Overall, the use of EV analysis as a diagnostic and prognostic tool has prodigious clinical potential.

Extracellular vesicles (EVs) have a demonstrated involvement in modulating the immune system. It has been proposed that EVs could be used as biomarkers for detection of inflammatory and immunological disorders. Consequently, it is of great interest to investigate EVs in more detail with focus on immunological markers. In this study, five major leukocyte subpopulations and the corresponding leukocyte-derived EVs were phenotyped with focus on selected immunological lineage-specific markers and selected vesicle-related markers. The leukocyte-derived EVs displayed phenotypic differences in the 34 markers investigated. The majority of the lineage-specific markers used for identification of the parent cell types could not be detected on EVs released from monocultures of the associated cell types. In contrast, the vesicular presentation of CD9, CD63, and CD81 correlated to the cell surface expression of these markers, however, with few exceptions. Furthermore, the cellular expression of CD9, CD63, and CD81 varied between leukocytes present in whole blood and cultured leukocytes. In summary, these data demonstrate that the cellular and vesicular presentation of selected lineage-specific and vesicle-related markers may differ, supporting the accumulating observations that sorting of molecular cargo into EVs is tightly controlled.

Extracellular vesicles (EVs) are a heterogeneous population of membrane-enclosed vesicles. EVs are recognized as important players in cell-to-cell communication and are described to be involved in numerous biological and pathological processes. The fact that EVs are involved in the development and progression of several diseases has formed the basis for the use of EV analysis in a clinical setting. As the interest in EVs has increased immensely, multiple techniques have been developed aiming at characterizing these vesicles. These techniques characterize different features of EVs, like the size distribution, enumeration, protein composition, and the intravesicular cargo (e.g., RNA). This review focuses on techniques that exploit the specificity and sensitivity associated with antibody-based assays to characterize the protein phenotype of EVs. The protein phenotype of EVs can provide information on the functionality of the vesicles and may be used for identification of disease-related biomarkers. Thus, protein profiling of EVs holds great diagnostic and prognostic potential.

The human major histocompatibility complex class II isotype HLA-DR is currently used as an activation marker for T cells. However, whether an endogenous protein expression or a molecular acquisition accounts for the presence of HLA-DR on T cells remains undetermined and still controversial. To further characterize this phenomenon, we compared several aspects of the presence of the HLA-DR protein to the presence of associated mRNA (HLA-DRB1), focusing on human T cells from peripheral blood of healthy individuals. Using a flow cytometric approach, we determined that the HLA-DR observed on CD4 + T cells was almost exclusively cell surface-associated, while for autologous CD19 + B cells, the protein could be located in the plasma membrane as well as in the cytoplasm. Moreover, negligible expression levels of HLA-DRB1 were found in CD4 + T cells, using an HLA-DRB1

Although the observation of major histocompatibility complex II (MHCII) receptors on T cells is longstanding, the explanation for this occurrence remains enigmatic. Reports of an inducible, endogenous expression exist, as do studies demonstrating a protein acquisition from other cells by mechanisms including vesicle transfer. Irrespective of origin, the presence of the human MHCII isotype, human leukocyte antigen DR (HLA-DR), potentially identifies a regulatory T cell population. Using an allogeneic mixed lymphocyte culture (MLC) to induce an antigen-specific immune response, the role of antigen-presenting cells (APCs) for the presence of HLA-DR on cluster of differentiation 3(CD3)+ CD4+ T cells was evaluated. Moreover, a functional phenotype was established for these T cells. It was demonstrated that APCs were essential for HLA-DR on CD3+ CD4+ T cells. Additionally, a regulatory T cell phenotype was induced in CD3+ CD4+ HLA-DR+ responder T cells with an expression of CD25, CTLA-4, CD62L, PD-1, and TNFRII. This phenotype was induced both with and without physical T cell:APC contact, which could reveal novel indications about its functionality. To further investigate contact-independent communication, a phenotype of the small cell-derived vesicles from the MLCs was determined. Yet heterogeneous, this vesicle phenotype displayed contact-dependent differences, providing clues about their intended function in cellular communication.