Generation of ULBP-expressing tumor cell lines. EL4 (A) and RMA (B) tumor cells expressing either empty vector (mock), ULBP1, ULBP2, ULBP3, or the murine NKG2D ligand RAE-1β were generated by retroviral-mediated gene transfer. Cells were single-cell sorted to obtain clones. Mock-transduced clones were identified by reverse-transcription (RT)–PCR. ULBP-expressing clones were stained with anti-ULBP1, anti-ULBP2, anti-ULBP3–specific (black histograms), or M230 isotype control antibody (white histograms), followed by anti–mouse IgG-PE. RAE-1β–expressing clones were stained with murine NKG2D-Fc (black histograms) or p7.5-Fc control (white histograms), followed by anti–human IgG Fc. Clones were analyzed by flow cytometry. Twenty positively expressing clones for each construct were pooled and used for future experiments. Staining of the pooled clones is shown.