Research Unit, Division of Health Research, Saskatchewan Cancer Agency, Department of Oncology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Cancer Biother Radiopharm. 2010 Feb;25(1):21-8. doi: 10.1089/cbr.2009.0714.
Exosomes (EXOs) derived from tumor cells have been used to stimulate antitumor immune responses. It has been demonstrated that EXO released by tumor cells engineered to express cytokines are of enhanced stimulatory effect on CD8(+) cytotoxic T-lymphocyte (CTL) responses and antitumor immunity. J558 is a mouse myeloma cell line expressing tumor antigen P1A. In this study, we purified EXO(TNF-a), EXO(IL-2), and EXO(IFN-gamma) released by three cytokine-gene (TNF-alpha, IL-2 and IFN-gamma)-engineered J558 (J558(TNF-a), J558(IL-2) and J558(IFN-gamma)) tumor cell lines from their culture supernatants, respectively, by differential ultracentrifugation. These EXOs showed a "saucer" or round shape with a diameter between 50 and 90 nm by electron microscopy and contained EXO-associated proteins, such as LAMP-1 and AIP1, but not lysate-associated protein galectin, by Western blot analysis. EXO displayed expression of molecules (H-2K(d), CD54, and P1A) similarly to, but to a lesser extent to, J558 tumor cells. We then compared the stimulatory effect of these EXOs on P1A-specific CD8(+) CTL responses and antitumor immunity 6 days subsequent to intravenous (i.v.) EXO immunization (30 microg/each BALB/c mouse). We demonstrated that EXO(TNF-alpha) immunization was able to induce more efficient P1A-specific CD8(+) T-cell response accounting for 0.62% of the total CD8(+) T-cell population, using PE-H-2K(d)/P1A peptide and FITC-CD8 staining by flow cytometric analysis then EXO(IL-2) (0.31%) and EXO(IFN-gamma) (0.22%) immunization (P < 0.05), respectively, at day 6 after immunization. EXO(IL-2) and EXO(IFN-gamma) vaccine (i.v. 30 microg/each mouse) only protected 3 of 8 (38%) and 2 of 8 (25%) mice from tumor growth after subcutaneous (s.c.) challenging of immunized mice with J558 tumor cells (0.5 x 10(6) cells/each mouse), whereas EXO(TNF-alpha) immunization protected all 8 of 8 (100%) mice from tumor growth (P < 0.05). Taken together, we demonstrate that EXO(TNF-a) released by engineered J558(TNF-a) tumor cells more efficiently stimulate tumor antigen P1A-specific CD8(+) CTL responses and antitumor immunity than EXO(IL-2) and EXO(IFN-gamma) released by engineered J558(IL-2) and J558(IFN-gamma) tumor cells. Therefore, TNF-alpha-expressing tumor cell-released EXO may represent a more effective EXO-based vaccine in the induction of antitumor immunity.
肿瘤细胞来源的外泌体（EXOs）已被用于刺激抗肿瘤免疫反应。已经证明，工程化表达细胞因子的肿瘤细胞释放的 EXO 对 CD8(+)细胞毒性 T 淋巴细胞（CTL）反应和抗肿瘤免疫具有增强的刺激作用。J558 是一种表达肿瘤抗原 P1A 的小鼠骨髓瘤细胞系。在这项研究中，我们通过差速超速离心分别从三种细胞因子基因（TNF-α、IL-2 和 IFN-γ）工程化的 J558（J558(TNF-α)、J558(IL-2)和 J558(IFN-γ)）肿瘤细胞系的培养上清液中纯化了 TNF-α、IL-2 和 IFN-γ 基因工程 J558 细胞释放的 EXO(TNF-a)、EXO(IL-2)和 EXO(IFN-gamma)。电子显微镜下这些 EXO 呈现“碟形”或圆形，直径在 50 到 90nm 之间，通过 Western blot 分析显示含有 EXO 相关蛋白，如 LAMP-1 和 AIP1，但不含溶酶体相关蛋白半乳糖凝集素。EXO 显示出与 J558 肿瘤细胞相似的分子（H-2K(d)、CD54 和 P1A）表达，但程度较低。然后，我们比较了这些 EXO 在静脉（i.v.）EXO 免疫（30μg/每只 BALB/c 小鼠）后 6 天对 P1A 特异性 CD8(+)CTL 反应和抗肿瘤免疫的刺激作用。我们证明，EXO(TNF-alpha)免疫能够诱导更有效的 P1A 特异性 CD8(+)T 细胞反应，占总 CD8(+)T 细胞群体的 0.62%，使用 PE-H-2K(d)/P1A 肽和 FITC-CD8 染色通过流式细胞术分析，然后是 EXO(IL-2)（0.31%）和 EXO(IFN-gamma)（0.22%）免疫（P<0.05），分别在免疫后第 6 天。EXO(IL-2)和 EXO(IFN-gamma)疫苗（i.v. 30μg/每只小鼠）仅保护 8 只中的 3 只（38%）和 8 只中的 2 只（25%）经皮下（s.c.）接种 J558 肿瘤细胞后免疫的小鼠免受肿瘤生长（每只小鼠 0.5 x 10(6)个细胞），而 EXO(TNF-alpha)免疫则保护所有 8 只中的 8 只（100%）免受肿瘤生长（P<0.05）。综上所述，我们证明，与工程化的 J558(IL-2)和 J558(IFN-gamma)肿瘤细胞释放的 EXO(IL-2)和 EXO(IFN-gamma)相比，工程化的 J558(TNF-a)肿瘤细胞释放的 EXO(TNF-a)更有效地刺激肿瘤抗原 P1A 特异性 CD8(+)CTL 反应和抗肿瘤免疫。因此，表达 TNF-α 的肿瘤细胞释放的 EXO 可能代表一种更有效的基于 EXO 的疫苗，可诱导抗肿瘤免疫。
