Cytotoxicity of NK cells towards target cells is governed by signals transmitted through multiple receptor-ligand interactions. Activation of primary NK cells, freshly isolated from human blood, is tightly regulated and requires signaling by combinations of synergistic receptors, which are not activating on their own (Bryceson et al., 2009
; Bryceson et al., 2006a
). The main question addressed here was how signals from complementary receptors synergize. Is the requirement for synergy due to complementation of distinct and necessary signals, or to a convergence of signals that have to override an activation threshold? Using co-activation by receptors NKG2D and 2B4 as a model, we have found that they synergize for PLC-γ activation by a pathway that is completely dependent on Vav1 and under negative regulation by the E3 ubiquitin ligase c-Cbl. Synergy is not achieved by signals that complement Vav1-dependent signals, nor by inhibition of c-Cbl-mediated ubiquitylation, but is required to overcome c-Cbl-mediated inhibition of Vav1-dependent signals. These results were obtained by stimulation of receptors either with Abs, or in the more physiological context of receptor-ligand interaction between NK cells and target cells.
The system used here for activation of resting NK cells did not involve signaling through ITAM- containing transmembrane adaptors, nor integrin-dependent signaling. ITAM-independent cytotoxicity has been demonstrated with mouse NK cells that lacked ZAP-70 and Syk kinases (Colucci et al., 2002
). Synergistic signaling by NKG2D and 2B4 induced degranulation but not granule polarization, which is dependent on the β2
integrin LFA-1 (Bryceson et al., 2005
). Therefore, our experimental approach avoided potential complications due to the contribution of Vav1 to “inside-out” signaling for LFA-1 (Ardouin et al., 2003
; Krawczyk et al., 2002
). Using Abs to crosslink receptors NKG2D and 2B4, or S2 cells expressing receptor ligands in the absence of LFA-1 ligands, we have shown that Vav1 has an indispensable role in synergistic signaling by NKG2D and 2B4 for PLC-γ2 activation, cytokine secretion, degranulation, and cytotoxicity by NK cells. Vav1 was also required for synergistic signaling by the combination of receptors 2B4 and DNAM-1.
Our results revealed that synergy does not occur by mutual amplification of NKG2D and 2B4 proximal signaling, but occurs downstream of 2B4 phosphorylation and recruitment of the adapter SAP, which were not enhanced by co-crosslinking with NKG2D. Conversely, PI3K-dependent phosphorylation of Akt induced by NKG2D crosslinking was not enhanced by co-crosslinking with 2B4. NKG2D signaling was sufficient to induce phosphorylation of Vav1 and its association with PLC-γ2. The C-terminal SH2 domain of PLC-γ can bind to a phosphorylated tyrosine in the regulatory, acidic domain of Vav1 (Miletic et al., 2006
). 2B4 signaling was sufficient to induce a low amount of PLC-γ phosphorylation. Synergistic phosphorylation of PLC-γ by the co-engagement of NKG2D, which can recruit Vav1 through the adapter Grb2 (Graham et al., 2006
; Upshaw et al., 2006
), and 2B4 may be due to a combination of Vav1 recruitment and phosphorylation. In support of this hypothesis, a membrane-targeted form of Vav1 synergized with 2B4 signals to overcome the dependence on NKG2D signaling.
