IL-2和IL-15激活供者自然杀伤细胞在异基因造血干细胞移植应用的实验研究

Experimental study on IL-2- and IL-15 application in allogeneic hematopoietic stem cell transplantation

目的探讨IL-2和IL-15激活的供者自然杀伤（NK）细胞在异基因造血干细胞移植（allo—HSCT）中减轻移植物抗宿主病（GVHD）发挥移植物抗白血病效应方面的作用。方法采用免疫磁珠分选小鼠脾NK细胞，采用添加IL-2和IL-15的培养基扩增NK细胞并测定NK细胞杀伤活力。C57BL／6小鼠作为供鼠，BALB／c小鼠作为受鼠，部分小鼠移植前8天静脉接种EL9611白血病细胞。异基因移植小鼠输注骨髓细胞5×10^6和脾细胞5×10^6。NK细胞治疗组输注骨髓细胞和脾细胞各5×10^6以及激活的NK细胞1×10^7，并且给予腹腔注射IL-2和IL-15。移植后观察GVHD发生、生存期、嵌合度、免疫重建。结果分选NK细胞纯度为95．7％～97．1％。培养后NK细胞杀伤活力较静息时增加3倍。单纯异基因骨髓细胞输注组小鼠未见GVHD发生，异基因骨髓及脾细胞移植对照组移植后1周起开始出现GVHD表现。实验组小鼠发生GVHD的严重程度明显低于脾细胞输注小鼠（P〈0．05）。单纯全身照射预处理小鼠生存期9．5～14．0d。白血病模型中对照组移植后100d生存率10％，其余死于白血病；实验组80％生存期超过100d，实验组生存期明显长于对照组（P〈0．01）。实验组小鼠移植后2周外周血NK细胞占4．8％，对照组外周血NK细胞占2．8％，实验组NK细胞恢复早于对照组（P〈0．05）。实验组TRBV基因重建比对照组快，而且TRBV家族基因表达数比对照组多，对照组小鼠多见单克隆及寡克隆表达。结论IL-2和IL-15在体外可以有效促进NK细胞增殖与激活。allo—HSCT时给予激活的供者NK细胞输注以及相关细胞因子处理可以促进免疫重建、减轻GVHD发生、降低白血病复发。

Objective To explore the impact of IL-2- and IL-15-activated donor natural killer（NK） cell infusion on graft-versus-host-disease （ GVHD ） and graft-versus-leukemia （GVL） effect post allogeneic hematopoietic stem cell transplantation （allo-HSCT）. Methods The C57BL/6 mice splenic NK cells were selected by microbeads, and then expanded in the media containing IL-2 and IL-15. The killing activity of NK cells was detected. In the leukemia mouse model, recipients （BALB/c） were intraveneously inoculated with EL9611 leukemia cells 8 days before transplantation. Lethally irradiated BALB/c recipient mice were transplanted with 5 × 10^6 bone marrow cells （BMCs） , or 5 × 10^6 BMCs plus 1 × 10^7 splenocyes with or without 1 × 10^7 activated NK cells. Additionally, NK cell infusion group mice were intraperitoneally injected with a mixture of IL-2 and IL-15 post transplant. Survival time, GVHD occurrence, lineage chimerism, TRBV spectratyping were observed post transplant. Results The purity of isolated splenic NK cells was 95.7% - 97.1%. The killing activity of NK cells after activation was increased by 3 times. GVHD did not occurred in allogeneic BMCs infusion group, whereas did from 1 week after transplant in allogeneic BMCs ＋ splenocytes infusion group. The severity of GVHD in total body irradiation （TBI） experimental group was significantly lower than in splenocytes infusion group （ P 〈 0.05）. The survival time was 9.5 - 14.0 d in TBI alone coditioning group. In leukemia mouse model, 100 day survival rate was 10% the rest of them were died of leukemia while in experimental group, the more than 100 days survival rate was 80% （P 〈0. 01 ）. PB NK cells at 2 week post-transplant were 4.8% in experimental group and 2.8% in cotrol group. NK cells recovery in experimental group was earlier than that in control group （ P 〈 0.05 ）. TRBV reconstitution was faster in experimental group than in control group, moreover, the number of TRBV family expression was more in experimental group than in control group which mainly expressed monoclone or oligoclone. Conclusions Donor alloreactive NK cells can be efficiently expanded and activated with IL-2 and IL-15. Donor activated NK cell infusion and IL- 2, IL-15 treatment can promote immune reconstitution, mitigate GVHD and reduce leukemia relapse.