Inhibitory receptors alter natural killer cell interactions with target cells yet allow simultaneous killing of susceptible targets

Abstract

Inhibitory receptors expressed on natural killer (NK) cells abrogate positive signals upon binding corresponding major histocompatibility complex (MHC) class I molecules on various target cells. By directly micromanipulating the effector-target cell encounter using an optical tweezers system which allowed temporal and spatial control, we demonstrate that Ly49-MHC class I interactions prevent characteristic cellular responses in NK cells upon binding to target cells. Furthermore, using this system, we directly demonstrate that an NK cell already bound to a resistant target cell may simultaneously bind and kill a susceptible target cell. Thus, although Ly49-mediated inhibitory signals can prevent many types of effector responses, they do not globally inhibit cellular function, but rather the inhibitory signal is spatially restricted towards resistant targets.

Figure 1

Changes in target cell morphology during NK cell cytotoxicity. Light microscopy images of NK cell–YB target cell interaction at various time points after target cell binding (0 min). (A) The NK cell had some moving filaments and began to round up (1.5 min). (B) The NK cell had rounded (11 min). (C) The target began to swell (14 min). (D) The target cell continued to increase in volume (22 min). (E) The YB target had completely lost membrane integrity (28 min). Scale bar, 10 μm.

Figure 2

Videomicroscopic analysis of NK cell interactions with single target cells (type II interaction). Bright field microscopy images taken at different time points after contact of an RNK.Ly-49A cell with a susceptible (YB) tumor target cell (00 min:00 s). (A) An NK cell (below) has bound a YB tumor target cell (middle), and a second NK cell (right) approached the first NK cell (02:27). (B) The second NK cell contacted the first NK cell and scanned its surface (04:18). (C) The second NK cell contacted the YB target cell, and extensive membrane interaction could be seen to form between effector and target (05:34). (D) The second NK cell was rounded, and the first NK cell formed lamellipodia on the opposite side of the target contact point (08:27). (E) The first NK cell pulled away from the target (10:33). (F) The target cell had undergone cellular disruption, and the second NK cell had started to leave the target (26:36). Scale bar, 10 μm. Video available at http://www.jem.org/cgi/content/full/190/7/1005/F2/DC1.

Figure 3

Videomicroscopic analysis of NK cell interactions with single target cells (type IV interaction). Bright field microscopy images taken at different time points after contact of an RNK.Ly-49A cell with a resistant (YB-D^d) tumor target cell. (A) The NK cell contacted the YB-D^d target cell (00:00). (B) The target cell was lifted up from the surface of the dish (02:20). (C) The effector cell continued to crawl under the target (03:24). (D) The target attached at the back of the NK cell as the NK cell moved forward (06:35). (E and F) The YB-D^d target cell was carried along and finally released into the surrounding medium (13:54–14:20). Scale bar, 10 μm. Video available at http://www.jem.org/cgi/content/full/190/7/1005/F3/DC1.

Figure 4

Duration of NK cell–target cell binding. Scatter plot representing interaction times between NK cells and susceptible (YB) and resistant (YB-D^d) tumor target cells. Conjugations that lasted for 25 min or less are shown. For each target, six conjugations lasted >25 min (not shown). The total number of effector–target observations that included YB or YB-D^d were 19 and 21, respectively. Horizontal bars indicate mean values of interaction times (YB: 10.7 ± 5.1 [mean ± SD], n = 13; YB-D^d: 7.6 ± 5.3, n = 15).

Figure 6

Directional inhibition of NK cell cytotoxicity. Images of NK cell–dual target cell interactions at various time points after contact with a resistant tumor target cell (NK/YB-D^d; −02:22) and a susceptible target cell (NK/YB; 00:00). (A) The NK cell was elongated just after first contact with both YB and YB-D^d (00:39). (B) The NK cell started to round up (05:47). (C) The effector cell was almost completely rounded (07:19). (D) The NK cell was elongated, and the YB target started to swell (23:19). (E and F) The YB target underwent loss of cell integrity (36:33–43:15). Scale bar, 10 μm. Video available at http://www.jem.org/cgi/content/full/190/7/1005/F6/DC1.

Figure 5

YB-D^d cells do not compete for YB killing. Lysis of ⁵¹Cr-labeled YB cells by RNK.Ly-49A NK cells in the presence of increasing amounts of unlabeled YB, YB-D^d, or RMA target cells. E/T ratio was 40:1. The results are representative of three independent experiments.