The NK cell receptor KIR2DL4 emerges as an activation receptor specific for soluble HLA-G. Constitutive endocytosis of KIR2DL4 occurs in freshly isolated, resting NK cells, in IL-2–activated NK cells, in NK cell lines, and in 293T cells transfected with KIR2DL4. At steady state, most of KIR2DL4 is intracellular, which explains the difficulty in detecting cell surface receptor by flow cytometry. Endocytosis explains also the unusual property of KIR2DL4 to induce cytokine and chemokine secretion in response to soluble mAbs and to soluble HLA-G, in the absence of further cross-linking, but not in response to solid-phase mAbs (either plate-coated or bead-bound). Therefore, lack of detectable KIR2DL4 at the cell surface should not be taken as evidence for a lack of function. Endocytosed KIR2DL4 showed extensive co-localization with Rab5, a GTPase that marks a subset of early endosomes.
Endosomes have emerged recently as a specialized signaling compartment [45
]. Some endocytosed receptors, such as the epidermal growth factor receptor and Toll-like receptor 9, signal from endosomes [45
]. It is thought that endocytic vesicles carry signaling platforms where endocytosed receptors link with downstream signaling molecules and provide sustained signaling [45
]. Our data strongly support signal transduction by KIR2DL4 in endosomes. First, KIR2DL4 is actively internalized into endosomes, where most of KIR2DL4 resides at steady state. Second, solid-phase Abs to KIR2DL4 did not induce IFN-γ secretion, indicating that cross-linking KIR2DL4 at the cell surface does not result in signaling. Third, expression of KIR2DL4 and of KIR2DL4 mutants in 293T cells resulted in secretion of IL-8 only for those receptors that were targeted to endosomes. A chimeric receptor with the transmembrane region and cytoplasmic tail of KIR2DL4 that remained at the cell surface did not activate IL-8 secretion, even after cross-linking at the cell surface. Extensive co-localization of endocytosed KIR2DL4 with a subset of early endosomes that carry the small GTPase Rab5 is interesting, as Rab5 was recently assigned a role in endosomal signaling by the epidermal growth factor receptor and by Toll-like receptor 9 [45
]. The signaling pathway triggered by KIR2DL4 requires further study. Ligand-bound KIR2DL4 must somehow be distinguished from free, endogenous KIR2DL4 in NK cells, as resting NK cells do not respond to KIR2DL4 until soluble ligand is added. This distinction may not be made in transfected 293T cells, which appear to respond to KIR2DL4 in the absence of a ligand. Thus, KIR2DL4 signaling is subject to tight regulation in NK cells.
Signaling by KIR2DL4 in 293T cells also implies that immunoreceptor tyrosine-based activation motif (ITAM)-containing subunits, such as the FcRI γ chain, are unlikely to be necessary. This conclusion is further supported by the lack of requirement for the arginine residue in the transmembrane region of KIR2DL4 for signaling in 293T cells. Unlike other NK cell activation receptors with charged residues in the transmembrane region, such as NKG2D and NKp46, which require a partner chain for transport out of the endoplasmic reticulum, KIR2DL4 is readily transported to the cell surface and to endosomes, even in the absence of a known adapter. The position of the arginine at the fourth amino acid position from the N-terminus of the transmembrane region, rather than deeper into the lipid bilayer, suggests that membrane insertion may not be compromised [48
]. However, induction of cytotoxicity by IL-2–activated NK cells through KIR2DL4 in a redirected lysis assay had been dependent on the arginine-tyrosine motif in the transmembrane region of KIR2DL4 [8
]. It is therefore likely that KIR2DL4 can signal in different ways, for cytokine secretion independent of an arginine-mediated association (this study), and for cytotoxicity through an arginine-mediated association with the FcRI γ chain [8
]. The biological context in which KIR2DL4-mediated cytotoxicity would occur is not clear. On the other hand, stimulation of a proinflammatory or proangiogenic response by soluble HLA-G could be a useful property in various physiological contexts. In particular, our study supports a new model for the role of NK cells in early pregnancy.
