异基因反应性NK细胞在供者淋巴细胞输注治疗单倍体相合造血干细胞移植后肺癌复发中的作用研究

Alloreactive NK Cells Enhance the Effect of Donor Lymphocyte Infusion in the Management of Relapsed Lung Cancer after Haploidentical Hematopoietic Stem Cell Transplantation

本研究旨在探讨预处理中异基因反应性NK细胞(allo-reactive NK cells,Allo-NK)在供者淋巴细胞输注(DLI)治疗单倍体相合造血干细胞移植后肺癌复发中的作用。采用免疫磁珠富集F1供鼠(H-2d/b)脾脏allo-NK,流式细胞术和LDH法检测其异体反应性。建立小鼠单倍体相合造血干细胞移植后肺癌复发模型,采用放射性核素标记法追踪DLI的体内分布。比较不同处理组小鼠的肿瘤大小、淋巴细胞浸润程度,采用RayBio(小鼠细胞因子抗体芯片检测血清中22种细胞因子的变化。结果表明:在移植后24-48小时,allo-NK组的回输供者淋巴细胞在受者肺脏、脾脏、肾脏中明显蓄积,浓度最高,时间最长。化疗+DLI组与化疗+PBS组的肿瘤体积相比无明显区别,但allo-NK+DLI组的肿瘤体积显著小于化疗+DLI组(p<0.05)和allo-NK+PBS组(p<0.01)组,且镜下可观察到肿瘤局部较多的淋巴细胞浸润。allo-NK+DLI组中MCP-1、IL-17、IL-12和MCP-5较对照组分别升高1.56、1.36、1.20、1.17倍;而IL-10降低42.8%。结论:allo-NK可以延长DLI在宿主体内停留时间,促进炎性因子和Th1类细胞因子分泌,抑制肿瘤生长,提高DLI治疗移植后肺癌复发的疗效。

This study was aimed to study the potential effects of alloreactive NK cells （allo-NKs） in therapy of relapsed lung cancer after haploidenfical hematopoietic stem cell transplantation using donor lymphocyte infusion （DLI）. The FI donors derived-NK cells were purified with MACS magnetic separation system, in which the proportion of the alloreactive Ly49A^＋ cells was detected by flowcytometry and alloreactivity was measured by LDH method. The relapse model of lung cancer after haploideutical-HSCT was estabished. The distribution kinetic of infused donor lymphocytes in vivo was analyzed. The inhibition of relapse tumor, infiltration of lymphocytes in situ and fluctuation of 22 kinds of cytokines in serum after DLI were compared among different groups. The results showed that the infused donor cells of allo- NK group were accumulated mostly in lung, spleen and kidney for more than 48 hours with considerable higher levels according to the distribution kinetic curve. The sizes of relapse tumors between chemotherapy ＋ PBS group and chemotherapy ＋ DLI group showed no difference. However, the relapsed tumors in allo-NK ＋ DLI group were significantly smaller than that in chemotherapy ＋ DLI group or allo-NK ＋ PBS group, in which increased infiltration of lymphocytes were defined in situ. The levels of cytokines such as MCP-1, IL-17, IL-12 and MCP-5 in serum of allo-NK ＋ DLI group ascended compared with control group, though the level of IL-10 declined simultaneously. It is concluded that allo-NKs prolong the survival time of infused donor lymphocytes in vivo, promote the secretion of inflammatory cytokines and Th1-type of cytokines, and further improve the antitumor effects of DLI against relapse after transplantation.