Human CD56bright NK Cells Acquire Potent Anti-Leukemia Functionality Following IL-15 Priming

Natural killer (NK) cells are innate lymphoid cells that mediate anti-tumor responses via cytotoxicity and effector cytokine production. Human NK cells are divided into two subsets based on relative expression of CD56 (CD56 bright and CD56 dim ) that classically participate in distinct functions. Cytotoxic CD56 dim NK cells respond to tumor targets without prior stimulation, resulting in target cell death and transient secretion of effector cytokines (e.g. IFN-γ). In contrast, immunoregulatory CD56 bright NK cells secrete abundant IFN-γ and other cytokines in response to cytokine receptor stimulation, but respond minimally to tumor target-based triggering. As a result of this dichotomy, translational strategies to enhance NK cell function for cancer immunotherapy have focused exclusively on the CD56 dim subset. Based upon studies in mouse NK cells, we hypothesized IL-15 priming would enhance CD56 bright anti-tumor functionality. Primary human NK cells from healthy donors were purified (u003e95% CD56 + CD3 - ), cultured overnight in medium alone (control) or medium with 5 ng/mL rhIL-15 (primed), washed, and assayed for anti-tumor responses. IL-15 priming significantly enhanced multiple CD56 bright NK cell functional responses to the prototypical AML target cell line K562 (CD107a+: control 20% vs. primed 59%, p bright NK cells was evident after 1 hour, and peaked after only 6 hours. In addition, flow-sorted IL-15-primed CD56 bright NK cells exhibited markedly increased killing of leukemia target cells. Similar results for functional comparisons were observed using primary NK cells from AML patients. Unexpectedly, IL-15-primed CD56 bright NK cell anti-leukemia responses significantly exceeded those of IL-15-primed CD56 dim NK cells. Multidimensional CyTOF analyses revealed that the maturity status of CD56 dim NK cells did not determine the extent to which they could be primed by IL-15. In response to IL-15, we observed selective activation of the PI3K/Akt/mTOR (4.2 fold increase CD56 bright NK cells, 1.2 CD56 dim NK cells, p bright NK cells, 1.2 CD56 dim NK cells, p bright NK cells. The Jak/STAT pathway was strongly activated in both CD56 bright and CD56 dim subsets. These data suggested a signaling mechanism for preferential CD56 bright NK cell priming by IL-15. Indeed, small molecule inhibitors of PI3K/Akt/mTOR and Ras/Raf/MEK/ERK pathways abrogated the anti-tumor responses of IL-15-primed CD56 bright NK cells, supporting this idea. Several NK cell effector mechanisms were enhanced in IL-15-primed CD56 bright NK cells, likely contributing to their augmented anti-tumor responsiveness. These included increased cytotoxic effector protein levels (granzyme B and perforin), as well as improved conjugate formation with tumor targets. Furthermore, blocking experiments demonstrated that IL-15-primed CD56 bright NK cell anti-tumor responses depended on LFA-1, CD2, and NKG2D receptor interactions with target cells. Finally, since IL-15-based therapeutics are being investigated in clinical trials, we tested whether the IL-15 super-agonist complex ALT-803 primes CD56 bright NK cells in vivo in cancer patients. There was an increase in leukemia target cell-triggered degranulation (CD107a+ unprimed 7% vs. primed 30%, p bright NK cells 24 hours post-ALT-803 administration to multiple myeloma patients, compared to unprimed, pre-therapy values. Collectively, these results suggest that CD56 bright NK cells play an under-appreciated anti-tumor role in settings of abundant IL-15, such as following lymphodepleting chemotherapy, during preparation for stem cell transplantation, at sites of inflammation, or after exogenous IL-15 administration. Since CD56 bright NK cells have different in vivo tissue localization (secondary lymphoid organs), distinct inhibitory, activating, and chemokine receptor expression compared to CD56 dim NK cells, and are thought to be the most abundant NK cell subset when considering all human tissues, this study identifies a promising NK cell subset to harness for cancer immunotherapy. Disclosures No relevant conflicts of interest to declare.