Phenotypic, proteomic, and functional analyses of cytokine-induced memory-like NK cells show two distinct subsets based on CD16 expression.

Carreira-Santos, SofíaGonzález-Sánchez, MarinaLópez-Sejas, NelsonHassouneh, FakhriGonzález-Fernández, LauroJorge, InmaculadaDurán, EstherPera, AlejandraVázquez, JesúsSolana, RafaelTarazona, RaquelCasado, Javier G2025-12-152025-12-152025-10-23Sci Rep. 2025 Oct 23;15(1):37053.https://hdl.handle.net/20.500.12105/27026NK cells are innate lymphoid cells that can acquire a memory-like phenotype in vitro when stimulated with IL-12, IL-15, and IL-18. These cytokine-induced memory-like (CIML) NK cells exhibit prolonged lifespan and increased cytotoxicity, making them ideal for immunotherapy. This study characterizes two CIML NK cell subsets based on CD16 expression. NK cells were isolated from the peripheral blood of healthy donors and stimulated overnight to induce a memory-like phenotype. After seven days, we analyzed the phenotype and degranulation potential of CD16-/CD56 + and CD16+/CD56 + cells. The subsets were purified by fluorescence-activated cell sorting (FACS) and examined using high-throughput multiplexed quantitative proteomics. CD16 - cells showed higher levels of activating receptors, increased Granulysin expression, and lower inhibitory receptor expression compared to CD16 + cells. Functionally, CD16 - cells exhibited greater degranulation capacity, as determined by CD107a/b expression, when co-incubated with K562 and melanoma cells. Proteomic profiling identified 35 differentially expressed proteins out of 4,750, with 22 downregulated and 13 upregulated in the CD16 - subset. Key proteins included Granzyme family proteins, NCAM1, CALM1, CD247, and Fc receptors. This study provides a detailed characterization of CIML NK cells based on CD16 expression. Our findings highlight the molecular and functional diversity of CIML NK cells and may guide improved cancer immunotherapy strategies.The author(s) acknowledge financial support for the research, authorship, and/or publication of this article. This work was supported by grants GR21178 and GR24081 to group “Inmunopatologı́a Tumoral” of Junta de Extremadura, Ministerio de Hacienda and co-funded by the European Union (to RT and JGC). Research project IB20132 funded by Consejería de Economía, Ciencia y Agenda Digital (now Consejería de Educación, Ciencia y Formación Profesional) of Junta de Extremadura and co-funded by the European Union (to RT); Project PI21/01125 (to RS), and PI19/00075 (to AP) funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union, and PECART 0060-2020 (to RS) from Secretarı́a General de Investigación, Desarrollo e Innovación en Salud, Junta de Andalucı́a, Spain. All co-financed by the European Union, European Regional Development Fund (FEDER) “Una manera de hacer Europa”. Employment promotion program for the hiring of research support personnel (reference TE-0032-21 to MGS) from Consejerı́a de Educación y Empleo, Junta de Extremadura and postdoctoral fellowship (reference DOC_01421 to FH) from Regional Ministry of Economic Transformation, Industry, Knowledge, and Universities of the Junta de Andalucı́a, both co-financed by the European Social Fund (ESF) under the Youth Employment Operational Programme 2014-2020. “The ESF invests in your future”. Grant EQC2018-004308-P funded by MCIN/ AEI/ https://doi.org/10.13039/501100011033 and by ‘ERDF A way of making Europe’.engVoRhttp://creativecommons.org/licenses/by/4.0/CD16Cancer immunotherapyCytokine-induced memory-like NK cellsDegranulation capacityEnrichment analysisNK cellsProteomicsPhenotypic, proteomic, and functional analyses of cytokine-induced memory-like NK cells show two distinct subsets based on CD16 expression.Attribution 4.0 International41131029SCIENTIFIC REPORTSopen access