Cytokine activation induces human memory-like NK cells

Abstract

Natural killer (NK) cells are lymphocytes that play an important role in the immune response to infection and malignancy. Recent studies in mice have shown that stimulation of NK cells with cytokines or in the context of a viral infection results in memory-like properties. We hypothesized that human NK cells exhibit such memory-like properties with an enhanced recall response after cytokine preactivation. In the present study, we show that human NK cells preactivated briefly with cytokine combinations including IL-12, IL-15, and IL-18 followed by a 7- to 21-day rest have enhanced IFN-γ production after restimulation with IL-12 + IL-15, IL-12 + IL-18, or K562 leukemia cells. This memory-like phenotype was retained in proliferating NK cells. In CD56(dim) NK cells, the memory-like IFN-γ response was correlated with the expression of CD94, NKG2A, NKG2C, and CD69 and a lack of CD57 and KIR. Therefore, human NK cells have functional memory-like properties after cytokine activation, which provides a novel rationale for integrating preactivation with combinations of IL-12, IL-15, and IL-18 into NK cell immunotherapy strategies.

Figure 1

Preactivation with IL-12 + IL-18 results in human memory-like NK cells with enhanced IFN-γ production after restimulation. (A) Schema of experimental approach with IL-12 + IL-18 preactivation. In later experiments, preactivated was varied as indicated. (B-C) PBMCs (containing 13% ± 5% CD56⁺CD3⁻ NK cells) were preactivated for 16 hours with control (IL-15, 1 ng/mL), or IL-12 (10 ng/mL) + IL-18 (50 ng/mL) + IL-15 (1 ng/mL). Cells were then washed 3 times to remove preactivating cytokines and cultured with low-dose IL-15 (1 ng/mL) to support survival for 7, 14, or 21 days. After each time point, cells were washed, restimulated for 6 hours with IL-12 (10 ng/mL) + IL-15 (100 ng/mL), and assayed for NK cell IFN-γ production by intracellular flow cytometry. (B) Representative bivariate flow plots from control and IL-12 + IL-18–preactivated NK cells showing IFN-γ production after IL-12 + IL-15 restimulation on days 7, 14, or 21 (gated on live CD45⁺CD56⁺CD3⁻ NK cells). (C) Summary data shown as means ± SEM percentage of IFN-γ⁺ NK cells. NK cells were assessed without restimulation immediately after preactivation (16 hours; n = 6; 3 independent experiments) to confirm that NK cells were preactivated or after 7 days of rest to demonstrate a return to a resting state (n = 8; 4 independent experiments). After 7 (n = 8; 4 independent experiments), 14 (n = 4; 2 independent experiments), or 21 (n = 4; 2 independent experiments) days, NK cells were restimulated with IL-12 + IL-15 for 6 hours and assayed for IFN-γ production. (D) Purified NK cells exhibit memory-like properties. Experiments were performed as in panels A-C, but with purified NK cells without restimulation (n = 6, 3 independent experiments for 16 hours; n = 12, 4 independent experiments for 7 days) and after IL-12 + IL15 restimulation for 7 days (n = 12; 4 independent experiments) or 14 days (n = 6; 3 independent experiments). Purified NK cells were ≥ 95% CD56⁺CD3⁻ with < 0.5% CD3⁺ T cells. *P < .05; **P < .01; ***P < .001.

