Lotte Hatting Pugholm

Phenotyping of Leukocytes and Leukocyte-Derived Extracellular Vesicles

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.

Antibody-Based Assays for Phenotyping of Extracellular Vesicles

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.

Enhanced Humoral Responses Induced by Targeting of Antigen to Murine Dendritic Cells

Dendritic cells (DCs) are superior in their ability to induce and control adaptive immune responses. These qualities have motivated the hypothesis that targeted delivery of antigen to DCs in vivo may be an effective way of enhancing immunization. Recent results show that antigen targeted to certain DC surface molecules may indeed induce robust immune responses. Targeting of antigen to DCs can be accomplished by the means of monoclonal antibodies. This study compared the humoral responses induced in mice by in vivo targeting of DCs using monoclonal antibodies specific for CD11c, CD36, CD205, Clec6A, Clec7A, Clec9A, Siglec‐H and PDC‐TREM. The results demonstrate that antigen delivery to different targets on DCs in vivo gives rise to humoral responses that differ in strength. Targeting of antigen to CD11c, CD36, CD205, Clec6A, Clec7A and PDC‐TREM induced significantly stronger antibody responses compared to non‐targeted isotype‐matched controls. Targeting of Clec9A and Siglec‐H did not lead to efficient antibody responses, which may be due to unfavourable properties of the targeting antibody, in which case, other antibodies with the same specificity might elicit a different outcome. Anti‐CD11c was additionally used for elucidating the impact of the route of vaccination, and the results showed only minor differences between the antibody responses induced after immunization either s.c., i.v. or i.p. Altogether, these data show that targeting of different surface molecules on DCs result in very different antibody responses and that, even in the absence of adjuvants, strong humoral responses was induced.

Presence of HLA-DR molecules and HLA-DRB1 mRNA in circulating CD4+ T cells

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 allele‐specific qPCR assay. Finally, the presence of HLA‐DR was not confined to activated CD4+ and CD8+ T cells, as evaluated by the co‐expression of CD25. The functional role of the HLA‐DR molecule on T cells remains enigmatic; however, this study presents evidence of fundamental differences for the presence of HLA‐DR on T cells from HLA‐DR in the context of antigen‐presenting cells, which is a well‐known phenomenon. Although an inducible endogenous protein expression cannot be excluded for the T cells, our findings suggest that a re‐evaluation of the HLA‐DR as a T cells activation marker is warranted.

Antibody-Mediated Delivery of Antigen to Dendritic Cells

Dendritic cells (DCs) are specialized antigen-presenting cells that control T cell responses. DCs play a dual role in inducing and orchestrating adaptive immune responses upon infection but also maintaining T cell tolerance. The superior capacity of DCs to control immunogenicity has initiated the development of DC-targeted vaccines that aim at inducing potent, durable and adjustable immune responses that could be clinically favourable in various human disorders. Specific delivery of antigen to DCs has been assayed in a research setting for a couple of decades and these efforts have now enabled implementation of DC-targeted vaccines in a clinical setting. The present review discusses targeting of DCs with special focus on antibody-mediated delivery of antigen.

In vitro assay for screening of optimal targets for antigen-delivery to murine dendritic cells

Targeting of antigen to dendritic cells (DCs) increase the efficiency of immunization procedures and may facilitate the development of more effective vaccines. Several surface molecules on DCs have shown to be useful for antigen targeting, but many more deserves investigation for their efficacy in this respect. With this end in mind, a simple in vitro assay for screening of optimal targets for antigen‐delivery to murine DCs was established. Splenocytes from mice immunized with rat IgG were targeted in vitro with a panel of different rat monoclonal antibodies (mAbs) directed against surface markers on murine DCs. The resulting T‐cell activation was analysed by determining the number of IFN‐γ and IL‐4 secreting cells by ELISPOT. A positive effect of targeting was evident with several of the mAbs. Thus, mAbs against CD11c, CD36, CD205 and Clec7A all induced IFN‐γ responses that were significantly higher than those induced by non‐targeting control mAbs. Anti‐CD36 also induced IL‐4 responses that were significantly higher than the control. The assay described here allows simultaneous analysis of a large number of potential target structures and facilitates direct comparison between the different targets regarding the strength of the T‐cell responses induced by the targeted DCs. The assay could be useful as a first‐line screening of potential target structures on murine DCs.

Oxygen-Related Differences in Cellular and Vesicular Phenotypes Observed for Ovarian Cell Cancer Lines

Extracellular vesicles (EVs) are one of several tools that cells use to communicate with each other. This communication is facilitated by a number of surface-associated proteins and the cargo of the vesicles. For several cancer types, the amount of EVs is observed to be up-regulated in patients compared to healthy individuals, possibly signifying the presence of an aberrant process. The hypoxia-induced release of EVs from cancer cells has been hypothesized to cause the malignant transformation of healthy recipient cells. In this study, the phenotype of cells and EVs from the ovarian cancer cell lines, COV504, SKOV3, and Pt4, were quantified and analysed under normoxic and hypoxic conditions. It was shown that both cells and EVs express common markers and that the EV phenotype varies more than the cellular phenotype. Additionally, cells subjected to 24 hours of hypoxia compared to normoxia produced more EVs. The phenotyping of EVs from cancer cell lines provides information about their molecular composition. This information may be translated to knowledge regarding the functionality of EVs and lead to a better understanding of their role in cancer.

The phenotypical changes of plasma EVs over time in healthy donors

Book: ISEV2017 To cite this article: (2017) Abstract Book: ISEV2017, Journal of Extracellular Vesicles, 6:sup1, 1310414, DOI: 10.1080/20013078.2017.1310414 To link to this article: https://doi.org/10.1080/20013078.2017.1310414

Phenotyping and quantification of cascade-primed immune cells (CAPRI) and their EVs

Book: ISEV2017 To cite this article: (2017) Abstract Book: ISEV2017, Journal of Extracellular Vesicles, 6:sup1, 1310414, DOI: 10.1080/20013078.2017.1310414 To link to this article: https://doi.org/10.1080/20013078.2017.1310414

Phenotyping of leukocytes and leukocyte-derived extracellular vesicles

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 inwhole blood and cultured leukocytes. In summary, these data demonstrate that the cellular and vesicular presentation of selected lineage-specific and vesicle-relatedmarkersmay differ, supporting the accumulating observations that sorting of molecular cargo into EVs is tightly controlled.ISEV2016 is organized by The Local Organizing Committee Chair Edit I Buzás (Hungary), Aled Clayton (United Kingdom), Dolores Di Vizio (USA), Juan Manuel Falcon-Perez (Spain), Guido Jenster (The Netherlands), Lorraine O’Driscoll (Ireland), Yong Song Gho (South Korea), Marjolein van Driel (The Netherlands), Hans van Leeuwen (The Netherlands), Guillaume van Niel (France), Marca HM Wauben (The Netherlands), Kenneth W Witwer (USA), María Yáñez-Mó (Spain) Together with the Executive ISEV Board (2014 – 2016) President: Jan Lötvall Secretary General: Clotilde Théry Interim Treasurer: Kenneth W Witwer Executive Chair Science / Meetings: Marca Wauben Executive Chair Education: Yong Song Gho Executive Chair Communication: Andrew Hill Members at Large: Peter Quesenberry, Kenneth W Witwer, Susmita Sahoo, Dolores Di Vizio, Chris Gardiner, Edit I Buzás, Hidetoshi Tahara, Suresh Mathivanan, Igor Kurochkin