The Reorientation of T-Cell Polarity and Inhibition of Immunological Synapse Formation by CD46 Involves Its Recruitment to Lipid Rafts

Many infectious agents utilize CD46 for infection of human cells, and therapeutic applications of CD46-binding viruses are now being explored. Besides mediating internalization to enable infection, binding to CD46 can directly alter immune function. In particular, ligation of CD46 by antibodies or by measles virus can prevent activation of T cells by altering T-cell polarity and consequently preventing the formation of an immunological synapse. Here, we define a mechanism by which CD46 reorients T-cell polarity to prevent T-cell receptor signaling in response to antigen presentation. We show that CD46 associates with lipid rafts upon ligation, and that this reduces recruitment of both lipid rafts and the microtubule organizing centre to the site of receptor cross-linking. These data combined indicate that polarization of T cells towards the site of CD46 ligation prevents formation of an immunological synapse, and this is associated with the ability of CD46 to recruit lipid rafts away from the site of TCR ligation

Spatial proteomics identifies a spectrum of immune dysregulation in acquired bone marrow failure syndromes

Poor graft function (PGF), manifested by multilineage cytopenias and complete donor chimerism post-allogeneic stem cell transplantation (alloSCT), and acquired aplastic anaemia (AA) are immune-mediated acquired bone marrow (BM) failure syndromes with a similar clinical presentation. In this study, we used spatial proteomics to compare the immunobiology of the BM microenvironment and identify common mechanisms of immune dysregulation under these conditions. Archival BM trephines from patients exhibited downregulation of the immunoregulatory protein VISTA and the M2 macrophage marker and suppressor of T-cell activation ARG1 with increased expression of the immune checkpoint B7-H3 compared to normal controls. Increased CD163 and CD14 expression suggested monocyte/macrophage skewing, which, combined with dysregulation of STING and VISTA, is indicative of an environment of reduced immunoregulation resulting in the profound suppression of hematopoiesis in these two conditions. There were no changes in the immune microenvironment between paired diagnostic AA and secondary MDS/AML samples suggesting that leukaemic clones develop in the impaired immune microenvironment of AA without the need for further alterations. Of the eight proteins with dysregulated expression shared by diagnostic AA and PGF, the diagnostic AA samples had a greater fold change in expression than PGF, suggesting that these diseases represent a spectrum of immune dysregulation. Unexpectedly, analysis of samples from patients with good graft function post-alloSCT demonstrated significant changes in the immune microenvironment compared to normal controls, with downregulation of CD44, STING, VISTA, and ARG1, suggesting that recovery of multilineage haematopoiesis post-alloSCT does not reflect recovery of immune function and may prime patients for the development of PGF upon further inflammatory insult. The demonstrable similarities in the immunopathology of AA and PGF will allow the design of clinical interventions that include both patient cohorts to accelerate therapeutic discovery and translation

Evolutionarily conserved bias of amino-acid usage refines the definition of PDZ-binding motif

<p>Abstract</p> <p>Background</p> <p>The interactions between PDZ (PSD-95, Dlg, ZO-1) domains and PDZ-binding motifs play central roles in signal transductions within cells. Proteins with PDZ domains bind to PDZ-binding motifs almost exclusively when the motifs are located at the carboxyl (C-) terminal ends of their binding partners. However, it remains little explored whether PDZ-binding motifs show any preferential location at the C-terminal ends of proteins, at genome-level.</p> <p>Results</p> <p>Here, we examined the distribution of the type-I (x-x-S/T-x-I/L/V) or type-II (x-x-V-x-I/V) PDZ-binding motifs in proteins encoded in the genomes of five different species (human, mouse, zebrafish, fruit fly and nematode). We first established that these PDZ-binding motifs are indeed preferentially present at their C-terminal ends. Moreover, we found specific amino acid (AA) bias for the 'x' positions in the motifs at the C-terminal ends. In general, hydrophilic AAs were favored. Our genomics-based findings confirm and largely extend the results of previous interaction-based studies, allowing us to propose refined consensus sequences for all of the examined PDZ-binding motifs. An ontological analysis revealed that the refined motifs are functionally relevant since a large fraction of the proteins bearing the motif appear to be involved in signal transduction. Furthermore, co-precipitation experiments confirmed two new protein interactions predicted by our genomics-based approach. Finally, we show that influenza virus pathogenicity can be correlated with PDZ-binding motif, with high-virulence viral proteins bearing a refined PDZ-binding motif.</p> <p>Conclusions</p> <p>Our refined definition of PDZ-binding motifs should provide important clues for identifying functional PDZ-binding motifs and proteins involved in signal transduction.</p

