No abstract

Immunosuppression is a typical hallmark of cancer and frequently includes perturbations of the NKG2D tumor recognition system as well as impaired signaling by other activating NK cell receptors. Several in vitro studies suggested that sustained engagement of the NKG2D receptor, as it is occurring in the tumor microenvironment, not only impairs expression and function of NKG2D but also impacts signaling by other activating NK receptors. Here, we made use of a transgenic mouse model of ubiquitous NKG2D ligand expression (H2-Kb-MICA mice) to investigate consequences of chronic NKG2D engagement in vivo for functional responsiveness by other activating NK receptors such as NKp46 and Ly49D. Unexpectedly, we found no evidence for an impairment of NKp46 expression and function in H2-Kb-MICA mice, as anticipated from previous in vitro experiments. However, we observed a marked downregulation and dysfunction of the activating receptor Ly49D in activated NK cells from H2-Kb-MICA mice. Ly49D shares the adaptor proteins DAP10 and DAP12 with NKG2D possibly explaining the collateral impairment of Ly49D function in situations of chronic NKG2D engagement. Altogether, our results demonstrate that persistent engagement of NKG2D in vivo, as often observed in tumors, can selectively impair functions of unrelated NK receptors and thereby compromise NK responsiveness to third-party antigens.

The mouse gut epithelium represents a constitutively challenged environment keeping intestinal commensal microbiota at bay and defending against invading enteric pathogens. The complex immunoregulatory network of the epithelial barrier surveillance also involves NK gene complex (NKC)-encoded C-type lectin-like molecules such as NKG2D and Nkrp1 receptors. To our knowledge, in this study, we report the first characterization of the orphan C-type lectin-like molecule Clr-a encoded by the Clec2e gene in the mouse NKC. Screening of a panel of mouse tissues revealed that Clec2e transcripts are restricted to the gastrointestinal tract. Using Clr-a-specific mAb, we characterize Clr-a as a disulfide-linked homodimeric cell surface glycoprotein. Of note, a substantial fraction of Clr-a molecules are retained intracellularly, and analyses of Clr-a/Clr-f hybrids attribute intracellular retention to both the stalk region and parts of the cytoplasmic domain. Combining quantitative PCR analyses with immunofluorescence studies revealed exclusive expression of Clr-a by intestinal epithelial cells and crypt cells throughout the gut. Challenge with polyinosinic-polycytidylic acid results in a rapid and strong downregulation of intestinal Clr-a expression in contrast to the upregulation of Clr-f, a close relative of Clr-a, that also is specifically expressed by the intestinal epithelium and acts as a ligand of the inhibitory Nkrp1g receptor. Collectively, we characterize expression of the mouse NKC-encoded glycoprotein Clr-a as strictly associated with mouse intestinal epithelium. Downregulation upon polyinosinic-polycytidylic acid challenge and expression by crypt cells clearly distinguish Clr-a from the likewise intestinal epithelium-restricted Clr-f, pointing to a nonredundant function of these highly related C-type lectin-like molecules in the context of intestinal immunosurveillance.

of MICA-specific antibodies. Such immunotolerance for the xenoantigen MICA ideally suits MICAgen mice for anti-MICA-based immunotherapies. Altogether, MICAgen mice represent a valuable model to study regulation, function, disease relevance, and therapeutic targeting of MICA in vivo.

AICL glycoproteins are cognate activation-induced ligands of the C-type lectin-like receptor NKp80, which is expressed on virtually all mature human NK cells, and NKp80-AICL interaction stimulates NK cell effector functions such as cytotoxicity and cytokine secretion. Notably, AICL and NKp80 are encoded by adjacent genes in the NK gene complex and are coexpressed by human NK cells. Whereas AICL is intracellularly retained in resting NK cells, exposure of NK cells to proinflammatory cytokines results in AICL surfacing and susceptibility to NKp80-mediated NK fratricide. In this study, we characterize molecular determinants of AICL glycoproteins that cause intracellular retention, thereby controlling AICL surface expression. Cys residing within the C-type lectin-like domain not only ensures stable homodimerization of AICL glycoproteins by disulfide bonding, but Cys is also required for efficient cell surface expression of AICL homodimers and essential for AICL-NKp80 interaction. In contrast, cytoplasmic lysines act as negative regulators targeting AICL for proteasomal degradation. One atypical and three conventional -linked glycosylation sites in the AICL C-type lectin-like domain critically impact maturation and surfacing of AICL, which is strictly dependent on glycosylation of at least one conventional glycosylation site. However, although the extent of conventional-linked glycosylation positively correlates with AICL surface expression, the atypical glycosylation site impairs AICL surfacing. Stringent control of AICL surface expression by glycosylation is reflected by the pronounced interaction of AICL with calnexin and the impaired AICL expression in calnexin-deficient cells. Collectively, our data demonstrate that AICL expression and surfacing are tightly controlled by several independent cellular posttranslational mechanisms.