固相MICA协同s4-1BBL、IL-15体外高效扩增CD3^+CD56^+细胞的研究

High expansion of CD3 and CD56 double positive cells with immobilized MHC class Ⅰ chain-related protein A,soluble 4-1BBL,and IL-15 in vivo

目的:观察固相MHCⅠ类相关抗原A(iMICA)是否协同s4-1BBL、IL-15促进外周血CD3+CD56+细胞的增殖并增强其活性。方法:首先将iMICA联合s4-1BBL、IL-15分别刺激外周血单个核细胞或纯化的CD3+CD56+细胞,共培养19天,动态观察CD3+CD56+细胞频率变化及记录其增殖曲线;其次利用LDH释放法和ELISA分别检测长期培养的CD3+CD56+细胞杀伤活性及IFNγ-、IL-4的分泌;最后检测CD3+CD56+细胞表面活性相关受体的表达。结果:iMICA联合s4-1BBL、IL-15促进CD3+CD56+细胞的频率自2%升高至21.7%,绝对细胞数平均增长32倍;扩增后的CD3+CD56+细胞杀伤K562活性明显增高,IFNγ-分泌能力增强,不分泌IL-4;其表面活化性受体表达上调,抑制性受体表达下调。结论:iMICA联合s4-1BBL、IL-15能高效扩增CD3+CD56+细胞,体外大量增殖后可用于肿瘤的过继免疫治疗。

Objective：To investigate whether immobilized MHC class Ⅰ chain-related protein A（MICA） could synergize with soluble 4-1BBL and IL-15 to promote expansion of peripheral CD3 and CD56 double positive cells ex vivo.Methods：Peripheral blood mononuclear cells or purified CD3＋CD56＋ cells were stimulated with immobilized MICA,s4-1BBL,and IL-15 for 19 days.Frequencies of CD3＋CD56＋ cells or the absolute cell numbers were observed during this long-term culture period.Next cytotoxicity and IFN-γ,IL-4 production of the expanded CD3 ^＋ CD56 ^＋ cells were detected by the LDH releasing assay and ELISA, respectively. Lastly, expression of function-associated receptors on CD3^＋ CD56^＋ cells was analyzed. Results：With 19-day culture,frequency of CD3^＋ CD56^＋ cells arrived from 2% to 21.7% on the average, and CD3 ^＋ CD56 ^＋ ceils could expand with 32 folds meanly. In addition, the long-term cultured CD3 ^＋ CD56 ^＋ cells were showed higher cytotoxicity and more production of IFN-γ, and could not secrete IL-4. Activating receptors were np-regalated to be present on the cultured CD3^＋ CD56 ^＋ cells whereas inhibitory receptors were decreased. Conclusion：Immobilized MICA in＇combination with soluble 4-1 BBL and IL-15 not only promotes high expansion of CD3 ^＋ CD56^＋ cells, but also enhances function of CD3 ^＋ CD56^ ＋ cells. The culture system we developed here may be used in tumor adoptive immunotherapy.