Optimized tetramer analysis reveals Ly49 promiscuity for MHC ligands.

Murine Ly49 receptors, which are expressed mainly on NK and NKT cells, interact with MHC class I (MHC-I) molecules with varying specificity. Differing reports of Ly49/MHC binding affinities may be affected by multiple factors, including cis versus trans competition and species origin of the MHC-I L chain (ß2-microglobulin). To determine the contribution of each of these factors, Ly49G, Ly49I, Ly49O, Ly49V, and Ly49Q receptors from the 129 mouse strain were expressed individually on human 293T cells or the mouse cell lines MHC-I-deficient C1498, H-2(b)-expressing MC57G, and H-2(k)-expressing L929. The capacity to bind to H-2D(b)- and H-2K(b)-soluble MHC-I tetramers containing either human or murine ß2-microglobulin L chains was tested for all five Ly49 receptors in all four cell lines. We found that most of these five inhibitory Ly49 receptors show binding for one or both self-MHC-I molecules in soluble tetramer binding assays when three conditions are fulfilled: 1) lack of competing cis interactions, 2) tetramer L chain is of mouse origin, and 3) Ly49 is expressed in mouse and not human cell lines. Furthermore, Ly49Q, the single known MHC-I receptor on plasmacytoid dendritic cells, was shown to bind H-2D(b) in addition to H-2K(b) when the above conditions were met, suggesting that Ly49Q functions as a pan-MHC-Ia receptor on plasmacytoid dendritic cells. In this study, we have optimized the parameters for soluble tetramer binding analyses to enhance future Ly49 ligand identification and to better evaluate specific contributions by different Ly49/MHC-I pairs to NK cell education and function.

Ly49Q positively regulates type I IFN production by plasmacytoid dendritic cells in an immunoreceptor tyrosine-based inhibitory motif-dependent manner.

Plasmacytoid dendritic cells (pDC) are the major producers of type I IFN during the initial immune response to viral infection. Ly49Q, a C-type lectin-like receptor specific for MHC-I, possesses a cytoplasmic ITIM and is highly expressed on murine pDC. Using Ly49Q-deficient mice, we show that, regardless of strain background, this receptor is required for maximum IFN-α production by pDC. Furthermore, Ly49Q expression on pDC, but not myeloid dendritic cells, is necessary for optimal IL-12 secretion, MHC-II expression, activation of CD4(+) T cell proliferation, and nuclear translocation of the master IFN-α regulator IFN regulatory factor 7 in response to TLR9 agonists. In contrast, the absence of Ly49Q did not affect plasmacytoid dendritic cell-triggering receptor expressed on myeloid cells expression or pDC viability. Genetic complementation revealed that IFN-α production by pDC is dependent on an intact tyrosine residue in the Ly49Q cytoplasmic ITIM. However, pharmacological inhibitors and phosphatase-deficient mice indicate that Src homology 2 domain-containing phosphatase 1 (SHP)-1, SHP-2, and SHIP phosphatase activity is dispensable for this function. Finally, we observed that Ly49Q itself is downregulated on pDC in response to CpG exposure in an ITIM-independent manner. In conclusion, Ly49Q enhances TLR9-mediated signaling events, leading to IFN regulatory factor 7 nuclear translocation and expression of IFN-I genes in an ITIM-dependent manner that can proceed without the involvement of SHP-1, SHP-2, and SHIP.

Impaired natural killer cell self-education and "missing-self" responses in Ly49-deficient mice.

Ly49-mediated recognition of MHC-I molecules on host cells is considered vital for natural killer (NK)-cell regulation and education; however, gene-deficient animal models are lacking because of the difficulty in deleting this large multigene family. Here, we describe NK gene complex knockdown (NKC(KD)) mice that lack expression of Ly49 and related MHC-I receptors on most NK cells. NKC(KD) NK cells exhibit defective killing of MHC-I-deficient, but otherwise normal, target cells, resulting in defective rejection by NKC(KD) mice of transplants from various types of MHC-I-deficient mice. Self-MHC-I immunosurveillance by NK cells in NKC(KD) mice can be rescued by self-MHC-I-specific Ly49 transgenes. Although NKC(KD) mice display defective recognition of MHC-I-deficient tumor cells, resulting in decreased in vivo tumor cell clearance, NKG2D- or antibody-dependent cell-mediated cytotoxicity-induced tumor cell cytotoxicity and cytokine production induced by activation receptors was efficient in Ly49-deficient NK cells, suggesting MHC-I education of NK cells is a single facet regulating their total potential. These results provide direct genetic evidence that Ly49 expression is necessary for NK-cell education to self-MHC-I molecules and that the absence of these receptors leads to loss of MHC-I-dependent "missing-self" immunosurveillance by NK cells.

Effect of Ly49 haplotype variance on NK cell function and education.