Figure 2

CD56^bright and CD56^dim NK cell subsets exhibit memory-like properties after IL-12 + IL-18 preactivation. (A-B) CD56^bright and CD56^dim NK subsets in purified NK cell (≥ 95% CD56⁺CD3⁻) cultures exhibit memory-like IFN-γ production. (A) Representative data from control and IL-12 + IL-18–preactivated NK cells demonstrating the NK subset analysis gating strategy and differential IFN-γ production after the 7-day rest and a 6-hour IL-12 + IL-15 restimulation. (B) Summary data demonstrating that IL-12 + IL-18–preactivated CD56^bright and CD56^dim NK cells exhibit a memory-like IFN-γ response after restimulation with IL-12 + IL-15, IL-12 + IL-18, or K562 leukemia cells. Purified (≥ 95% CD56⁺CD3⁻) NK cells were cultured as in Figure 1A for 7 days and restimulated with cytokines or K562 (4:1 effector: target ratio) as indicated for 6 hours and then assayed for IFN-γ. Data shown are the means ± SEM percentage of IFN-γ⁺ NK cells, gated on CD56^bright or CD56^dim subsets, with n = 12 donors (4 independent experiments) for IL-12 + IL-15 restimulation, n = 6 donors (3 independent experiments) for IL-12 + IL-18 restimulation, and n = 9 donors (4 independent experiments) for K562 restimulation. (C-D) Flow-sorted CD56^bright and CD56^dim NK cells cultures exhibit memory-like IFN-γ production. (C) NK cell subsets were flow sorted (≥ 99% purity) and then preactivated as per Figure 1A, rested for 7 days, restimulated (6 hours) with IL-12 + IL-15, and stained for intracellular IFN-γ. The NK cell subset phenotype was retained in each sorted population after 7 days of culture. (D) Summary data are shown as means ± SEM of the percentage of IFN-γ⁺ sorted CD56^bright and CD56^dim NK cells after 7 days of rest followed by 6 hours of restimulation with IL-12 + IL-15 (n = 6; 3 independent experiments). *P < .05; **P < .01; ***P < .001.

Figure 3

Preactivation dose response of IL-12 and IL-18 for memory-like NK cell induction. Purified (≥ 95% CD56⁺CD3⁻) NK cells were preactivated for 16 hours with the indicated concentrations of IL-12 + IL-18 or control conditions (C; 1 ng/mL of IL-15 only). After preactivation, cells were washed as in Figure 1A and then rested for 7 days in 1 ng/mL of IL-15 before restimulation with IL-12 + IL-15 for 6 hours to assess IFN-γ production (n = 4; 2 independent experiments). Summary data are shown as means ± SEM percentage of IFN-γ⁺ CD56^bright (A) and CD56^dim (B) NK cells. Statistical comparisons are made between the indicated dose of IL-12 and IL-18 and the control cells. *P < .05; **P < .01

Figure 4

Cell surface marker changes after preactivation and induction of human memory-like NK cells. Purified NK cells (≥ 95% CD56⁺CD3⁻) were cultured for 7 days as in Figure 1A and assayed without restimulation for CD94, NKG2A, NKG2C, NKp46, CD69, KIR, and CD57. Representative flow histograms are shown in pre-gated CD56^bright and CD56^dim NK cell subsets, overlaying control cells (open lines) and preactivated cells(gray filled lines). Summary data are shown as means ± SEM percentage of positive or MFI for the marker indicated in CD56^bright and CD56^dim NK cells. CD56^dim NK cells had significantly increased expression of CD94 and NKG2A 7 days after preactivation compared with controls. Both CD56^dim and CD56^bright NK cells had increased CD69 and NKp46 surface expression 7 days after preactivation (n = 4-6; 2 independent experiments) *P < .05; **P < .01; ***P < .001.

