Extracellular vesicles (EVs) can be harvested from cell culture supernatants and from all body fluids. EVs can be conceptually classified based on their size and biogenesis as exosomes and microvesicles. Nowadays, it is however commonly accepted in the field that there is a much higher degree of heterogeneity within these two subgroups than previously thought. For instance, the surface marker profile of EVs is likely dependent on the cell source, the cell’s activation status, and multiple other parameters. Within recent years, several new methods and assays to study EV heterogeneity in terms of surface markers have been described; most of them are being based on flow cytometry. Unfortunately, such methods generally require dedicated instrumentation, are time-consuming and demand extensive operator expertise for sample preparation, acquisition, and data analysis. In this study, we have systematically evaluated and explored the use of a multiplex bead-based flow cytometric assay which is compatible with most standard flow cytometers and facilitates a robust semi-quantitative detection of 37 different potential EV surface markers in one sample simultaneously. First, assay variability, sample stability over time, and dynamic range were assessed together with the limitations of this assay in terms of EV input quantity required for detection of differently abundant surface markers. Next, the potential effects of EV origin, sample preparation, and quality of the EV sample on the assay were evaluated. The findings indicate that this multiplex bead-based assay is generally suitable to detect, quantify, and compare EV surface signatures in various sample types, including unprocessed cell culture supernatants, cell culture-derived EVs isolated by different methods, and biological fluids. Furthermore, the use and limitations of this assay to assess heterogeneities in EV surface signatures was explored by combining different sets of detection antibodies in EV samples derived from different cell lines and subsets of rare cells. Taken together, this validated multiplex bead-based flow cytometric assay allows robust, sensitive, and reproducible detection of EV surface marker expression in various sample types in a semi-quantitative way and will be highly valuable for many researchers in the EV field in different experimental contexts.

Bietti9s crystalline corneoretinal dystrophy (BCD) is a recessive degenerative eye disease caused by germline mutations in the CYP4V2 gene. More than 80% of mutant alleles consist of three mutations, that is, two splice-site alterations and one missense mutation, c.992C>A, which translates to p.H331P. In the present study, we analyzed the expression of CYP4 family members in human tissues and conducted functional studies with the wild-type and p.H331P enzymes, to elucidate the link between CYP4V2 activity and BCD. Expression analysis of 17 CYP1 to CYP4 genes showed CYP4V2 to be a major cytochrome P450 in ARPE-19 cells (a human cell line spontaneously generated from normal human retinal pigmented epithelium) and the only detectable CYP4 transcript. Immunohistochemical analyses demonstrated that CYP4V2 protein was present in epithelial cells of the retina and cornea and the enzyme was localized to endoplasmic reticulum. Recombinant reconstituted CYP4V2 protein metabolized eicosapentaenoic acid and docosahexaenoic acid (an important constituent of the retina) to their respective ω-hydroxylated products at rates similar to those observed with purified CYP4F2, which is an established hepatic polyunsaturated fatty acid (PUFA) hydroxylase. The disease-associated p.H331P variant was undetectable in Western blot analyses of HepG2 cells stably transduced with lentiviral expression vectors. Finally, overexpression of functional CYP4V2 in HepG2 cells altered lipid homeostasis. We demonstrated that CYP4V2 protein is expressed at high levels in ocular target tissues of BCD, that the enzyme is metabolically active toward PUFAs, and that the functional deficit among patients with BCD who carry the H331P variant is most likely a consequence of the instability of the mutant protein.

Activated and in vitro expanded natural killer (NK) cells have substantial cytotoxicity against many tumor cells, but their in vivo efficacy to eliminate solid cancers is limited. Here, we used chimeric antigen receptors (CARs) to enhance the activity of NK cells against Ewing sarcomas (EwS) in a tumor antigen-specific manner. Expression of CARs directed against the ganglioside antigen G_D2 in activated NK cells increased their responses to G_D2+ allogeneic EwS cells in vitro and overcame resistance of individual cell lines to NK cell lysis. Second-generation CARs with 4-1BB and 2B4 co-stimulatory signaling and third-generation CARs combining both co-stimulatory domains were all equally effective. By contrast, adoptive transfer of G_D2-specific CAR gene-modified NK cells both by intratumoral and intraperitoneal delivery failed to eliminate G_D2-expressing EwS xenografts. Histopathology review revealed upregulation of the immunosuppressive ligand HLA-G in tumor autopsies from mice treated with NK cells compared to untreated control mice. Supporting the relevance of this finding, in vitro co-incubation of NK cells with allogeneic EwS cells induced upregulation of the HLA-G receptor CD85j, and HLA-G1 expressed by EwS cells suppressed the activity of NK cells from three of five allogeneic donors against the tumor cells in vitro. We conclude that HLA-G is a candidate immune checkpoint in EwS where it can contribute to resistance to NK cell therapy. HLA-G deserves evaluation as a potential target for more effective immunotherapeutic combination regimens in this and other cancers.

Background: Fanconi anaemia (FA) is a heterogeneous inherited disorder clinically characterised by progressive bone marrow failure, congenital anomalies and a predisposition to malignancies. Objective: Determine, based on correction of cellular phenotypes, whether XRCC2 is a FA gene. Methods: Cells (900677A) from a previously identified patient with biallelic mutation of XRCC2, among other mutations, were genetically complemented with wild-type XRCC2. Results: Wild-type XRCC2 corrects each of three phenotypes characteristic of FA cells, all related to the repair of DNA interstrand crosslinks, including increased sensitivity to mitomycin C (MMC), chromosome breakage and G2–M accumulation in the cell cycle. Further, the p.R215X mutant of XRCC2, which is harboured by the patient, is unstable. This provides an explanation for the pathogenesis of this mutant, as does the fact that 900677A cells have reduced levels of other proteins in the XRCC2–RAD51B-C-D complex. Also, FANCD2 monoubiquitination and foci formation, but not assembly of RAD51 foci, are normal in 900677A cells. Thus, XRCC2 acts late in the FA–BRCA pathway as also suggested by hypersensitivity of 900677A cells to ionising radiation. These cells also share milder sensitivities towards olaparib and formaldehyde with certain other FA cells. Conclusions: XRCC2/FANCU is a FA gene, as is another RAD51 paralog gene, RAD51C/FANCO. Notably, similar to a subset of FA genes that act downstream of FANCD2, biallelic mutation of XRCC2/FANCU has not been associated with bone marrow failure. Taken together, our results yield important insights into phenotypes related to FA and its genetic origins.