Figure 5.

NK cells conjugate simultaneously to target cells and to other NK cells and provide CD8 ligation. Freshly isolated NK cells (CD8⁺ and CD8^–) were co-incubated with K562 cells for 30–240 min. At 60 min NK–NK and NK–target cell conjugates formed (a). (b) shows a 90-minute conjugate of a single K562 target cell (i) and a CD8⁺ NK cell (ii) with capping of the CD8 molecule (Leu2 FITC) at the cell–cell synapse between two CD8⁺ NK cells (ii and iii). Furthermore, the second CD8⁺ NK cell has formed synapses with two CD8^– NK cells (iv). The insert (v) shows the homogeneous expression of CD8 on a non-conjugated NK cell. Smaller conjugates were apparent involving a CD8⁺ NK cell (ii) and a target cell (i) with CD8 ligation by a single CD8^– NK cell (iii) (c). Apoptosis was measured at 240 min by flow cytometric assessment of annexin V expression on CD8⁺ and CD8^– NK cells in the presence or absence of W632 (d). CD8⁺ NK showed low expression of annexin V (shaded histogram) in the absence of W632 in contrast to the high expression on the CD8^– cells (dashed histogram) and coincident reduction in forward angle light scatter (not shown) indicative of apoptosis. In the presence of the MHC Class I blocking mAb W632 the CD8⁺ NK cells showed equivalent levels of apoptosis to the CD8^– cells (solid line). In 1-hr K562 conjugation assays with five normal donors (e), blockade of the CD8–MHC Class I interaction with W632 (solid bars) increased the activation-induced apoptosis within the CD8⁺ NK subset significantly (P < 0·001; paired t-test; n = 5) to levels equivalent to those of CD8^– NK cells in the same culture and this could be prevented by direct ligation of CD8 on the NK-cell surface with RPA-T8 mAb (hatched bars). Neither mAb had any effect on the CD8^– NK cells in the culture (e).