NK细胞预防移植复发及治疗老年白血病临床安全性观察

Clinical Safety of NK Cell in the Prevention of Leukemia Relapse Post-transplantation and in Treatment of the Elderly Leukemia Patients

目的:观察急性髓性白血病(AML)患者异基因造血干细胞移植(allo-HSCT)时、移植后及老年AML患者巩固化疗后,供者NK细胞输注的安全性。方法:入组40例AML患者,其中年龄>60岁AML完全缓解(CR)患者21例,allo-HSCT患者19例。采集健康直系亲属或供者外周血,密度梯度离心法分离单个核细胞,将细胞接种于NK细胞激活剂包被的培养瓶中,以重组人IL-15及IL-2细胞因子组合,优势扩增人NK细胞14 d。质检合格后进行静脉输注。台盼蓝拒染法计数细胞总数和活力,流式细胞术分析免疫细胞表型。AML患者巩固化疗后接受NK细胞输注,共78次;allo-HSCT患者移植时输注NK细胞11次,移植后3个月内输注NK细胞共32次。输注期间及之后48 h,记录患者体温、血压等,以观察细胞治疗的急性毒性,并记录急性移植物抗宿主病(GVHD)发生情况。结果:免疫表型分析显示,细胞扩增前外周血中NK细胞(CD3^(-)CD56^(+))占单个核细胞的比例为(14.10±4.22)%(n=121),培养14 d后CD3^(-)CD56^(+)细胞比例为(87.29±8.75)%(n=121),NK细胞增殖倍数为753.47±140.13倍(n=121)。NK细胞输注量为每次(7.58±2.50)×10^(7)/kg,其中3例患者各有1次输注出现低至中度发热,且均为多次输注后出现,对症处理后当天体温均恢复正常。1例4次输注均有发热,最高体温在38.5-39.0℃,给予对症处理后,体温在1-2 h内降至正常,后无任何不适。老年AML患者均未发生GVHD,移植患者6例发生急性GVHD,其中I度5例,Ⅱ度为1例。结论:采用该体系可获得高纯度的NK细胞,用于白血病造血干细胞移植及老年AML患者,细胞输注是安全的。

Objective:To observe the safety of donor NK cell infusions in the settings of hematopoietic stem cell transplantation and after consolidation chemotherapy in elderly patients with acute myeloid leukemia(AML).Methods:Forty patients with AML were included,in which 21 patients aged over 60 years were at the stage of complete remission(CR)and 19 patients that received allogeneic hematopoietic stem cell transplantation(allo-HSCT).Mononucleated cells were isolated from peripheral blood from the donors(for allo-HSCT)or healthy immediate family members(elderly AML).The cells were seeded into the flasks pre-coated with NK cell specific activators,and expanded in media containing recombinant human IL-15 and IL-2 for 14 days.The cells were transfused intravenously after the identification of quality control.Trypan blue exclusion test was used for the determination of cell viability and counting.Flow cytometry analysis was performed to assess the surface antigenic profile.Seventy-eight infusions of the cell products were received by the elderly patients with AML after consolidation chemotherapy,11 infusions were received by the patients during allo-HSCT and 32 infusions 3 moths after transplantation.The safety of cell therapy,body temperature,blood pressure and other indexes were observe during and 48 hours after cell transfusion.Meanwhile,the occurrence and severity of acute graft-versus-host disease(GVHD)were documented.Results:Flow cytometry analysis showed that the proportion of NK cells(CD3^(-)CD56^(+))in the mononucleated cells before culture was(14.10±4.22)%(n=121),and the proportion increased dramatically up to(87.29±8.75)%(n=121)after culture for 14 days,the number of NK cells increased to 753.47±140.13 times(n=121).The doses of the infused NK cells was(7.58±2.50)×10^(7)/kg per infusion.Moderate fever occurred in three cases after multiple infusions,and the temperature restored to normal on the same day after treatment.Fever was observed in one patient after every infusion of four times in total.The temperature reached to 38.5-39.0℃and returned to normal within 1-2 hours after adequate antipyretic treatment,and then there was no discomfort.No GVHD was observed in the elderly AML patients,while 6 cases that received allo-HSCT developed moderate acute GVHD,among them grade I in 5 cases and grade II in 1 case.No other severe toxicities were observed.Conclusion:NK cell products with a high-purity could be obtained by ex vivo expansion with this protocol.The transfusion of these expanded cells is generally safe in the elderly patients with AML that have received chemotherapy or patients that received hematopoietic stem cell transplantation.