Characterization of human thymic exosomes

Abstract

Exosomes are nanosized membrane-bound vesicles that are released by various cell types and are capable of carrying proteins, lipids and RNAs which can be delivered to recipient cells. Exosomes play a role in intercellular communication and have been described to mediate immunologic information. In this article we report the first isolation and characterization of exosomes from human thymic tissue. Using electron microscopy, particle size determination, density gradient measurement, flow cytometry, proteomic analysis and microRNA profiling we describe the morphology, size, density, protein composition and microRNA content of human thymic exosomes. The thymic exosomes share characteristics with previously described exosomes such as antigen presentation molecules, but they also exhibit thymus specific features regarding surface markers, protein content and microRNA profile. Interestingly, thymic exosomes carry proteins that have a tissue restricted expression in the periphery which may suggest a role in T cell selection and the induction of central tolerance. We speculate that thymic exosomes may provide the means for intercellular information exchange necessary for negative selection and regulatory T cell formation of the developing thymocytes within the human thymic medulla.

Figure 1. Morphological characteristics of human thymic EVs.

(A) EVs from human thymic cultures visualized with electron microscopy. Arrow heads point toward EVs with a typical exosomal cup-shaped morphology and a size range of 50–100 nm. Samples from 3 individuals were analyzed with electron microscopy. (B) Size distribution of isolated EVs observed in a NanoSight LM10. The data was analyzed with Nanoparticle tracking analysis software, with a minimum expected particle size setting of 30 nm and the number of tracks analyzed for each sample exceeding 200. In agreement with exosome characteristics, most of the isolated EVs have a size of less than 100 nm. Data is presented as mean ± SEM as a result of 5 analyzed samples. (C) Density profile of isolated EVs. EVs were layered on top of a D₂O/sucrose gradient and centrifuged at 100,000 g for 14 hours. Fractions were collected and their density and protein concentration was measured. The density of the isolated EVs peaked at 1.18–1.19 g/ml, which is within the density range of exosomes. Data is presented as mean ± SEM as a result of 5 density gradients.

Figure 2. Human thymic EV surface markers analyzed by flow cytometry.

(A) Histograms represent density gradient samples red (1.19 g/ml) and orange (1.15 g/ml) stained for the exosomal marker TSG101 (EVs+beads+antibodies) and blue represent negative control (beads+antibodies). (B) Red histograms represent samples (EVs+beads+antibodies) and blue represent negative controls (beads+antibodies). Hence, the isolated EVs were positive for the markers shown in the figure. A gate was created covering the latex bead population and from the bead population the histogram was created. Representative data in terms of median fluorescence intensity is shown (MFI_TSG101 = 77 600, MFI_CD81 = 7287, MFI_CD9 = 4000, MFI_TGF-β = 34 500, MFI_HLA-DR = 209 000, MFI_MFG-E8 = 33 800). (C) Experimental replicates of the flow cytometry analysis evaluated statistically with the Mann-Whitney test. P_TSG101<0.0001, P_CD81 = 0.0286, P_CD9 = 0.0571, P_TGF-β = 0.0022, P_HLA-DR = 0.0079, P_MFG-E8 = 0.0022. Data is presented as median with interquartile range.

Figure 3. Cellular localization of EV proteins.

Protein annotation was retrieved from UniProt and subcellular distribution was assigned based on gene ontology cellular component reduced to generic terms to give a broad overview of the localization. The pie chart is constructed from the shared proteins of two analyzed EV samples.

Figure 4. Heat-map illustrating a hierarchical cluster analysis of tissue expression of proteins identified in thymic EVs.

The heat map is constructed from the proteins shared between two analyzed EV samples (proteins not yet investigated in the HPA database are not included). Proteins and tissues are hierarchally clustered according to biological function on the y- and x-axis, respectively. The expression level is graded 0–3 and illustrated by colour shift from blue to red. Encircled and enlarged is a cluster of proteins with an expression pattern in the HPA concentrated to immune-tissues. Also note the high frequency of TRAs, only expressed in a few tissues, in the left panel.

Figure 5. miRNAs from exosomes of different origin compared in a Venn-diagram.

Green circle: shared miRNAs isolated from human thymic EVs (n = 4). Red circle: miRNAs isolated from exosomes descending from Raji B cells. Blue circle: miRNAs isolated from exosomes descending from Jurkat T cells. 40 miRNAs were shared between all three sources while 38 miRNAs were exclusively found in EVs from human thymic tissue.