CRTAM expression on NK cells is induced by NK cell conjugation with tumor targets or engagement of NK cell–activating receptors. (A) CRTAM expression was analyzed with the CRTAM mAb Cr24.1 on resting NK cell bulk cultures (unst), NK cells stimulated with PMA/ionomycin for 4 hours, or NK cells conjugated for 20 hours with the NK-susceptible targets A549 (lung carcinoma cell line) and MZ-2 (melanoma cell line). Percentages of CRTAM-positive cells (indicated) varied in different experiments from 25% to 50% of total cells. One of 6 concordant experiments is shown. NKp30 was used as a NK cell–specific marker for counterstaining. (B) CRTAM expression is induced upon cross-linking of some NK cell–activating receptors. CRTAM expression was determined 20 hours after stimulation with plastic coated CD16, NKp44, NKp46, NKp30, and NKG2D mAbs (all mouse IgG1). NKp30 and CD16 mAbs always induced the strongest up-regulation of CRTAM. NKG2D cross-linking reproducibly did not up-regulate CRTAM expression in at least 3 separate experiments. CRTAM was detected with Cr24.1 followed by biotinylated anti–mouse IgG2a and streptavidin allophycocyanin. Shaded histograms represent staining with Cr24.1; open histograms, second- and third-step reagents alone. Comparable results were obtained at 20 and 42 hours of incubation.