CD47 cross-dressing by extracellular vesicles expressing CD47 inhibits phagocytosis without transmitting cell death signals.

Transgenic CD47 overexpression is an encouraging approach to ameliorating xenograft rejection and alloresponses to pluripotent stem cells, and the efficacy correlates with the level of CD47 expression. However, CD47, upon ligation, also transmits signals leading to cell dysfunction or death, raising a concern that overexpressing CD47 could be harmful. Here, we unveiled an alternative source of cell surface CD47. We showed that extracellular vesicles, including exosomes, released from normal or tumor cells overexpressing CD47 (transgenic or native) can induce efficient CD47 cross-dressing on pig or human cells. Like the autogenous CD47, CD47 cross-dressed on cell surfaces is capable of interacting with SIRPα to inhibit phagocytosis. However, ligation of the autogenous, but not cross-dressed, CD47 induced cell death. Thus, CD47 cross-dressing provides an alternative source of cell surface CD47 that may elicit its anti-phagocytic function without transmitting harmful signals to the cells. CD47 cross-dressing also suggests a previously unidentified mechanism for tumor-induced immunosuppression. Our findings should help to further optimize the CD47 transgenic approach that may improve outcomes by minimizing the harmful effects of CD47 overexpression.

Genetic barcoding with fluorescent proteins for multiplexed applications.

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery of fluorescent proteins and their genes have enabled the engineering of protein fusions for localization, the analysis of transcriptional activation and translation of proteins of interest, or the general tracking of individual cells and cell populations. The use of fluorescent protein genes in combination with retroviral technology has further allowed the expression of these proteins in mammalian cells in a stable and reliable manner. Shown here is how one can utilize these genes to give cells within a population of cells their own biosignature. As the biosignature is achieved with retroviral technology, cells are barcoded 'indefinitely'. As such, they can be individually tracked within a mixture of barcoded cells and utilized in more complex biological applications. The tracking of distinct populations in a mixture of cells is ideal for multiplexed applications such as discovery of drugs against a multitude of targets or the activation profile of different promoters. The protocol describes how to elegantly develop and amplify barcoded mammalian cells with distinct genetic fluorescent markers, and how to use several markers at once or one marker at different intensities. Finally, the protocol describes how the cells can be further utilized in combination with cell-based assays to increase the power of analysis through multiplexing.

A Multiplexed Cell-Based Assay for the Identification of Modulators of Pre-Membrane Processing as a Target against Dengue Virus.

The DenV pre-membrane protein (prM) is a crucial chaperone for the viral envelope protein, preventing premature fusion with vesicles during viral export. prM molecules in immature particles are cleaved by host proteases, leading to mature fusogenic virions. Blockade of prM cleavage would restrict fusion and represents a novel druggable opportunity against DenV. We have thus established a cell-based platform to monitor prM processing that relies on an engineered two-tag scaffold that travels to the cell surface through the secretory pathway. The assay discriminates between a single cell-surface tag when prM is cleaved and two tags when it is not, as detected through fluorescent-coupled antibodies by flow cytometry. The assay, miniaturized into a 96-well plate format, was multiplexed with the HIV-1 envelope boundary, also cleaved in the same pathway. A pilot screen against 1280 compounds was executed, leading to the identification of a potential active and corroborating the robustness of our assay for large-scale screening. We describe for the first time a cell-based assay that monitors DenV prM processing within the classical secretory pathway, which was exploited to identify a potential novel drug against DenV.