IL-15 Primes a Highly Potent Anti-Leukemia Response By CD56bright NK Cells

Romee, Rizwan; Leong, Jeffrey W; Schneider, Stephanie E; Sullivan, Ryan P; Fehniger, Todd A.

doi:10.1182/blood.V122.21.2283.2283

Abstract

Background

NK cells are innate lymphocytes that are important for host defense against infections, and are potent anti-cancer immune effectors. In peripheral blood, human NK cells are categorized into developmentally related, but functionally distinct, subsets. CD56^dim NK cells, thought to be developmentally more mature, are the major subset in peripheral blood (80-95%), express perforin and granzyme B at rest and exhibit degranulation, cytotoxicity and IFN-γ responses against tumor targets without prior stimulation. In contrast, CD56^bright NK cells, are less mature, are the major subset in secondary lymphoid tissues, lack expression of perforin and granzyme B and are associated with minimal degranulation, cytotoxicity, and IFN-γ responses to tumor targets. IL-15 has been shown to support the survival and proliferation of CD56^bright NK cells, but its impact on the anti-leukemia response has not been reported. Here we investigate the impact of brief exposure to human recombinant IL-15 on functional responses of CD56^bright and CD56^dim NK cells to leukemia target cells including primary AML blasts.

Methods

Normal human donor NK cells (>95% purity) were cultured with cytokine free media (control) or with 5 ng/ml of rhIL-15 (primed) for 16 hours, washed and then tested for functional responses after co-incubation with K562 cells or primary AML blasts for 6 hours. NK cell functional responses assessed include degranulation, cytokine production and cytotoxicity (using flow based killing assays). For the tumor:nk cell conjugate analyses, pre-stained NK cells were co-incubated with CFSE labeled K562 cells and then CD56^bright conjugate formation assessed by gating on CFSE⁺CD56⁺CD16^- cells.

Results

Anti-leukemia effector functions of human NK cells are classically attributed to the CD56^dim subset, however after priming for 16 hours with rhIL-15 (5 ng/mL, a concentration that stimulates via the IL-2/15Rβγ_c), surprisingly, we observed that IL-15-primed CD56^bright NK cells exhibited significantly greater degranulation (CD107a), cytokine (IFN-γ and TNF-α) and cytotoxic responses to both K562 leukemia cells (Figure 1) and to primary AML blasts (figure 2), compared to IL-15 primed CD56^dim NK cells from the same donor. Further, we found a marked increase in the expression of perforin (70 ± 5% vs. 12 ± 6%, P< 0.0001), granzyme B (64 ± 5% vs. 12 ± 2.5%, P< 0.0001), and TRAIL (89 ± 2.5% vs. 6 ± 1%, P< 0.0001) in IL-15 primed CD56^bright NK cells.

We found an increased number of tumor conjugates with the IL-15 primed, compared to control, CD56^bright cells at 5 minutes (19 ± 3% vs. 3.5 ± 1%, P= 0.02), 15 minutes (22 ± 3% vs. 8 ± 2%, P= 0.0003) or at 30 minutes (13 ± 2% vs. 3.5 ± 1%, P= 0.008) from the same donors. Further, there was a significant increase in the expression of NKG2D (MFI of 8.5 ± 2 vs. 3 ± 0.5, P= 0.03), NKp30 (65 ± 4% vs. 21 ± 3%, P< 0.0001), NKp44 (57 ± 3% vs. 15 ± 3%, P< 0.0001), CD2 (MFI of 25 ± 1.5 vs. 13 ± 1, P=0.004) and LFA-1/CD11a (MFI of 45 ± 1 vs. 29 ± 2, P=0.006) in the IL-15 primed CD56^bright NK cells. Due to their known role in activating anti-tumor target responses by NK cells, NKG2D, NKp44, NKp30, CD2, and LFA-1 were evaluated for a non-redundant contribution to the anti-leukemia response of IL-15 primed CD56^bright NK cells. Simultaneous blockade of these receptors caused almost complete abrogation of the enhanced anti-leukemic response by the IL-15 primed CD56^bright NK cells (Figure 3).

Conclusions

CD56^bright NK cells are traditionally considered to poorly respond to leukemia targets. Here we show that stimulation with IL-15 for a few hours markedly enhances their anti-leukemia properties including degranulation and cytotoxicity, as well as IFN-γ and TNF-α production, to a level significantly exceeding CD56^dim NK cells. These functional enhancements are explained by multiple mechanisms, including increased cytotoxic effector proteins (perforin, granzyme B, TRAIL), improved leukemia cell conjugation, and enhanced activation requiring LFA-1, CD2 and NKG2D. These results suggest that CD56^bright NK cells may play an under-appreciated anti-tumor role in settings of abundant IL-15, such as following lymphodepleting chemotherapy, preparation for stem cell transplantation, or exogenous IL-15 administration.