UL16-Binding Proteins, Novel MHC Class I-Related Proteins, Bind to NKG2D and Activate Multiple Signaling Pathways in Primary NK Cells

Abstract

The UL16-binding proteins (ULBPs) are a novel family of MHC class I-related molecules that were identified as targets of the human CMV glycoprotein, UL16. We have previously shown that ULBP expression renders a relatively resistant target cell sensitive to NK cytotoxicity, presumably by engaging NKG2D, an activating receptor expressed by NK and other immune effector cells. In this study we show that NKG2D is the ULBP counterstructure on primary NK cells and that its expression is up-regulated by IL-15 stimulation. Soluble forms of ULBPs induce marked protein tyrosine phosphorylation, and activation of the Janus kinase 2, STAT5, extracellular signal-regulated kinase, mitogen-activated protein kinase, and phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signal transduction pathways. ULBP-induced activation of Akt and extracellular signal-regulated kinase and ULBP-induced IFN-γ production are blocked by inhibitors of PI 3-kinase, consistent with the known binding of PI 3-kinase to DAP10, the membrane-bound signal-transducing subunit of the NKG2D receptor. While all three ULBPs activate the same signaling pathways, ULBP3 was found to bind weakly and to induce the weakest signal. In summary, we have shown that NKG2D is the ULBP counterstructure on primary NK cells and for the first time have identified signaling pathways that are activated by NKG2D ligands. These results increase our understanding of the mechanisms by which NKG2D activates immune effector cells and may have implications for immune surveillance against pathogens and tumors.

Comparative analysis of human NK cell activation induced by NKG2D and natural cytotoxicity receptors

“…ULBP-1 and MICA) induces CD25 expression, cytokine secretion and proliferation. Previous studies also reported the effect of soluble recombinant ULBP on the induction of GM-CSF and IFN-+ production by activated NK cells [3,27].…”

“…Indeed, the binding of MICA and ULBP-1 to NKG2D-DAP10 and to an activating receptor complex that includes ITAM-bearing adaptors would reconcile the fact that triggering of the NKG2D-DAP10 complex by specific mAb is insufficient to induce IFN-+ secretion in human and mouse NK cells [12][13][14]21], while stimulation with NKG2D ligands induces IFN-+ secretion in human NK cells [3,27]. Yet, one should take into account that anti-NKG2D mAb abolish the binding of MICA-Fc and ULBP-Fc to NK cells (Fig.…”

“…Among NK cell activating receptors, NKG2D is a lectin-like homodimeric receptor that recognizes a variety of stress-induced self-ligands, such as MICA, MICB and ULBP molecules in humans as well as H60, Rae1 and MULT molecules in the mouse [2]. Besides its expression on NK cells, NKG2D expression is also detected in CD8 + T cells and +ˇT cells, and can be up-regulated by IL-15 [2,3]. TNF- § and IL-15 are also associated with induction of NKG2D expression in CD4 + CD28 -autoreactive T cells from patients with rheumatoid arthritis [4].…”

ULBP6/RAET1L is an additional human NKG2D ligand

“…Differences in binding affinities to NKG2D have been reported for both mouse and human NKG2D ligands [34][35][36]. It has been proposed that molecular competition for receptor binding may exist between NKG2D ligands, with one ligand being favoured over another by NKG2D, due to more favourable binding kinetics [35].…”

“…The extracellular domains of NKG2D ligands share as little as 20-25% sequence identity [33]. In mouse and human different NKG2D ligands have been reported to have different affinities of binding to NKG2D [16,[34][35][36], although it is unknown whether this translates into functional differences in NK-cell stimulation. NKG2D ligands also differ in the nature of their association to cell membranes; some NKG2D ligands are glycosyl-phosphatidylinositol (GPI)-anchored to membranes, whereas others have transmembrane and cytoplasmic domains of variable length and sequence [9].…”