Ly49 cluster sequence analysis in a mouse model of diabetes: an expanded repertoire of activating receptors in the NOD genome

Abstract

The mouse Ly49 and human killer cell immunoglobulin-like receptors (KIR) gene clusters encode activating and inhibitory class I MHC receptors on natural killer (NK) cells. A direct correlation between the presence of multiple activating KIR and various human autoimmune diseases including diabetes has been shown. Previous studies have implicated NK cell receptors in the development of diabetes in the non-obese diabetic (NOD) inbred mouse strain. To assess the contribution of Ly49 to NOD disease acceleration the Ly49 gene cluster of these mice was sequenced. Remarkably, the NOD Ly49 haplotype encodes the largest haplotype and the most functional activating Ly49 of any known mouse strain. These activating Ly49 include three Ly49p-related and two Ly49h-related genes. The NOD cluster contains large regions highly homologous to both C57BL/6 and 129 haplotypes, suggesting unequal crossing over as a mechanism of Ly49 haplotype evolution. Interestingly, the 129-like region has duplicated in the NOD genome. Thus, the NOD Ly49 cluster is a unique mix of elements seen in previously characterized Ly49 haplotypes resulting in a disproportionately large number of functional activating Ly49 genes. Finally, the functionality of activating Ly49 in NOD mice was confirmed in cytotoxicity assays.

Effect of Ly49 Haplotype Variance on NK Cell Function and Education

“…It has long been known that different inbred mouse strains exhibit variation in NK cytotoxicity and cytokine production (25). For example, B6 NK cells appear to be more active than 129S1 NK cells (20), despite possessing a smaller Ly49 haplotype (26). One explanation is that activating Ly49 from the B6 strain deliver stronger DAP12-derived signals than similar Ly49 from the 129S1 strain, as evidenced in studies of Ly49D function (27).…”

“…Early RFLP experiments led to the observation that different inbred mouse strains contain variable Ly49 haplotypes (19). For instance, NOD/ShiLtJ mice have the largest Ly49 repertoire with 21 genes, 129S1/SvImJ (129S1) mice possess 19 Ly49 genes, B6 mice possess 15 Ly49 genes, and BALB/c mice have the smallest known Ly49 repertoire with only 8 genes (17,20). The consequences to innate immunity of Ly49 haplotype variation are most obviously manifested in the absence of activating Ly49 receptor function.…”

Impaired natural killer cell self-education and “missing-self” responses in Ly49-deficient mice

“…15 ConA blast target cells were prepared by culturing 2.5 ϫ 10 6 cells/mL of splenocytes for 2 days in cRPMI containing 5 g/mL ConA (SigmaAldrich). To measure production of IFN-␥, 1 ϫ 10 6 splenocytes isolated from poly(I:C)-treated mice (150 g; 18 hours earlier) were incubated for 5 hours with target cells (1:1 ratio) or in plates coated with 1 g/mL anti-NKp46 antibody.…”

“…Adherent lymphokine-activated killer (LAK) cells were generated as previously described. 15 Semiquantitative RT-PCR and genomic PCR RNA was isolated from LAK cells with the use of TRIzol reagent (Invitrogen). cDNA was synthesized with the Superscript First-Strand cDNA synthesis kit (Invitrogen).…”

Optimized Tetramer Analysis Reveals Ly49 Promiscuity for MHC Ligands

“…Additionally, two Ly49, Ly49B and Ly49Q, are expressed on myeloid-derived cells, including macrophages, neutrophils, and DCs (6,7). Initial cDNA screening of inbred mouse strains revealed extreme genetic diversity and polymorphism in different mouse strains (8), which was later revealed to be a result of genomic plasticity within the Ly49 region resulting in cluster sizes ranging from 8 to 22 Ly49 genes (9,10). Most Ly49 receptors identified to date are inhibitory and contain a cytoplasmic ITIM domain encoded in exon 2 that becomes phosphorylated during ligand binding.…”