The activating receptor NKp65 is selectively expressed by human ILC3 and demarcates ILC3 from mature NK cells.

Innate lymphocytes comprise cytotoxic natural killer (NK) cells and tissue-resident innate lymphoid cells (ILC) that are subgrouped according to their cytokine profiles into group 1 ILC (ILC1), ILC2, and ILC3. However, cell surface receptors unambiguously defining or specifically activating such ILC subsets are scarcely known. Here, we report on the physiologic expression of the human activating C-type lectin-like receptor (CTLR) NKp65, a high-affinity receptor for the CTLR keratinocyte-associated C-type lectin (KACL). Tracking rare NKp65 transcripts in human blood, we identify ILC3 to selectively express NKp65. NKp65 expression not only demarcates "bona fide" ILC3 from likewise RORγt-expressing ILC precursors and lymphoid tissue inducer cells but also from mature NK cells which acquire the NKp65-relative NKp80 during a Notch-dependent differentiation from NKp65+ precursor cells. Hence, ILC3 and NK cells mutually exclusively and interdependently express the genetically coupled sibling receptors NKp65 and NKp80. Much alike NKp80, NKp65 promotes cytotoxicity by innate lymphocytes which may become relevant during pathophysiological reprogramming of ILC3. Altogether, we report the selective expression of the activating immunoreceptor NKp65 by ILC3 demarcating ILC3 from mature NK cells and endowing ILC3 with a dedicated immunosensor for the epidermal immune barrier.

Trefoil factor 3 shows anti-inflammatory effects on activated microglia.

Microglial cells are a major source of pro-inflammatory cytokines during central nervous system (CNS) inflammation. They can develop a pro-inflammatory M1 phenotype and an anti-inflammatory M2 phenotype. Shifting the phenotype from M1 to M2 might be an important mechanism to overcome CNS inflammation and to prevent or reduce neuronal damage. Here, we demonstrate that the anti-inflammatory protein trefoil factor 3 (TFF3) is secreted by astrocytes and that its transcription is significantly reduced after incubation with lipopolysaccharide (LPS). Moreover, we demonstrate that microglial cells cultured in the presence of TFF3 show reduced expression and secretion of pro-inflammatory cytokines after LPS stimulation.

Key residues at the membrane-distal surface of KACL, but not glycosylation, determine the functional interaction of the keratinocyte-specific C-type lectin-like receptor KACL with its high-affinity receptor NKp65.

Keratinocyte-associated C-type lectin (KACL) is a peculiar C-type lectin-like receptor (CTLR) due to its selective expression by human keratinocytes and cognate interaction with the genetically coupled CTLR NKp65. KACL and NKp65 are members of the CLEC2 and NKRP1 subfamilies of natural killer gene complex (NKC)-encoded CTLR, respectively. Most NKRP1 molecules are expressed on NK cells and T cells and act as receptors of CLEC2 glycoproteins with their genes being intermingled in a certain sub-region of the mammalian NKC. The reasons for the tight genetic linkage of these dedicated receptor/ligand pairs are unknown, as is the physiological expression of NKp65. Recently, we reported that the CTLR NKp65 and KACL interact with high affinity, resulting in activation of NKp65-expressing NK-92MI cells. Here, we address the molecular basis of this high-affinity interaction by analysing KACL mutants with KACL-specific monoclonal antibodies (mAb), soluble NKp65 (sNKp65) and NK-92MI-NKp65 cells. We find that none of the three N-linked carbohydrates of KACL glycoproteins significantly contributes to KACL surface expression and NKp65 interaction. However, KACL mutants with non-conservative amino acid substitutions of arginine 158 or isoleucine 161 abrogated binding of both KACL-specific mAb OMA1 and sNKp65, well in line with the blockade of NKp65-KACL interaction by OMA1. Accordingly, functional recognition of these KACL mutants by NK-92M-NKp65 cells was completely abolished. Arginine 158 and isoleucine 161 located at the membrane-distal surface of KACL were defined as residues, decisively determining functional KACL-NKp65 interaction that is independent of KACL glycosylation.

Interaction of C-type lectin-like receptors NKp65 and KACL facilitates dedicated immune recognition of human keratinocytes.

Many well-known immune-related C-type lectin-like receptors (CTLRs) such as NKG2D, CD69, and the Ly49 receptors are encoded in the natural killer gene complex (NKC). Recently, we characterized the orphan NKC gene CLEC2A encoding for KACL, a further member of the human CLEC2 family of CTLRs. In contrast to the other CLEC2 family members AICL, CD69, and LLT1, KACL expression is mostly restricted to skin. Here we show that KACL is a non-disulfide-linked homodimeric surface receptor and stimulates cytotoxicity by human NK92MI cells. We identified the corresponding activating receptor on NK92MI cells that is encoded adjacently to the CLEC2A locus and binds KACL with high affinity. This CTLR, termed NKp65, stimulates NK cytotoxicity and release of proinflammatory cytokines upon engagement of cell-bound KACL. NKp65, a distant relative of the human activating NK receptor NKp80, possesses an amino-terminal hemITAM that is required for NKp65-mediated cytotoxicity. Finally, we show that KACL expression is mainly restricted to keratinocytes. Freshly isolated keratinocytes express KACL and are capable of stimulating NKp65-expressing cells in a KACL-dependent manner. Thus, we report a unique NKC-encoded receptor-ligand system that may fulfill a dedicated function in the immunobiology of human skin.

