Optimized Tetramer Analysis Reveals Ly49 Promiscuity for MHC Ligands

Abstract

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 (β2-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-2b–expressing MC57G, and H-2k–expressing L929. The capacity to bind to H-2Db– and H-2Kb–soluble MHC-I tetramers containing either human or murine β2-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-2Db in addition to H-2Kb 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.

Ly49 receptors: evolution, genetic diversity, and impact on immunity

“…Ly49 molecules are able to interact with MHC‐I molecules in cis (MHC‐I molecules expressed on the same cell) or in trans (MHC‐I molecules expressed on another cell) . Recent studies using mouse‐derived MHC‐I tetramers to evaluate Ly49:MHC‐I binding in the absence of cis interactions have revealed a broader binding specificity between Ly49 and MHC‐I molecules than previously appreciated . Ly49:MHC‐I binding specificity has also been evaluated in mice which express single and multiple MHC‐I molecules to circumvent the limitations of soluble tetramer binding studies .…”

Structural Basis for Recognition of Cellular and Viral Ligands by NK Cell Receptors

“…In mice, the Ly49 family includes at least 23 members (Ly49A–W), along with multiple allelic variants (96, 97). Most Ly49s inhibit NK-cell-mediated cytolysis upon recognizing one or more H-2D or H-2K alleles (96, 98, 99). However, some Ly49s are activating.…”

Mutagenesis of Ly49B Reveals Key Structural Elements Required for Promiscuous Binding to MHC Class I Molecules and New Insights into the Molecular Evolution of Ly49s

“…Such cis interactions with cI molecules expressed on the same cells have been reported for several Ly49s, and they can strongly inhibit the binding of these Ly49s to soluble cI multimers (8,46,47). Although in one of these studies it was reported that Ly49B does not engage in cis interactions with mouse cI molecules (8), the possibility that Ly49B engages in cis interactions with rat cI molecules on YB2 cells was directly addressed by exposing Ly49B-transfected cells to acid under conditions previously demonstrated to selectively disrupt cI molecules and liberate Ly49s from sequestration by endogenous cIs (46).…”