Maturation of human natural killer cells (NK cells) as defined by accumulation of cell surface expression of CD57 is associated with increased cytotoxic character and TNF and IFN-γ production upon target cell recognition. Notably, multiple studies point to a unique role for CD57+ NK cells in cancer immunosurveillance, yet there is scant information about how they mature. In this study, we show that pharmacological inhibition of GSK3 kinase in peripheral blood NK cells expanded ex vivo with IL-15 greatly enhances CD57 upregulation and late-stage maturation. GSK3 inhibition elevated the expression of several transcription factors associated with late-stage NK cell maturation including T-BET, ZEB2 and BLIMP-1 without affecting viability or proliferation. When exposed to human cancer cells, NK cell expanded ex vivo in the presence of a GSK3 inhibitor exhibited significantly higher production of TNF and IFN-γ, elevated natural cytotoxicity, and increased antibody-dependent cellular cytotoxicity (ADCC). In an established mouse xenograft model of ovarian cancer, adoptive transfer of NK cells conditioned in the same way also displayed more robust and durable tumor control. Our findings show how GSK3 kinase inhibition can greatly enhance the mature character of NK cells most desired for effective cancer immunotherapy.

Natural killer (NK) cells with adaptive immunological properties expand and persist in response to human cytomegalovirus. Here, we explored the metabolic processes unique to these cells. Adaptive CD3CD56CD57NKG2C NK cells exhibited metabolic hallmarks of lymphocyte memory, including increased oxidative mitochondrial respiration, mitochondrial membrane potential, and spare respiratory capacity. Mechanistically, we found that a short isoform of the chromatin-modifying transcriptional regulator, AT-rich interaction domain 5B (ARID5B), was selectively induced through DNA hypomethylation in adaptive NK cells. Knockdown and overexpression studies demonstrated that ARID5B played a direct role in promoting mitochondrial membrane potential, expression of genes encoding electron transport chain components, oxidative metabolism, survival, and IFN-γ production. Collectively, our data demonstrate that ARID5B is a key regulator of metabolism in human adaptive NK cells, which, if targeted, may be of therapeutic value.

Cerebral malaria is a deadly complication of Plasmodium infection and involves blood brain barrier (BBB) disruption following infiltration of white blood cells. During experimental cerebral malaria (ECM), mice inoculated with Plasmodium berghei ANKA-infected red blood cells develop a fatal CM-like disease caused by CD8 T cell-mediated pathology. We found that treatment with interleukin-15 complex (IL-15C) prevented ECM, whereas IL-2C treatment had no effect. IL-15C-expanded natural killer (NK) cells were necessary and sufficient for protection against ECM. IL-15C treatment also decreased CD8 T cell activation in the brain and prevented BBB breakdown without influencing parasite load. IL-15C induced NK cells to express IL-10, which was required for IL-15C-mediated protection against ECM. Finally, we show that ALT-803, a modified human IL-15C, mediates similar induction of IL-10 in NK cells and protection against ECM. These data identify a regulatory role for cytokine-stimulated NK cells in the prevention of a pathogenic immune response.

Conflict of interest: JSM serves on the Scientific Advisory Board (SAB), consults for GT BioPharma and Fate Therapeutics, and has received research funds from these relationships. He also serves on the SAB for CytoSen and Onkimmune. F. Cichocki consults for Fate Therapeutics and has received research funds from these relationships. BRB declares a financial conflict with Tmunity and Kadmon Corp and receives research funds from Fate Therapeutics. He also serves on the SAB for GT Biopharma, Magenta Therapeutic, and Five Prime Therapeutics and consults for Regeneron and Equillium Inc. DJD is the Chair of the SAB of Helocyte Inc., from which he receives royalties and research funding via a sponsored research agreement. None of these relationships had a role in funding this research. All conflicts are managed per institutional policies.

<p>This supplementary file contains Supplemental Figures 1-6. Supplemental Figure 1 shows that 7-day ex vivo culture of NK cells with IL-15 and 5 μM CHIR99021 leads to an increase in the frequencies of NK cells with heterogeneous adaptive NK cell phenotypes (defined by expression of CD57, PLZF, SYK and FcεR1γ) relative to DMSO controls. Supplemental Figure 2 contains a detailed phenotypic characterization of receptor expression and cytotoxic granule component levels in sorted CD3-CD56dimCD57- and CD3-CD56dimCD57+ NK cells cultured for 7 days with IL-15 and either DMSO or 5 μM CHIR99021. Supplementary Figure 3 shows an analysis of NK cell phenotype, viability and proliferation after 7-day culture with IL-15 and either DMSO or CHIR99021 at several concentrations (1 μM, 3 μM and 5 μM). Supplemental Figure 4 shows an analysis of NK cell function (CD107a and IFN-γ) against K562 cells. NK cells were cultured for 7 days in IL-15 and either DMSO or 5 μM CHIR99021. Function was determined for individual CD3-CD56+ NK cell subsets gated on CD57 and NKG2C. Supplemental Figure 5 shows an analysis of NK cell function (CD107a and IFN-γ) against K562 cells. NK cells were cultured for 7 days in IL-15 and either DMSO or 5 μM CHIR99021. Function was determined for individual CD3-CD56+ NK cell subsets gated on CD57 and KIR. Supplemental Figure 6 shows the frequency of ex vivo expanded NK cells expressing CD57 and/or NKG2C 14 days after adoptive transfer into NSG mice.</p>