SHP-1- and Phosphotyrosine-Independent Inhibitory Signaling by a Killer Cell Ig-Like Receptor Cytoplasmic Domain in Human NK Cells

Abstract

Killer cell Ig-like receptors (KIR) are MHC class I-binding immunoreceptors that can suppress activation of human NK cells through recruitment of the Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) to two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic domains. KIR2DL4 (2DL4; CD158d) is a structurally distinct member of the KIR family, which is expressed on most, if not all, human NK cells. 2DL4 contains only one ITIM in its cytoplasmic domain and an arginine in its transmembrane region, suggesting both inhibitory and activating functions. While 2DL4 can activate IFN-γ production, dependent upon the transmembrane arginine, the function of the single ITIM of 2DL4 remains unknown. In this study, tandem ITIMs of KIR3DL1 (3DL1) and the single ITIM of 2DL4 were directly compared in functional and biochemical assays. Using a retroviral transduction method, we show in human NK cell lines that 1) the single ITIM of 2DL4 efficiently inhibits natural cytotoxicity responses; 2) the phosphorylated single ITIM recruits SHP-2 protein tyrosine phosphatase, but not SHP-1 in NK cells; 3) expression of dominant-negative SHP-1 does not block the ability of 2DL4 to inhibit natural cytotoxicity; 4) surprisingly, mutation of the tyrosine within the single ITIM does not completely abolish inhibitory function; and 5) this correlates with weak SHP-2 binding to the mutant ITIM of 2DL4 in NK cells and a corresponding nonphosphorylated ITIM peptide in vitro. These results reveal new aspects of the KIR-inhibitory pathway in human NK cells, which are SHP-1 and phosphotyrosine independent.

Recognition of HLA‐G by the NK cell receptor KIR2DL4 is not essential for human reproduction

“…The ubiquitous transcription of the KIR2DL4 gene in the NK cell clones of all analyzed individuals and its conservation in evolution suggest that the product of this gene could essentially contribute to the biology of human NK cells, a view further supported by its distinct structure and signaling properties [11,14,16,17,22,23]. Preferential expression of the KIR2DL4 ligand, HLA-G, in the placenta pointed to a possible participation of KIR2DL4 in the poorly understood function of uterine NK cells in the maternal-fetal interface.…”

“…For instance, HLA-G and, perhaps, HLA-C can be recognized directly by uterine NK cells through LILRB1; in addition, the leader peptides of those MHC molecules can be presented by HLA-E to NK cell clones expressing CD94/NKG2 heterodimers [9,10,[45][46][47]. However, none of the aforementioned receptors has signaling properties or distributions similar to those reported for KIR2DL4 [11,14,[22][23][24]. By contrast, KIR3DL3, whose ligand and functionality are unknown, shares with KIR2DL4 having a single ITIM and being well conserved in different individuals of several primate species [32][33][34][48][49][50].…”

“…However, KIR2DL4 is the only MHC receptor whose gene is transcribed in all NK cells, in contrast with the clonal distribution seen in all other KIR, in LILRB1 and in CD94:NKG2 heterodimers [17,[19][20][21]. The structure of KIR2DL4 is also divergent and transmits a complex signal to NK cells: it inhibits their cytotoxicity but induces secretion of IFN-+ [14,[22][23][24], a cytokine that contributes to placental development in mice [25]. Finally, whereas other KIR genes are represented in only some individuals from a few primate species, KIR2DL4 has orthologs in all those species and is remarkably conserved in different humans [18,26,27].…”

The CD155/poliovirus receptor enhances the proliferation of ras‐mutated cells

“…22 However, it has been found that ITIM might associate with SHP-2 regardless of its phosphorylation status. 31 SHP-2 is a likely mediator of the cellular functions of CD155, since a dominant negative SHP-2 inhibits cell migration and FAK dephosphorylation after ligand stimulation of cellular CD155. 22 In addition, SHP-2 has been implicated in activation of Ras/MAPK signaling pathways and cell proliferation.…”

Bispecific Minibodies Targeting HER2/neu and CD16 Exhibit Improved Tumor Lysis When Placed in a Divalent Tumor Antigen Binding Format

“…CD16 ECD-The CD16A (CD16) extracellular domain was subcloned from the retroviral vector pBMN-IRES-EGFP (a kind gift of Dr. Sei-ich Yusa and Dr. Kerry Campbell, Fox Chase Cancer Center, Philadelphia, PA) (50) and into the pSec Tag2/Hygro A mammalian expression vector (Invitrogen, Corp). This plasmid contained an Ig -chain leader sequence located 5Ј to the CD16 cDNA as a means to induce secretion of the expressed CD16 ECD protein into the cell supernatant.…”