Peptides isolated from HLA-Cw*0304 confer different degrees of protection from natural killer cell-mediated lysis

Abstract

HLA class I molecules bind peptides derived from proteins degraded in the cytoplasm and display them for surveillance by the immune system. The recognition of HLA class I molecules by natural killer (NK) cells generally inhibits the lytic process. To investigate the role of peptides in the interaction between HLA class I molecules and NK receptors, we first had to identify representative endogenous peptides. Individual peptides bound to HLA-Cw*0304 were isolated and sequenced by tandem mass spectrometry. These peptides ranged in length from 8 to 11 residues and shared an alanine at position 2 and a C-terminal leucine. The murine transporters associated with antigen processing (TAP)-deficient cell line RMA-S was transfected with HLA-Cw*0304 to test whether HLA molecules loaded with a single peptide could deliver the inhibitory signal to NK cells expressing p58.2, which is a killer cell inhibitory receptor known to interact with HLA molecules bearing the HLA-Cw3 public epitope. We found that, in the absence of exogenous peptides, the HLA-Cw*0304 transfectants were killed at levels comparable to untransfected RMA-S cells whereas protection from lysis required both HLA-Cw*0304 heavy chain expression and an exogenously added HLA-Cw*0304-binding peptide. Importantly, not only were HLA-Cw*0304-binding peptides required for protection, but the ability of individual peptides to provide protection differed widely. These studies indicate that the ability to distinguish between subsets of peptides may be a general feature of HLA class I recognition by NK cells.

Figure 1

CAD mass spectrum of peptide ions at m/z 869 (peptide 8). Predicted masses for fragment ions of types b and y (29) are shown above and below the deduced sequence, respectively. Ions observed in the spectrum are underlined. Interpretation of CAD spectra is fully explained elsewhere (29). Because Ile and Leu are of identical mass, they cannot be differentiated on the triple quadrupole instrument and are specified here as Lxx.

Figure 2

Cell surface stability of HLA-Cw*0304. RMA-S cells, and RMA-S-HLA-Cw*0304 transfectants were assayed for cell surface expression at t = 0 min (open bar) and after 3 h at 37°C (filled bar). β₂m alone or β₂m plus the indicated peptide was added to the RMA-S-HLA-Cw*0304 transfectants as described.

Figure 4

A subset of endogenous peptides confers protection from lysis by p58.2⁺ NK clones. The same NK clones as in Fig. 2 (JS14 is not shown) were assayed in a cytotoxicity assay against RMA-S-HLA-Cw*0304 cells plus β₂m and with different HLA-Cw*0304-specific peptides. The data are expressed as relative lysis (see Methods) at E/T ratio of 6:1. JS1, JS3, JS10, JS14, and JS19 NK clones were also assayed at E/T ratio 3:1 with similar results.

Figure 3

Peptides are required to induce protection from lysis by p58.2⁺ NK clones. p58.2⁺ (JS1, JS3, JS10, JS19, JS56, and JS63) and p58.2⁻ (JS14 and JS44) NK clones were assayed in a cytotoxicity assay against RMA-S (filled bar), RMA-S-HLA-Cw*0304 (striped bar), RMA-S-HLA-Cw*0304 plus β₂m (shaded bar), and RMA-S-HLA-Cw*0304 plus β₂m and peptide GAVDPLLAL (open bar). The data are expressed as the percentage of specific lysis at a E/T ratio of 6:1. JS1, JS3, JS10, JS14, and JS19 NK clones were also assayed at E/T ratio 3:1 with similar results (data not shown).