprisingly, we found that the time it takes for maximal induction of IFITM3 is relatively slow for an interferon-stimulated gene at around 36 hours. Low basal expression and slow induction of IFITM3 could increase the risk of influenza virus infection in selected immune cells. ImportanceInfluenza virus infection remains one of the top ten threats to global health, causing significant deaths and hospitalisations across the world each year. Understanding mechanisms for controlling influenza virus infection remain a priority. The interferon-induced transmembrane protein IFITM3 can restrict influenza infection by limiting replication of the virus. The precise mechanisms of how IFITM3 reduced replication of influenza are unknown, although it is predicted to prevent release of viral contents into the cytosol by preventing pore formation on the endosomal compartments where it is suggested to reside. Here we have shown that the expression level of IFITM3 is important in determining the control of influenza virus infection. We find an expression pattern for IFITM3 that varies based on cell type, tissue locality, differentiation state and cell naivety, all of which highlights cells that may be at the highest risk of influenza infection.

immunology↗

bioRxiv2019

Broadly inhibiting anti-neuraminidase monoclonal antibodies induced by trivalent influenza vaccine and H7N9 infection in humans

The majority of antibodies induced by influenza neuraminidase (NA), like those against hemagglutinin (HA), are relatively specific to viruses isolated within a limited time-window as seen in serological studies and the analysis of many murine monoclonal antibodies. We report three broadly reactive human monoclonal antibodies (mAbs) targeting N1 NA. Two were isolated from a young adult vaccinated with trivalent influenza vaccine (TIV), which inhibited N1 NA from viruses isolated from human over a period of a hundred years. The third antibody isolated from a child with acute mild H7N9 infection inhibited both group 1 N1 and group 2 N9 NAs. In addition, the antibodies cross-inhibited the N1 NAs of highly pathogenic avian H5N1 influenza viruses. These antibodies are protective in prophylaxis against seasonal H1N1 viruses in mice. This study demonstrates that human antibodies to N1 NA with exceptional cross-reactivity can be recalled by vaccination and highlights the importance of standardizing the NA antigen in seasonal vaccines to offer optimal protection.\n\nImportanceAntibodies to the influenza NA can provide protection against influenza disease. Analysis of human antibodies to NA lags behind that for HA. We show that human monoclonal antibodies against NA induced by vaccination and infection can be very broadly reactive and able to inhibit a wide spectrum of N1 NAs between 1918 and 2018. This suggests that antibodies to NA may be a useful therapy, and that efficacy of influenza vaccines could be enhanced by ensuring appropriate content of NA antigen.\n\nHighlights of the paperO_LIAntibodies that inhibit influenza viruses with N1 neuraminidase (NA), with broad reactivity for viruses isolated between 1918-2018, can be isolated from human recipients of seasonal influenza vaccine\nC_LIO_LIAntibodies targeting N1 NA of human seasonal H1N1 viruses can cross-react with a variety of avian N1 neuraminidases\nC_LIO_LIAcute H7N9 infection can recall memory B cells to N1 NA and elicit cross-reactive antibodies to the group 1 N1 and group 2 N9 NAs\nC_LIO_LIAntibodies to N1 NA with this broad reactivity protect against lethal virus challenge\nC_LI

immunology↗

bioRxiv2019

Identification of peptide coatings that enhance diffusive transport of nanoparticles through the tumor microenvironment

In solid tumors, increasing drug penetration promotes their regression and improves the therapeutic index of compounds. However, the heterogeneous extracellular matrix (ECM) acts a steric and interaction barrier that hinders effective transport of therapeutics, including nanomedicines. Specifically, the interactions between the ECM and surface physicochemical properties of nanomedicines (e.g. charge, hydrophobicity) impedes their diffusion and penetration. To address the challenges using existing surface chemistries, we used peptide-presenting phage libraries as a high-throughput approach to screen and identify peptides as coatings with desired physicochemical properties that improve diffusive transport through the tumor microenvironment. Through iterative screening against the ECM and identification by next-generation DNA sequencing and analysis, we selected individual clones and measured their transport by diffusion assays. Here, we identified a net-neutral charge, hydrophilic peptide P4 that facilitates significantly higher diffusive transport of phage than negative control through in vitro tumor ECM. Through alanine mutagenesis, we confirmed that the hydrophilicity, charge, and their spatial ordering impact diffusive transport. P4 phage clone exhibited almost 200-fold improved uptake in ex vivo pancreatic tumor xenografts compared to the negative control. Nanoparticles coated with P4 exhibited [~]40-fold improvement in diffusivity in pancreatic tumor tissues, and P4-coated particles demonstrated less hindered diffusivity through the ECM compared to particles functionalized with gold standard poly(ethylene) glycol or iRGD peptide ligand. By leveraging the power of molecular diversity using phage display, we can greatly expand the chemical space of surface chemistries that can improve the transport of nanomedicines through the complex tumor microenvironment to ultimately improve their efficacy.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC=\"FIGDIR/small/659524v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (23K):\norg.highwire.dtl.DTLVardef@18bc04forg.highwire.dtl.DTLVardef@1feb597org.highwire.dtl.DTLVardef@14403e5org.highwire.dtl.DTLVardef@5bbf6c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2019

M13 phage display to identify a permeating peptide against hyperconcentrated mucin

