Ligand Binding to Inhibitory Killer Cell Ig-Like Receptors Induce Colocalization with Src Homology Domain 2-Containing Protein Tyrosine Phosphatase 1 and Interruption of Ongoing Activation Signals

Abstract

Interaction of NK cells with target cells leads to formation of an immunological synapse (IS) at the contact site. NK cells form two distinctly different IS, the inhibitory NK cell IS (NKIS) and the cytolytic NKIS. Cognate ligand binding is sufficient to induce clustering of inhibitory killer cell Ig-like receptors (KIR) and phosphorylation of both the receptor and the phosphatase Src homology domain 2-containing protein tyrosine phosphatase 1 (SHP-1). Recruitment and activation of SHP-1 by a signaling competent inhibitory receptor are essential early events for NK cell inhibition. We have in the present study used three-dimensional immunofluorescence microscopy to analyze distribution of inhibitory KIR, SHP-1, LFA-1, and lipid rafts within the NKIS during cytolytic and noncytolytic interactions. NK clones retrovirally transduced with the inhibitory KIR2DL3 gene fused to GFP demonstrate colocalization of KIR2DL3 with SHP-1 in the center of early inhibitory NKIS. Ligand binding translocates the receptor to the center of the IS where activation signals are accumulating and provides a docking site for SHP-1. SHP-1 and rafts cluster in the center of early inhibitory NKIS and late cytolytic NKIS, and whereas rafts continue to increase in size in cytolytic conjugates, they are rapidly dissolved in inhibitory conjugates. Furthermore, rafts are essential only for cytolytic, not for inhibitory, outcome. These results indicate that the outcome of NK cell-target cell interactions is dictated by early quantitative differences in cumulative activating and inhibitory signals.

Simulations of the NK Cell Immune Synapse Reveal that Activation Thresholds Can Be Established by Inhibitory Receptors Acting Locally

“…2) showed increasing KIR, HLA, and ICAM-1 numbers in the initial contact area because of their binding to their ligands, which retained these molecules in the center of the synapse. This is consistent with the observation that in the inhibitory synapse, SH2 domain-containing phosphatase 1 molecules that associate with inhibitory receptors are more concentrated in the initial contact area than in the periphery (3,16). In the activating synapse, these graphs show the expected: activating receptors, ligands, and membrane microdomains move to the center, and LFA-1 molecules move to the surrounding ring.…”

“…This increase of KIR2DL numbers in the initial contact area is probably due to random diffusion and binding to HLA molecules because we do not assume here a directed movement of inhibitory molecules. This is consistent with the observations on SH2 domain-containing phosphatase 1 molecules, which associate with the inhibitory receptors and are more concentrated in the initial contact area than in the periphery in the inhibitory NKIS (3,16). NKG2D molecule numbers increase in the surrounding ring, probably due to random diffusion from outside this area, whereas in the initial contact area the number of NKG2D remains constant during the simulation, as there is no binding of activating ligands.…”

“…The scenario of NKIS assembly has been observed experimentally in detail (15)(16)(17). Activating signals lead to directed movement of molecules (14,18,19), and of several types of membrane microdomains (20)(21)(22)(23)(24)(25)(26)(27)(28)(29), toward the center of the contact area.…”

Stable masking by H-2Ddcis ligand limits Ly49A relocalization to the site of NK cell/target cell contact

“…This redistribution of inhibitory KIR occurs independent of receptor signaling (20,21). In contrast, KIR signaling via Src homology region 2 domain-containing phosphatase-1 recruitment is required to prevent the sustained transduction of NK cell activation signals and protect target cells from lysis (20,(23)(24)(25). Although these events have been extensively studied by using human NK cells, very little information is available about the behavior of murine Ly49 receptors.…”

Activating Ly-49 Receptors Regulate LFA-1-Mediated Adhesion by NK Cells

“…It remains to be determined whether LFA-1 adhesion is regulated in a similar fashion on resting human NK cells. However, given that LFA-1-mediated adhesion in human NK cells can be negatively regulated by inhibitory receptors, it seems likely that LFA-1 adhesion is modulated in human NK cells as well (30,40).…”