冻存后脐血NK细胞的培养及CAR-NK的功能初探

Culture of NK Cells in Umbilical Cord Blood after Cryopreservation and Preliminary Study on the Function of CAR-NK

该研究优化冻存后脐血NK细胞的培养条件,构建了靶向CD19的CAR-NK细胞,并探究了CD19-CAR-NK对CD19+B细胞淋巴瘤的杀伤作用。首先将冻存的脐血单个核细胞复苏并静置18 h,通过CD56磁珠纯化NK细胞,使其在CBMC中的比例从13.8%提高至96.6%。然后比较使用不同细胞因子及其组合对NK细胞增殖、分化、凋亡及功能的影响,发现IL-2和IL-15均能活化NK细胞,但IL-2培养时NK细胞增殖优于IL-15(P<0.05),IL-2+15下CD56dim NK亚群占比[(74.33±5.03)%]最多,凋亡[(4.6±0.14)%]最少,累计增殖(62.67±3.21)倍,显著优于其他细胞因子组合(P<0.05)。该研究使用PDK1抑制剂BX795提高CAR-19慢病毒载体对NK细胞的转导效率,发现6 μmol/L BX795可将病毒转导效率提高3.6倍,且在此浓度下病毒转导效率[(36.4±1.91)%]最大。最后在体外验证CD19-CAR-NK对CD19~+肿瘤细胞的杀伤能力,发现CD19-CAR-NK可增强NK细胞对Nalm6的杀伤作用,但不影响NK对Nalm6-CD19KO的杀伤。综上所述,该研究得出IL-2+15是冻存后脐血NK细胞的最佳细胞因子组合,被CAR修饰的NK细胞能特异性识别CD19抗原并杀伤CD19~+肿瘤细胞。

This study was to optimize the culture conditions of cord blood NK cells after cryopreservation,construct CD19-targeting CAR-NK cells,and explore the killing effect of CD19-CAR-NK on CD19+B-cell lymphoma.First,the cryopreserved umbilical cord blood mononuclear cells were recovered and allowed to stand for 18 h,NK cells were purified by CD56 magnetic beads,and the proportion of NK cells in CBMC increased from 13.8% to 96.6% after purification by magnetic beads.And then the effects of different cytokines and their combinations on NK proliferation,differentiation,apoptosis and function were compared,both IL-2 and IL-15 could activate NK cells,but NK cells proliferated better when cultured with IL-2 compared with IL-15 (P<0.05).NK cells under IL-2+15 accounted for the largest proportion of CD56dim (74.33±5.03)%,the least apoptosis (4.6±0.14)%,and the cumulative proliferation was (62.67±3.21) times,which was significantly better than other cytokine combinations (P<0.05).In this study,the PDK1 inhibitor BX795 was used to improve the transduction efficiency of CAR-19 lentiviral vector on NK cells,6 μmol/L BX795 could increase the viral transduction efficiency of NK cells by 3.6 times,and the transduction efficiency (36.4±1.91)% was the highest at this concentration.Finally this study verified the killing ability of CD19-CAR-NK on CD19+tumor cells in vitro.CD19-CAR-NK could enhance the killing effect of NK cells on Nalm6,but did not affect the killing of Nalm6-CD19KO.In conclusion,this study concluded that IL-2+15 was the best cytokine combination for umbilical cord blood NK cells after cryopreservation.NK cells modified by CAR can specifically recognize CD19 antigen and kill CD19+tumor cells.