ULBP6/RAET1L is an additional human NKG2D ligand

Abstract

To date five ULBP/RAET (UL16-binding protein, also known as retinoic acid early transcript) genes, encoded on human chromosome 6q24.2-q25.3, have been shown to encode ligands of the activating immunoreceptor NKG2D. Here, we show that a sixth gene, ULBP6/RAET1L, is a polymorphic locus that expresses a functional transcript. ULBP6 had a more restricted expression profile in cell lines and primary human tissues than other NKG2D ligands, but expression was detected in several human papillomavirus-positive cervical carcinoma cell lines and was inducible on infection with human CMV. ULBP6 bound to recombinant NKG2D as well as the human CMV immune evasion molecule UL16. By confocal microscopy we show that UL16 retains ULBP6 inside the cell, preventing it from reaching the cell surface. Expression of ULBP6 on target cells induced a significant increase in NK-cell killing. Comparison of ULBP6 with ULBP4 and ULBP5 indicated that differences in recombinant NKG2D binding correlated with differences in NK-cell activation.Key words: Immune evasion . Infectious diseases . Innate immunity . NK cells . NK-cell ligandsSupporting Information available online IntroductionThe stimulatory immune receptor NKG2D (NK group 2, member D) plays an important role in anti-pathogen and anti-cancer immune responses [1]. In humans, NKG2D is expressed by all NK cells and almost all abT cells and gdT cells [1]. It is normally absent from CD4 1 T cells but can be induced in some circumstances, such as in rheumatoid arthritis patients [2] and on exposure to human CMV (HCMV) [3]. NKG2D signalling function is mediated exclusively through the adaptor molecule DAP10 in humans [4], whereas mouse NKG2D can associate with both DAP10 and DAP12 [5,6]. The downstream effects of NKG2D ligation are now recognised to depend on synergy with other cell surface receptors and cytokines, with IL-15 signalling recently being shown to have a particularly close association with the NKG2D-DAP10 complex [7,8].NKG2D ligands are a diverse array of cellular proteins, all of which have structural similarity to MHC class I molecules [9]. In humans NKG2D ligands are encoded in two gene families, MIC (MHC-class-I-polypeptide related sequence) and ULBP/RAET (UL16-binding protein, also known as retinoic acid early transcript) [10][11][12][13]. We will follow the ULBP nomenclature from this point. In mouse, NKG2D ligands comprise three members of the H60 family, five members of the Rae1 (retinoic acid early inducible transcript-1) family, and Mult-1 (murine ULBP-like transcript) [14][15][16][17].According to the initial models of NKG2D function, its ligands were thought to be largely absent from healthy cells, but their expression could be induced on viral infection and on tumourigenesis [1]. Ectopic expression of NKG2D ligand leads to anticancer immune responses to transplanted tumours in mice, and the NKG2D knockout mouse has an increased susceptibility to certain types of cancer [18][19][20]. The importance of NKG2D in antiviral immune responses is illustrated by the fact that ...

Intracellular Sequestration of the NKG2D Ligand ULBP3 by Human Cytomegalovirus

“…Expression of UL142 causes intracellular retention of ULBP3 in the cis-Golgi complex UL16 mediates decreased surface expression of MICB and ULBP1, 2, and 6 by intracellular retention in the ER or cis-Golgi (27,28). More recently, we showed that UL142 similarly retains full-length alleles of MICA in the cis-Golgi (38).…”

“…Because expression of these ligands is induced by infection (26), HCMV must prevent their upregulation to minimize their effects. The viral UL16 protein binds ULBP1, 2, and 6, as well as MICB (but not the related ULBP3 and MICA) (27,28); thus, it mediates their intracellular retention in the endoplasmic reticulum (ER) or Golgi to inhibit surface upregulation (29,30). MICB expression is also controlled by an HCMV-encoded microRNA (UL112-1) that reduces MICB translation (31).…”

“…rUL16 protein was shown to have no binding to ULBP3 (27) or RAET1E/ULBP4 and only very weak or marginal binding to RAET1G/ULBP5 (28,48); therefore, no HCMV genes that are able to prevent the surface expression of ULBP3 or RAET1E/G have been described. Because UL16 and MCMV m138 (45,46) downregulate multiple NKG2DLs, we sought to determine whether HCMV UL142 had a similar property.…”

Post-translational Modification of the NKG2D Ligand RAET1G Leads to Cell Surface Expression of a Glycosylphosphatidylinositol-linked Isoform

“…An alternative hypothesis is that RAET1G functions as a decoy in viral infection. RAET1G protein interacts weakly with NKG2D compared with ULBP2 (7,10). RAET1G protein might sequester viral proteins that target ULBP2, for example, although it binds UL-16 very weakly if at all.…”

“…Both MIC family proteins, MICA and MICB, consist of three ␣ domains, transmembrane domains, and cytoplasmic tail. In contrast, ULBP/RAET1 proteins have two ␣ domains and, of the six molecules, four (ULBP1, ULBP2, ULBP3, and ULBP6 (RAET1L)), are GPI-anchored proteins (4,7). The other two, ULBP4 (RAET1E) and ULBP5 (RAET1G) have been considered to have transmembrane domains (4,8).…”