Identification and characterization of a mouse monoclonal antibody (M10) directed against canine (dog) CD8⁺ lymphocytes

Monoclonal antibodies (mAb) were produced by immunizing BALB/c mice with non-adherent dog lymphocytes. M10 was specific for a subset of dog lymphocytes. M10 belonged to the IgG1 subclass and reacted with 26% of dog peripheral blood lymphocytes, 24% of spleen lymphocytes, 81% of thymus cells, 1.2% of bone marrow cells (5.8% of bone marrow lymphocytes) and 23% of PHA-stimulated lymphocytes. Immunohistology of snap-frozen thymus and spleen showed that the spleen B-cell area stained negative, whereas the spleen T-cell area and the thymus medulla exhibited positive reaction in 20-30%. The thymus cortex was strongly positive. M10 diminished cell lysis by 58% in cell mediated lysis assays (CML). Immunoblot assays revealed that M10 recognized an antigen with a molecular weight of 76 kD under non-reducing and 33 kD under reducing conditions. Finally, M10 bound to a canine CD8α transfected rat T-cell line (NB2). These findings characterize M10 as an antibody directed against the dog CD8 antigen.