A ligand of KIR2DL4 is the nonclassical MHC class I molecule HLA-G, which is produced naturally in soluble form by alternative splicing [41
] and by proteolytic cleavage of membrane-bound HLA-G [42
]. In vitro studies have shown that HLA-G induces apoptosis of T cells, inhibition of alloproliferation of T cells and unfractionated mononuclear cells, and inhibition of NK cell cytotoxicity [19
]. In contrast, HLA-G stimulated proliferation and cytokine secretion in uterine NK cells [29
]. In this study, four different soluble forms of HLA-G have been internalized into KIR2DL4-containing endosomes. HLA-G shed from the cell membrane and the naturally secreted form of HLA-G, each produced in transfected 721.221 cells, were endocytosed into KIR2DL4-containing compartments of co-cultured cells. In addition, recombinant HLA-G, produced in E. coli
and refolded with β2-microglobulin and peptide, was also internalized, as was an sHLA-G produced in transfected CHO cells. Competition for HLA-G uptake with a soluble, recombinant KIR2DL4-Ig fusion protein established, for the first time, a direct interaction between KIR2DL4 and HLA-G. So far, evidence for KIR2DL4 binding to HLA-G had been indirect. Binding of soluble KIR2DL4-Ig fusion proteins to cells expressing high levels of HLA-G [8
] had been detected, but binding of soluble HLA-G tetramers to NK cells or soluble HLA-G to KIR2DL4 in surface plasmon resonance studies had not been detected [35
]. The detection of KIR2DL4–HLA-G interaction may have been possible here due to accumulation of soluble HLA-G into KIR2DL4-containing endosomes.
In pregnancy, tolerance of the semiallogeneic fetus by the maternal immune system has long presented an immunological paradox [52
]. To avoid rejection of the fetus, several factors must regulate maternal responsiveness to fetal alloantigens. Regulatory mechanisms may include inhibitory receptors on lymphocytes that bind ligands on fetal cells [5
] and suppression of T lymphocytes by tryptophan catabolism via indoleamine-2,3-dioxygenase activity [55
]. An alternate and nonexclusive view is that interactions of fetal cells with immune cells at the maternal–fetal interface play a role in proper implantation by shaping vascular adaptations of the surrounding maternal tissue via cytokine secretion [25
]. Uterine tissue in early human pregnancy is characterized by extensive vascular remodeling, by invasion of HLA-G–expressing trophoblast cells, and by an abundance of maternal NK cells. Engagement of KIR2DL4 by soluble ligand induced a profile of gene transcription and protein secretion that is consistent with a proangiogenic response. The controversial role of HLA-G expression on extravillous cytotrophoblast cells—be it for inhibition or activation of maternal NK cells—may be settled in favor of activation of NK cells through soluble HLA-G for the purpose of vascular remodeling. Successful implantation and placentation require vascular endothelial growth factor, an angiogenic growth factor that is secreted by trophoblast cells throughout gestation [57
]. The products of several genes up-regulated on NK cells by KIR2DL4 engagement, including TNF-α, IL-1β, and IFN-γ, induce vascular endothelial growth factor production in trophoblast cells, thereby influencing uterine angiogenesis [58
]. Another gene induced by KIR2DL4 in NK cells, COX-2, is a rate-limiting enzyme in prostaglandin biosynthesis, which is required for uterine angiogenesis during implantation in mice [59
]. In addition, IL-8 is known to induce neovascularization in tumor and viral models [60
]. Secretion of IL-23, which is encoded by IL12B
genes, was also up-regulated by KIR2DL4 engagement with soluble ligands. Selective induction of these two genes occurs in mouse uterine NK cells at gestation day 6 [62
]. In summary, the spectrum of proangiogenic mediators that are induced in resting NK cells by soluble HLA-G and by anti-KIR2DL4 mAb supports a role for KIR2DL4 in facilitating implantation and neovascularization during pregnancy. Our data, together with evidence that secretion of soluble HLA-G by in vitro fertilized human embryos is associated with a higher pregnancy rate [63
] and that reduced levels of soluble HLA-G during early pregnancy correlate with a higher incidence of preeclampsia [65
], suggest that KIR2DL4 has a positive role in reproductive success.
The description of soluble HLA-G expression in other cell types and tissues, such as erythroblasts during erythropoiesis [66
], suggests the interesting possibility that a KIR2DL4-mediated signal in NK cells could serve a proinflammatory or proangiogenic function in other contexts as well. In this study, we show that constitutive internalization of KIR2DL4 results in uptake of soluble HLA-G and accumulation into endocytic compartments where the bulk of KIR2DL4 resides. Binding of soluble agonist by KIR2DL4 has the potential advantage of promoting specific NK cell responses without the complicating contribution of multiple receptor–ligand interactions that occur during cell–cell contacts, which engage a wide array of activating and inhibitory NK cell receptors.