LMP-1–derived peptides are a potent inhibitor of NKG2A⁺NK cells. (A) HLA-E stabilization assay: K562-HLA-E∗0103/0103 or K562-HLA-E∗0101/0101 cells were incubated together with 300 μM of the positive control (VMAPRTLIL) or the LMP-1–derived GGDPHLPTL, GSDPHLPTL, GGDPHLPPL, GGDPPLPTL, GCDPHLPTL, GIDPHLPTL, GAGPHLPTL, GGDTPLPTL, GDDPHLPTL, GGDPHVPTL, and GTDPHLPTL peptides. The surface expression of HLA-E was then assessed after 16 hours of coculture by flow cytometry. Box plot represents the mean (±SD) of 3 independent replicates. Each peptide was compared with the negative control (dashed black line) (ie, K562-HLA-E∗0103/0103 cells or K562-HLA-E∗0101/0101 cells without peptides) using the Mann-Whitney test. (B) NKG2A⁺ inhibition assay: K562-HLA-E∗0103/0103 or K562-HLA-E∗0101/0101 cells were first incubated together with 300 μM of the positive control (VMAPRTLIL) or the LMP-1–derived GGDPHLPTL, GSDPHLPTL, GGDPHLPPL, GGDPPLPTL, GCDPHLPTL, GIDPHLPTL, GAGPHLPTL, GGDTPLPTL, GDDPHLPTL, GGDPHVPTL, and GTDPHLPTL peptides and then incubated together with preactivated enriched CD56⁺ NK cells. The percentage of CD107-expressing NKG2A⁺ NK cells was assessed by flow cytometry. Plots represent the mean (±SD) of 12 independent biological replicates. Each peptide was compared with the negative control (dashed black line) (ie, K562-HLA-E∗0103/0103 cells or K562-HLA-E∗0101/0101 cells without peptides) using the Mann-Whitney test. (C-D) EBV dissemination assay: K562-CR2-HLA-E∗0103/0103 or K562-CR2-HLA-E∗0101/0101 cells were infected with the EBV B95-8 isolate and cultured together with sorted NKG2A⁺ NK cells for 2, 4, 8, 12, or 14 days. The percentage of BZLF1⁺ K562-CR2-HLA-E∗0103/0103 or K562-CR2-HLA-E∗0101/0101 cells (C) or granzyme B–expressing NKG2A⁺ NK cells (D) was assessed by flow cytometry after 2, 4, 8, 12, or 14 days of coculture. The dashed black line indicates the percentage of granzyme B–expressing NKG2A⁺ NK cells in the absence of any peptides. (C) Plots represent the mean (±SD) of 12 independent biological replicates. Repeated measures 1-way analysis of variance (with the Geisser-Greenhouse correction) was used to analyze differences between the peptides. (D) Box plot represents the mean (±SD) of 12 independent replicates. Each peptide was compared with the negative control (dashed black line) (ie, K562-HLA-E∗0103/0103 cells or K562-HLA-E∗0101/0101 cells without peptides) using the Mann-Whitney test. P < .05 was considered significant. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. MFI, mean fluorescence intensity; ns, not significant; Pos. Ctrl., positive control.