摘要
目的研究人胸腺球蛋白(TG)在临床治疗级培养体系中,对细胞因子诱导的杀伤细胞(CIK细胞)增殖、免疫细胞表型和细胞毒作用的影响。方法分离11例健康正常人外周血单个核细胞(PBMC),γ干扰素预处理;1 d后,TG或CD3 mAb刺激;随后,每3 d补充IL-2等添加物,培养至21 d。台盼蓝拒染法计数细胞总数和活力;流式细胞术分析CD3、CD4、CD8、CD16/CD56和NK细胞活化/抑制受体表达情况以及CD25+Foxp3+调节T细胞(Treg)的变化;乳酸脱氢酶释放法检测细胞毒活性。结果 TG和CD3 mAb均能刺激CIK细胞生长,但CD3 mAb作用弱于TG。培养7 d,2组CIK细胞中,CD3+CD16+CD56+细胞、CD3-CD16+CD56+细胞及NK细胞活化/抑制受体表达均出现一过性减少;培养14 d,开始恢复,并持续增加至21 d;在7 d、14 d和21 d等3个检测点,CD3 mAb组的三者均低于TG组(P<0.05),而且,细胞毒活性也低于TG组。值得注意的是,培养7 d,Treg一过性增加,且TG组明显高于CD3 mAb组(P<0.05);另外,在整个培养期间,2组CIK细胞中,CD3+CD4+细胞持续减少,而CD3+CD8+细胞在7 d,即迅速增加,并维持该水平至21 d,这种改变在2组间无差异。结论 TG能选择性地促进CIK细胞中主要效应细胞增殖、分化和成熟,提高其细胞毒活性,因此,其具有替代CD3 mAb用于制备临床级CIK细胞制品的可行性;CD3 mAb和TG培养体系均可一过性地扩增负相调控细胞Treg,剔除或减少Treg可能有助于提高主要效应细胞产率。
Objective To study the effect of thymoglobulin （TG） on proliferation, immune cell phenotype and cytotoxicity of cytokine-induced killer （CIK） cells in a clinical-grade culture protocol. Methods Peripheral blood mononuclear cells （PBMCs） isolated from 11 healthy donors were primed with IFN-γ on day 0 and treated with either TG or CD3 mAb on day l. Thereafter, the cells were fed with IL-2 every 3 days until day 21. Aliquots of cells were harvested weekly. The cell number and viability were measured using trypan blue exclusion. The expressions of CD3, CD4, CD8, CD16/CD56, NK activating/ inhibitory receptor, and the CD25 ＋ Foxp3 ＋ regulatory T cells （Tregs） were analyzed with flow cytometry. The cytotoxicity of CIK cells against K562 cells were determined by lactate dehydrogenase （LDH） release assay on day 16 and day 21. Results Both TG and CD3 mAb stimulated the growth of CIK cells. However, the effect of CD3 mAb was weaker than that of TG. Flow cytometric analysis showed that the percentages of CD3 ＋ CD16 ＋ CD56 ＋ cells and CD3 - CD16 ＋ CD56 ＋ cells and the expression of NK activating/inhibitory receptor recovered and increased continuously until the end of culture （day 21 ） following a transient decrease at day 7. Noticeably, on day 7, 14 and 21, the percentages of CD3＋ CDI6＊ CD56＋ cells and CD3 - CD16 ＋ CD56 ＋ cells as well as the expression of NK activating/inhibitory receptor were higher in TG-induced CIK cells than those in CD3 mAb-induced CIK cells （ P 〈 0.05 ） ; Moreover, LDH release assay revealed that the cytotoxicity of CIK cells against K562 cells in TG-induced CIK cells was significantly higher than that of CD3 mAb-induced CIK cells （ P 〈 0.05）. In both CD3 mAb-induced CIK cell culture system and TG-induced CIK cell culture system, Tre＋ increased transiently at day 7;moreover, the percentage of Treg in TG-induced CIK cells was significantly higher than that of CD3 mAb-induced CIK cells （P〈 0.05 ）. In addition, both CD3 mAb and TG reduced the percentage of CD3 ＋ CD4＋ cells continuously, meanwhile increased the percentage of CD3 ＋ CD8 ＋ cells. There was no significant difference in the changes of CD3 ＋ CD4 ＋ cells and CD3 ＋ CD8 ＋ cells between the two CIK cell culture systems. Conclusion Compared with CD3 mAb, TG more selectively expanded CD3 ＋ CD16 ＋ CD56 ＋ cells and CD3 - CD16 ＋ CD56＋ cells （ CIK effector cells） and promoted the differentiation and maturation of these CIK effector cells with more powerful cytotoxic activity. Therefore, it is feasible for TG to substitute CD3 mAb to prepare the clinical grade products of CIK cells. Both CD3 mAb and TG increased negative regulatory cells, Tregs, transiently in CIK culture system and depleting or reducing Tregs might be helpful for increasing the production efficacy of the main effector cells in CIK cells.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2014年第7期681-685,690,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
"十二五"重大新药创制国家科技重大专项"综合性新药研发技术大平台-生物技术药物规模化制备技术平台"(2012ZX09301003-001-005-037)
