Two Novel NKG2D Ligands of the Mouse H60 Family with Differential Expression Patterns and Binding Affinities to NKG2D

Abstract

H60, originally described as a dominant minor histocompatibility Ag, is an MHC class I-like molecule that serves as a ligand for the NKG2D receptor. In the present study, we identified two novel mouse chromosome 10-encoded NKG2D ligands structurally resembling H60. These ligands, which we named H60b and H60c, encode MHC class I-like molecules with two extracellular domains. Whereas H60b has a transmembrane region, H60c is a GPI-anchored protein. Recombinant soluble H60b and H60c proteins bound to NKG2D with affinities typical of cell–cell recognition receptors (Kd = 310 nM for H60b and Kd = 8.7 μM for H60c). Furthermore, expression of H60b or H60c rendered Ba/F3 cells susceptible to lysis by NK cells, thereby establishing H60b and H60c as functional ligands for NKG2D. H60b and H60c transcripts were detected only at low levels in tissues of healthy adult mice. Whereas H60b transcripts were detectable in various tissues, H60c transcripts were detected mainly in the skin. Infection of mouse embryonic fibroblasts with murine cytomegalovirus induced expression of H60b, but not H60c or the previously known H60 gene, indicating that transcriptional activation of the three types of H60 genes is differentially regulated. The present study adds two new members to the current list of NKG2D ligands.

ULBP6/RAET1L is an additional human NKG2D ligand

“…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].…”

“…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].…”

NKG2D-Based Cancer Immunotherapy

“…The identified mouse NKG2D ligands include family members of: the MHC I-related family members of retinoic acid early transcript RAE-1(α, β, γ, δ, and ε) and H60 (a, b, c), and the murine ULBP-like transcript 1 (MULT1) (Cerwenka et al, 2000;Diefenbach et al, 2003;O'Callaghan et al, 2001;Takada et al, 2008). All of these ligands only have the MHC class I-like α1 and α2 extracelluar domains.…”

“…H60a, the first ligand of the family was initially identified as a minor histocompatibility antigen by immunizing C57BL⁄6 mice with MHC-identical BALB.B cells (Malarkannan et al, 1998). Two novel members of H60 family were identified, and named as H60b and H60c (Takada et al, 2008). MULT1 is the unique member of the ULBP-like family of mouse NKG2D ligands and was found by database searching for mouse sequences with similarities to human ULBP (Carayannopoulos et al, 2002).…”

Posttranslational regulation of the NKG2D ligand Mult1 in response to cell stress

“…These data suggested that Mult1 cell surface expression is controlled posttranscriptionally, either at the translational or protein level. To test for protein level regulation, the open reading frames of Mult1 or of a distantly related NKG2D ligand, H60c ( 18 ), were N-terminally FLAG tagged and cloned into a retroviral expression vector in the absence of their untranslated regions, which might contain cis-acting elements that regulate mRNA stability. The vector used for this analysis encoded a downstream IRES-GFP cassette, such that GFP expression could be used as a marker to indicate that cells had been successfully transduced and expressed the bicistronic Mult1-GFP mRNA.…”