Functional Analysis of the Molecular Factors Controlling Qa1-Mediated Protection of Target Cells from NK Lysis

Abstract

CD94/NKG2 receptors on mouse NK cells recognize the nonclassical class I molecule Qa1 and can deliver inhibitory signals that prevent NK cells from lysing Qa1-expressing cells. However, the exact circumstances under which Qa1 protects cells from NK lysis and, in particular, the role of the dominant Qa1-associated peptide, Qdm, are unclear. In this study, we examined in detail the lysis of Qa1-expressing cells by fetal NK cells that express CD94/NKG2 receptors for Qa1 but that lack receptors for classical class I molecules. Whereas mouse L cells and human C1R cells transfected with Qa1 were resistant to lysis by these effectors, Qa1-transfected TAP-deficient human T2 cells showed no resistance despite expressing high levels of surface Qa1. However, these cells could be efficiently protected by exposure to low concentrations of Qdm peptide or certain Qdm-related peptides. By contrast, even prolonged exposure of TAP-deficient RMA/S cells to high doses of Qdm peptide failed to induce levels of surface Qa1 detectable with a Qa1-specific mAb or to protect them from NK lysis, although such treatment induced sensitivity to lysis by Qa1-specific CTL. Collectively, these findings indicate that high surface expression of Qa1 is necessary but not sufficient for protection, and that effective protection requires the expression of sufficient levels of suitable Qa1-peptide complexes to overcome activatory signals. Results obtained with a series of substituted Qdm peptides suggest that residues at positions 3, 4, 5, and 8 of the Qdm sequence, AMAPRTLLL, are important for recognition of Qa1-Qdm complexes by inhibitory CD94/NKG2 receptors.

A Peptide from Heat Shock Protein 60 Is the Dominant Peptide Bound To Qa-1 in the Absence of the MHC Class Ia Leader Sequence Peptide Qdm

“…Although the cells used to identify this peptide, namely C1R cells, express very low levels of MHC class Ia molecules overall, they do express HLA-B35 at 60-fold reduced levels and HLA-Cw4 at normal levels (44), relative to the parental cell line. Peptides derived from the leader sequences of both of these proteins (VTAPRTVLL and VMAPRTLIL, respectively) have been shown to bind to Qa-1 b with a similar affinity to Qdm (8,9,21,39). However, these two MHC class Ia leader peptides were not detected in the extracts from MHC class I immunoprecipitates.…”

“…Most of these GroEL/Qa-1-reactive T cells cross-react with a peptide derived from the homologous murine protein, hsp60 (GMKFDRGYI), in a Qa-1-restricted fashion (35,36). These two peptides are not able to inhibit NK cell lysis of Qa-1-expressing target cells (21).…”

“…CD94/NKG2 receptors are believed to modulate this process by surveying class Ia expression indirectly. They perform this function by monitoring the TAP-dependent presentation of the dominant leader peptide by Qa-1 and HLA-E (1, 20) in a peptide-specific fashion (9,(21)(22)(23). NK receptors, such as CD94/NKG2 receptors, are also thought to finely regulate the threshold for T cell activation (24,25).…”

Cutting Edge: HLA-E Binds a Peptide Derived from the ATP-Binding Cassette Transporter Multidrug Resistance-Associated Protein 7 aSnd Inhibits NK Cell-Mediated Lysis

“…The alanine in P1 and P3 of the MRP7 peptide are in excellent agreement with amino acids that are accommodated by HLA-E as evidenced by the fact that the P1 and P3 residues of all leaderderived inhibitory peptides are also alanine. Although crystallographic studies indicate that the B pocket of HLA-E may be optimized for a P2 methionine, (10) the data presented here together with the observations from other groups using a soluble refolding assay, NK inhibition, and random peptide libraries clearly substantiate that leucine in P2 is also well-suited for both HLA-E and Qa-1-binding peptides that inhibit NK cell lysis (15,16,26). The presence of a positively charged amino acid within the middle region of an HLA-E-binding peptide is a key component of the sequence selectivity imposed by the HLA-E structure (10).…”

“…Qa-1 b is the murine MHC class Ib functional homologue of HLA-E and several reports have found that both molecules bind a nearly identical repertoire of leader-derived peptides (1)(2)(3)(12)(13)(14)(15)(16). Early studies using L cells transfected with the Qa-1 b -encoding gene T23 showed that the level of surface Qa-1 b is selectively increased following elevated temperature at 42°C (17).…”