TCRαβ+NK1.1+ (NKT) cells are known to express various NK cell-associated molecules including the Ly49 family of receptors for MHC class I, but its functional significance has been unclear. Here, we examined the expression of Ly49A, C/I and G2 on various NKT cell populations from normal and MHC class I-deficient C57BL/6 mice as well as their responsiveness to α-galactosylceramide (α-GalCer), a potent stimulator of CD1d-restricted NKT cells. The frequency and the level of Ly49 expression varied among NKT cells from different tissues, and were regulated by the expression of MHC class I and CD1d in the host. Stimulation of various NKT cells with α-GalCer suggested that Ly49 expression inversely correlates with the responsiveness of NKT cells to α-GalCer. Moreover, α-GalCer presented by normal dendritic cells stimulated purified Ly49−, but not Ly49+, splenic NKT cells, whereas MHC class I-deficient dendritic cells presented α-GalCer to both Ly49+ and Ly49− NKT cells equally well. Therefore, MHC class I on APCs seems to inhibit activation of NKT cells expressing Ly49. To further characterize CD1d-restricted NKT cells, we generated an α-GalCer-responsive NKT cell line from thymocytes. The line could only be generated from Ly49−NK1.1+CD4+ thymocytes but not from other NKT cell subsets, and it lost expression of NK1.1 and CD4 during culture. Together, these results indicate the functional significance of Ly49 expression on NKT cells.

Murine natural killer (NK) cells express two families of MHC class I-specific receptors, namely the Ly49 family and CD94/NKG2 heterodimers. Stochastic co-expression of these receptors generates diverse receptor repertoires in adult NK-cell populations, whereas fetal NK cells have much more limited receptor diversity as they mostly express CD94/NKG2A but not Ly49. These receptors are also expressed on CD8-T cells and NK1.1+ T cells and regulate their functions, but their expression pattern on NK cells is significantly different from those on T cells. Thus, expression of Ly49 and CD94/NKG2 is developmentally regulated. NK cells acquire the Ly49 family of receptors in an orderly manner as they differentiate from bone marrow progenitors in vitro. Similarly, acquisition of CD94 and NKG2 by NK cells as they differentiate from embryonic stem cells is also orderly To gain insight into the mechanisms regulating Ly49 expression, potential regulatory regions of several Ly49 genes have been examined. Ly49 genes with different expression patterns have remarkably similar sequences in the putative regulatory regions. Finally, a functional Ly49 gene has been identified in baboon, and primate comparisons suggest that functional extinction of the Ly49 gene in the human lineage seems to have been a relatively recent event.

In mice there are two families of MHC class I-specific receptors, namely the Ly49 and CD94/NKG2 receptors. The latter receptors recognize the nonclassical MHC class I Qa-1b and are thought to be responsible for the recognition of missing-self and the maintenance of self-tolerance of fetal and neonatal NK cells that do not express Ly49. Currently, how NK cells acquire individual CD94/NKG2 receptors during their development is not known. In this study, we have established a multistep culture method to induce differentiation of embryonic stem (ES) cells into the NK cell lineage and examined the acquisition of CD94/NKG2 by NK cells as they differentiate from ES cells in vitro. ES-derived NK (ES-NK) cells express NK cell-associated proteins and they kill certain tumor cell lines as well as MHC class I-deficient lymphoblasts. They express CD94/NKG2 heterodimers, but not Ly49 molecules, and their cytotoxicity is inhibited by Qa-1b on target cells. Using RT-PCR analysis, we also report that the acquisition of these individual receptor gene expressions during different stages of differentiation from ES cells to NK cells follows a predetermined order, with their order of acquisition being first CD94; subsequently NKG2D, NKG2A, and NKG2E; and finally, NKG2C. Single-cell RT-PCR showed coexpression of CD94 and NKG2 genes in most ES-NK cells, and flow cytometric analysis also detected CD94/NKG2 on most ES-NK cells, suggesting that the acquisition of these receptors by ES-NK cells in vitro is nonstochastic, orderly, and cumulative.

Multiple NK cell receptors for MHC class I have been identified. They include killer inhibitory receptors and CD94/NKG2 heterodimers in humans and the Ly49 family in mice. Here we report the cloning of murine NKG2A, B and C. The deduced amino acid sequence of mouse NKG2A contains only one consensus cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM). NKG2A from B6 and BALB/c mice differ by six amino acid residues in the extracellular domain. Murine NKG2B, like its human conterpart, appears to be a splice variant of NKG2A and lacks a large portion of the stalk region. Murine NKG2C lacks an ITIM in its cytoplasmic domain, a feature shared by human and rat NKG2C. However, unlike the human counterpart, the transmembrane domain of mouse NKG2C does not contain a charged amino acid residue. Mouse NKG2A mRNA was detected in IL-2-activated NK cells and spleen cells but not in other tissues. The NKG2A gene was localized on the distal portion of chromosome 6 where the NK complex has been located. These results further extend the repertoire of C-type lectin receptors on murine NK cells.

The CD94/NKG2A heterodimer is an inhibitory receptor expressed on a subset of mouse NK cells. CD94/NKG2A recognizes the non-classical MHC class I (class Ib) molecule Qa-1(b) and inhibits NK cytotoxicity. Qa-1(b) presents a peptide derived from the leader sequence of classical MHC class I molecules. Here, we examined the role of CD94/NKG2A in T cell-mediated cytotoxicity. Soluble tetrameric Qa-1(b) bound to almost all CD8(+), but not CD4(+), T cells. This binding seems to be mediated by CD8, because COS cells transfected with CD8 also bound Qa-1(b) tetramer. Therefore, the expression of CD94/NKG2 in T cells was further examined by single-cell RT-PCR. Most murine CD8(+) T cells constitutively expressed CD94 and NKG2A transcripts, whereas they were not detected in CD4(+) T cells. Co-expression of Qa-1(b) and D(k) on target cells significantly inhibited cytotoxicity of D(k)-specific cytotoxic T lymphocytes generated by mixed lymphocyte reaction, indicating that Qa-1(b) on antigen-presenting cells interacts with CD94/NKG2A on CD8 T cells and regulates classical MHC class I-restricted cytotoxic T cells. These results suggest a significant role of CD94/NKG2A as an inhibitory receptor on CD8(+) T cells.