髓系抑制性细胞对神经母细胞瘤抗原特异性细胞毒性T淋巴细胞体外增殖和杀伤抑制作用

Myeloid-derived Suppressor Cells Inhibit Proliferation and Killing Activity of Neuroblastoma Antigen-specifi c Cytotoxic T Lymphocyte in vitro

目的探讨髓系抑制性细胞(myeloid-derived suppressor cell,MDSC)对神经母细胞瘤抗原特异性细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)体外增殖和杀伤活性的影响。方法体外培养神经母细胞瘤SK-N-SH细胞、自BALB/c小鼠分离培养树突状细胞(dendritic cell,DC)和CD3+T细胞,制备DC诱导的神经母细胞瘤抗原特异性CTL。分离纯化小鼠MDSC,将CTL与MDSC混合培养,采用CFSE荧光染色和流式细胞学方法,检测MDSC对CTL增殖抑制情况。将CTL与SK-N-SH、MDSC混合培养,ELISA法检测不同组CTL对SK-N-SH杀伤率及上清液中IL-2和IFN-γ分泌情况。结果磁珠分选纯化后Gr-1+CD11b+MDSC细胞比例为84.6%。负载抗原的CTL细胞上清液中IL-2和IFN-γ含量较单纯培养T细胞上清液中IL-2和IFN-γ含量明显增高(P<0.05)。与MDSC共培养的CTL细胞增殖明显受抑;而单独培养CTL随时间延长细胞增殖明显。MDSC+CTL+SK-N-SH组杀伤率较CTL+SK-N-SH组明显降低(t=6.506,P<0.001);两组上清液中IL-2和IFN-γ分泌量差异亦有统计学意义(均P<0.01)。结论 MDSC可抑制神经母细胞瘤抗原特异性CTL的体外增殖和活性而产生免疫耐受,抑制CTL对神经母细胞瘤细胞的杀伤作用。

Objective To explore the inhibitory role of myeloid-derived suppressor cell（MDSC） in the proliferation and killing activity of neuroblastoma antigen-specifi c cytotoxic T lymphocyte（CTL） in vitro. Methods The neuroblastoma antigen specifi c CTLs were successfully prepared on the basis of cultivation of neuroblastoma SK-N-SH cells and separation of BALB/c mice myeloid-derived dendritic cell（DC） and CD3＋T cells in vitro. MDSCs were purifi ed and cultivated with CTLs, then the inhibitory role of MDSC in the proliferation of CTL was detected by fl uorescence staining of 5,6-carboxyfl uorescein diacetate succinimidyl ester（CFSE） and fl ow cytometry. Furthermore, CTL, SK-N-SH and MDSC were mixed and cultivated, the killing rate of CTL on SK-N-SH and the secretion of IL-2, IFN-γ in supernatant of the different groups were detected by ELISA. Results After magnetic cell sorting, the rate of Gr-1＋CD11 b＋MDSC reached to 84.6% by fl ow cytometry test. The levels of IL-2 and IFN-γ in supernatant of antigen-loaded CTLs were signifi cantly higher than those in supernatant of T cells（P〈0.05）. The proliferation of CTLs cultivated with MDSC was significantly inhibited, with strong fluorescence in view： however, CTLs cultivated alone proliferated obviously, with weak fl uorescence intensity. The killing rate of CTLs to SK-N-SH in MDSC＋CTL＋SK-NSH group was signifi cant lower than that in CTL＋SK-N-SH group（t=6.506, P〈0.001）. Signifi cant difference existed in the secretion levels of IL-2 and IFN-γ in the supernatant between the two groups（all P〈0.01）. Conclusion MDSC inhibite the proliferation and activity of neuroblastoma antigen-specific CTLs in vitroresult in immune tolerance and reduced the killing effect of CTL on neuroblastoma cells.