KIR2DS1+ NK cells alloreactivity against HLA-C2 group targets after inhibitory KIR blockades in vitro

Abstract

Objective To study the killing effects of the killer cell immunoglobutin-like receptors (KIR) KIR2DS1-positive natural killer (NK) cells against acute, myeloid leukemia (AML) target cells, and to explore the KIR, human leucocyte antigen-Cw, and Cw loci (HLA-Cw) of NK cells, and, HLA-Cw of target cells mismatches destruction mechanism. Methods High-purified NK cells separated by DNYAL bead negative selection from healthy donor peripheral blood were taken as effector cells, and the freshly isolated bone marrow mononuclear cells from newly diagnosed AML patients as target cells. The anti-CD158a, CD158b monoclonal antibody was used to block inhibitory KLR receptors of NK cells (such as: KIR2DL1, KIR2DL2, KIR2DL3). The NK cells cytotoxicities against target cells before and after KIR blockades were detected by MTT reduction assay in vitro. Polymerase chain reaction and sequence specific primers (PCR-SSP) genotyping techniques were used to detect HLA-Cw, KIR gene of the healthy donors and patients. NK cells and target cells were divided into group C1 (expressing HLA-Cw 01, 03, 07, 08, 12, 14, 16 alleles), C2 group (expressing HLA-Cw 02, 04, 05, 06, 15, 17, 18 alleles) and the C1/C2 group (co-expressing the alleles in C1 group and C2 group) based on HLA-Cw. Results The purity of NK cells analyzed by flow cytometry was (90.8±6.08)%, and the killing effects of NK cells were significantly enhanced after inhibitory KIR blockades (t=-3.00, P=0.005). The cell lysis in C1 group, KIR2DS1+ NK cells against C2 group target cells (57.37±1.40)% was significantly higher than that against C1 group (44.19±4.67) % and C1/C2 group (36.77±6.56)% target cells (F= 11.87, P = 0.021), furthermore the differences in cell lysis among the above three groups become more evident after the inhibitory KIR blockades (F = 18. 72, P=0. 009). Conclusions The activity of NK cells was increased after the inhibitory KIR blockades, and the killing activities of KIR2DS1+ NK cells were higher than KIR2DS1 NK cells. The incompatibility between KIR, HLA-Cw of NK cells and HLA-Cw of target cells mediated NK alloactivation, realizing missing self recognition, and enabling NK cells cytotoxieity against targets. Key words: Hematopoietic stem cell transplantation; Killer cells, natural; Receptors,polymeric immunoglobulin