Costimulation of NK cells leads to enhanced activation of Syk and ERK, but not p38, Jnk, or Akt. (A) Purified human NK cells (more than 97% CD56+/CD3−) underwent FcγRIIIa cross-linking by sequential treatment with an F(ab′)2 fragment of a mouseanti–human FcγRIIIa Ab (clone 3G8) and a secondary F(ab′)2 goat anti-mouse Ab. Recombinant human IL-12 was added at a concentration of 10 ng/mL at 37°C. Control conditions consisted of control Ab-treated NK cells (−), NK cells activated via FcR ligation alone (FcR), or NK cells activated with IL-12 alone (IL-12). Following stimulation, NK cells were lysed at the indicated time points for immunoblot analysis of p-Syk, p-ERK, p-STAT4, or p-Akt. Numbers below immunoblots represent the fold increase in phosphoprotein levels for each condition, normalized to total protein and expressed in relative densitometric units. (B-D) Time course for the activation of Syk, ERK, and STAT4 in NK cells stimulated as described in panel A. Levels of each phosphoprotein were quantified via densitometry and plotted as fold increase in phosphoprotein normalized for total protein versus time for each treatment condition. Immunoblots depict results from one representative donor. Graphs below immunoblots represent the mean plus or minus SEM from all 5 donors tested. *P < .05 versus the fold induction with IL-12 or FcR cross-linking alone at the time point.
