Cancer Research Unit, Saskatchewan Cancer Agency, Division of Oncology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 4H4, Canada.
Biochem Biophys Res Commun. 2013 Aug 16;438(1):152-5. doi: 10.1016/j.bbrc.2013.07.044. Epub 2013 Jul 20.
Natural CD4(+)25(+) and CD8(+)25(+) regulatory T (Tr) cells have been shown to inhibit autoimmune diseases. Immune cells secrete exosomes (EXOs), which are crucial for immune regulation. However, immunomodulatory effect of natural Tr cell-secreted EXOs is unknown. In this study, we purified natural CD8(+)25(+) Tr cells from C57BL/6 mouse naive CD8(+) T cells, and in vitro amplified them with CD3/CD28 beads. EXOs (EXO(Tr)) were purified from Tr cell's culture supernatants by differential ultracentrifugation and analyzed by electron microscopy, Western blot and flow cytometry. Our data showed that EXO(Tr) had a "saucer" or round shape with 50-100 nm in diameter, contained EXO-associated markers LAMP-1 and CD9, and expressed natural Tr cell markers CD25 and GITR. To assess immunomodulatory effect, we i.v. immunized C57BL/6 mice with ovalbumin (OVA)-pulsed DCs (DC(OVA)) plus Tr cells or EXO(Tr), and then assessed OVA-specific CD8(+) T cell responses using PE-H-2K(b)/OVA tetramer and FITC-anti-CD8 antibody staining by flow cytometry and antitumor immunity in immunized mice with challenge of OVA-expressing BL6-10OVA melanoma cells. We demonstrated that DC(OVA)-stimulated CD8(+) T cell responses and protective antitumor immunity significantly dropped from 2.52% to 1.08% and 1.81% (p<0.05), and from 8/8 to 2/8 and 5/8 mice DC(OVA) (p<0.05) in immunized mice with co-injection of Tr cells and EXO(Tr), respectively. Our results indicate that natural CD8(+)25(+) Tr cell-released EXOs, alike CD8(+)25(+) Tr cells, can inhibit CD8(+) T cell responses and antitumor immunity. Therefore, EXOs derived from natural CD4(+)25(+) and CD8(+)25(+) Tr cells may become an alternative for immunotherapy of autoimmune diseases.
天然的 CD4(+)25(+)和 CD8(+)25(+)调节性 T(Tr)细胞已被证明可以抑制自身免疫性疾病。免疫细胞分泌外泌体(EXOs),这对于免疫调节至关重要。然而,天然 Tr 细胞分泌的 EXOs 的免疫调节作用尚不清楚。在这项研究中,我们从 C57BL/6 小鼠幼稚 CD8(+)T 细胞中纯化了天然的 CD8(+)25(+)Tr 细胞,并通过 CD3/CD28 珠粒在体外扩增它们。通过差速超速离心从 Tr 细胞的培养上清液中纯化外泌体(EXO(Tr)),并通过电子显微镜、Western blot 和流式细胞术进行分析。我们的数据显示,EXO(Tr)呈“碟形”或圆形,直径为 50-100nm,含有外泌体相关标记物 LAMP-1 和 CD9,并表达天然 Tr 细胞标记物 CD25 和 GITR。为了评估免疫调节作用,我们通过静脉注射用卵清蛋白(OVA)脉冲的树突状细胞(DC(OVA))加 Tr 细胞或 EXO(Tr)免疫 C57BL/6 小鼠,然后通过流式细胞术使用 PE-H-2K(b)/OVA 四聚体和 FITC-抗-CD8 抗体染色评估 OVA 特异性 CD8(+)T 细胞反应,并在免疫接种的小鼠中用表达 OVA 的 BL6-10OVA 黑色素瘤细胞进行挑战,评估抗肿瘤免疫。我们证明,DC(OVA)刺激的 CD8(+)T 细胞反应和保护性抗肿瘤免疫显著下降,从 2.52%降至 1.08%和 1.81%(p<0.05),从 8/8 降至 2/8 和 5/8 只小鼠(p<0.05),在共注射 Tr 细胞和 EXO(Tr)的免疫小鼠中。我们的结果表明,天然的 CD8(+)25(+)Tr 细胞释放的 EXOs,与 CD8(+)25(+)Tr 细胞一样,可以抑制 CD8(+)T 细胞反应和抗肿瘤免疫。因此,天然的 CD4(+)25(+)和 CD8(+)25(+)Tr 细胞衍生的 EXOs 可能成为自身免疫性疾病免疫治疗的一种替代方法。