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. 2022 Dec 9;141(13):1560–1573. doi: 10.1182/blood.2022017650

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Figure 3. — BZLF1-dervied SQAPLPCVL peptides are a potent inhibitor of NKG2A⁺ cells. (A-D) NKG2A⁺ inhibition assays: 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 BZLF1-dervied SQAPLPCVL peptide and then incubated together with (A-B) preactivated enriched CD56⁺ NK or (C-D) preactivated enriched CD8⁺ T cells. The percentage of CD107-expressing NKG2A⁺ NK cells (A) or IFN-γ–expressing NKG2A⁺ CD8⁺ T cells (C) was assessed by flow cytometry. Each peptide was compared with the negative control (dashed black line) (ie, K562-HLA-E∗0103/0103 or K562-HLA-E∗0101/0101 cells without peptides) using the Mann-Whitney test. (B,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 (B) or sorted NKG2A⁺ CD8⁺ T cells (D) for 2, 4, 8, 12, or 14 days. The percentage (B,D) of BZLF1+ K562-CR2-HLA-E∗0103/0103 or K562-CR2-HLA-E∗0101/0101 cells was assessed after 2, 4, 8, 12, or 14 days, and granzyme B–expressing NKG2A⁺ NK cells (B) or NKG2A⁺ CD8⁺ T cells (D) were assessed by flow cytometry after 14 days of coculture. Repeated measures 1-way analysis of variance (ANOVA) (with the Geisser-Greenhouse correction) was used to analyze differences between the peptides and the granzyme B–expressing NKG2A⁺ NK (B) or NKG2A⁺ CD8⁺ T cells (D) without peptides. (A,C) Plot represents the mean (±SD) of 12 independent biological replicates. (E-F) Analysis of the percentage (E) and the expression level (mean fluorescence intensity [MFI]) (F) of NKG2A⁺ expressing non–SQAPLPCVL-specific CD8⁺ T cells and NKG2A⁺ expressing SQAPLPCVL-specific T cells. The NKG2A MFI was assessed in both NKG2A⁺ CD8⁺ T-cell subsets in 12 EBV-seropositive blood donors by flow cytometry and compared using the Wilcoxon matched t-test. (G) 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⁺ expressing SQAPLPCVL-specific T cells or sorted NKG2A^–SQAPLPCVL-specific T 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 was assessed by flow cytometry after 2, 4, 8, 12, or 14 days of coculture. RM 1-way ANOVA (with the Geisser-Greenhouse correction) was used to analyze differences between the cell subsets. P < .05 was considered significant. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. Pos. Ctrl., positive control.

Figure 3. — BZLF1-dervied SQAPLPCVL peptides are a potent inhibitor of NKG2A⁺ cells. (A-D) NKG2A⁺ inhibition assays: 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 BZLF1-dervied SQAPLPCVL peptide and then incubated together with (A-B) preactivated enriched CD56⁺ NK or (C-D) preactivated enriched CD8⁺ T cells. The percentage of CD107-expressing NKG2A⁺ NK cells (A) or IFN-γ–expressing NKG2A⁺ CD8⁺ T cells (C) was assessed by flow cytometry. Each peptide was compared with the negative control (dashed black line) (ie, K562-HLA-E∗0103/0103 or K562-HLA-E∗0101/0101 cells without peptides) using the Mann-Whitney test. (B,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 (B) or sorted NKG2A⁺ CD8⁺ T cells (D) for 2, 4, 8, 12, or 14 days. The percentage (B,D) of BZLF1+ K562-CR2-HLA-E∗0103/0103 or K562-CR2-HLA-E∗0101/0101 cells was assessed after 2, 4, 8, 12, or 14 days, and granzyme B–expressing NKG2A⁺ NK cells (B) or NKG2A⁺ CD8⁺ T cells (D) were assessed by flow cytometry after 14 days of coculture. Repeated measures 1-way analysis of variance (ANOVA) (with the Geisser-Greenhouse correction) was used to analyze differences between the peptides and the granzyme B–expressing NKG2A⁺ NK (B) or NKG2A⁺ CD8⁺ T cells (D) without peptides. (A,C) Plot represents the mean (±SD) of 12 independent biological replicates. (E-F) Analysis of the percentage (E) and the expression level (mean fluorescence intensity [MFI]) (F) of NKG2A⁺ expressing non–SQAPLPCVL-specific CD8⁺ T cells and NKG2A⁺ expressing SQAPLPCVL-specific T cells. The NKG2A MFI was assessed in both NKG2A⁺ CD8⁺ T-cell subsets in 12 EBV-seropositive blood donors by flow cytometry and compared using the Wilcoxon matched t-test. (G) 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⁺ expressing SQAPLPCVL-specific T cells or sorted NKG2A^–SQAPLPCVL-specific T 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 was assessed by flow cytometry after 2, 4, 8, 12, or 14 days of coculture. RM 1-way ANOVA (with the Geisser-Greenhouse correction) was used to analyze differences between the cell subsets. P < .05 was considered significant. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001. Pos. Ctrl., positive control.