The Cbl proteins c-Cbl and Cbl-b are multifunctional adaptor molecules with ubiquitin ligase activity, which regulate signaling by diverse receptors (Duan et al., 2004
; Huang and Gu, 2008
; Liu, 2004
; Swaminathan and Tsygankov, 2006
; Thien and Langdon, 2005
). Cbl-mediated ubiquitylation of receptors can result in internalization from the cell surface and delivery to lysosomal compartments. Ubiquitylation of cytosolic signaling molecules can lead to degradation by proteasomes. In our study, c-Cbl-dependent ubiquitylation of Vav1 was detected. However, the total amount of cellular Vav1 did not change appreciably under the different stimulation conditions, consistent with the fact that only a small fraction of total Vav1 was phosphorylated and ubiquitylated. Furthermore, as inhibition of proteasomal degradation had a very small effect on the half-life of phosphorylated Vav1, ubiquitylation may inhibit Vav1 signaling independently of degradation, as was shown for the inhibition of PI3K in primary T cells through Cbl-b-mediated ubiquitylation of p85 in the absence of degradation (Fang and Liu, 2001
c-Cbl is required for T cell development past the double-negative stage (Naramura et al., 1998
). c-Cbl can inhibit TCR signals through ubiquitylation of the TCR ζ chain (Naramura et al., 1998
; Wang et al., 2001
) and Vav1 (Miura-Shimura et al., 2003
), and by promoting internalization of LAT (Balagopalan et al., 2007
). Work in mice with single and double deficiencies in c-Cbl and the adapter SLP76 showed that c-Cbl inhibits a bypass signaling pathway that is independent of SLP76, as a double SLP76 and c-Cbl deficiency restored development (Chiang et al., 2009
; Chiang et al., 2004
). In contrast, in our study with NK cells, c-Cbl knockdown did not rescue the Vav1 defect, implying that c-Cbl regulates a Vav1-dependent signaling pathway. Overexpression of c-Cbl and Vav1 led to further mechanistic insights into the synergy-dependent activation of NK cells. If c-Cbl were to regulate a Vav1-independent pathway, c-Cbl overexpression would not overcome Vav1-dependent synergistic activation signals. However, inhibition of synergistic activation by overexpression of c-Cbl provided independent evidence that c-Cbl regulates Vav1-dependent signaling. If c-Cbl were to control an obligate complement to Vav1 signals, Vav1 overexpression would not overcome inhibition by c-Cbl. However, Vav1 overexpression resulted in permissive NK cell degranulation after stimulation with S2 cells expressing either ULBP1 or CD48, and in phosphorylation of PLC-γ2 after stimulation with NKG2D or 2B4 alone. These results exclude the possibility of an obligate complement to Vav1 signals that is controlled by c-Cbl. Synergistic signals from NKG2D and 2B4 did not block c-Cbl phosphorylation or Vav1 ubiquitylation, but were required to overcome inhibition by c-Cbl. Whereas the mechanism by which c-Cbl regulates activation signals in NK cells is very different from that by which c-Cbl regulates signaling for T cell development, it exhibits similarities to inhibition by Cbl-b in naïve T cells, which has to be overcome by CD28 through activation of Vav1 (Acuto and Michel, 2003
; Bachmaier et al., 2000
; Chiang et al., 2000
; Huang and Gu, 2008
; Krawczyk et al., 2000
). The assignment of specific functions to Cbl isoforms is therefore different in T cells and NK cells.
Inhibition of NK cell responses by ITIM-containing receptors occurs at a proximal step, upstream of Ca2+
mobilization and of actin polymerization-dependent processes (Long, 2008
). The identification of Vav1 as a primary substrate for dephosphorylation by SHP-1 during inhibitory receptor engagement by MHC class I on target cells (Peterson and Long, 2008
; Stebbins et al., 2003
), and as an essential component in the NKG2D and 2B4 synergy, as shown here, provides an explanation for the dominance of inhibitory receptors over synergistic activation signals. Our data revealed a hierarchy of inhibitory signals to control NK cell activation: a Vav1-dependent synergy was required to overcome inhibition by c-Cbl but was dominantly inhibited by CD94-NKG2A, even when inhibition by c-Cbl had been lifted.
The expression of ligands for synergistic natural cytotoxicity receptors correlated with the sensitivity of primary tumor cells to killing by freshly isolated, primary NK cells (Carlsten et al., 2007
). As the co-activation receptor NKG2D provides innate protection from spontaneous tumor cells (Guerra et al., 2008
; Smyth et al., 2005
), and downregulation of Cbl-b in mice results in spontaneous tumor rejection by CD8 T cells (Chiang et al., 2007
; Loeser et al., 2007
), our new understanding of the regulation of NK cell responses through c-Cbl, and the synergy-independent activation through individual receptors after c-Cbl downmodulation, may open ways to optimize NK cell effector functions.