Fig. 2

ADCC bystander killing in cocultures containing AQP4-expressing and null CHO cells. a Cocultures at 1:50 CHO-AQP4:CHO-null cell ratio were pre-incubated for 30 min with AQP4-IgG (or control IgG) then washed followed by addition of NK cells with a fixable dead cell stain at 30 min prior to fixation. b AQP4 immunofluorescence (green) with dead cell stain (red) at low magnification (left) in cocultures incubated with 5 μg/ml AQP4-IgG and NK cells at an effector:target cell ratio of 5:1. Three fields at high magnification are shown (right). c Fraction of red-stained, dead CHO-null cells as a function of distance from dead CHO-AQP4 cells (mean ± S.E.M., 5 slides with > 80 dead cells analyzed, ** P<0.01, *P<0.05 comparing AQP4-IgG + NK vs. control IgG + NK or AQP4-IgG or pure CHO-null cells by two-way ANOVA). d Fraction of dead bystander cells, as in (c), as a function of incubation time with NK cells (mean ± S.E.M., 5 slides, ** P<0.01 by unpaired t test). Inset shows representative fields at 15 min and 30 min. White filled arrow indicates dead CHO-AQP4 cell at 30 min. e Control studies including CHO-AQP4 and CHO-null cocultures incubated with 5 μg/ml control IgG and NK cells at an effector:target cell ratio of 5:1, with AQP4-IgG alone, and pure CHO-null cells incubated with AQP4-IgG and NK cells. f Cocultures were incubated with NK cells and 1% serum from two seropositive NMO patients, and immunostained as in panel (b)