FIGURE 4.

The altered triacidic motif within the NKp65 hemITAM does not affect cellular activation. A, degranulation of NK-92MI-NKp65 or NK-92MI-NKp65-N3D cells after 2-h co-culture with P815 loaded with the anti-NKp65 mAb OMAR1. For control, NK-92MI cells were treated with PMA plus ionomycin (P/I). The percentage of CD107a⁺ NK-92MI cells is shown. Viable (DAPI⁻) CD56⁺ cells were gated for analysis by flow cytometry. Depicted are the means ± S.D. of triplicates from one representative of at least three independent experiments. B, the calcium flux of NK-92MI-NKp65 (top panel) or NK-92MI-NKp65-N3D cells (bottom panel) was assessed by flow cytometry upon NKp65 cross-linking. OMAR1 (solid line) or an irrelevant IgG1 (dashed line) was added to NK-92MI cells and, after baseline recording for 30 s, cross-linked by addition of anti-mouse IgG antibodies (vertical dashed line). The ratio of median fluorescence intensities (fluo-4/fura red) was recorded as a function of time by flow cytometry. One representative of three independent experiments is depicted. C, the calcium flux of Jurkat-NKp65, Jurkat-NKp65-N3D, or Jurkat-NKp80-DG cells was assessed upon cross-linking with OMAR1 (NKp65) or 5D12 (NKp80-DG) as described in B.