Abstract
The spleen is a secondary lymphoid organ with multiple functions including the removal of senescent red blood cells and the coordination of immune responses against blood-borne pathogens, such as malaria parasites. Despite the major role of the spleen, the study of its function in humans is limited by ethical implications to access human tissues. Here, we employed multiparameter flow cytometry combined with cell purification techniques to determine human spleen cell populations from transplantation donors. Spleen immuno-phenotyping showed that CD45+ cells included B (30%), CD4+ T (16%), CD8+ T (10%), NK (6%) and NKT (2%) lymphocytes. Myeloid cells comprised neutrophils (16%), monocytes (2%) and DCs (0.3%). Erythrocytes represented 70%, reticulocytes 0.7% and hematopoietic stem cells 0.02%. Extracellular vesicles (EVs) are membrane-bound nanoparticles involved in intercellular communication and secreted by almost all cell types. EVs play several roles in malaria that range from modulation of immune responses to vascular alterations. To investigate interactions of plasma-derived EVs from Plasmodium vivax infected patients (PvEVs) with human spleen cells, we used size-exclusion chromatography (SEC) to separate EVs from the bulk of soluble plasma proteins and stained isolated EVs with fluorescent lipophilic dyes. The integrated cellular analysis of the human spleen and the methodology employed here allowed in vitro interaction studies of human spleen cells and EVs that showed an increased proportion of T cells (CD4+ 3 fold and CD8+ 4 fold), monocytes (1.51 fold), B cells (2.3 fold) and erythrocytes (3 fold) interacting with PvEVs as compared to plasma-derived EVs from healthy volunteers (hEVs). Future functional studies of these interactions can contribute to unveil pathophysiological processes involving the spleen in vivax malaria.
Highlights
The spleen is a secondary lymphoid organ with multiple functions in physiology and immunity, including the removal of senescent red blood cells (RBCs) from circulation, the recycling of iron and the coordination of innate and adaptive immune responses against blood-borne pathogens (Bowdler, 2002; Mebius and Kraal, 2005)
In this study we have performed an integrated characterization of human spleen cells using multiparameter flow cytometry, a powerful technique that has been systematically applied in immunophenotyping in clinical and research settings (O’Donnell et al, 2013)
Our work allowed the quantification of several immune cells including neutrophils, monocytes, DCs, T and B lymphocytes, NK and NKT cells as well as resident reticulocytes, HSCs and mature RBCs (Figure 1)
Summary
The spleen is a secondary lymphoid organ with multiple functions in physiology and immunity, including the removal of senescent red blood cells (RBCs) from circulation, the recycling of iron and the coordination of innate and adaptive immune responses against blood-borne pathogens (Bowdler, 2002; Mebius and Kraal, 2005). A recent study using a large cohort of organ transplantation donors as source of lymphoid organs, including the spleen, has shown the most complete characterization of human spleen immune cells including myeloid populations as monocytes and neutrophils as well as lymphoid cells at different activation states (Carpenter et al, 2018a). These studies highlighted the relevance of organ transplantation donors as a source of physiological human tissue to conduct immunological studies
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