Figure 5

Agonistic anti-CD134 mAb promote NK associated cytokine release in an Fc:FcγR dependent manner. (A) PBMCs were stimulated with SEB (1 ng/mL) for 24 hours to upregulate CD134 expression on NK cells and then incubated with an isotype control, CD134 agonist mAb (5 μg/mL), or multimeric CD134 ligand (1 μg/mL) for 6 hours in the presence of brefeldin A or monensin. Intracellular IFNγ and TNFα, and surface CD107a expression were examined on NK cells by flow cytometry. Representative plots are shown from at least 5 experiments. (B–D) PBMCs were treated as described in (A). In addition, deglycosylated agonistic CD134 mAb and a multimeric control ligand was used. The graphs show the percentages of IFNγ⁺ (B), TNFα⁺ (C) and CD107a⁺ (D) NK cells after deduction of baseline expression on untreated cells. Paired t test, n = 5–6, *P < 0.05, **P < 0.01, ns = not significant. (E) BCL₁-bearing Fcγ chain −/− mice were treated as in Fig. 1A. A Kaplan Meier survival curve is shown. n = 5/group, representative of duplicate experiments.