Background T cell tolerance of allergic cutaneous contact sensitivity (CS) induced in mice by high doses of reactive hapten is mediated by suppressor cells that release antigen-specific suppressive nanovesicles. Objective To determine the mechanism(s) of immune suppression mediated by the nanovesicles. Methods T cell tolerance was induced by i.v. injections of hapten conjugated to self antigens of syngeneic erythrocytes and subsequent contact immunization with the same hapten. Lymph node and spleen cells from tolerized or control donors were harvested and cultured to produce a supernatant containing suppressive nanovesicles that were isolated for testing in active and adoptive cell transfer models of CS. Results Tolerance was shown due to exosome-like nanovesicles in the supernatant of CD8+ suppressor T cells that were not Treg. Antigen specificity of the suppressive nanovesicles was conferred by a surface coat of antibody light chains, or possibly whole antibody, allowing targeted delivery of selected inhibitory miRNA-150 to CS effector T cells. Nanovesicles also inhibited CS in actively sensitized mice after systemic injection at the peak of the responses. The role of antibody and miRNA-150 was established by tolerizing either panimmunoglobulin deficient JH-/- or miRNA-150-/- mice that produced non-suppressive nanovesicles. These nanovesicles could be made suppressive by adding antigen-specific antibody light chains or miRNA-150, respectively. Conclusions This is the first example of T cell regulation via systemic transit of exosome-like nanovesicles delivering a chosen inhibitory miRNA to target effector T cells in an antigen-specific manner by a surface coating of antibody light chains.

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Rationale Endothelial-to-mesenchymal transition (EndoMT) is implicated in myofibroblast-like cell-mediated damage to the coronary arterial wall in acute Kawasaki disease (KD) patients, as evidenced by positive staining for connective tissue growth factor (CTGF) and EndoMT markers in KD autopsy tissues. However, little is known about the molecular basis of EndoMT involved in KD. Objective We investigated the microRNA (miRNA) regulation of CTGF and the consequent EndoMT in KD pathogenesis. As well, the modulation of this process by statin therapy was studied. Methods and Results Sera from healthy children and KD subjects were incubated with human umbilical vein endothelial cells (HUVECs). Cardiovascular disease-related miRNAs, CTGF, and EndoMT markers were quantified using RT-qPCR, ELISA, and Western blotting. Compared to healthy controls, HUVEC incubated with sera from acute KD patients had decreased miR-483, increased CTGF, and increased EndoMT markers. Bioinformatics analysis followed by functional validation demonstrated that Krüppel-like factor 4 (KLF4) transactivates miR-483, which in turn targets the 3′ untranslated region of CTGF mRNA. Overexpression of KLF4 or pre-miR-483 suppressed, whereas knockdown of KLF4 or anti-miR-483 enhanced, CTGF expression in ECs in vitro and in vivo. Furthermore, atorvastatin, currently being tested in a Phase I/IIa clinical trial in KD children, induced KLF4-miR-483, which suppressed CTGF and EndoMT in ECs. Conclusions KD sera suppress the KLF4-miR-483 axis in ECs leading to increased expression of CTGF and induction of EndoMT. This detrimental process in the endothelium may contribute to coronary artery abnormalities in KD patients. Statin therapy may benefit acute KD patients, in part through the restoration of KLF4-miR-483 expression. Clinical Trial Registration NCT01431105