Differential NKG2D binding to highly related human NKG2D ligands ULBP2 and RAET1G is determined by a single amino acid in the alpha2 domain.

The activating NK cell receptor NKG2D binds to numerous stress-induced cell surface glycoproteins with MHC class I-related ectodomains. In humans, NKG2D ligands (NKG2DL) are members of the MHC-encoded MIC and non-MHC-encoded ULBP families of proteins. The redundancy of NKG2DL raises questions about unique features associated with individual NKG2DL. The ULBP family member RAET1G contains an ectodomain highly related to ULBP2, but is unique by virtue of an extended cytoplasmic domain. Since RAET1G is poorly characterized, we studied expression and functional interactions of RAET1G in comparison to ULBP2. RAET1G transcripts were detected in most human tissues with an overall expression pattern similar to ULBP2. However, although ULBP2 strongly binds both NKG2D and the immunoevasive human cytomegalovirus glycoprotein UL16, RAET1G only weakly interacts with NKG2D and does not bind UL16. Differential binding capacities of the two highly related ectodomains are mainly due to a substitution of a conserved amino acid in the alpha2 domain of RAET1G. In functional terms, the reduced apparent avidity of RAET1G for NKG2D results in a less-efficient NKG2D down-regulation and NK degranulation. Altogether, RAET1G, like ULBP2, appears broadly expressed, but exhibits a lower apparent avidity for NKG2D due to a mutation in the center of the MHC-like fold.

Glial Cell Line-Derived Neurotrophic Factor Family Members Reduce Microglial Activation via Inhibiting p38MAPKs-Mediated Inflammatory Responses.

Previous studies have shown that glial cell line-derived neurotrophic factor (GDNF) family ligands (GFL) are potent survival factors for dopaminergic neurons and motoneurons with therapeutic potential for Parkinson's disease. However, little is known about direct influences of the GFL on microglia function, which are known to express part of the GDNF receptor system. Using RT-PCR and immunohistochemistrym we investigated the expression of the GDNF family receptor alpha 1 (GFR alpha) and the coreceptor transmembrane receptor tyrosine kinase (RET) in rat microglia in vitro as well as the effect of GFL on the expression of proinflammatory molecules in LPS activated microglia. We could show that GFL are able to regulate microglia functions and suggest that part of the well known neuroprotective action may be related to the suppression of microglial activation. We further elucidated the functional significance and pathophysiological implications of these findings and demonstrate that microglia are target cells of members of the GFL (GDNF and the structurally related neurotrophic factors neurturin (NRTN), artemin (ARTN), and persephin (PSPN)).

CLEC2A: a novel, alternatively spliced and skin-associated member of the NKC-encoded AICL-CD69-LLT1 family.

The human natural killer gene complex (NKC) encodes for numerous C-type lectin-like receptors (CTLR), which are expressed on various immune cells including natural killer (NK) cells and myeloid cells. Certain activation-induced, NKC-encoded CTLR are grouped into the C-type lectin domain family 2 (CLEC2 family) which, in humans, comprises AICL (CLEC2B), CD69 (CLEC2C), and LLT1 (CLEC2D). In this paper, we characterize a novel member of the CLEC2 family, the human orphan gene CLEC2A. The C-type lectin-like domain (CTLD) of CLEC2A is most similar to the CTLD of LLT1 ( approximately 60% similarity). Like mouse CLEC2 family members Clr-b and Clr-g, CLEC2A lacks two highly conserved cysteines (Cys4 and Cys5), which form an intramolecular bond in the CTLD of most CTLR. Alternative splicing of exon 2 and of two distinct terminal exons (exon 5A/B), respectively, gives rise to four CLEC2A variants differing in the usage of the transmembrane domain and/or in the carboxyterminal portion of the CTLD. CLEC2A transcripts were detected primarily in myeloid cell lines, but not in epithelial cell lines. In tissues, CLEC2A is selectively expressed in the skin and, at lower abundance, in hematopoietic and gonadal tissues. Finally, we show that the CLEC2A1 variant is readily expressed at the cell surface, where it may serve as a ligand for NKC-encoded NK receptors.

Human cytomegalovirus-encoded UL16 discriminates MIC molecules by their alpha2 domains.

Human CMV infection results in MHC class I down-regulation and induction of NKG2D ligand expression favoring NK recognition of infected cells. However, human CMV-encoded UL16 counteracts surface expression of several NKG2D ligands by intracellular retention. Interestingly, UL16 interacts with MICB, but not with the closely related MICA, and with UL16-binding proteins (ULBP) ULBP1 and ULBP2, which are only distantly related to MICB, but not with ULPB3 or ULBP4, although all constitute ligands for NKG2D. Here, we dissected the molecular basis of MICA-MICB discrimination by UL16 to elucidate its puzzling binding behavior. We report that the UL16-MICB interaction is independent of glycosylation and demonstrate that selective MICB recognition by UL16 is governed by helical structures of the MICB alpha2 domain. Transplantation of the MICB alpha2 domain confers UL16 binding capacity to MICA, and thus, diversification of the MICA alpha2 domain may have been driven by the selective pressure exerted by UL16.