Study Of The Killing Effects Of Donor KIR2DS4~+ NK Cells On Recipient DC

Objective:The emergence of haploidentical stem cell transplantations brought a trace of dawn for patients without HLA-matched donors,while the severe infection caused by intensive conditioning regimens and the occurrence of GVHD have limited its widespread use.At present,in order to reduce the incidence of GVHD,mainly through the removal of T cells before transplantation and immunosuppressants after transplantation.Although GVHD is controllable,it will significantly increase graft failure and the risk of leukemia recurrence.Studies have shown that GVHD is mainly due to DC processing and delivery antigens to donor T cells,DC play a key role in the occurrence of donor T cell-mediated GVHD.Alloreactive NK cells can kill the recipient antigen presenting cell(mainly DC),blocking the antigen presenting pathway,thus preventing the occurrence of GVHD.In this study,we investigated the killing effect of NK cells with different KIR phenotype and the killing effect of KIR2DS4~+ NK cells to different phenotypes of DC,and provided the basis for KIR guided clinical selection of the optimal donor.Methods:1.Purification and culture of NK cells: Adopt lymphocyte separation and Rosettesep NK cell enrichment mixture,the peripheral blood of healthy donors were negative selection to isolate the NK cells,then cultured for 14 days in a medium;2.Purification and culture of DC: Mononuclear cells were gathered by gradient density method using lymphocyte separation solution.After incubation for 2 h in the incubator,the suspension cells were discarded and the adherent cells were cultured inmedium for 7 days;3.The killing activity of different groups of NK cells against DC: CCK-8colorimetry was used to detect the cytotoxicity of different groups of NK cells to DC;4.Analysis of clinical data of patients who underwent allo-HSCT and observe the incidence of GVHD.Results:1.Identification of NK cells: The proportion ofCD3-CD16+CD56+cells in peripheral blood was increased from(21.90±7.25)% to(92.80±2.42)%;2.Killing activity of DCs with different KIR combined NK cells: Compared with KIR2DL1+2DS4~+NK cells,the killing activity of KIR2DL1+2DS1+2DS4~+and KIR2DL1+2DS1+NK cells were strong(P<0.05);3.Killing activity of DCs with different KIR haplotype NK cells: The killing effect of KIR B haplotype NK cells on DC was stronger than that of KIR A haplotype,and the difference was statistically significant(t=2.19;P<0.05);4.In three patients who underwent allo-HSCT,two of them receiving the graft for KIR B haplotype occurred mild GVHD;one patient receiving the graft for KIR A haplotype occurred severe GVHD.Conclusion:1.KIR2DS4~+ NK cells can mediate the killing of DC;2.KIR2DS4 and 2DS1 may play a synergistic role in the killing of NK cells against DC.