NK cells are innate immune lymphocytes important for host defense against infection, and also mediate anti-tumor responses. NK cell effector functions are triggered by ligand-mediated engagement of activating receptors and by cytokine stimulation. Human NK cells may be divided into developmental and functional CD56bright and CD56dim subsets with distinct biology. Both NK subsets have constitutive expression of CD122 (IL-2/15Rβ) and CD132 (γc), allowing for responsiveness to IL-15 and high doses (1 nM) of IL-2. The high affinity heterotrimeric IL-2 receptor is formed by CD122, CD132, and CD25 (IL-2Rα), allowing for responses to very low (10 pM) concentrations of IL-2. Previous studies demonstrated that resting CD56bright, but not CD56dim, NK cells constitutively express a functional high-affinity IL-2Rαβγ, as picomolar concentrations of IL-2 result in proliferation and co-stimulate IFN-γ production. We hypothesized that cytokine activation, which occurs at the site of an inflammatory response or interaction with a priming dendritic cell, may induce the expression of CD25 on CD56dimNK cells, allowing for enhanced responsiveness to IL-2.
Purified normal donor NK cells (>95% CD56+CD3-) were cultured for 16h in media containing various cytokines, including IL-15 (100ng/mL) + IL-18 (50ng/mL), IL-15 (100ng/mL) + IL-12 (10ng/mL) or low dose IL-15 (1ng/mL) as a control (to support survival). Following stimulation, cells were washed and analyzed for surface expression of CD25 by antibody staining and flow cytometry. To assess the functional capacity of an induced IL-2Rα chain, NK cells were purified and treated with cytokines as above, washed extensively and replated in media devoid of all cytokines. At 3d after initial stimulation, cultures were briefly (15min) stimulated with IL-2 (10pM, 100pM and 1nM) or IL-15 (100ng/mL) to induce phosphorylation of STAT5. Cells were immediately fixed, permeablized, and assessed for intracellular phosphoSTAT5 by flow cytometry.
Pretreatment with IL-15 + IL-18 or IL-12 + IL-18, but no single cytokine, induced marked upregulation of CD25 on both CD56bright as well as CD56dim NK cells, with 93.6 ±1.9% of CD56bright and 97.7 ±1.2% (n=4) of CD56dim NK cells positive for CD25 expression immediately following the initial 16h stimulation with IL-15+18, compared to 20.7 ±6.8 and 3.6 ±1.4% for control treated (low dose IL-15) CD56bright and CD56dim NK. In preliminary experiments, CD25 appears to be markedly upregulated at the mRNA level, following treatment with IL-15+18. While kinetic analyses revealed that the absolute surface receptor expression was maximal at time points early after stimulation, 75.8 ±4.1% of CD56bright and 84.9 ±5.7% (n=4) of CD56dimNK cells pretreated with IL-15+18 retained CD25 surface expression at 7d post-stimulation.
Importantly, CD25 induced on both CD56dim and CD56bright NK cells resulted in a signaling-competent high affinity IL-2 receptor, as stimulation with low dose IL-2 at 3d following initial cytokine pre-activation revealed increased production of phosphoSTAT5, versus control treated NK cells. The greatest enhancement was noted in CD56dim NK cells, showing an 8-fold increase in responsiveness to low dose (10pM) IL-2 stimulation (48.2±7.9% pSTAT5+ in IL-15+18 pretreated cells vs. 5.8±2.4% in control treated cells, p<0.02). Cytokine-pretreated CD56bright NK cells demonstrated no enhancement in phosphoSTAT5 following stimulation with low dose IL-2 (10pM) (27.6 ±8.3% in IL-15+18 pretreated vs. 26.0 ±6.4% in control) at this time-point. However, these results are in agreement with published data, which describe expression of CD25 on resting CD56bright but not CD56dimNK cells.
Here, we report the induction of CD25, and a signal-competent high-affinity component of the IL-2 receptor, on CD56dim human NK cells, following cytokine pre-activation. These results have implications for the function of both CD56bright and CD56dim NK cells in the context of inflammation and potential for cross-talk with IL-2-producing T cells during an adaptive immune response. In addition, since rhIL-2 is clinically available and used following NK cell adoptive transfer in leukemia patients, these data provides a rationale for low dose IL-2 following allogeneic NK cell pre-activation with combinations of IL-15, IL-12, and IL-18.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.