van Ostaijen-ten Dam Monique M, Prins Henk-Jan, Boerman Gerharda H, Vervat Carly, Pende Daniela, Putter Hein, Lankester Arjan, van Tol Maarten J D, Zwaginga Jaap J, Schilham Marco W
*Pediatric Immunology Laboratory, Department of Pediatrics Departments of ‡Medical Statistics §Immunohematology and Blood Bank, Leiden University Medical Center, Leiden, The Netherlands †IRCCS AOU San Martino-IST, Genoa, Italy.
J Immunother. 2016 Feb-Mar;39(2):90-100. doi: 10.1097/CJI.0000000000000110.
Cell-based immunotherapy using donor-derived natural killer (NK) cells after allogeneic hematopoietic stem cell transplantation may be an attractive treatment of residual leukemia. This study aimed to optimize clinical grade production of a cytokine-activated NK-cell product. NK cells were isolated either by double depletion (CD3(-), CD19(-)) or by sequential depletion and enrichment (CD3(-,) CD56(+)) via CliniMACS from leukapheresis material and cultured in vitro with interleukin (IL)-2 or IL-15. Both NK cell isolation procedures yielded comparable recovery of NK cells and levels of T-cell contamination. After culture with cytokines, the CD3(-)CD56(+) procedure resulted in NK cells of higher purity, that is, less T cells and monocytes, higher viability, and a slightly higher yield than the CD3(-)CD19- procedure. CD69, NKp44, and NKG2A expression were higher on CD3(-)CD56(+) products, whereas lysis of Daudi cells was comparable. Five days of culture led to higher expression of CD69, NKp44, and NKp30 and lysis of K562 and Daudi cell lines. Although CD69 expression and lysis of Daudi cells were slightly higher in cultures with IL-2, T-cell contamination was lower with IL-15. Therefore, further experiments were performed with CD3(-)CD56(+) products cultured with IL-15. Cryopreservation of IL-15-activated NK cells resulted in a loss of cytotoxicity (>92%), whereas thawing of isolated, uncultured NK cells followed by culture with IL-15 yielded cells with about 43% of the original lytic activity. Five-day IL-15-activated NK cells lysed tumor target cell lines and primary leukemic blasts, providing the basis for NK cell–based immunotherapeutic strategies in a clinical setting.
异基因造血干细胞移植后使用供体来源的自然杀伤（NK）细胞进行基于细胞的免疫治疗可能是一种有吸引力的残留白血病治疗方法。本研究旨在优化细胞因子激活的NK细胞产品的临床级生产。通过CliniMACS从白细胞分离材料中通过双重去除（CD3(-)，CD19(-)）或通过顺序去除和富集（CD3(-)，CD56(+)）分离NK细胞，并在体外与白细胞介素（IL）-2或IL-15一起培养。两种NK细胞分离程序产生的NK细胞回收率和T细胞污染水平相当。在用细胞因子培养后，CD3(-)CD56(+)程序产生的NK细胞纯度更高，即T细胞和单核细胞更少，活力更高，产量略高于CD3(-)CD19(-)程序。CD3(-)CD56(+)产品上的CD69、NKp44和NKG2A表达更高，而对Daudi细胞的杀伤作用相当。培养五天导致CD69、NKp44和NKp30表达更高以及对K562和Daudi细胞系的杀伤作用增强。尽管在IL-2培养物中CD69表达和对Daudi细胞的杀伤作用略高，但IL-15培养物中的T细胞污染更低。因此，对用IL-15培养的CD3(-)CD56(+)产品进行了进一步实验。IL-15激活的NK细胞冷冻保存导致细胞毒性丧失（>92%），而分离的未培养NK细胞解冻后用IL-15培养产生的细胞具有约43%的原始裂解活性。五天IL-15激活的NK细胞裂解肿瘤靶细胞系和原发性白血病母细胞，为临床环境中基于NK细胞的免疫治疗策略提供了基础。