The class I MHC-specific receptors expressed by murine NK cells exhibit remarkable variation. Specific activating killer Ig-related receptor/Ly49 have major effects on autoimmune and infectious disease induction and outcome in humans and mice. However, these studies are greatly affected by individual background genetics. Furthermore, the educational impact of variable inhibitory KIR/Ly49 gene numbers on NK cell development and the subsequent ability to survey for MHC class I (MHC-I) expression remain unknown. To address these questions, Ly49 congenic mice were generated that maintain a 129-derived Ly49 gene cluster on a C57BL/6 genetic background (B6.Ly49(129) mice), and the in vitro and in vivo NK cell function of these mice was compared with their inbred parental 129S1 and C57BL/6 counterparts. Notably, target cell recognition directed by activating Ly49 receptors was profoundly affected by allelic variation in B6.Ly49(129) congenic cells versus C57BL/6 NK cells. Furthermore, when assessing NK cell function based on education and subsequent recognition of the C57BL/6 MHC-I haplotype by inhibitory Ly49 receptors, B6.Ly49(129) congenic mice exhibited robust NK cell activity, demonstrating efficient NK cell education by the 129S1 Ly49 cluster during development. The responsiveness of NK cells expressing 129S1 Ly49 was shown to be mediated by subsets expressing one or more self-MHC receptors, including Ly49I, Ly49O, Ly49V, and NKG2A. These findings demonstrate that the genetically segregating and diverse MHC-I and Ly49 loci in mice exhibit independent and epistatic effects on NK cell education that can be uncoupled during the intercrossing of inbred strains.

Positive regulation of plasmacytoid dendritic cell function via Ly49Q recognition of class I MHC.

Plasmacytoid dendritic cells (pDCs) are an important source of type I interferon (IFN) during initial immune responses to viral infections. In mice, pDCs are uniquely characterized by high-level expression of Ly49Q, a C-type lectin-like receptor specific for class I major histocompatibility complex (MHC) molecules. Despite having a cytoplasmic immunoreceptor tyrosine-based inhibitory motif, Ly49Q was found to enhance pDC function in vitro, as pDC cytokine production in response to the Toll-like receptor (TLR) 9 agonist CpG-oligonucleotide (ODN) could be blocked using soluble monoclonal antibody (mAb) to Ly49Q or H-2K(b). Conversely, CpG-ODN-dependent IFN-alpha production by pDCs was greatly augmented upon receptor cross-linking using immobilized anti-Ly49Q mAb or recombinant H-2K(b) ligand. Accordingly, Ly49Q-deficient pDCs displayed a severely reduced capacity to produce cytokines in response to TLR7 and TLR9 stimulation both in vitro and in vivo. Finally, TLR9-dependent antiviral responses were compromised in Ly49Q-null mice infected with mouse cytomegalovirus. Thus, class I MHC recognition by Ly49Q on pDCs is necessary for optimal activation of innate immune responses in vivo.

Recognition of H-2K(b) by Ly49Q suggests a role for class Ia MHC regulation of plasmacytoid dendritic cell function.

Ly49Q is a member of the polymorphic Ly49 family of NK cell receptors that displays both a high degree of conservation and a unique expression pattern restricted to myeloid lineage cells, including plasmacytoid dendritic cells (pDC). The function and ligand specificity of Ly49Q are unknown. Here, we use reporter cell analysis to demonstrate that a high-affinity ligand for Ly49Q is present on H-2(b), but not H-2(d), H-2(k), H-2(q), or H-2(a)-derived tumor cells and normal cells ex vivo. The ligand is peptide-dependent and MHC Ia-like, as revealed by its functional absence on cells deficient in TAP-1, beta(2)m, or H-2K(b)D(b) expression. Furthermore, Ly49Q is specific for H-2K(b), as the receptor binds peptide-loaded H-2K(b) but not H-2D(b) complexes, and Ly49Q recognition can be blocked using anti-K(b) but not anti-D(b) mAb. Greater soluble H-2K(b) binding to ligand-deficient pDC also suggests cis interactions of Ly49Q and H-2K(b). These results demonstrate that Ly49Q efficiently binds H-2K(b) ligand, and suggest that pDC function, like that of NK cells, is regulated by classical MHC Ia molecules. MHC recognition capability by pDC has important implications for the role of this cell type during innate immune responses.

Molecular and genetic basis for strain-dependent NK1.1 alloreactivity of mouse NK cells.

NK1.1 alloantigen expression can be used to define NK cells in certain mouse strains, such as B6 (NKR-P1C) and SJL (NKR-P1B). However, BALB/c NK cells do not react with the anti-NK1.1 mAb, PK136. To investigate the NK1.1(-) phenotype of BALB/c NK cells, we have undertaken NK1.1 epitope mapping and genomic analysis of the BALB/c Nkrp1 region. Bacterial artificial chromosome library analysis reveals that, unlike the Ly49 region, the Nkrp1-Ocil/Clr region displays limited genetic divergence between B6 and BALB/c mice. In fact, significant divergence is confined to the Nkrp1b and Nkrp1c genes. Strikingly, the B6 Nkrp1d gene appears to represent a divergent allele of the Nkrp1b gene in BALB/c mice and other strains. Importantly, BALB/c NK cells express abundant and functional Nkrp1 transcripts, and the BALB/c NKR-P1B receptor functionally binds Ocil/Clr-b ligand. However, the BALB/c NKR-P1B/C sequences differ from those of the known NK1.1 alloantigens, and epitope mapping demonstrates that directed mutation of a single amino acid in the NKR-P1B(BALB) protein confers NK1.1 reactivity. Thus, PK136 mAb recognizes, in part, a distal C-terminal epitope present in NKR-P1B(Sw/SJL) and NKR-P1C(B6), but absent in NKR-P1A/D/F(B6) and NKR-P1B/C(BALB). Allelic divergence of the Nkrp1b/c gene products and limited divergence of the BALB/c Nkrp1-Ocil/Clr region explain a longstanding confusion regarding the strain-specific NK1.1 alloantigen reactivity of mouse NK cells.