Abstract
The C-type lectin-like receptor NKG2D contributes to the immunosurveillance of virally infected and malignant cells by cytotoxic lymphocytes. A peculiar and puzzling feature of the NKG2D-based immunorecognition system is the high number of ligands for this single immunoreceptor. In humans, there are a total of eight NKG2D ligands (NKG2DL) comprising two members of the MIC (MICA, MICB) and six members of the ULBP family of glycoproteins (ULBP1 to ULBP6). While MICA has been extensively studied with regard to its biochemistry, cellular expression and function, very little is known about the NKG2DL ULBP4. This is, at least in part, due to its rather restricted expression by very few cell lines and tissues. Recently, constitutive ULBP4 expression by human monocytes was reported, questioning the view of tissue-restricted ULBP4 expression. Here, we scrutinized ULBP4 expression by human peripheral blood mononuclear cells and monocytes by analyzing ULBP4 transcripts and ULBP4 surface expression. In contrast to MICA, there was no ULBP4 expression detectable, neither by freshly isolated monocytes nor by PAMP-activated monocytes. However, a commercial antibody erroneously indicated surface ULBP4 on monocytes due to a non-ULBP4-specific binding activity, emphasizing the critical importance of validated reagents for life sciences. Collectively, our data show that ULBP4 is not expressed by monocytes, and likely also not by other peripheral blood immune cells, and therefore exhibits an expression pattern rather distinct from other human NKG2DL.
Highlights
The human UL16-binding proteins (ULBPs) are a family of six MHC class I-related glycoproteins (ULBP1-6) encoded in the RAET1 gene cluster of the long arm of human chromosome 6 [1,2]
No DUMO1 binding was detected to any of the immune cell subsets of any of the donors. These results suggested that there are no ULBP4 molecules on the cell surface of T cells, NK cells, and monocytes
To test whether ULBP4 surface expression by monocytes may be inducible upon activation, as reported for other human NKG2D ligand (NKG2DL) [35], we broadly enriched monocytes from freshly isolated PBMC using a pan monocyte isolation kit (S1 Fig), and cultured these in vitro for 16 h prior to flow cytometric analysis
Summary
The human UL16-binding proteins (ULBPs) are a family of six MHC class I-related glycoproteins (ULBP1-6) encoded in the RAET1 gene cluster of the long arm of human chromosome 6 [1,2]. They consist of an MHC class I-like α1α2 domain anchored in the cell membrane either by a GPI-anchor (ULBP1-3, ULBP6) or via a transmembrane domain (ULBP4, ULBP5) [1,2,3,4]. ULBPs together with the MHC-encoded MHC class I chain-related glycoproteins A and B (MICA and MICB) constitute the ligands of the activating immunoreceptor NKG2D [1,2,3,4,5]. NKG2D ligation promotes cytolysis of NKG2D ligand (NKG2DL)-expressing target cells and cytokine
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