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Their ability to engage in killing is critical for efficient target elimination and depends on tightly regulated Ca2+ signaling controlling granzyme degranulation. Toll-like receptor (TLR) engagement has been linked to Ca2+ signaling in other immune cell types, while a specific role in NK cells remains unresolved. Given that NK cells express a broad range of TLRs, and that presence of TLR ligands\u2014including damage- and pathogen-associated molecular patterns\u2014can influence clinical scenarios including adoptive NK cell therapy, investigating TLR-driven Ca2+ signaling in NK cells is particularly relevant. Here, we examined how stimulation with selected TLR ligands influences Ca2+ signaling and NK cell activity. Immediate stimulation induced a rapid elevation of cytosolic Ca2+ in expanded NK cells. Functionally, TLR stimulation increased degranulation and enhanced cytotoxicity at high effector-to-target ratios. Under conditions of target excess, however, TLR-treated NK cells displayed impaired killing, likely due to unbalanced Ca2+ levels. These findings demonstrate that TLR signaling directly modulates Ca2+ flux in NK cells and can either potentiate or impair cytotoxic activity depending on context. Although clinical implications remain hypothetical, such TLR-driven dysregulation affects NK cell killing activity in the inflammatory or pathogen-rich environments frequently encountered after chemotherapy. Our findings suggest that TLR\u2013Ca2+ impact on cytotoxicity should be considered in adoptive transfer therapies where previous treatment affects systemic levels of TLR ligands, such as in patients with acute myeloid leukemia, where NK cell immunotherapy is frequently tested in trials.<\/jats:p>","DOI":"10.1093\/jleuko\/qiag092","type":"journal-article","created":{"date-parts":[[2026,7,20]],"date-time":"2026-07-20T11:25:06Z","timestamp":1784546706000},"source":"Crossref","is-referenced-by-count":0,"title":["TLR-mediated calcium signaling regulates killing capacity of expanded NK cells: challenges for potential AML immunotherapy"],"prefix":"10.1093","volume":"118","author":[{"given":"Vojt\u011bch","family":"Graman","sequence":"first","affiliation":[{"name":"Department of Modern Immunotherapy Research, Institute of Hematology and Blood Transfusion , U Nemocnice 1, Prague 2 128 00 ,","place":["Czechia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ad\u00e9la","family":"J\u00edlkov\u00e1","sequence":"additional","affiliation":[{"name":"Department of Modern Immunotherapy Research, Institute of Hematology and Blood Transfusion , U Nemocnice 1, Prague 2 128 00 ,","place":["Czechia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marek","family":"Jedli\u010dka","sequence":"additional","affiliation":[{"name":"Department of Modern Immunotherapy Research, Institute of Hematology and Blood Transfusion , U Nemocnice 1, Prague 2 128 00 ,","place":["Czechia"]},{"name":"Department of Cell Biology, Faculty of Science, Charles University , Albertov 6, Prague 2 128 00 ,","place":["Czechia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Veronika","family":"\u0160vubov\u00e1","sequence":"additional","affiliation":[{"name":"Department of Modern Immunotherapy Research, Institute of Hematology and Blood Transfusion , U Nemocnice 1, Prague 2 128 00 ,","place":["Czechia"]},{"name":"Department of Cell Biology, Faculty of Science, Charles University , Albertov 6, Prague 2 128 00 ,","place":["Czechia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zuzana","family":"Tom\u00e1\u0161ikov\u00e1","sequence":"additional","affiliation":[{"name":"Cellular and Molecular Immunoregulation, International Clinical Research Center, St.Anne's University Hospital Brno , Peka\u0159sk\u00e1 53, Brno 656 91 ,","place":["Czechia"]},{"name":"Department of Experimental Biology, Faculty of Science, Masaryk University , Kamenice 753\/5, Brno 625 00 ,","place":["Czechia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mat\u00fa\u0161","family":"Otruba","sequence":"additional","affiliation":[{"name":"Department of Modern Immunotherapy Research, Institute of Hematology and Blood Transfusion , U Nemocnice 1, Prague 2 128 00 ,","place":["Czechia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kate\u0159ina","family":"Kuglerov\u00e1","sequence":"additional","affiliation":[{"name":"Department of Modern Immunotherapy Research, Institute of Hematology and Blood Transfusion , U Nemocnice 1, Prague 2 128 00 ,","place":["Czechia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-5954-5191","authenticated-orcid":false,"given":"Eva","family":"Ma\u0161\u00ednov\u00e1","sequence":"additional","affiliation":[{"name":"Department of Modern Immunotherapy Research, Institute of Hematology and Blood Transfusion , U Nemocnice 1, Prague 2 128 00 ,","place":["Czechia"]},{"name":"Department of Cell Biology, Faculty of Science, Charles University , Albertov 6, Prague 2 128 00 ,","place":["Czechia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jana","family":"Szabov\u00e1","sequence":"additional","affiliation":[{"name":"Department of Modern Immunotherapy Research, Institute of Hematology and Blood Transfusion , U Nemocnice 1, Prague 2 128 00 ,","place":["Czechia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2657-1664","authenticated-orcid":false,"given":"Michal","family":"Santocki","sequence":"additional","affiliation":[{"name":"Cellular and Molecular Immunoregulation, International Clinical Research Center, St.Anne's University Hospital Brno , Peka\u0159sk\u00e1 53, Brno 656 91 ,","place":["Czechia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tereza","family":"Fiedlerov\u00e1","sequence":"additional","affiliation":[{"name":"Department of Modern Immunotherapy Research, Institute of Hematology and Blood Transfusion , U Nemocnice 1, Prague 2 128 00 ,","place":["Czechia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marcela Hortov\u00e1","family":"Kohoutkov\u00e1","sequence":"additional","affiliation":[{"name":"Cellular and Molecular 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