Monoclonal antibodies with various reactivity to p58 killer inhibitory receptors

Abstract

Human natural killer (NK) cells have receptors that bind to HLA class I molecules. Among these receptors, p58 and p50 bind to HLA-C. P58 belongs to the killer inhibitory receptor (KIR) and p50 belongs to the killer activatory receptor (KAR). Previously, we obtained three recombinant p58 KIR or p50 KAR proteins, KAR-K1 (KIR2DS4), KIR-K6 (KIR2DL1), and KIR-K7 (KIR2DL3). In this study, we produced and characterized seven monoclonal antibodies (MAbs) to the p58 KIR and p50 KAR proteins. The MAbs were classified in three groups according to their antigen-binding specificity: 5IB103 and 26ID707 were KAR-K1-specific; A809, 190IIC311, and 197IIC611 were KIR-K7-specific; while A210 and A803g bound to all three recombinant proteins. The MAbs reactive to KIR-K7 bound to the gamma3 domain among two immunoglobulin (Ig) domains of KIR-K7. Immunofluorescence staining and flow cytometric analysis with A803g showed reactivity to about 10% of peripheral blood mononuclear cells and 35% of purified natural killer cells. Double immunofluorescence staining with A803g and anti-CD56 Ab showed that CD56 and p58 or p50 were expressed on NK cells in a mutually exclusive way. We also investigated T-cell markers in A803g+ cells. A803g+ T cells were almost CD8+ cells. MAbs produced in this study can be utilized practically in the investigation of biological characteristics of p58 KIR and p50 KAR.