Functional properties of CD2- and CD2+ bovine NK cell subsets. (A) Intracellular IFN-γ response in bovine heparinized whole blood, stimulated for18 hrs with 200U/ml rbIL-2 and 5U/ml rbIL-12. Cells were surface labelled and gated for NKp46+ and CD2+ or CD2-, and bars represent net per cent cells positive for cytoplasmatic IFN-γ (stimulated samples minus unstimulated controls). Each of five tested animals is shown individually; error bars represent SD of repeated assays (n = 1–3). (B) IFN-γ measured in supernatants of bovine NK cells separated with respect to CD2 expression, cultured for around one week in the presence of 100U/ml rbIL-2 and stimulated for the last 24 hours with 0.5U/ml rbIL-12. Responses were measured by an ELISA and are shown as stimulated samples minus unstimulated controls for individual animals. (C) Cytotoxicity of NK cell subsets obtained likewise, against the bovine kidney cell line MDBK, in a 4 hrs 51Cr release assay. Bars represent four individual animals and show lytic units (LU) defined as stated in Methods. (D) Redirected lysis of FcR-bearing murine P815 target cells by similar NK cell subsets, in the presence of α-NKp46 mAb, calculated and presented as in (C).