Huang Yong, Jiang Er-lie, Zhou Zheng, He Yi, Wang Mei, Liu Qing-guo, Zhai Wen-jing, Han Ming-zhe
Laboratory of Stem Cell Transplantation, Institute of Hematology, CAMS and PUMC, Tianjin 300020, China.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2005 Jun;27(3):315-20.
To evaluate the method for expanding Valpha24 natural killer T (NKT) cells with alpha-galactosylceramide (alpha-GalCer) ex vivo.
Mononuclear cells (MNCs) isolated from adult peripheral blood or umbilical cord blood (UCB) were divided into three groups. In Group A1 (n = 5), CD34+ progenitorderived dendritic cells were differentiated in a cytokine-supplemented culture system from cord blood and acted as antigen presenting cells (APC) to induce the expansion of cord blood Valpha24 NKT cells in presence of alpha-GalCer; in Group A2 (n = 5), adult peripheral monocyte-derived dendritic cells (Mo-DC) were used as APC to induce the expansion of adult peripheral NKT cells in presence of alpha-GalCer; whereas in Group B (n = 16), alpha-GalCer was added into adult peripheral MNCs culture system without additional DCs. Cytokine-produce were measured by ELISA, and NKT cells' proliferation ability, cytotoxicity, and suppressive effect on mixed lymphocyte reaction (MLR) were examined by MTT assays.
Valpha24 NKT cells in Group A1, A2, and B were expanded up to 128 (95-207), 250.5 (179.6-790.6), and 326 (101-2 136) -fold by day 12, respectively. Adult NKT cells expanded in Group B were markedly better than those in Group A1 (P = 0.038). When stimulating by PMA, the NKT cells had a 3-day stimulate index of 1.80 +/- 0.41; and the secretion ratio of IL-4 to IFN-gamma of UCB or adult peripheral blood NKT cells were 0.30 +/- 0.13 and 0.28 +/- 0.18; and the ex vivo antitumor effect of expanded NKT cells were found in cell line HL60, KG1a, and Raji except for K562; and the suppressive effect of expanded NKT cells or the culture supernatant on MLR were confirmed.
Alpha-GalCer can facilitate the rapid shorttime expansion of Valpha24 NKT cells in presence of IL-2 and IL-15. These expanded NKT cells, kill tumor cell lines, and inhibit can massively excret IL-4 and IFN-gamma allogeneic T-cell response.
评估体外利用α-半乳糖神经酰胺（α-GalCer）扩增Vα24自然杀伤T（NKT）细胞的方法。
从成人外周血或脐带血（UCB）中分离的单核细胞（MNC）分为三组。A1组（n = 5），在细胞因子补充培养体系中，由脐血分化出CD34⁺祖细胞来源的树突状细胞作为抗原呈递细胞（APC），在α-GalCer存在的情况下诱导脐血Vα24 NKT细胞扩增；A2组（n = 5），成人外周血单核细胞来源的树突状细胞（Mo-DC）作为APC，在α-GalCer存在的情况下诱导成人外周血NKT细胞扩增；而B组（n = 16），将α-GalCer加入成人外周血MNC培养体系中，不添加额外的树突状细胞。通过ELISA检测细胞因子产生情况，通过MTT法检测NKT细胞的增殖能力、细胞毒性以及对混合淋巴细胞反应（MLR）的抑制作用。
到第12天时，A1组、A2组和B组的Vα24 NKT细胞分别扩增至128（95 - 207）倍、250.5（179.6 - 790.6）倍和326（101 - 2136）倍。B组扩增的成人NKT细胞明显优于A1组（P = 0.038）。经佛波酯（PMA）刺激时，NKT细胞的3天刺激指数为1.80±0.41；脐血或成人外周血NKT细胞的IL-4与IFN-γ分泌比值分别为0.30±0.13和0.28±0.18；扩增的NKT细胞对细胞系HL60、KG1a和Raji有体外抗肿瘤作用，但对K562无作用；同时证实了扩增的NKT细胞或培养上清液对MLR的抑制作用。
α-GalCer在IL-2和IL-15存在的情况下可促进Vα24 NKT细胞快速短期扩增。这些扩增的NKT细胞可大量分泌IL-4和IFN-γ，杀伤肿瘤细胞系，并抑制同种异体T细胞反应。