Figure 5

Enhanced IFN-γ production by human memory-like NK cells is associated with a CD94⁺NKG2A⁺CD57⁻KIR⁻CD69⁺ phenotype. Purified NK cells (≥ 95% CD56⁺CD3⁻) were cultured as in Figure 1A for 7 days and assessed for IFN-γ in concert with the indicated cell surface marker after 6 hours of IL-12 + IL-15 restimulation. (A) Representative bivariate flow plots from control and IL-12 + IL-18–preactivated NK cells showing correlation of IFN-γ production (all were restimulated with IL-12 + IL-15) in populations positive or negative for CD94, NKG2A, NKG2C, KIR, CD57, and CD69. (B) Because CD56^bright NK cells had > 95% or < 10% expression of CD94, NKG2A, KIR, and CD57 (Figure 6), IFN-γ correlative data are shown gated on CD56^dim NK cells. There were a greater percentage of IFN-γ⁺ NK cells in the CD94⁺, NKG2A⁺, NKG2C⁺, and CD69⁺ fractions of CD56^dim NK cells. In contrast, there were a significantly greater percentage of IFN-γ⁺ NK cells in CD57⁻ and KIR⁻ CD56^dim NK cells. There were no significant correlations of NKG2C or CD69 expression with IFN-γ production in CD56^bright NK cells (not shown). Statistical comparisons are between marker positive and negative preactivated or control NK cell association with IFN-γ. A larger number of donors were assessed for CD94, NKG2A, and CD57 correlations to assess the more subtle differences between marker positive and negative control cells (n = 4-17 donors; 2-6 independent experiments). *P < .05; **P < .01; ***P < .001.

Figure 6

Memory-like NK cells proliferate and retain enhanced IFN-γ production after cell division. Purified NK cells were labeled with CFSE to track cell division, sorted into CD56^bright and CD56^dim NK subsets (≥ 99% purity), and preactivated and rested as per Figure 1A. After 7 days, cells were restimulated with IL-12 + IL-15 and analyzed simultaneously for IFN-γ and CFSE to track cell divisions. (A) Representative bivariate flow plots of CD56^bright and CD56^dim NK cells demonstrating that preactivation results in both increased cell division (CFSE dilution) and enhanced IFN-γ production. (B) Summary results showing the increased proliferation 7 days after preactivation (p.a.) compared with control (c) in both CD56^bright and CD56^dim NK cell subsets. (C) Summary results are shown as the means ± SEM percentage of IFN-γ⁺ by NK cell generation (n = 4 donors, 2 independent experiments). Similar findings were observed in purified NK cells that were then gated on CD56^bright and CD56^dim NK cells (n = 6 donors; 2 independent experiments; supplemental Figure 5). *P < .05; **P < .01; ***P < .001.

Figure 7

Additional preactivation conditions lead to enhanced memory-like NK cell IFN-γ production. (A-B) Purified (≥ 95% CD56⁺CD3⁻) NK cells were stimulated for 16 hours with either low-dose IL-15 (1 ng/mL, control), IL-12 (10 ng/mL), high-dose IL-15 (100 ng/mL), IL-18 (50 ng/mL), or the indicated IL-12/IL-15/IL-18 combinations. After 16 hours of activation, the cells were washed as in Figure 1A and then rested for 7 days before restimulation with IL-12 + IL-15 for 6 hours to assess IFN-γ production. In all conditions in which high-dose IL-15 was not used, low-dose IL-15 was included to support survival during preactivation. These data show that preactivation with all combinations of IL-12, IL-15, and IL-18 lead to memory-like NK cell IFN-γ production on restimulation (n = 4; 2 independent experiments). (C) In a separate set of donors, purified (≥ 95% CD56⁺CD3⁻) NK cells were preactivated for 16 hours by cross-linking CD16 with plate-bound anti-CD16 mAb (or control mouse IgG1) or CD16 cross-linking in combination with IL-12 (10 ng/mL), IL-18 (50 ng/mL), or IL-12 (10 ng/mL) + IL-18 (50 ng/mL). The preactivated cells were washed as in Figure 1A, rested for 7 days, and then restimulated with IL-12 + IL-15 for 6 hours to assess IFN-γ production. Summary data are shown as means ± SEM percentage of IFN-γ⁺ CD56^dim NK cells. These data show that CD16 cross-linking in combination with cytokines (IL-12 and IL-12 + IL-18) results in enhanced IFN-γ production on restimulation (n = 6; 3 independent experiments). Statistical comparisons are with control conditions, except in panel C as indicated. *P < .05; **P < .01; ***P < .001.