wrote the manuscript. Notes Competing Interests The authors declare that they have no competing interests. Footnotes Electronic supplementary material Supplementary information accompanies this paper at 10.1038/s41598-017-14000-z. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.. cells to produce interferon- (IFN) and granzyme B (GZB) in the absence of antigens, whereas control exosomes derived Carotegrast from antigen-stimulated CTLs did not. In addition, IL-12 induced exosomes are able to strengthen the effects of poor antigen stimulation on CTLs. Proteomic analysis demonstrates that IL-12 stimulation alters catalytic and binding activities of proteins in CTL exosomes. Our findings indicate that the biological function and morphology of exosomes secreted by CTLs can be influenced by the type of stimulation CTLs receive. Thus, a fully functional, ongoing, antigen-specific CTL response may influence bystander CD8+ T cells through secretion of exosomes. Introduction One of the bodys primary responses to contamination is the activation of cytotoxic T lymphocytes (CTLs), which undergo drastic expansion and become effectors that eliminate pathogen-infected cells. Communication between antigen-specific and non-specific immune cells is critical to the ability of the immune system to mount a vigorous adaptive immune response while maintaining functional innate and adaptive Rabbit polyclonal to AMDHD1 immunity against Carotegrast other pathogens. While much important knowledge has been uncovered1,2, understanding of CTL intercellular communication mechanisms remains incomplete. Advancing this understanding may lead to improvement in the design of immunotherapies in a variety of applications, such as chronic infections and cancers. One validated mechanism of CTL intercellular communication is usually via extracellular vesicles, particularly exosomes3. Exosomes are membrane-bound vesicles secreted by somatic cells4C8, including T cells and B cells, that range in size from 30 to 150 nm3. Exosome formation can be driven by two pathways; exosomal sorting complex required for transport (ESCRT)-dependent9,10, and ESCRT-independent3,11,12. Exosome secretion may be constitutive, as in most cancer cells, or regulated, as in T and B cells, which require receptor stimulation3,13C15. Exosomes are effective immune regulators based on their unique features: small size enabling rapid and unadulterated horizontal transfer of materials between cells; enclosed environment to protect cargo (proteins and RNAs) from degradation during transport; and ability to fuse with biological membranes. Production of exosomes by T cells only occurs following T cell activation13C16. The biological function of exosomes is usually thought to be related to the proteins3 and/or RNAs17 contained therein. Exosomes from CD8+ T cells have been shown to inhibit HIV transcription that enhances IL-2-mediated immune responses in na?ve CD8+ T cells, suggesting that activated T cells (both CD4+ and CD8+) may specifically communicate with resting, bystander T cells via exosomes19. In mice, antigen-stimulated CD8+ T cells secrete exosomes that enhance the metastasis of melanoma cells to the lung via Fas signaling brought on by the exosome protein FasL20. However, it remains unknown if and how variations in CTL-derived exosome functions are associated with differences in CTL stimulation. Full activation of CTLs requires three discrete signals: antigen (1), costimulation (2), and inflammatory cytokines (3), such as IL-1221. Here, we investigate the hypothesis that exosomes secreted by activated CTLs differ based upon the stimulation from signal 3. Specifically, we focused on CTL stimulation by the cytokine IL-12, which has been shown to be an important third signal cytokine in murine models21,22. To test this hypothesis, we used OT-I transgenic CD8+ T cells in an system. Our results demonstrate that IL-12 induces structurally and functionally distinct activated CTL-derived exosomes, which can thereby activate bystander CD8+ T cells without the presence of antigen. Results IL-12 stimulation impacts activated CTL-derived vesicle size Purified na?ve CD8+ T cells from OT-I mice were stimulated with antigen and costimulation (2 signals-2SI) or 2SI plus IL-12 (3 signals-3SI) in vesicle-depleted media23,24. Extracellular vesicles were purified from supernatant three days following stimulation25C27 and exhibited size ranges (Fig.?1A) and morphology (Fig.?1C) consistent with exosomes. The vesicles derived from 2SI-activated CTLs (cont-exo) were larger than 3SI-conditioned CTL-derived vesicles (IL-12-exo), with mean sizes of 144 and 77?nm, respectively (Fig.?1A). In both populations, protein content was elevated at 12.2 and 11.0?g/mL (Fig.?1B) as compared to supernatant from non-activated cells (~0.1?g/mL)25,28C30. Vesicle concentrations from the two activated cell populations were also comparable, at 1.55 and 1.71??109/mL in supernatant, respectively, as detected by a NanoSight LM1031 (Fig.?1B), consistent with comparable data from other cell types25,28,31,32. Therefore, the morphology of extracellular vesicles from antigen-stimulated CTLs appears to resemble that of exosomes. Open in a separate window Physique 1 Characterization of CTL-derived vesicles. Na?ve CD8+ T cells purified from OT-I mice were stimulated with 2SI (cont) or 3SI (IL-12) for three days and rather than data, which should be further confirmed in the future. The variations in CTL exosomes secreted under differential stimulation may be related Carotegrast to exosome biogenesis itself. Exosome formation is usually driven by two alternate pathways,.