人外周血自然杀伤T细胞体外扩增及其功能的初步研究

A preliminary study on the in vitro expansion of the natural killer T cells from human peripheral blood cells and its functional studies

为了建立人自然杀伤T(NKT)细胞在体外扩增的方法并对其功能进行初步的研究,通过不同的方法从人外周血单个核细胞(MNC)和纯化T淋巴细胞中扩增TCRVα24+/Vβ11+NKT细胞,并采用流式细胞术测定NKT细胞中IL-4、IFN-γ、TNF-α分泌水平。采用CD4/CD8免疫磁珠去除CD4+和CD8+细胞进一步纯化NKT细胞,并用DIOC18染色及流式细胞术测定NKT细胞杀伤活性。α半乳糖神经酰胺(α-Galcer)和IL-2可以使NKT细胞在体外扩增。扩增19d后,TCRVα24+/Vβ11+细胞比率最高上升到25.5%±7.2%,NKT细胞最大扩增倍数达到(1.51±0.91)×104倍。体外扩增的NKT细胞高表达TCRVα24、Vβ11、CD3、CD161,低表达CD56。在CD3单克隆抗体和IL-2刺激下,TCRVβ11+细胞分泌IL-4和IFN-γ的细胞比例均高于TCRVβ11-细胞(P<0.05)。去除CD4+和CD8+细胞后NKT细胞含量上升到80%。NKT细胞对肿瘤细胞株U937和HL60以及树突状细胞具有较强的细胞毒效应。NKT细胞可以通过α-Galcer直接从人外周血MNC扩增获得,从而简化实验步骤。

To establish a method to expand the natural killer T cells （NKT） in vitro and to investigate its functions, the TCRVα24^＋/Vβ1 1^＋NKT cells from human peripheral blood mononuclear cells （MNC） and purified T lymphocytes were expanded by several kinds of methods, The levels of IL-4, IFN-γ/and TNF-α secretions from NKT cells were determined by flow cytometry, and the CD4^＋ and CD8^＋ cells were deleted by using CD4/CD8 Dynal beads to further purify NKT cells. Meanwhile, the cytotoxic activity of NKT cells was measured by means of DIOC18 staining and flow cytometry. It was found that the NKT ceils could be expanded in vitro through the actions of α-galactosylceramide（α-Galcer） and IL-2, and the ratio of TCRVα24/TCRVβ11 elevated up to 25.5%±7.2% after 19 days of expansion. The highest multiplicity in expansion of NKT cells might reach up to （1.51±0.91）×10^4 times. The expanded NKT cells demonstrated a high level of expression on TCRVα24, TCRVβ11, CD3 and CD161 and a low level of expression on CD56, CD14 and CD19. Under the stimulation of CD3 monoclonal antibody and IL-2, the proportion of TCRVβ11^＋ cells secreting IL-4 and IFN-γ/was higher than that of the TCRVβ11^- cells （P〈0.05）. In addition, when the CD4^＋ and CD8^＋ cells were deleted, the frequency of NKT cells increased to 80%. An intense cytotoxic effect of the NKT cells to tumor cell lines U937 and HL60 as well as to dendritic ceils could be demonstrated. In conclusion, it is evident that the NKT cells can be expanded from human peripheral blood mononuclear cells directly, thus making simplification of the experimental procedures.