Background: Clinical studies demonstrated the immune modulation of cord blood-derived stem cells (CB-SC) for the treatment of type 1 diabetes and other autoimmune diseases, with long-lasting clinical efficacy. To determine the molecular mechanisms underlying the immune modulation of CB-SC, the actions of exosomes released from CB-SC were explored in this study.

Methods: Exosomes were isolated from CB-SC cultures using ultracentrifugation and confirmed with different markers. The activated T cells and purified monocytes from peripheral blood mononuclear cells (PBMC) were treated with CB-SC in the presence or absence of the purified exosomes, followed by functional and flow cytometry analysis of phenotypic changes with different immune cell markers.

Results: CB-SC-derived exosomes displayed the exosome-specific markers including CD9, CD63, and Alix, at the size of 85.95 ± 22.57 nm. In comparison with the treatment of CB-SC, functional analysis demonstrated that the CB-SC-derived exosomes inhibited the proliferation of activated PBMC, reduced the production of inflammatory cytokines, downregulated the percentage of activated CD4⁺ T and CD8⁺ T cells, and increased the percentage of naive CD4⁺ T and CD8⁺ T cells. Using the fluorescence dye DiO-labeled exosomes, flow cytometry revealed that exosomes preferably bound to the monocytes in the PBMC, leading to an improvement of mitochondrial membrane potential of treated monocytes. Further study indicated that the purified monocytes gave rise to spindle-like macrophages displaying type 2 macrophage (M2) surface markers and upregulating an expression of immune tolerance-related cytokines after the treatment with exosomes.

Conclusions: CB-SC-derived exosomes display multiple immune modulations and primarily on monocytes, contributing to the immune education of CB-SC in the clinical treatment of autoimmune diseases.

背景：临床研究表明，脐带血干细胞（CB-SC）的免疫调节可用于治疗1型糖尿病和其他自身免疫性疾病，并具有长期的临床疗效。为了确定CB-SC免疫调节基础的分子机制，本研究探讨了从CB-SC释放的外来体的作用。

方法：使用超速离心从CB-SC培养物中分离外泌体，并用不同的标记物进行确认。在有或没有纯化外泌体的情况下，用CB-SC处理活化的T细胞和来自外周血单核细胞（PBMC）的纯化单核细胞，然后用不同的免疫细胞标记对表型变化进行功能和流式细胞术分析。

结果：CB-SC衍生的外泌体显示了外泌体特异性标记，包括CD9，CD63和Alix，大小为85.95±22.57 nm。与CB-SC的治疗相比，功能分析表明CB-SC衍生的外泌体抑制了活化的PBMC的增殖，减少了炎性细胞因子的产生，下调了活化的CD4 ⁺ T和CD8 ⁺ T细胞的百分比，并且增加了原始CD4 ⁺ T和CD8 ⁺的百分比T细胞。使用荧光染料DiO标记的外泌体，流式细胞仪显示外泌体优选与PBMC中的单核细胞结合，从而提高了处理过的单核细胞的线粒体膜电位。进一步的研究表明，纯化的单核细胞产生了纺锤状的巨噬细胞，该巨噬细胞显示了2型巨噬细胞（M2）表面标记并在外泌体处理后上调了免疫耐受相关细胞因子的表达。

结论： CB-SC衍生的外来体显示多种免疫调节，并且主要在单核细胞上发挥作用，有助于CB-SC在自身免疫性疾病的临床治疗中进行免疫教育。