The cell biological basis of ITIM receptor signaling in NK cells has remained largely unexplored because of difficulties in visualizing inhibitory and activating signals simultaneously. Using a photoinducible ligand for KIR2DL2, we were able to separate the stimulation of activating and inhibitory pathways in time and establish a spatial and temporal window of sufficient size to actually observe interactions between them. Our work provides insight into the mechanisms of ITIM receptor signaling and signal integration in NK cells.
Photostimulation of KIR2DL2 during ongoing activating responses is admittedly an imperfect model for the simultaneous triggering of activating and inhibitory receptors that presumably occurs in many NK cell–target cell synapses. Nevertheless, we feel that our results reflect a biologically relevant mechanism for ITIM receptor signaling for the following reasons. First, the retraction response we observe requires ITIM signaling and SHP-1/2 activity, the same molecular determinants necessary for KIR-mediated inhibition in other systems. Second, our observation that NK cells remain responsive to inhibitory stimulation well after the onset of activating signals is consistent with previous experiments showing that NK cells modify the morphology of a growing synapse upon encountering ITIM receptor ligands (Culley et al., 2009
). In vivo, the ability to respond to fresh inhibitory stimulation in this manner is likely important for keeping growing cytolytic synapses with bona fide targets from spilling over onto adjacent bystander cells.
Pioneering imaging studies in T cells have indicated that activating signals are initiated by antigen receptor microclusters at the periphery of the synapse and that, in most cases, signaling is down-regulated as these clusters approach the center (Campi et al., 2005
; Mossman et al., 2005
; Yokosuka et al., 2005
; Varma et al., 2006
). Thus, sustained signaling is dependent on the continuous formation of new peripheral microclusters. Using DAP10 as a probe, we observed that the NKG2D receptor complex forms two kinds of clusters, a mobile variety that is generated in the periphery and migrates centripetally and an immobile variety that accumulates closer to the center. Interestingly, NKG2D-DAP10 does not coalesce into a central supramolecular activation cluster over time, possibly because NKG2D signaling is mediated by Tyr-Ile-Met-Asn motifs in DAP10 rather than the immunotyrosine-based activation motifs contained in antigen receptors. Nevertheless, peripheral DAP10 microclusters contain higher levels of phosphotyrosine, suggesting that they mediate most of the NKG2D-dependent signaling. Strikingly, it is in this peripheral zone that we observed UV-induced accumulation of KIR2DL2 microclusters, suppression of DAP10 microclusters, and actin remodeling.
It is well established that filamentous actin at the synapse is required for receptor-proximal activating signals in NK cells (Orange et al., 2003
; Watzl and Long, 2003
; Barber et al., 2004
; Endt et al., 2007
). Consistent with these studies, we observed that disrupting synaptic actin with latrunculin blocked DAP10 microcluster formation and movement. Interestingly, when cells were subjected to KIR2DL2 photostimulation, suppression of activating microclusters occurred within seconds, before the dissolution of the peripheral actin ring, indicating that cytoskeletal retraction per se is not responsible for microcluster suppression. It is possible, however, that the dramatic actin reorganization induced by KIR2DL2 signaling is preceded by a period of actin destabilization that is more difficult to detect and that this initial actin destabilization is sufficient to suppress activating microclusters.
The pathway linking inhibitory receptors to actin remodeling remains unclear but is likely to involve Vav-1, a guanine nucleotide exchange factor that is phosphorylated and activated during synapse formation (Bustelo, 2000
; Riteau et al., 2003
). Vav-1 stimulates the Rho family GTPase Rac1, which is thought to promote cytolytic function and target cell adhesion by triggering actin polymerization and the up-regulation of integrins (Billadeau et al., 1998
; Galandrini et al., 1999
; Riteau et al., 2003
; Nolz et al., 2008
). It is known that SHP-1 directly dephosphorylates Vav-1 downstream of ITIM receptors (Stebbins et al., 2003
), which could conceivably lead to the actin remodeling we have observed.
