Despite the promise of RNA interference (RNAi) and its potential, e.g. for use in cancer therapy, several technical obstacles must first be overcome. The major hurdle of RNAi-based therapeutics is to deliver nucleic acids across the cell’s plasma membrane. This study demonstrates that exosome vesicles derived from humans can deliver short interfering RNA (siRNA) to human mononuclear blood cells. Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication, i.e. antigen presentation, tolerance development and shuttle RNA (mainly mRNA and microRNA). Having tested different strategies, an optimized method (electroporation) was used to introduce siRNA into human exosomes of various origins. Plasma exosomes (exosomes from peripheral blood) were used as gene delivery vector (GDV) to transport exogenous siRNA to human blood cells. The vesicles effectively delivered the administered siRNA into monocytes and lymphocytes, causing selective gene silencing of mitogen-activated protein kinase 1. These data suggest that human exosomes can be used as a GDV to provide cells with heterologous nucleic acids such as therapeutic siRNAs.

It has previously been shown that nano-meter sized vesicles (30–100 nm), exosomes, secreted by antigen presenting cells can induce T cell responses thus showing the potential of exosomes to be used as immunological tools. Additionally, activated CD3+ T cells can secrete exosomes that have the ability to modulate different immunological responses. Here, we investigated what effects exosomes originating from activated CD3+ T cells have on resting CD3+ T cells by studying T cell proliferation, cytokine production and by performing T cell and exosome phenotype characterization. Human exosomes were generated in vitro following CD3+ T cell stimulation with anti-CD28, anti-CD3 and IL-2. Our results show that exosomes purified from stimulated CD3+ T cells together with IL-2 were able to generate proliferation in autologous resting CD3+ T cells. The CD3+ T cells stimulated with exosomes together with IL-2 had a higher proportion of CD8+ T cells and had a different cytokine profile compared to controls. These results indicate that activated CD3+ T cells communicate with resting autologous T cells via exosomes.

No abstract

SummaryGut mesenchymal fibroblasts form complex phenotypical and functional populations. They participate actively in homeostatic maintenance of the extracellular matrix, epithelial barrier function, repair mechanisms and leucocyte migration. In inflammation, they become activated, change matrix expression and synthesize proinflammatory mediators. Subpopulations of mucosal fibroblasts express CD40 and the aim of this study was to define its role in their proinflammatory function. Stable primary fibroblast lines derived from normal mouse colon and inflamed colon from CD4 + CD45RB high -transplanted SCID mice were used as models to explore the role of mucosal fibroblast CD40 in the inflammatory process. Phenotype correlated with in situ fibroblast phenotype in the tissues of origin. Lines from both sources co-expressed CD40 and Thy1·2 independently of a-smooth muscle actin. A subpopulation of CD40 + fibroblasts from normal colon expressed CD40 at high levels and expression was enhanced by interferon (IFN)-g treatment, whereas all CD40 + fibroblasts from colitis expressed at low levels and expression was unaffected by IFN-g treatment. Despite lower-level expression of CD40 by cells from colitis, they secreted constitutively interleukin (IL)-6 and C-C chemokine (CCL)2. Ligation of CD40 enhanced secretion of these mediators and induced secretion of CCL3. CD40 in cells from colitis was more responsive to ligation than CD40 on cells from normal tissue and this sensitivity was amplified selectively by the action of IFN-g. We conclude that the inflammatory milieu in colitis induces long-lasting changes in phenotype and proinflammatory function in colonic fibroblasts. In particular, proinflammatory signalling from fibroblast CD40 is amplified synergistically by the Th1 effector T cell cytokine, IFN-g.

Indoleamine 2,3‐dioxygenase (IDO), a tryptophan‐metabolizing enzyme expressed by dendritic cells (DC), has the potential to inhibit T cell responses and to promote tolerance. In contrast, cholera toxin (CT), the enterotoxin produced by Vibrio cholerae, promotes T cell responses, partly through its ability to induce DC maturation and promote antigen presentation. We hypothesized that the adjuvant activity of CT is associated with a lack of induction of IDO in DC. To test this hypothesis, monocyte‐derived DC were pulsed with CT, and the IDO mRNA expression, IDO functional activity and cytokine production were measured as well as the ability of DC to induce T cell responses in vitro. Cholera toxin exposure induced enhanced levels of IDO mRNA in DC but no functional IDO protein activity. Cholera toxin pulsing however primed DC for CD40L‐induced IDO protein activity. CD40L stimulation of CT‐pulsed DC induced a modest IL‐12p40 production, but not IL‐12p70 or IL‐23 secretion. Furthermore, CT‐pulsed DC induced strong allogeneic and autologous T cell responses in vitro, which were not affected by the IDO‐specific inhibitor 1‐methyl tryptophan. Our results show that CT per se does not induce the expression of functional IDO protein, although it primes DC for CD40L‐mediated IDO production and IL‐12p40 secretion. Furthermore, CT‐treated DC were equally powerful in their T cell stimulatory capacity as cytokine‐matured DC.