Extracellular vesicles (EVs) play a crucial role in the intercellular crosstalk. Mesenchymal stem cell-derived EVs (MSC-EVs), displaying promising therapeutic roles, contribute to the strong rationale for developing EVs as an alternative therapeutic option. EV analysis still represents one of the major issues to be solved in order to translate the use of MSC-EV detection in clinical settings. Even if flow cytometry (FC) has been largely applied for EV studies, the lack of consensus on protocols for FC detection of EVs generated controversy. Standard FC procedures, based on scatter measurements, only allows the detection of the “tip of the iceberg” of all EVs. We applied an alternative FC approach based on the use of a trigger threshold on a fluorescence channel. The EV numbers obtained by the application of the fluorescence triggering resulted significantly higher in respect to them obtained from the same samples acquired by placing the threshold on the side scatter (SSC) channel. The analysis of EV concentrations carried out by three different standardized flow cytometers allowed us to achieve a high level of reproducibility (CV < 20%). By applying the here-reported method highly reproducible results in terms of EV analysis and concentration measurements were obtained.

Extracellular vesicles (EVs) are a heterogenous population of plasma membrane-surrounded particles that are released in the extracellular milieu by almost all types of living cells. EVs are key players in intercellular crosstalk, both locally and systemically, given that they deliver their cargoes (consisting of proteins, lipids, mRNAs, miRNAs, and DNA fragments) to target cells, crossing biological barriers. Those mechanisms further trigger a wide range of biological responses. Interestingly, EV phenotypes and cargoes and, therefore, their functions, stem from their specific parental cells. For these reasons, EVs have been proposed as promising candidates for EV-based, cell-free therapies. One of the new frontiers of cell-based immunotherapy for the fight against refractory neoplastic diseases is represented by genetically engineered chimeric antigen receptor T (CAR-T) lymphocytes, which in recent years have demonstrated their effectiveness by reaching commercialization and clinical application for some neoplastic diseases. CAR-T-derived EVs represent a recent promising development of CAR-T immunotherapy approaches. This crosscutting innovative strategy is designed to exploit the advantages of genetically engineered cell-based immunotherapy together with those of cell-free EVs, which in principle might be safer and more efficient in crossing biological and tumor-associated barriers. In this review, we underlined the potential of CAR-T-derived EVs as therapeutic agents in tumors.

Chimeric antigen receptor (CAR) T-cell–derived extracellular vesicles (EVs) might represent a new therapeutic tool for boosting CAR T-cell antileukemic effects. Here, a cohort of 22 patients who received infusion with CD19 CAR T cells were monitored for the presence of circulating CD19 CAR+ T-cell–derived EVs (CD19.CAR+EVs), which were then separated and functionally characterized for their killing abilities. A good manufacturing practice (GMP)-compliant separation method was also developed. Results demonstrated that CD19.CAR+EVs were detectable in peripheral blood up to 2 years after infusion, indicating long-lasting persistence of their parental cells. Notably, early decreases of circulating CD19.CAR+EV concentrations correlated with failure of CAR T-cell therapy. Circulating CD19.CAR+EVs displayed a median size (standard deviation) of 133.1 ± 65.5 nm and carried a proapoptotic protein cargo. These EVs expressed higher CAR levels than their parental cells. Furthermore, CD19.CAR+EVs did not activate heterologous T cells and produced significant, specific, and dose-dependent cytotoxic effects on CD19+ cell lines and primary cells. The new GMP-compliant EV isolation method allowed for a recovery of 63% ± 5.7% of CD19.CAR+EVs. A deeper analysis of the different protein cargoes carried by EVs derived from different CAR T-cell subpopulations identified a proapoptotic functional pathway linked to CD8+LAG-3+ EVs. Overall, our data indicate that CD19.CAR+EVs may be proposed as promising dynamic new biomarkers of CAR T-cell activity and, by contributing to the direct killing of leukemic targets, represent a new product with strong therapeutic potential that could be infused independently of CAR T cells.

