IL-2 activates NK cells as effector cells for targeting multiple myeloma in hypoxic environment.

<p>(A–B) Pre-incubation (16h) of NK- and OPM-1 MM cells were performed as indicated on the <i>x</i>-axis. IL-2 activation of indicated conditions was done by adding 1000 IU/ml of IL-2 during the 16 hour pre-incubation period. Upon pre-incubation NK cells and MM cells were cocultured in a 4.5 hour cytotoxicty (A) or CD107a (B) assay. In (A) each dot represents mean of triplicate cultures of individual donors. In (B) NK cell degranulation was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064835#pone-0064835-g005" target="_blank">figure 5</a> by quantification of the percentage of CD107a positive NK cells. Data in (B) show means of duplicate cultures of individual donors. Statistics were performed with one-way repeated measures ANOVA with Bonferroni correction * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001. (C) NK cells and RPMI-8226/S or L-363 MM cells were pre-incubated as described in 6A and subsequently combined in a 4.5 hour cytotoxicity assay. Plots show data obtained from one donor. (D) Flow cytometric analysis of surface expression of NK cell receptors and intracellular expression of perforin and granzyme B after 14–16 hours of incubation at 21% or 0% O₂ in the presence of IL-2. Graphs depict quantification of the % positive NK cells or MFI for the different receptors. Each dot represents one donor. Shown alongside the graphs is the <i>x</i>-axis oxygen parameter. (E) NKG2D surface expression on NK cells upon 14–16 hours incubation at 21% O₂ and 0% O₂ in the presence (solid black line) or absence (grey filled) of IL-2 and matched isotype (grey solid line). Each dot represents an individual donor. Statistics were performed with paired <i>t</i>- test. ** <i>p</i><0.01, *** <i>p</i><0.001.