The Scaffolding Protein Dlg1 Is a Negative Regulator of Cell-Free Virus Infectivity but Not of Cell-to-Cell HIV-1 Transmission in T Cells

Background: Cell-to-cell virus transmission of Human immunodeficiency virus type-1 (HIV-1) is predominantly mediated by cellular structures such as the virological synapse (VS). The VS formed between an HIV-1-infected T cell and a target T cell shares features with the immunological synapse (IS). We have previously identified the human homologue of the Drosophila Discs Large (Dlg1) protein as a new cellular partner for the HIV-1 Gag protein and a negative regulator of HIV-1 infectivity. Dlg1, a scaffolding protein plays a key role in clustering protein complexes in the plasma membrane at cellular contacts. It is implicated in IS formation and T cell signaling, but its role in HIV-1 cell-to-cell transmission was not studied before. Methodology/Principal Findings: Kinetics of HIV-1 infection in Dlg1-depleted Jurkat T cells show that Dlg1 modulates the replication of HIV-1. Single-cycle infectivity tests show that this modulation does not take place during early steps of the HIV-1 life cycle. Immunofluorescence studies of Dlg1-depleted Jurkat T cells show that while Dlg1 depletion affects IS formation, it does not affect HIV-1-induced VS formation. Co-culture assays and quantitative cell-to-cell HIV-1 transfer analyses show that Dlg1 depletion does not modify transfer of HIV-1 material from infected to target T cells, or HIV-1 transmission leading to productive infection via cell contact. Dlg1 depletion results in increased virus yield and infectivity of the viral particles produced. Particles with increased infectivity present an increase in their cholesterol content and during the first hours of T cell infection these particles induce higher accumulation of total HIV-1 DNA

Distinct functions of HTLV-1 Tax1 from HTLV-2 Tax2 contribute key roles to viral pathogenesis

While the human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia/lymphoma (ATL), to date, its close relative HTLV-2 is not associated with ATL or other types of malignancies. Accumulating evidence shows that HTLV-1 Tax1 and HTLV-2 Tax2 have many shared activities, but the two proteins have a limited number of significantly distinct activities, and these distinctions appear to play key roles in HTLV-1 specific pathogenesis. In this review, we summarize the functions of Tax1 associated with cell survival, cell proliferation, persistent infection as well as pathogenesis. We emphasize special attention to distinctions between Tax1 and Tax2

Polarization of excitation light influences molecule counting in single-molecule localization microscopy

Single-molecule localization microscopy has been widely applied to count the number of biological molecules within a certain structure. The percentage of molecules that are detected significantly affects the interpretation of data. Among many factors that affect this percentage, the polarization state of the excitation light is often neglected or at least unstated in publications. We demonstrate by simulation and experiment that the number of molecules detected can be different from -40 up to 100 % when using circularly or linearly polarized excitation light. This is determined mainly by the number of photons emitted by single fluorescent molecule, namely the choice of fluorescence proteins, and the background noise in the system, namely the illumination scheme. This difference can be further exaggerated or mitigated by various fixation methods, magnification, and camera settings We conclude that the final choice between circularly or linearly polarized excitation light should be made experimentally, based on the signal to noise ratio of the system

Three-dimensional localisation of fluorescence resonance energy transfer in living cells under two-photon excitation

Three-dimensional (3-D) imaging of fluorescence resonance energy transfer (FRET) in human cells under two-photon excitation was demonstrated in this study. A sample was prepared by expressing a donor and an acceptor in living cells and using an antibody to secure the proximity of contact between the donor and the acceptor. The quenching of fluorescence emission of a donor in the double-labelled cells indicates the presence of FRET that occurred in these living cells. Because of the quadratic relation of the excitation power, 3-D localisation of FRET becomes possible

Spectra and lifetimes of fluorescence resonance energy transfer flurophores under two-photon excitation

We show two-photon spectra and lifetimes acquired using conventional confocal microscopes equipped with an ultra-short pulsed laser and a time-gated intensified charge coupled device. We report on the two-photon spectra and lifetimes of Alexa350, enhanced green fluorescent protein (EGFP), EGFP-CD46, and Cy3 labelled antibodies. Cellular and extracellular EGFP two-photon spectra and lifetimes are compared