Arf6 is involved in the regulation of CD16-dependent cytotoxicity. (A) Human NK cells were left uninfected or infected with empty virus (WR) or with recombinant vaccinia viruses encoding HA-tagged Arf6 wild-type (Arf6-WT) or Arf6 mutant Q67L and were assessed in a 4-hour ⁵¹Cr release assay against P815 target cells in the presence of anti-CD16 mAb. No detectable cytotoxicity against P815 targets was observed in the absence of anti-CD16 mAb at the indicated E/T ratios (not shown). Data are expressed as lytic units/10⁶ cells ± SD referred to 3 independent experiments. (B) Cells were left uninfected (□) or infected with empty virus (WR, ▪), or with recombinant vaccinia viruses encoding HA-tagged Arf6 wild-type (Arf6-WT, •) or Arf6 mutant Q67L (○). Surface receptor levels were evaluated as described in “Materials and methods.” Data derived from 3 independent experiments are expressed as the percentage of the mean fluorescence intensity ± SD with respect to the control cells. Each experimental group has its own control sample. (C) NK cells were infected as indicated and stimulated by means of reverse ADCC as in panel A. The percentage of NK cells conjugated with target cells containing polarized granules is shown (see also Figure S1). Data from 3 independent experiments ± SD is shown. (D) NK cells were infected as indicated and stimulated with anti–MHCI-(control mAb), IgG F(ab′)₂-, IgG-, or anti–CD16-coated polystyrene beads. Cell supernatants were collected and assayed for BLT esterase release. Data represent the percentage ± SD of specific release (sample/total release) from 3 independent experiments. (E) Overexpressed Arf6 constructs of a representative experiments are shown.