Mucus is an impregnable barrier for drug delivery across the epithelia for treatment of mucosal-associated diseases. While current carriers are promising for mucus penetration, their surface chemistries do not possess chemical complexity to probe and identify optimal physicochemical properties desired for mucus penetration. As initial study, we use M13 phage display presenting random peptides to select peptides that can facilitate permeation through hyperconcentrated mucin. Here, a net-neutral charge, hydrophilic peptide was identified to facilitate transport of phage and fluorophore conjugates through mucin barrier compared to controls. This initial finding warrants further study to understand how composition and spatial distribution of physicochemical properties of peptides can be optimized to improve transport across the mucus barrier.

bioengineering↗

bioRxiv2019

Composition and structure of synaptic ectosomes exporting antigen receptor linked to functional CD40 ligand from helper T-cells

Cell communication through extracellular vesicles is an emerging topic in biology, including communication between cells of the immune system. Planar supported lipid bilayers (PSLBs) presenting T cell receptor (TCR) ligands and intercellular adhesion molecule-1 (ICAM-1) induce budding of extracellular microvesicles enriched in functional TCR, defined here as synaptic ectosomes (SE), from helper T cells. SE bind peptide-MHC directly exporting TCR into the synaptic cleft, but their ability to incorporate other effectors is unknown. Here, we utilized bead supported lipid bilayers (BSLB) to capture SE from single immunological synapses (IS), determined SE composition by immunofluorescence flow cytometry and enriched SE for proteomic analysis by particle sorting. Our results demonstrate selective enrichment of CD40 ligand (CD40L) and inducible T-cell costimulator (ICOS) in SE in response to addition of CD40 and ICOS ligand (ICOSL), respectively, to SLB presenting TCR ligands and ICAM-1. TCR triggering mobilized intracellular CD40L to the T cells surface at the IS, where it engaged CD40 to enable sorting into SE. SEs were enriched in tetraspanins and bone marrow stromal cell antigen 2 (BST-2) by immunofluorescence and TCR signalling and endosomal sorting complexes required for transport by proteomics. Super-resolution microscopy demonstrated that CD40L is present in microclusters within CD81 defined SE that are spatially segregated from TCR/ICOS/BST-2 microclusters. CD40L in SE retains the capacity to induce dendritic cell (DC) maturation and cytokine production. SE enabled helper T cells to release effectors physically linked to TCR. One Sentence SummaryTCR and CD40L microclusters can be linked in synaptic ectosomes (extracellular vesicles) that are released in the immunological synapse by helper T cells and induce dendritic cell maturation and cytokine production.

immunology↗

bioRxiv2019

Use of a pandemic H1N1 strain with updated haemagglutinin and neuraminidase results in increased nasopharyngeal shedding and improved immunogenicity to Russian-backbone live attenuated influenza vaccine among children aged 2 - 4 years old: an open label, prospective, observational, phase 4 study in The Gambia.

BackgroundPoor efficacy and effectiveness of the pandemic H1N1 (pH1N1) component in intranasal live attenuated influenza vaccine (LAIV) has been demonstrated in several studies. The reasons for this are unclear, but may be due to impaired replicative fitness of pH1N1 A/California/07/2009-like (Cal09) LAIV strains. MethodsIn an open-label, prospective, observational, phase 4 study, we evaluated the impact of updating the pH1N1 component in the Russian-backbone trivalent LAIV from Cal09 in 2016-17 (n=118) to an A/Michigan/45/2015-like strain (A/17/New York/15/5364, NY15) in 2017-18 (n=126), on shedding and immunogenicity in Gambian children aged 2-4 years old. The study was nested within a larger randomised controlled trial investigating LAIV-microbiome interactions (ClinicalTrials.gov NCT02972957). FindingsCal09 showed impaired nasopharyngeal shedding compared to H3N2 and influenza B, along with sub-optimal serum antibody and T-cell responses. Following the switch to NY15, a significant increase in pH1N1 shedding was seen, along with improvements in seroconversion and influenza-specific CD4+ T-cell responses. Viral kinetics in vitro mirrored these findings, with NY15 showing greater replicative ability than Cal09 in human nasal epithelial cells. Persistent shedding to day 7 was independently associated with both seroconversion and CD4+ T cell response in multivariable logistic regression. InterpretationOur results suggest that the pH1N1 component switch in LAIV may have overcome problems in prior formulations. LAIV effectiveness against pH1N1 should therefore improve in upcoming influenza seasons. Our data also highlight the importance of evaluating replicative fitness, in addition to antigenicity, when selecting annual LAIV components and design of potentially more effective vaccines. FundingThe Wellcome Trust.

clinical trials↗

bioRxiv2018

A New Method for Primary Culture of Mouse Dorsal Root Ganglion Neurons

In order to establish a simple and highly purified method for primary culture of mouse dorsal root ganglion neurons(DRGn), in this study, the DRGn of young mice were obtained by collagenase type I and trypsin digestion. Then the DRGn were obtained by immunocytochemical staining of mouse neuron specific enolase (NSE) monoclonal antibody, while using flow cytometry to further detect the positive rates of DRGn. The cultured primary DRGn grew well and had a purity of about 83.72%. The DRGn survival time was 60 days when cultured in DMEM medium containing nerve growth factor (NGF). The culture scheme is simple and stable, and a large number of high purity DRGn could be cultured, which provides a reliable model for further study of nerve cells.

neuroscience↗