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Activation of natural killer (NK) cells with the cytokines interleukin-12 (IL-12), IL-15, and IL-18 induces their differentiation into memory-like (ML) NK cells; however, the underlying epigenetic and transcriptional mechanisms are unclear. By combining ATAC-seq, CITE-seq, and functional analyses, we discovered that IL-12/15/18 activation results in two main human NK fates: reprogramming into enriched memory-like (eML) NK cells or priming into effector conventional NK (effcNK) cells. eML NK cells had distinct transcriptional and epigenetic profiles and enhanced function, whereas effcNK cells resembled cytokine-primed cNK cells. Two transcriptionally discrete subsets of eML NK cells were also identified, eML-1 and eML-2, primarily arising from CD56 bright or CD56 dim mature NK cell subsets, respectively. Furthermore, these eML subsets were evident weeks after transfer of IL-12/15/18-activated NK cells into patients with cancer. Our findings demonstrate that NK cell activation with IL-12/15/18 results in previously unappreciated diverse cellular fates and identifies new strategies to enhance NK therapies.

Metastatic (m) colorectal cancer (CRC) is an incurable disease with a poor prognosis and thus remains an unmet clinical need. Immune checkpoint blockade (ICB)-based immunotherapy is effective for mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) mCRC patients, but it does not benefit the majority of mCRC patients. NK cells are innate lymphoid cells with potent effector responses against a variety of tumor cells but are frequently dysfunctional in cancer patients. Memory-like (ML) NK cells differentiated after IL-12/IL-15/IL-18 activation overcome many challenges to effective NK cell anti-tumor responses, exhibiting enhanced recognition, function, and in vivo persistence. We hypothesized that ML differentiation enhances the NK cell responses to CRC. Compared to conventional (c) NK cells, ML NK cells displayed increased IFN-γ production against both CRC cell lines and primary patient-derived CRC spheroids. ML NK cells also exhibited improved killing of CRC target cells in vitro in short-term and sustained cytotoxicity assays, as well as in vivo in NSG mice. Mechanistically, enhanced ML NK cell responses were dependent on the activating receptor NKG2D as its blockade significantly decreased ML NK cell functions. Compared to cNK cells, ML NK cells exhibited greater antibody-dependent cytotoxicity when targeted against CRC by cetuximab. ML NK cells from healthy donors and mCRC patients exhibited increased anti-CRC responses. Collectively, our findings demonstrate that ML NK cells exhibit enhanced responses against CRC targets, warranting further investigation in clinical trials for mCRC patients, including those who have failed ICB., Competing Interests: M.M.B.-E. and T.A.F. are inventors on patent/patent applications (15/983,275, 62/963,971, and PCT/US2019/060005) licensed to Wugen Inc. and held/submitted by Washington University that involve ML NK cells. This results in potential royalties to M.M.B.-E., T.A.F., and Washington University from Wugen Inc. M.M.B.-E. has equity, consulting, and royalty interest in Wugen Inc. T.A.F. reports research funding from HCW Biologics Inc., Wugen, Affimed and the NIH during the conduct of the study. T.A.F. has equity, research funding, consulting, and royalty interest in Wugen Inc. Unrelated to this work, T.A.F. also reports consulting for Affimed, AI Proteins, Smart Immune, and advises (equity interest) Indapta and OrcaBio. Unrelated to this work, J.A.F. is an inventor on patent/patent application (WO 2019/152387, US 63/018,108) licensed to Kiadis Inc. and held/submitted by Nationwide Children’s Hospital on TGF-β resistant, expanded NK cells. Unrelated to this work, J.A.F. has a monoclonal antibody unrelated to the present work licensed to EMD Millipore. Unrelated to this work, C.C.C. reports equity in Pionyr Immunotherapeutics. Unrelated to this work, D.A.R.-G. receives consulting fees from Cartography Inc. All other authors declare that they have no competing interests., (© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Purpose: Treatment of advanced melanoma is a clinical challenge. Natural killer (NK) cells are a promising cellular therapy for T cell-refractory cancers, but are frequently deficient or dysfunctional in patients with melanoma. Thus, new strategies are needed to enhance NK-cell antitumor responses. Cytokine-induced memory-like (ML) differentiation overcomes many barriers in the NK-cell therapeutics field, resulting in potent cytotoxicity and enhanced cytokine production against blood cancer targets. However, the preclinical activity of ML NK against solid tumors remains largely undefined., Experimental Design: Phenotypic and functional alterations of blood and advanced melanoma infiltrating NK cells were evaluated using mass cytometry. ML NK cells from healthy donors (HD) and patients with advanced melanoma were evaluated for their ability to produce IFNγ and kill melanoma targets in vitro and in vivo using a xenograft model., Results: NK cells in advanced melanoma exhibited a decreased cytotoxic potential compared with blood NK cells. ML NK cells differentiated from HD and patients with advanced melanoma displayed enhanced IFNγ production and cytotoxicity against melanoma targets. This included ML differentiation enhancing melanoma patients' NK-cell responses against autologous targets. The ML NK-cell response against melanoma was partially dependent on the NKG2D- and NKp46-activating receptors. Furthermore, in xenograft NSG mouse models, human ML NK cells demonstrated superior control of melanoma, compared with conventional NK cells., Conclusions: Blood NK cells from allogeneic HD or patients with advanced melanoma can be differentiated into ML NK cells for use as a novel immunotherapeutic treatment for advanced melanoma, which warrants testing in early-phase clinical trials., (©2021 The Authors; Published by the American Association for Cancer Research.)