Cytokine Activation and CD16 Cross-Linking Leads to the Generation of Human Memory-Like NK Cells

How NK cells acquire enhanced functionality against infection or malignancy remains an important aspect of NK cell biology. Recent studies in mice have shown that activation of NK cells can lead to memory-like properties. The NK cell memory-like response is characterized by an initial activation event, a subsequent return to the resting state, and then enhanced functionality (e.g. IFN-γ production) upon re-stimulation. The discovery of memory-like NK cells has important implications for NK cell-based therapeutics by providing new strategies to enhance and prolong NK cell responses in patients. We hypothesized that cytokines can induce human memory-like NK cells with enhanced ‘recall’ function.

Normal donor PBMCs, purified NK cells (>95% purity) and flow-sorted NK cell subsets (>99% purity) were cultured with low dose (1 ng/mL) IL-15 alone (control) or activated with combinations of IL-12 (10 ng/ml), IL-15 (100 ng/ml) and IL-18 (50 ng/ml) or by cross-linking CD16 (plate-bound anti-CD16 mAb) ± cytokines for 16 hours. After washing, the cells were then cultured for an additional 7–42 days in low dose IL-15 (to support survival). Following this prolonged rest period in vitro, NK cell responses (IFN-γ, degranulation) were assessed after 6-hour re-stimulation with cytokines or K562 leukemia cells. For proliferation experiments, NK cells were labeled with CFSE (5 uM, Sigma) for 5 minutes.

Initial stimulation with IL-12 + IL-18 for 16 hours resulted in the majority of NK cells producing IFN-γ protein, returning to baseline with no spontaneous IFN-γ production after several days of rest. However, after 7 days of rest the IL-12 + IL-18 pre-activated NK cells exhibited significantly increased IFN-γ response upon re-stimulation with IL-12 + IL-15 compared to controls, by both CD56^bright (68 ± 6% vs. 38 ± 5%, p<0.0005) and CD56^dim (30 ± 6% vs. 5 ± 2%, p<0.0005) subsets, suggestive of a memory-like phenotype. Similar results were demonstrated after IL-12 + IL-18 or K562 restimulation (p<0.005). Experiments with flow-sorted NK cell subsets demonstrated similar results after IL-12 + IL-15 restimulation, 45 ± 5% vs.15 ± 2% in CD56^bright NK cells (p<0.005) and 30 ± 6.5% vs. 3 ± 1% in CD56^dim NK cells (p<0.05), confirming that the human NK cell memory-like response is NK cell intrinsic. Pre-activation of NK cells with IL-15 + IL-18 or IL-12 + IL-15 resulted in significantly enhanced IFN-γ function after restimulation in both CD56^bright (p<0.0005) and CD56^dim (p<0.05) cells on restimulation with IL-12 + IL-15. Interestingly, pre-activation using CD16 cross-linking in combination with IL-12 also resulted in a memory-like IFN-γ response in CD56^dim NK cells after IL-12 + IL-15 restimulation (p<0.05) or IL-12 + IL-18 restimulation (p<0.005). IL-12 + IL-18 pre-activated CD56^bright and CD56^dim NK cells proliferated extensively, and demonstrated similar enhanced IFN-γ production after cell division, indicating heritable memory-like properties (p<0.05). Enhanced IFN-γ responses by IL-12 + IL-18 pre-activated NK cells (within PBMC) were durable, since similar results were obtained after 6 weeks of rest in vitro (p<0.05). In CD56^dimNK cells, the enhanced IFN-γ production after IL-12 + IL-15 restimulation was associated with expression of CD94, NKG2A, NKG2C, CD69, and lack of CD57 and KIR. There was a modest increase in the expression of IL12Rb1 and IL-12Rb2 level by MFI (p<0.05) that did not result in increased pSTAT4 levels on restimulation. Notably, we did not observe any significant differences in the IFN-γ mRNA transcript levels between pre-activated and control cells suggesting that the predominant mechanism mediating enhanced IFN-γ response occurs at the post-transcriptional and/or post-translational level.

Human NK cells pre-activated with combinations of IL-12, IL-15, IL-18, and CD16 cross-linking have memory-like properties evidenced by enhanced function (IFN-γ production) upon re-stimulation with leukemia targets and cytokines. This memory-like phenotype persisted after extensive proliferation and was evident at least 6 weeks after initial pre-activation. To our knowledge, this is the first report of human memory-like NK cells induced by cytokines and/or CD16 pre-activation. This provides a new rationale for investigating short-term pre-activation with combinations of IL-12, IL-15, and IL-18 in NK cell-based immunotherapy.

No relevant conflicts of interest to declare.