The Anti-Human P2X7 Monoclonal Antibody (Clone L4) Can Mediate Complement-Dependent Cytotoxicity of Human Leukocytes

Abstract

P2X7 is an extracellular adenosine 5′-triphosphate (ATP)-gated cation channel that plays various roles in inflammation and immunity. P2X7 is present on peripheral blood monocytes, dendritic cells (DCs), and innate and adaptive lymphocytes. The anti-human P2X7 monoclonal antibody (mAb; clone L4), used for immunolabelling P2X7 or blocking P2X7 activity, is a murine IgG2_b antibody, but its ability to mediate complement-dependent cytotoxicity (CDC) is unknown. In this study the functionality of this mAb was confirmed by inhibition of ATP-induced Ca²⁺ responses in HEK-293 cells expressing P2X7 (HEK-P2X7). Spectrophotometric measurements of lactate dehydrogenase release demonstrated that the anti-P2X7 mAb mediated CDC in HEK-P2X7 but not HEK-293 cells. Further, flow cytometric measurements of the viability dye, 7-aminoactinomycin D, showed that this mAb mediated CDC in human RPMI 8226 but not mouse J774 cells. Immunolabelling with this mAb and flow cytometry revealed that relative amounts of cell surface P2X7 varied between human peripheral blood leukocytes. As such, the anti-P2X7 mAb preferentially mediated CDC of leukocytes that displayed relatively high cell surface P2X7, namely monocytes, DCs, natural killer T cells, myeloid-derived suppressor cells, and T helper 17 cells. Together, this data highlights a novel approach to target cellular P2X7 and to limit unwanted P2X7 functions.