A Study On Vitro Cytotoxicity Mediated By NK Cells With Different KIR Haplotype Groups

BackgroundKiller cell immunoglobulin-like receptors(KIRs)expressed on the surface of NK cells and a subset of T cells regulate function of NK cells through interacting with class I HLA(human leukocyte antigen)which play an important role in tumor immunity.Previous studies showed discrepant results in the studies of association between KIR-HLA diversity and leukemia.In our previous studies,we found that KIR AA haplotype group showed an increased frequency in healthy controls than that in acute lymphoblastic leukemia(55.3%vs 45.8%,P=0.004)and acute myeloid leukemia patients(55.3%vs 48.4%,P=0.034)in southern Chinese Han population.These results implied that KIR haplotype groups play a critical role in risk/protection of leukemia.Thus,the present study investigated the effect of KIR haplotype groups on vitro cytotoxicity mediated by NK cells.SubjectPeripheral blood samples were randomly collected from 32 volunteer blood donors of southern Chinese Han individuals.Thirteen bone marrow samples were collected from primary leukemia patients.K562 cells(human leukemia cell line)and leukemic blast cells separated from bone marrow samples were served as target cells in vitro cytotoxicity test.MethodPeripheral blood mononuclear cells(PBMC)separated from healthy blood donors were used in vitro NK cell expansion for 13-14 days to obtain a large quantity of NK cells with high purity.Two flow cytometry detecting methods were compared to establish and select a more sensitive,stable method for detecting death rate of target cells in cytotoxicity of NK cells against K562 cells or leukemic blast cells.DNA samples of volunteer blood donors and leukemia patients were isolated.The presence or absence of KIR genes in blood donors were detennined by polymerase chain reaction with sequence specific primers(PCR-SSP).Class I HLA genes in leukemia patients were detected by polymerase chain reaction with reverse sequence specific oligonucleotide(PCR-rSSO).NK cells were co-incubated with class I HLA deficient K562 cells and leukemic blast cells identified class I HLA genes,respectively.The death rate of target cells representing the killing capacity of NK cells was detected by flow cytometry.NK cells were co-incubated with K562 cells.The ratio of NK cells expressed CD107α was detected by flow cytometry.Statistical analysis was performed by using IBM SPSS 22.0 Statistics software.Result1.After vitro expansion,the quantity and purity of NK cells increased during the culture period.The purity of NK cell in PBMC inclines from 11.76±3.55%to 83.65±3.18%after expansion.2.Thirty-two volunteer blood donors(donors of NK cells)were recruited in this study.Three KIR haplotype groups and ten KIR haplotype profiles were determined in these donors.Thirteen leukemic patients were enrolled.Ten different HLA-A alleles,seventeen different HLA-B alleles and thirteen HLA-C alleles were detected.3.NK cells with KIR AA haplotype group showed higher cytotoxicity against K562 cells than those with KIR Bx haplotype group(59.51 ± 14.59%vs 42.47± 14.90%,P=0.001).NK cells with KIR2DL5,2DS1 and 3DS1 genes showed significantly lower cytotoxicity against K562 cells than those without these genes.4.In the cytotoxicity assay of NK cells against leukemic blast cells,NK cells with KIR AA haplotype group(20.57±10.30%vs 15.27±8.19%,P=0.01)showed stronger killing effect than those with KIR Bx haplotype group,as well.NK cells with KIR2DL5,2DS1 and 3DS1 genes showed significantly lower cytotoxicity against leukemic cells than those without these genes.5.While detected the CD107α expression of NK cells activated by class I HLA deficient K562 cells,NK cells with KIR AA haplotype group showed higher ratio of CD 107α expression than those with KIR Bx haplotype group(24.98±10.22%vs 17.11±7.53%,P=0.028).Conclusions and significanceCompared with the cytotoxicity of NK cells in individuals with KIR Bx haplotype group,NK cells in individuals with KIR AA haplotype group carrying more inhibitory KIR genes showed stronger killing effect in vitro cytotoxicity against K562 cells and class I HLA down-regulated leukemic blast cells.Moreover,NK cells with KIR AA haplotype group showed higher ratio of CD 107α expression than those with KIR Bx haplotype group.The function of KIRs on NK cells depends on the presence of class I HLA ligands.Upon abnormal conditions,HLA class I is differentially down-regulated or deficient on leukemic cells.Therefore,the suppression meditated by inhibitory KIRs is reduced,leading to the activation of NK cells with KIR AA haplotype group which consists of predominantly inhibitory KIRs.The greater the number of inhibitory receptors on NK cells,the stronger the cytotoxic responsiveness against leukemic cells.The results of present study can provide valuable information and clues for donor selection for hematopoietic stem cell transplantation and personal immunotherapy in leukemia patients.