Characterization of a Novel Killer Cell Lectin-Like Receptor (KLRH1) Expressed by Alloreactive Rat NK Cells

Abstract

NK cells have the ability to recognize and kill MHC-mismatched hemopoietic cells. In the present study, strain-specific differences in the rat NK allorecognition repertoire were exploited to generate Abs against receptors that may be involved in allogeneic responses. A mAb termed STOK9 was selected, and it reacted with subsets of NK cells and NKR-P1+ T cells from certain rat strains possessing highly alloreactive NK cells. The STOK9+ NK subset was broadly alloreactive and lysed Con A lymphoblast targets from a range of MHC-mismatched strains. The mAb STOK9 precipitated a 75-kDa dimeric glycoprotein from NK lysates. Expression cloning revealed that each monomer consisted of 231 aa with limited homology to other previously characterized killer cell lectin-like receptors (KLRs). This glycoprotein therefore constitutes a novel KLR branch, and it has been termed KLRH1. A gene in the central region of the natural killer gene complex on rat chromosome 4 encodes KLRH1. A mouse homolog appears to be present as deduced from analyses of genomic trace sequences. The function of KLRH1 is unknown, but it contains an immunoreceptor tyrosine-based inhibitory motif, suggesting an inhibitory function. The MHC haplotype of the host appears to influence KLRH1 expression, suggesting that it may function as an MHC-binding receptor on subsets of NK cells and T lymphocytes.

Ly49B Is Expressed on Multiple Subpopulations of Myeloid Cells

“…Initial attempts to reveal inhibitory activity using reverse Ab-dependent cellular cytotoxicity assays were unsuccessful, but negative results in such assays have been reported previously with receptors of known inhibitory capability (see Discussion in Ref. 64), and alternative approaches will need to be explored. Additional priorities for future work include the determination of the three-dimensional structure of Ly49B and identification of its ligands.…”

Two Structurally Related Rat Ly49 Receptors with Opposing Functions (Ly49 Stimulatory Receptor 5 and Ly49 Inhibitory Receptor 5) Recognize Nonclassical MHC Class Ib-Encoded Target Ligands

“…Ly49i5 was cloned using the ZAP-Express system (Stratagene) and a previously described cDNA library from KLRH1 ϩ NK cells (30), except that the primary cDNA was ligated into the ZAP-Express vector instead of into pMET7. Complexity of the primary phage library comprised Ͼ2 ϫ 10 6 clones.…”

“…In short, a monolayer of DT381 cells was transiently transfected with the cDNA expression library referred to above in the pMET7 vector (30). After 2 days, cells were stained with mAb Fly5, washed, and panned on petri dishes precoated with rabbit anti-mouse IgG (Valeant Pharmaceuticals/Cappel).…”

Ly-49s3 Is a Promiscuous Activating Rat NK Cell Receptor for Nonclassical MHC Class I-Encoded Target Ligands

“…The cDNA library used to clone Ly-49s3 was generated from an NK cell subset (KLRH1 ϩ ) from PVG rats, and has been described previously (23). In short, template mRNA from IL-2-activated KLRH1 ϩ NK cells was used for the production of adapted cDNA, which was unidirectionally ligated into the EcoRI (5Ј) and XhoI (3Ј) sites of the expression vector pMET7 (DNAX, Palo Alto, CA).…”

“…Thus, cDNAs for DAP12-associated receptors can be identified in 293T cells stably transfected with DAP12, by their ability to induce cell surface expression of DAP12. We used this expression requirement of DAP12 to screen a cDNA library derived from a highly alloreactive NK cell subset (KLRH1 ϩ ) from the PVG rat strain (23). By transiently transfecting the cDNA library into 293T cells stably expressing FLAG-tagged mouse DAP12 (293T-DAP12/FLAG cells), we were able to isolate several clones that induced DAP12/FLAG expression on the cell surface.…”