Nano-sized vesicles, so called extracellular vesicles (EVs), from regenerative cardiac cells represent a promising new therapeutic approach to treat cardiovascular diseases. However, it is not yet sufficiently understood how cardiac-derived EVs facilitate their protective effects. Therefore, we investigated the immune modulating capabilities of EVs from human cardiac-derived adherent proliferating (CardAP) cells, which are a unique cell type with proven cardioprotective features. Differential centrifugation was used to isolate EVs from conditioned medium of unstimulated or cytokine-stimulated (IFNγ, TNFα, IL-1β) CardAP cells. The derived EVs exhibited typical EV-enriched proteins, such as tetraspanins, and diameters mostly of exosomes (< 100 nm). The cytokine stimulation caused CardAP cells to release smaller EVs with a lower integrin ß1 surface expression, while the concentration between both CardAP-EV variants was unaffected. An exposure of either CardAP-EV variant to unstimulated human peripheral blood mononuclear cells (PBMCs) did not induce any T cell proliferation, which indicates a general low immunogenicity. In order to evaluate immune modulating properties, PBMC cultures were stimulated with either Phytohemagglutin or anti-CD3. The treatment of those PBMC cultures with either CardAP-EV variant led to a significant reduction of T cell proliferation, pro-inflammatory cytokine release (IFNγ, TNFα) and increased levels of active TGFβ. Further investigations identified CD14+ cells as major recipient cell subset of CardAP–EVs. This interaction caused a significant lower surface expression of HLA-DR, CD86, and increased expression levels of CD206 and PD-L1. Additionally, EV-primed CD14+ cells released significantly more IL-1RA. Notably, CardAP-EVs failed to modulate anti-CD3 triggered T cell proliferation and pro-inflammatory cytokine release in monocultures of purified CD3+ T cells. Subsequently, the immunosuppressive feature of CardAP-EVs was restored when anti-CD3 stimulated purified CD3+ T cells were co-cultured with EV-primed CD14+ cells. Beside attenuated T cell proliferation, those cultures also exhibited a significant increased proportion of regulatory T cells. CardAP-EVs have useful characteristics that could contribute to enhanced regeneration in damaged cardiac tissue by limiting unwanted inflammatory processes. It was shown that the priming of CD14+ immune cells by CardAP-EVs towards a regulatory type is an essential step to attenuate significantly T cell proliferation and pro-inflammatory cytokine release in vitro.

中文翻译：

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来自再生人类心脏细胞的细胞外囊泡通过引发单核细胞充当有效的免疫调节剂

来自再生心脏细胞的纳米大小的囊泡，即所谓的细胞外囊泡（EVs），代表了一种有前途的治疗心血管疾病的新治疗方法。然而，尚未充分了解心脏源性电动车如何促进其保护作用。因此，我们研究了人类心脏衍生的粘附增生（CardAP）细胞对EV的免疫调节能力，这是具有经验证的心脏保护功能的独特细胞类型。差异离心法用于从未刺激或细胞因子刺激（IFNγ，TNFα，IL-1β）CardAP细胞的条件培养基中分离出EV。衍生的EV表现出典型的富含EV的蛋白质（例如四跨膜蛋白），并且直径大多为外泌体（<100 nm）。细胞因子刺激导致CardAP细胞释放较小的EV，而整合素ß1表面表达较低，而两个CardAP-EV变体之间的浓度均不受影响。两种CardAP-EV变体暴露于未刺激的人外周血单核细胞（PBMC）均不会诱导任何T细胞增殖，这表明免疫原性普遍较低。为了评估免疫调节特性，用植物血凝素或抗CD3刺激PBMC培养物。用任一种CardAP-EV变体处理这些PBMC培养物，可显着降低T细胞增殖，促炎性细胞因子释放（IFNγ，TNFα）并增加活性TGFβ的水平。进一步的研究确定CD14 +细胞是CardAP–EV的主要受体细胞亚群。这种相互作用导致HLA-DR，CD86的表面表达明显降低，并且CD206和PD-L1的表达水平增加。此外，由EV引发的CD14 +细胞释放出更多的IL-1RA。值得注意的是，CardAP-EV在纯化的CD3 + T细胞单培养中未能调节抗CD3触发的T细胞增殖和促炎性细胞因子释放。随后，当抗CD3刺激的纯化CD3 + T细胞与EV引发的CD14 +细胞共培养时，CardAP-EV的免疫抑制特征得以恢复。除了减毒的T细胞增殖外，这些培养物还显示出调节性T细胞的比例显着增加。CardAP-EV具有有用的特性，可通过限制有害的炎症过程来促进受损心脏组织中的再生。