Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-G

Received March Activation of NK Cells by an Endocytosed Receptor for Soluble HLA-G Sumati Rajagopalan Yenan T. Bryceson Shanmuga P. Kuppusamy Daniel E. Geraghty Arnold van der Meer Irma Joosten Eric O. Long Hidde Ploegh, Harvard Medical School, United States of America Signaling from endosomes is emerging as a mechanism by which selected receptors provide sustained signals distinct from those generated at the plasma membrane. The activity of natural killer (NK) cells, which are important effectors of innate immunity and regulators of adaptive immunity, is controlled primarily by receptors that are at the cell surface. Here we show that cytokine secretion by resting human NK cells is induced by soluble, but not solid-phase, antibodies to the killer cell immunoglobulin-like receptor (KIR) 2DL4, a receptor for human leukocyte antigen (HLA)-G. KIR2DL4 was constitutively internalized into Rab5-positive compartments via a dynamin-dependent process. Soluble HLA-G was endocytosed into KIR2DL4-containing compartments in NK cells and in 293T cells transfected with KIR2DL4. Chemokine secretion induced by KIR2DL4 transfection into 293T cells occurred only with recombinant forms of KIR2DL4 that trafficked to endosomes. The profile of genes up-regulated by KIR2DL4 engagement on resting NK cells revealed a proinflammatory/proangiogenic response. Soluble HLA-G induced secretion of a similar set of cytokines and chemokines. This unique stimulation of resting NK cells by soluble HLA-G, which is endocytosed by KIR2DL4, implies that NK cells may provide useful functions at sites of HLA-G expression, such as promotion of vascularization in maternal decidua during early pregnancy. - Natural killer (NK) cells are a subset of lymphocytes that mediate innate immunity and regulate adaptive immunity via cytokine secretion and cytotoxic activity. The activation of NK cell function is a result of the integration of activating and inhibitory signals delivered by NK cell receptors [1,2]. NK cell receptors recognize ligands that are either up-regulated or expressed constitutively on target cells. Cells can become sensitive to NK cellmediated cytotoxicity for various reasons, such as loss of major histocompatibility complex (MHC) class I expression or up-regulation of surface molecules in response to DNA damage or stress, which can occur as a result of infection or transformation [3,4]. Normal cells are resistant to NK cell-mediated cytotoxicity due to the presence of inhibitory receptors on the surface of NK cells that recognize major histocompatibility complex (MHC) class I molecules. The interaction between inhibitory receptors and their MHC ligands on target cells transduces a negative signal that blocks the lytic activity of NK cells. NK cells in peripheral blood represent 5%10% of circulating lymphocytes. In contrast, NK cells are the predominant lymphocyte subset in uterus. In early pregnancy, uterine NK cells proliferate and remain in the decidua basalis, which consists of uterine tissue at the maternalfetal interface [5]. The precise function of uterine NK cells is still unknown. Both peripheral blood and uterine human NK cells express killer cell immunoglobulin-like receptors (KIR), a family of NK cell receptors that recognize MHC class I molecules. KIR are type I transmembrane glycoproteins with two or three Iglike domains and cytoplasmic tails of varying lengths [6]. KIRs with long cytoplasmic domains (KIR2DL and KIR3DL) are inhibitory receptors that contain cytoplasmic immunoreceptor tyrosine-based inhibition motifs (ITIM). Those KIRs with short cytoplasmic domains (KIR2DS and KIR3DS) are activating receptors that associate with the adapter DAP12 via a lysine residue in their transmembrane region. With one exception, KIR genes are not expressed in all NK cells. Rather, each NK cell expresses its own repertoire of KIR genes. KIR2DL4 is an evolutionarily conserved, framework member of the KIR gene family that is expressed by all KIR haplotypes and in all NK cells. In contrast to other activating or inhibitory KIR family members, which regulate NK cell cytotoxicity and cytokine production, KIR2DL4 activates cytokine production, but not cytotoxicity, in resting NK cells from peripheral blood [7]. It is unique in its genomic organization and regulation and in its protein structure and function [712]. KIR2DL4 is polymorphic with two reported genetic variants, designated 10A and 9A [13]. While the product of the 10A allele is detectable on the cell surface, the 9A allele, encoding a protein with a truncated cytoplasmic tail, is not stable at the cell surface [14]. KIR2DL4 has a charged arginine residue in its transmembrane region; however, unlike other activating KIR2DS that pair with the adapter DAP-12, KIR2DL4 can associate with the FceRI c chain [15]. Engagement of KIR2DL4 results in activation despite the inhibitory potential conferred by the presence of an ITIM in its cytoplasmic tail [7,16,17]. In resting, peripheral blood NK cells, ligation of KIR2DL4 with monoclonal antibody (mAb) results in interferon (IFN)-c production but not cytotoxicity [7]. The very low cell surface expression of KIR2DL4 [14,17] has been difficult to reconcile with the functional outcome associated with this receptor. Data suggest that KIR2DL4 binds the nonclassical class I molecule HLA-G [8,9]. HLA-G, a nonclassical class I molecule of limited polymorphism, has a unique expression pattern restricted mainly to trophoblast cells that invade the maternal decidua during early pregnancy [18]. HLA-G expression may be inducible in other cell types, in response to inflammation, infection, and transformation [19]. Several isoforms of HLA-G are expressed in the placenta, including membrane-bound forms (HLA-G1, -G2, -G3, and -G4) and soluble forms (HLA-G5 and -G6) [20]. Whereas membrane HLA-G expression in trophoblast cells is restricted to extravillous trophoblast cells, which invade the maternal decidua, expression of soluble HLA-G was detected in all types of placental trophoblast cells [21]. To date, the precise role of HLA-G in the placenta remains unclear [22]. A common hypothesis proposes that expression of HLA-G on invading trophoblast cells is needed to prevent NK cell attack [9,23]. However, this might not be necessary as trophoblast cells are intrinsically resistant to NK cellmediated lysis [24]. An alternative hypothesis proposes that trophoblastNK cell interactions regulate expression of cytokines by NK cells to promote remodeling of the maternal vasculature, which is required to establish adequate blood supply to the fetus [5,25,26]. The benefit of NK cell activation during early pregnancy is supported by genetic studies on preeclampsia, a potentially fatal disease due to incomplete remodeling of spiral arteries by trophoblast cells. Resistance to preeclampsia correlated with combinations of fetal HLA genes and maternal KIR genes that seem to favor NK cell activation over NK cell inhibition [27]. In m (...truncated)