Cytomegalovirus reactivation after allogeneic transplantation promotes a lasting increase in educated NKG2C+ natural killer cells with potent function

Abstract

During mouse cytomegalovirus (CMV) infection, a population of Ly49H(+) natural killer (NK) cells expands and is responsible for disease clearance through the induction of a "memory NK-cell response." Whether similar events occur in human CMV infection is unknown. In the present study, we characterized the kinetics of the NK-cell response to CMV reactivation in human recipients after hematopoietic cell transplantation. During acute infection, NKG2C(+) NK cells expanded and were potent producers of IFNγ. NKG2C(+) NK cells predominately expressed killer cell immunoglobulin-like receptor, and self-killer cell immunoglobulin-like receptors were required for robust IFNγ production. During the first year after transplantation, CMV reactivation induced a more mature phenotype characterized by an increase in CD56(dim) NK cells. Strikingly, increased frequencies of NKG2C(+) NK cells persisted and continued to increase in recipients who reactivated CMV, whereas these cells remained at low frequency in recipients without CMV reactivation. Persisting NKG2C(+) NK cells lacked NKG2A, expressed CD158b, preferentially acquired CD57, and were potent producers of IFNγ during the first year after transplantation. Recipients who reactivated CMV also expressed higher amounts of IFNγ, T-bet, and IL-15Rα mRNA transcripts. Our findings support the emerging concept that CMV-induced innate memory-cell populations may contribute to malignant disease relapse protection and infectious disease control long after transplantation.

Figure 1

NKG2C⁺ NK cells increase after CMV reactivation after HCT and produce high levels of IFNγ. PBMCs from HCT recipients were studied before viral infection, at diagnosis of viral reactivation, and after 2, 4, and 8 weeks of antiviral therapy. Cells were incubated in either medium alone (not shown) or with K562 cells for 5 hours. (A) After incubation, NKG2C expression was measured on CD56⁺CD3⁻ NK cells and the absolute number of NKG2C⁺ NK cells/μL was calculated. Aggregate data from recipients in the indicated groups is shown. Points represent the means ± SEM. Previral samples were compared with samples after infection using the Student t test. *P ≤ .05. (B) CD107a expression (left panel) and IFNγ production (right panel) was measured on NKG2C⁺ (black bars) or NKG2C⁻ (white bars) NK cells. Bars represent the means ± SEM. Previral samples were compared with samples after infection using the Student t test. NKG2C⁺ NK cells were compared with NKG2C⁻ NK cells using the paired t test. *P ≤ .05, **P < .01, and ***P < .001.

Figure 2

NKG2C⁺ NK cells are predominantly KIR⁺ and education through self-KIR are required for robust IFNγ production. (A) NKG2C⁺ (black bars) and NKG2C⁻ (white bars) NK cells were gated as being KIR⁺ based on staining with a cocktail of Abs against CD158a/h, CD158b/j, and CD158e. Bars represent the means ± SEM. NKG2C⁺ cells were compared with NKG2C⁻ samples using the paired t test. *P ≤ .05, **P < .01, and ***P < .001. (B) NKG2C⁺ NK cells were gated as being either KIR⁺ or KIR⁻ and IFNγ production was measured after incubation in either medium alone (not shown) or with K562 cells for 5 hours. Bars represent the means ± SEM. KIR⁺ cells were compared with KIR⁻ samples using the paired t test. *P ≤ .05, **P < .01, and ***P < .001. (C) Single KIR⁺ NKG2C⁺ NK cells were grouped as expressing either self-KIR (black bars) or nonself KIR (white bars) based on the recipient KIR ligand status (left panel). IFNγ production was measured after incubation in either medium alone (not shown) or with K562 cells for 5 hours. Bars represent the means ± SEM. Self-KIR⁺ cells were compared with non-self-KIR⁺ samples using the Student t test. Bars represent the means ± SEM. **P < .01.

