Lytic granule convergence occurs despite inhibitory synapse formation and is not reversed by KIR clustering. 721.221 cells expressing HLA-Cw3 or HLA-Cw4 were used as susceptible or nonsusceptible target cells for YTS GFP-KIR2DL1, respectively. (A) Four-hour 51Cr release cytotoxicity assay of YTS GFP-KIR2DL1 cells against 721-Cw3 (red) or 721-Cw4 (blue) target cells. Time-lapse frames of lytic granule movement in YTS GFP-KIR2DL1 cells conjugated to (B) 721-Cw3 or (C) 721-Cw4 cells. The leftmost panel shows transmitted light images of the conjugate pictured, and each confocal image demonstrates immunofluorescence in the plane of converged lytic granules; green, GFP-KIR2DL1; red, LysoTracker-loaded acidified organelles. T = 0 refers to the time that image acquisition began, which was between 1 and 5 minutes after YTS GFP-KIR2DL1 cells were added to the imaging chamber. (D) Quantitative analyses of lytic granule distance from the centroid of the granules as a function of time as measured by mean centroid to granule distance in 9 cells; error bars show ±SD. Mean distance of lytic granules from the centroid of the granules in YTS GFP-KIR2DL1:721.221-Cw4 conjugates was not significantly different from that in YTS GFP-KIR2DL1:721.221-Cw3 conjugates.
