Cell biological steps and checkpoints in accessing NK cell cytotoxicity

Abstract

Natural killer (NK) cell-mediated cytotoxicity is governed by the formation of a lytic immune synapse in discrete regulated steps, which give rise to an extensive array of cellular checkpoints in accessing NK cell-mediated cytolytic defense. Appropriate progression through these cell biological steps is critical for the directed secretion of specialized secretory lysosomes and subsequent target cell death. Here we highlight recent discoveries in the formation of the NK cell cytolytic synapse as well as the molecular steps and cell biological checkpoints required for this essential host defense process.

Figure 1

Stepwise molecular progression through NK cell cytotoxicity. NK cell cytotoxicity can be broken down into three main stages: recognition (a–d), effector (d–p) and termination (q, r). During the recognition stage, the NK cell is particularly sensitive to inhibitory signaling (d). Key events include lytic granule convergence (c), the actin-dependent firm adhesion of the NK cell (e) and subsequent F-actin conduit formation (h), MTOC polarization (j) and LG fusion and exocytosis (o). This is followed by vesicle recycling (p), a period of relative inactivity (q) and detachment and subsequent serial killing (r). See text for further details.

Figure 2

Stepwise stages during NK cell-mediated cytotoxicity: YTS GFP-actin (green) cells were labeled with Lysotracker Red (red) to selectively label lytic granules and co-incubated with CellMask-labeled (Life Technologies, Carlsbad, CA, USA) (yellow) 721.221 target cells in the presence of SYTOX Blue (Life Technologies) (blue) for detection of cell death. Conjugates were imaged by spinning disk confocal microscopy for a total of 90 min. The images shown are frames acquired approximately every 15 min. (a) The NK cell is conjugated to the target cell and lytic granules are dispersed. (b) Granules are converged at the MTOC. (c) The MTOC along with converged lytic granules are polarized at the immune synapse. F-actin is evidently reorganized from a to c. (d) More F-actin reorganization is observed and lytic granules are docked at the plasma membrane. (e) Target cell death begins as SYTOX Blue enters the cell. (f) Target cell undergoes apoptosis and NK prepares for detachment as shown by reversal of shape change. Scale bar=4 μm.