Human thymic epithelial primary cells produce exosomes carrying tissue-restricted antigens - PubMed (original) (raw)

Human thymic epithelial primary cells produce exosomes carrying tissue-restricted antigens

Gabriel Skogberg et al. Immunol Cell Biol. 2015 Sep.

Abstract

Exosomes are nano-sized vesicles released by cells into the extracellular space and have been shown to be present in thymic tissue both in mice and in humans. The source of thymic exosomes is however still an enigma and hence it is not known whether thymic epithelial cells (TECs) are able to produce exosomes. In this work, we have cultured human TECs and isolated exosomes. These exosomes carry tissue-restricted antigens (TRAs), for example, myelin basic protein and desmoglein 3. The presence of TRAs indicates a possible role for thymic epithelium-derived exosomes in the selection process of thymocytes. The key contribution of these exosomes could be to disseminate self-antigens from the thymic epithelia, thus making them more accessible to the pool of maturing thymocytes. This would increase the coverage of TRAs within the thymus, and facilitate the process of positive and negative selection.

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Figures

Figure 1

Figure 1

Cultured cells display the phenotype, cell-surface markers and mRNAs of TECs. (a) Light microscopy and immunofluorescent stainings of cultured human TECs. Morphology of cells growing from a piece of thymic tissue (left), and formed HC-like structures (middle and right). (b) Immunofluorescent stainings for K5 (mTECs; green, × 25), K8 (mTECs, cTECs; green, × 25), EpCAM (TECs; green, × 25), HLA-DR (TECs; green, × 10). CD11c (DCs; green, × 10) was included to estimate DC contamination. No wash and wash: illustration of non-adherent cell (thymocyte) removal by wash of cultures (× 10). For applicable pictures: Hoescht (nuclei; blue) and cell mask (cell membranes; red). Negative controls were performed by replacement of primary antibodies with isotype-matched antibodies. Double immunostainings: EpCAM/K8 (TECs; yellow, cTECs; red × 25), EpCAM/K5 (TECs; yellow, mTECs; green × 25) and K8/K5 (cTECs; red, mTECs; green × 25). Representative pictures are shown. (c) mRNA profile of cultured cells include TEC-mRNAs and TRA-mRNAs. (d) mRNA profile for K5, K8, AIRE and MBP in cultured TECs over time analyzed by RT–qPCR. GAPDH was used as an endogenous control and expression was related to expression at day 3. Data are presented as mean±s.e.m. (_n_=3).

Figure 2

Figure 2

Characteristics of exosomes from cultured TECs. (a) Left panel: Size distribution of TEC derived exosomes was investigated using Nanosight LM10 instrument. Distribution peaked at 136 nm and the majority of particles were smaller than 200 nm. The number of tracks exceeded 200 in each measurement and the minimum expected particle setting was 30 nm. Data are presented as mean+s.e.m. as a result of four analyzed cultures. Right panel: An overlay comparison of exosomes isolated from human thymic tissue (bars+s.e.m.) and exosomes isolated from cultured TECs (bars+s.e.m.). (b) Flow-cytometry analysis with blue histograms representing samples (exosomes+beads+antibody) and red histograms negative controls (beads+antibody). The exosomes stained positive for TSG101 and HLA-DR, weakly positive for CD9 and CD81 and negative for CD45. The exosomes were close to negative for CD63. Gating strategy was: all beads with coupled exosomes were gated upon and investigated for stained markers. Representative stainings are shown.

Figure 3

Figure 3

Proteomic analysis of cells and exosomes compared in a Venn diagram (culture 1). The diagram illustrates the distribution of the identified proteins between cultured cells (left circle) and exosomes (right circle) as well as the numbers of TRAs (ellipse) in the different categories.

Figure 4

Figure 4

Schematic illustration of protein examples found in the TEC exosome proteome (culture 1). TEC exosomes contain TEC proteins, autoantigens and TRAs (light gray fields). TRAs shown in the figure are either exosome specific (left TRA circle) or detected in both exosomes and cultured cells (right TRA circle). Examples of several typical exosomal proteins are found within the TEC exosomes (dark gray fields). The depicted typical exosomal proteins are represented in the core exosomal protein list retrieved from the Exocarta database and presented with gene symbols.

Figure 5

Figure 5

Scheme of thymic culture approach, tissue removal and exosome-supernatant collection. Thymic tissue was washed, trimmed and cut into pieces. The pieces were washed, treated with enzymes by incubation for 90 min. The pieces were washed, and grown in complete media for 6 days. All pieces of tissue were removed and all media was discarded and replaced with fresh complete media. The remaining cells were cultured for another 72 h after which half of the media was collected for exosomal isolation and replaced with fresh complete media. Finally, another 72 h of culture took place and at the end all media was collected for exosomal isolation and cells were released by trypsinization for downstream analysis.

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