Visualization of inhibitory Ly49 receptor specificity with soluble major histocompatibility complex class I tetramers

Abstract

Murine natural killer (NK) cells are inhibited from killing their targets by the interaction between inhibitory, C-type lectin like Ly49 receptors and major histocompatibility complex (MHC) class I molecules. The receptors have overlapping specificity, and it has been difficult to analyze specific aspects of the interaction between different Ly49 receptors and their respective ligands. We have addressed this problem using tetramers of bacterially expressed, non-glycosylated, MHC class I molecules refolded with different peptides. Our results indicate that this technology is useful for analysis of Ly49 receptor specificity as well as for monitoring of NK cell subsets, with the following major conclusions emerging from this study: (1) tetramers of H-2D(d) bound the Ly49A receptor; the MHC associated glycan, previously suggested to be involved in recognition by this receptor, is thus not required for Ly49A receptor binding; (2) in support and extension of a recent report indicating peptide selectivity in the recognition of H-2K(b) by Ly49C(+) cells, H-2K(b) tetramer binding to Ly49C receptors was strongly influenced by the peptide presented by the MHC class I molecule; (3) tetramer binding allowed visualization of interactions that have not previously been detected in functional studies, such as the recognition of H-2D(b) by Ly49A and Ly49C.

Tracking the Response of Natural Killer T Cells to a Glycolipid Antigen Using Cd1d Tetramers

“…Finally, the affinity might be too low to detect binding, by analogy with the interaction of CD8 with most MHC class I tetramers. It is clear, however, that some NK receptor–class I interactions have a higher affinity 32 33 34 47 48. Further experiments will be required to uncover the basis for this CD1d-mediated inhibition of NK cells.…”

Activating and inhibitory receptors of natural killer cells

“…Other Ly49 receptors include activating Ly49P and Ly49W, which are also reported to interact with H‐2D d 33 , 34 . The ‘recognition’ of MHC class I by Ly49 receptors require the presence of a peptide bound in the groove of the MHC molecule, although the specificity of this peptide‐binding varies between Ly49 receptors 35 , 36 , 37 , 38 . Interestingly, it has been reported that Ly49 molecules can bind MHC molecules in cis (that is, on the same membrane), which can reduce the capacity for trans binding and hence reduce the required signaling threshold for activation 39 , 40 .…”

Influence of SHIP on the NK Repertoire and Allogeneic Bone Marrow Transplantation

“…Thus, the repertoire distortion in adult SHIP Ϫ/Ϫ mice leads to an NK compartment dominated by a subset of cells with the following repertoire: Ly49A ϩ C ϩ D -G2 -I -CD94 -. In vitro and in vivo studies indicated that Ly49C and Ly49A can bind ligands in the H2b haplotype of SHIP Ϫ/Ϫ mice; however, these two receptors also bind and transmit inhibitory signals from ligands in most or all H2 haplotypes (9)(10)(11)(12). Therefore, SHIP deficiency leads to an NK inhibitory repertoire that is both self-specific and promiscuous for other ligands.…”