Integrins play a critical role in NK cell function by promoting synapse formation and the polarization of cytolytic granules (Barber et al., 2004
; Bryceson et al., 2005
). Our observations that KIR2DL2 signaling induced retraction and that Mn2+
blocked this response are consistent with previous work suggesting a direct link between ITIM-dependent signaling and the regulation of integrins (Burshtyn et al., 2000
; Bryceson et al., 2009
). The formation and maintenance of integrin-mediated cell–cell contacts require Vav and the Rho family GTPases, and also depend on a strong physical linkage between ligand-bound integrins and the underlying actin cytoskeleton (Swat and Fujikawa, 2005
; Abram and Lowell, 2009
). KIR2DL2-induced actin remodeling would presumably weaken this adhesive network either by breaking contacts between integrins and the cytoskeleton or by somehow inducing affinity down-regulation of integrins. That Mn2+
treatment preserves the contact area in photostimulation experiments is consistent with the notion that outside-in signaling is affected by ITIM receptors, as previously suggested (Barber et al., 2004
). This result also implies that there is at least some down-regulation of integrin affinity taking place in response to ITIM signaling. That Mn2+
does not block the dissolution of the actin ring, however, indicates that outside-in signaling alone is insufficient to counteract the effects of KIR2DL2.
Actin remodeling and concomitant retraction are well suited as mechanisms for NK cell inhibition for two reasons. First, by targeting the integrity of the synapse, which is required by most, if not all, activating receptors, actin remodeling provides an elegant way to block effector responses that is independent of the specific activating pathways involved. Second, because retraction breaks cell–cell contact and hence the receptor–ligand interactions that drive the response, it is self-limiting and more easily constrained in space and time. This would presumably facilitate efficient scanning of potential target cells in vivo. In this context, retraction from inhibitory cells would not only prevent inappropriate killing responses but also play an important role in directing those responses to the correct targets.
Using photostimulation as well as antibody-mediated receptor cross-linking, we found that KIR2DL2 only weakly inhibits ongoing Ca2+
responses despite the fact that it blocks the initiation of Ca2+
flux when triggered concurrently with activating receptors. This result is intriguing, particularly because KIR2DL2-induced retraction was not diminished, in our hands, by prolonged exposure to activating signals. Collectively, our data suggest that strong adhesion to the target cell is required for the initiation, but not the maintenance, of Ca2+
signals. Precisely why KIR2DL2 stimulation does not block ongoing Ca2+
responses is unclear. It is possible that ITIM receptor signaling disrupts early events, such as the activation of phospholipase C, that are required for the initiation of Ca2+
flux but has less of an effect on store-operated calcium channels or other downstream components that have been implicated in the sustained phase of the response (Lewis, 2001
). Further studies will be required to explore this issue. From a functional perspective, however, limiting the scope of KIR2DL2 action could facilitate target cell killing in vivo. Elevated cytoplasmic Ca2+
is required for the secretion of lytic granules (Ostergaard et al., 1987
; Takayama and Sitkovsky, 1987
; Esser et al., 1998
). The insensitivity of ongoing Ca2+
responses to ITIM receptor signals would presumably allow NK cells to mount a cytolytic response at one cell–cell interface while receiving inhibitory signals at a distal contact site. This model is consistent not only with our data but also with recent experiments indicating that ITIM receptors are more effective at blocking lytic granule polarization toward the synapse than at disrupting degranulation (Das and Long, 2010
The extent to which the cellular consequences of KIR2DL2 signaling will apply to related signaling pathways in other cell types remains to be seen. It is worth noting, however, that the effectors of phosphorylated ITIMs, SHP-1 and particularly SHP-2, are broadly expressed, as is the tyrosine kinase Ableson, which is also required for inhibitory KIR function in NK cells (Peterson and Long, 2008
). Conceptually, localized retraction is an elegant mechanism for the inhibition of signals delivered by membrane-bound ligands, and it is conceivable that this mechanism would be useful in processes such as cell migration and neuronal path finding. In that regard, it is intriguing that the ITIM receptor PirB was recently shown to promote axonal collapse in sensory neurons (Atwal et al., 2008
). Future mechanistic studies will be required to determine whether or not cursory similarities like these reflect a shared mechanism for the inhibitory regulation of cell–cell interactions.