Analysis of Qa-1(b) peptide binding specificity and the capacity of CD94/NKG2A to discriminate between Qa-1-peptide complexes

Abstract

The major histocompatibility complex class Ib protein, Qa-1(b), serves as a ligand for murine CD94/NKG2A natural killer (NK) cell inhibitory receptors. The Qa-1(b) peptide-binding site is predominantly occupied by a single nonameric peptide, Qa-1 determinant modifier (Qdm), derived from the leader sequence of H-2D and L molecules. Five anchor residues were identified in this study by measuring the peptide-binding affinities of substituted Qdm peptides in experiments with purified recombinant Qa-1(b). A candidate peptide-binding motif was determined by sequence analysis of peptides eluted from Qa-1 that had been folded in the presence of random peptide libraries or pools of Qdm derivatives randomized at specific anchor positions. The results indicate that Qa-1(b) can bind a diverse repertoire of peptides but that Qdm has an optimal primary structure for binding Qa-1(b). Flow cytometry experiments with Qa-1(b) tetramers and NK target cell lysis assays demonstrated that CD94/NKG2A discriminates between Qa-1(b) complexes containing peptides with substitutions at nonanchor positions P4, P5, or P8. Our findings suggest that it may be difficult for viruses to generate decoy peptides that mimic Qdm and raise the possibility that competitive replacement of Qdm with other peptides may provide a novel mechanism for activation of NK cells.

Figure 1

Peptide binding to Qa-1^b. Soluble recombinant Qa-1^b (30 nM) was incubated overnight at room temperature in duplicate with 0.2 μM biotin-Qdm4C and the indicated concentrations of unlabeled competitor peptide. Qa-1^b was then captured with anti-β2m Ab and bound biotinylated peptide was detected with europium-streptavidin fluorescence, indicated as counts per second (cps). The dashed line indicates quantity of bound biotin-Qdm4C in the absence of competitor peptide. The polyoma virus peptide MT(389–397), RRLGRTLLL, binds Dk with high affinity (reference 41).

Figure 2

Pool sequencing of peptides eluted from soluble Qa-1^b refolded with a random nonameric library. Recombinant Qa-1^b heavy chain was refolded in the presence of a random nonameric library and human β2m. Free peptide and β2m were removed by gel filtration, and bound peptide was eluted and analyzed by Edman degradation. The yield of each amino acid in pmol (indicated by single letter code) is shown. Positions where the yield of amino acid is at least 150% of the previous cycle are underlined.

Figure 3

Selective incorporation of peptides during in vitro folding of Qa-1^b. Folding reactions were carried out in the presence of pools of Qdm peptides each randomized at a single position. After purification, peptide was eluted from the Qa-1 complexes and analyzed by reversed-phase HPLC, mass spectrometry, and Edman sequencing. Shown are HPLC profiles of the Qdm library randomized at P9 and the peptides eluted from Qa-1 that was folded in the presence of this library. *Peak identified as peptide with Leu at P9 (Qdm).

Figure 4

D^b is the only cellular source of peptide capable of binding to Qa-1^b and inhibiting NK lysis. IL-2–cultured CD3-NK1.1 plus NKG2 plus splenocytes from K^b−/− mice were used as effector cells. Spleen cells from B6 β2m^−/−, B6 K^b−/−, and B6 K^b−/−D^b−/− mice were cultured overnight with ConA (2.5 μg/ml), labeled for 1 h at 37°C with ⁵¹Cr, and used as targets in a standard 4-h ⁵¹Cr-release assay. Qdm peptide (50 μM) was included during culture with ConA, labeling, and lysis phases of the assay for the indicated groups of target cells.

Figure 6

Inhibition of NK cell killing by Qa-1^b peptide complexes. IL-2–cultured CD3⁻NK1.1⁺ NK cells were sorted into Qa-1^b–Qdm tetramer positive and negative populations. After further culture, the activated NK effector cells were tested for their ability to kill Cr⁵¹-labeled Qa-1^b–transfected T2 cell (T237) targets in the presence of the indicated peptides. Targets were incubated at room temperature overnight with 30 μM peptide and ⁵¹Cr. Peptide (30 μM) was included in the killing assay.

Figure 5

Binding of Qa-1^b tetramers containing substituted Qdm peptides to CD94/NKG2A. (A) Flow cytometry profiles were obtained using CD3⁻B220⁻ C57BL/10 splenocytes stained with anti-NK1.1 and Qa-1^b tetramers, prepared with Qdm or substituted Qdm peptides containing Lys at the indicated position. The Dk leader peptide differs from Qdm, containing a Val instead of Ala at P3. Numbers in the upper right hand corner correspond to percentage of NK1.1⁺ cells that are tetramer positive. (B) Chinese hamster ovary transfectants expressing CD94 and NKG2A were stained with Qa-1^b tetramers (solid line) or streptavidin-allophycocyanin (dotted line).