经抗CD158单克隆抗体修饰的供者自然杀伤细胞对受者树突状细胞的杀伤作用

目的 研究应用抗CD158a和CD158b单克隆抗体封闭供者杀伤细胞免疫球蛋白样受体(KIR)的抑制性受体KIR2DL1及KIR2DL3后,供者自然杀伤细胞(NK细胞)对受者树突状细胞(DC)的杀伤作用,及其减少移植物抗宿主病(GVHD)的作用.方法 对15例异基因全相合造血干细胞移植的供、受者进行HLA及KIR高分辨基因分析,采集供者骨髓及受者外周血单个核细胞,培养受者DC作为靶细胞,使用磁珠负选高纯度供者NK细胞作为效应细胞,用抗CD158a和CD158b单抗封闭供者KIR抑制性受体,并采用四甲基偶氮唑盐比色法分别检测抑制性KIR封闭前后NK细胞对靶细胞的杀伤作用.结果 在效靶比为10∶1下,供者NK细胞抑制性受体经抗体封闭后,对受者DC的杀伤活性明显提高,随着封闭抗体浓度的增加而增高,各浓度间比较,差异均有统计学意义(P＜0.05).当供、受者KIR配体匹配模式为宿主抗移植物(HVG)模式时,供者NK细胞对受者成熟DC的杀伤效果明显高于供、受者KIR基因全相合或移植物抗宿主(GVH)模式,在用不同浓度单抗封闭KIR抗体后,随着封闭抗体浓度的增加,杀伤效果增高更明显(P＜0.05).结论 利用抗CD158单抗封闭KIR可以提高供者来源NK细胞对受者DC的杀伤活性,输注异源反应性NK细胞可能作为一种新的治疗GVHD的方法.

Killing effects of anti-CD158 monoclonal antibody-modified NK cells against the allogeneic dendritic cells

Objective To investigate the killing effect of donor NK cells on recipient dentritic cells (DCs) by blocking KIR2DL1 and KIR2DL3 with monoclonal antibodies CD158a and CD158b,and the possible mechanism about donor NK cells reducing the incidence of graft versus host disease (GVHD).Methods SSP-PCR was used to examine the genotypic makeup of KIR in 15 pairs of patients and their HLA-identical sibling hematopoietic stem cell transplantation (HSCT) donors in our department.Peripheral blood mononuclear cells (PBMC) from 15 pairs of patients and their donors were extracted and preserved,and donor NK cells were isolated by DYNABEADS UNTOUCHED HUMAN NK sorting kit.Methyl thiazolyl tetrazolium (MTT) reduction assay was used to detect the killing activity of donor NK cells against recipients DCs.To observe the different NK killing activity before and after KIR receptors blockade,the anti-CD158a and CD158b monoclonal antibodies were used to block KIR inhibitory receptors.Results The killing effect of donor NK cells was significantly enhanced after inhibitory receptor KIRs blockades,the killing effect of NK cells in HVG KIR ligandmismatching pattern group was significantly higher than GVH or KIR receptor-ligand matched pattern group.The killing effect became stronger when the dose of antibody was increased.Conclusion It is demonstrated in vitro that the killing effect of alloreactive NK cells could be enhanced by KIR functional modification,and the infusion of alloreactive NK cells could be used as a new strategy of immunotherapy for GVHD.