SAMHD1 is a host restriction factor that functions to restrict both retroviruses and DNA viruses, based on its nuclear deoxynucleotide triphosphate (dNTP) hydrolase activity that limits availability of intracellular dNTP pools. In the present study, we demonstrate that SAMHD1 expression was increased following human cytomegalovirus (HCMV) infection, with only a modest effect on infectious virus production. SAMHD1 was rapidly phosphorylated at residue T592 after infection by cellular cyclin-dependent kinases, especially Cdk2, and by the viral kinase pUL97, resulting in a significant fraction of phosho-SAMHD1 being relocalized to the cytoplasm of infected fibroblasts, in association with viral particles and dense bodies. Thus, our findings indicate that HCMV-dependent SAMHD1 cytoplasmic delocalization and inactivation may represent a potential novel mechanism of HCMV evasion from host antiviral restriction activities. Human cytomegalovirus (HCMV) is among the most widespread viruses worldwide, causing severe systemic illness in individuals without a fully competent immune system (e.g., transplanted patients and infants). Therefore, host immune response is crucial to control HCMV replication and dissemination throughout the organism. Some frontline proteins belonging to the innate immune system, known as restriction factors, are already expressed and active even before a pathogen infects a cell and are able to mediate a cell-intrinsic resistance to infections. Among them is SAMHD1, a nuclear deoxynucleotide triphosphate (dNTP) hydrolase that limits the availability of intracellular dNTPs, essential components of growing DNA molecules, including those of replicating viruses. We thus investigated SAMHD1 ability to counteract HCMV infection. We show that SAMHD1 expression was upregulated after infection, but with only a modest effect in limiting viral replication. This was mainly due to HCMV-induced phosphorylation at Threonine-592 (the key residue known to negatively regulate viral restriction), to phosphoT592-SAMHD1 relocalization to the cytoplasm of infected cells, and to its association with viral particles. The unprecedented observation of relocation of phosphoT592-SAMHD1 outside the nucleus may affect SAMHD1 function upon HCMV infection, and thus might represent a novel HCMV-mediated immune evasion strategy that will need further investigations.

Extracellular Vesicles (EVs) are circulating particles surrounded by a plasma membrane carrying a cargo consisting of proteins, lipids, RNAs, and DNA fragments, stemming from the cells from which they originated. EV factors (i.e., miRNAs) play relevant roles in intercellular crosstalk, both locally and systemically. As EVs increasingly gained attention as potential carriers for targeted genes, the study of EV effects on the host immune response became more relevant. It has been demonstrated that EVs regulate the host immune response, executing both pro- and anti-inflammatory functions. It is also known that physical exercise triggers anti-inflammatory effects. This review underlines the role of circulating EVs as players in the anti-inflammatory events associated with the regulation of the host’s immune response to physical exercise.

In this review, we propose that paraganglioma is a fundamentally organized, albeit aberrant, tissue composed of neoplastic vascular and neural cell types that share a common origin from a multipotent mesenchymal-like stem/progenitor cell. This view is consistent with the pseudohypoxic footprint implicated in the molecular pathogenesis of the disease, is in harmony with the neural crest origin of the paraganglia, and is strongly supported by the physiological model of carotid body hyperplasia. Our immunomorphological and molecular studies of head and neck paragangliomas demonstrate in all cases relationships between the vascular and the neural tumor compartments, that share mesenchymal and immature vasculo-neural markers, conserved in derived cell cultures. This immature, multipotent phenotype is supported by constitutive amplification of NOTCH signaling genes and by loss of the microRNA-200s and -34s, which control NOTCH1, ZEB1, and PDGFRA in head and neck paraganglioma cells. Importantly, the neuroepithelial component is distinguished by extreme mitochondrial alterations, associated with collapse of the ΔΨm. Finally, our xenograft models of head and neck paraganglioma demonstrate that mesenchymal-like cells first give rise to a vasculo-angiogenic network, and then self-organize into neuroepithelial-like clusters, a process inhibited by treatment with imatinib.