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

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 cell 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

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

Detecting protein-protein interactions in cells using fluorescence energy transfer

Not supplied

A functional interaction between CD46 and DLG4: a role for DLG4 in epithelial polarization

Using a yeast two-hybrid screen, we identified a physical interaction between CD46 and DLG4. CD46 is a ubiquitous human cell-surface receptor for the complement components C3b and C4b and for measles virus and human herpesvirus 6. DLG4 is a scaffold protein important for neuronal signaling and is homologous to the Drosophila tumor suppressor DLG. We show that an interaction between CD46 and DLG4 is important for polarization in epithelial cells. Specifically, we show (i) biochemical evidence for an interaction between CD46 and DLG4, (ii) that this interaction is specific for the Cyt1 (but not Cyt2) domain of CD46, (iii) that both CD46 and an alternatively spliced isoform of DLG4 are polarized in normal human epithelial cells, and (iv) that the polarized expression of CD46 in epithelial cells requires the DLG4-binding domain and alters with expression of a truncated form of DLG4. This is the first identification of a direct and cytoplasmic domain-specific interaction between CD46 and an intracellular signaling molecule and provides a molecular mechanism for the polarization of CD46. These data also indicate that, in addition to the known role for DLG4 in neuronal cells, DLG4 may be important for polarization in epithelial cells

Ratio coefficients from experimental data are dependent on the threshold value.

<p>Fluorescence intensity ratios were extracted from images of dividing MLA cells expressing GFP, and for a random selection of 10 events, ratios plotted again threshold setting. Ratios were calculated using Proportion (<b>A</b>) and PR (<b>B</b>). Different colors represent different events.</p

Sensitivity test from simulations.

<p>Cell divisions were simulated in increasing ratios from 1 to 1.5 with even increments of 0.05, giving n = 1100 divisions in total. PR^major was plotted against PR^minor for non-clustered (<b>A</b>) and clustered (<b>B</b>) data. Data is showed as major/minor plot, under a range of thresholds from T = 0%, to T = 80% in increments of 20%. The magenta line shows gating exclusion of 10% of data with the highest PR^minor. The gate shifts right as T value increase. The colours in the figure legend represent different ratios and corresponding to the ratio colour of each data dot. Input for simulations: θ was chosen randomly varying from 0 to 90 degrees, distribution of parental radius and total intensity were selected randomly from a real distribution from real data, number of clusters in one of the daughter cells was 20 to 100, and the number of clusters in the other daughter cell was multiplied in the simulated ratio giving a possible range from 30 to 150.</p