NKG2A-Expressing Natural Killer Cells Dominate the Response to Autologous Lymphoblastoid Cells Infected with Epstein-Barr Virus

Abstract

Epstein-Barr virus (EBV) is a human γ-herpesvirus that establishes latency and lifelong infection in host B cells while achieving a balance with the host immune response. When the immune system is perturbed through immunosuppression or immunodeficiency, however, these latently infected B cells can give rise to aggressive B cell lymphomas. Natural killer (NK) cells are regarded as critical in the early immune response to viral infection, but their role in controlling expansion of infected B cells is not understood. Here, we report that NK cells from healthy human donors display increased killing of autologous B lymphoblastoid cell lines (LCLs) harboring latent EBV compared to primary B cells. Coculture of NK cells with autologous EBV⁺ LCL identifies an NK cell population that produces IFNγ and mobilizes the cytotoxic granule protein CD107a. Multi-parameter flow cytometry and Boolean analysis reveal that these functional cells are enriched for expression of the NK cell receptor NKG2A. Further, NKG2A⁺ NK cells more efficiently lyse autologous LCL than do NKG2A^- NK cells. More specifically, NKG2A⁺2B4⁺CD16^-CD57^-NKG2C^-NKG2D⁺ cells constitute the predominant NK cell population that responds to latently infected autologous EBV⁺ B cells. Thus, a subset of NK cells is enhanced for the ability to recognize and eliminate autologous, EBV-infected transformed cells, laying the groundwork for harnessing this subset for therapeutic use in EBV⁺ malignancies.

Keywords: B cells; Epstein–Barr virus; NK cells; NKG2A; lymphoblastoid cell lines.

Figure 1

NK cells kill autologous EBV⁺ LCL. (A–F) Expression of NK ligands CD48, HLA-A,B,C, and HLA-E on autologous CD19⁺ B cells and EBV⁺ LCL. (A–C) Representative flow cytometry shows isotype (light gray shaded) or NK ligand-stained (light gray outline) primary CD19⁺ B cells or isotype (dark gray shaded) or NK ligand-stained (dark gray outline) autologous LCLs. (D–F) Stain indices (34) of N = 6 donors. (G) NK killing of CFSE-labeled target cells (721.221, primary B cells and autologous LCL target cells). The percentage of dead target cells, as detected by 7-AAD and CFSE, are shown in representative samples. (H) Specific killing was calculated as described in Section “Materials and Methods.” Each of the N = 7 donors is labeled with a unique symbol. Error bars represent the minimum and maximum values for each condition. All p-values were calculated using the Wilcoxon matched-pairs signed rank test.

Figure 2

NK cells respond to autologous LCL and primary B cells with similar frequency. IFNγ (A), CD107a (B), or both (C) expression on NK cells (CD3⁻CD14⁻CD19⁻CD56⁺) alone (NK only) or after coculture with target cells (721.221 cells, primary B cells, or autologous LCLs). Error bars represent the minimum and maximum values for each condition. Each of the N = 10 donors is labeled with a unique symbol. All p-values were calculated using the Wilcoxon matched-pairs signed rank test.

Figure 3

Higher frequencies of NK cells responding to autologous LCL are NKG2A⁺. Frequency of 2B4⁺, CD16⁺, CD57⁺, NKG2A⁺, NKG2C⁺, and NKG2D⁺ cells within IFNγ⁻ or IFNγ⁺ (A), CD107a⁻ or CD107a⁺ (B), and CD107a⁻IFNγ⁺ or CD107a⁺IFNγ⁺ (C) NK populations after coculture with autologous LCL (N = 10). All p-values were calculated using the Wilcoxon matched-pairs signed rank test.

Figure 4

Higher frequencies of NK cells respond specifically to autologous LCL and not primary B cells are NKG2A⁺. Frequency of CD16⁺, NKG2A⁺, and NKG2C⁺ cells within IFNγ⁺ (A), CD107a⁺ (B), and CD107a⁺IFNγ⁺ (C) NK populations after coculture with primary B cells (open circles) or autologous LCL (filled circles) from N = 10 donors. All p-values were calculated using the Wilcoxon matched-pairs signed rank test.

Figure 5

Higher frequencies of NK cells responding specifically to autologous LCL are NKG2A⁺2B4⁺CD16⁻CD57⁻NKG2C⁻NKG2D⁺. Visualization of the frequencies of 64 different NK receptor combinations in each of N = 10 donors within CD107a⁺IFNγ⁺ NK populations cocultured with primary B cells (A) and autologous LCL (B). The 11 most frequent receptor combinations are shown in color, and the legend describes these combinations of 2B4 (2B), CD16 (16), CD57 (57), NKG2A (2A), NKG2C (2C), and NKG2D (2D). (C) The average difference in each receptor combination between CD107a⁺IFNγ⁺ NK cells from cocultures with primary B cells or autologous LCLs and (D–N) comparisons of the frequencies between CD107a⁺IFNγ⁺ NK populations from coculture with primary B cells (open circles) and autologous LCL (filled circles). All p-values were calculated using the Wilcoxon matched-pairs signed rank test. A Bonferroni correction was used to adjust for multiple comparisons.

Figure 5

Higher frequencies of NK cells responding specifically to autologous LCL are NKG2A⁺2B4⁺CD16⁻CD57⁻NKG2C⁻NKG2D⁺. Visualization of the frequencies of 64 different NK receptor combinations in each of N = 10 donors within CD107a⁺IFNγ⁺ NK populations cocultured with primary B cells (A) and autologous LCL (B). The 11 most frequent receptor combinations are shown in color, and the legend describes these combinations of 2B4 (2B), CD16 (16), CD57 (57), NKG2A (2A), NKG2C (2C), and NKG2D (2D). (C) The average difference in each receptor combination between CD107a⁺IFNγ⁺ NK cells from cocultures with primary B cells or autologous LCLs and (D–N) comparisons of the frequencies between CD107a⁺IFNγ⁺ NK populations from coculture with primary B cells (open circles) and autologous LCL (filled circles). All p-values were calculated using the Wilcoxon matched-pairs signed rank test. A Bonferroni correction was used to adjust for multiple comparisons.

Figure 6

NKG2A⁺ NK cells kill autologous LCL better than NKG2A⁻ NK cells. NKG2A⁺ and NKG2A⁻ NK cells were sorted and placed in a killing assay with CellTrace Violet-labeled autologous LCL targets at a 4:1 effector:target ratio. Specific killing was calculated as described in Section “Materials and Methods.” Each of the N = 6 donors is labeled with a unique symbol. All p-values were calculated using the Wilcoxon matched-pairs signed rank test.