Figure 3

CMV reactivation induces an increase of NKG2C⁺ NK cells and a more mature NK-cell phenotype in the first year after transplantation. NKG2C expression (left panel), mean fluorescence intensity (MFI, right panel, A), CD56^dim expression (B, left top panel), NKG2A expression (B, right top panel), and KIR expression based on a cocktail of Abs against CD158a/h, CD158b/j, and CD158e (B, left bottom panel) and CD57 expression (B, right bottom panel) were measured on CD56⁺ CD3⁻ NK cells from seronegative (○, n = 10), seropositive (no CMV reactivation; ■, n = 5) and seropositive (CMV reactivation; ▵, n = 8) recipients at day 28, day 60, day 100, 6 months, and 1 year after transplantation. Points represent the means ± SEM. The slope of time was compared between recipients who reactivated CMV and combined recipients who did not reactivate CMV using a linear mixed model.

Figure 4

Expanding NKG2C⁺ NK cells are more mature (KIR⁺ and NKG2A⁻) in the first year after CMV reactivation. NKG2C⁺ NK cells from seronegative (top panel), seropositive (no CMV reactivation, middle panel), and seropositive (CMV reactivation, bottom panel) recipients at the indicated time points were gated as being either NKG2A⁺ (○) or NKG2A⁻ (■, A) and either KIR⁺ (○) or KIR⁻ (■, B) based on a cocktail of Abs against CD158a/h, CD158b/j, and CD158e. Points represent the means ± SEM. NKG2A⁺ and NKG2A⁻ NK cells (or KIR⁺ and KIR⁻ NK cells) were compared for the same group of recipients at each time point using the paired t test. *P ≤ .05 and **P < .01.

Figure 5

CD158b⁺ is preferentially expressed on expanding NKG2C⁺ NK cells after CMV reactivation. NKG2C⁺ (○) and NKG2C⁻ (■) NK cells from recipients without CMV reactivation (A) and with CMV reactivation (B) at the indicated time points were further gated as being CD158a⁺ (top panel), CD158b⁺ (middle panel), or KIR3DL1⁺ (bottom panel). Points represent the means ± SEM. The slope of time for NKG2C⁺ NK cells was compared using a linear mixed model.

Figure 6

CD57 is preferentially acquired on NKG2C⁺ cells in the first year after CMV reactivation. CD57 co-expression was measured on NKG2C⁺ (○) and NKG2C⁻ (■) NK cells from recipients without CMV infection (top panel) and with CMV infection (bottom panel). The ratio of CD57⁺/CD57⁻ was plotted at the indicated time points. Points represent the means ± SEM. The slope of time for NKG2C⁺ NK cells was compared between recipients who reactivated CMV and those who did not using a linear mixed model, P = .0015.

Figure 7

IFNγ production, but not CD107a expression, is increased after CMV reactivation. PBMCs from seronegative (black bars, n = 10), seropositive (no CMV reactivation, white bars, n = 5), and seropositive (CMV reactivation, gray bars, n = 8) recipients at the indicated time points were incubated in either medium alone (not shown) or with K562 cells for 5 hours. (A) CD107a expression (left panel) and IFNγ production (right panel) was measured on NKG2C⁺ NK cells. Bars represent the means ± SEM. NKG2C⁺ cells from different groups of recipients were compared at each time point using the Student t test. *P ≤ .05. (B) NKG2C⁺ NK cells from seropositive (CMV reactivation) recipients were further gated as being KIR⁺ (black bars) or KIR⁻ (white bars) based on staining with a cocktail of Abs against CD158a/h, CD158b/j, and CD158e, and IFNγ production was measured for each subset. Bars represent the means ± SEM. KIR⁺ and KIR⁻ NK cells were compared at each time point using the paired t test. *P ≤ .05. (C) NKG2C⁺ NK cells from seropositive (CMV reactivation) recipients were further gated as being CD57⁺ (black bars) or CD57⁻ (white bars) and IFNγ production was measured for each subset. Bars represent the means ± SEM. CD57⁺ and CD57⁻ NK cells were compared at each time point using the paired t test. *P ≤ .05. (D) IFNγ, T-bet, and IL-15Rα mRNA expression were measured on resting PBMCs at 6 months and 1 year after HCT. Samples were normalized to 18S RNA. Bars represent the means ± SEM. Recipients who reactivated CMV and those who did not were compared at each time point using the t test. *P ≤ .05, **P < .01, and ***P < .001.