{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T16:01:13Z","timestamp":1787932873439,"version":"build-2784847793"},"reference-count":20,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2023,2,9]],"date-time":"2023-02-09T00:00:00Z","timestamp":1675900800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"INCA\/DGOS PRT-K program 2021","award":["2021-014"],"award-info":[{"award-number":["2021-014"]}]},{"name":"INCA\/DGOS PRT-K program 2021","award":["ANR-10-LABX-53"],"award-info":[{"award-number":["ANR-10-LABX-53"]}]},{"name":"Montpellier Universit\u00e9 d\u2019Excellence","award":["2021-014"],"award-info":[{"award-number":["2021-014"]}]},{"name":"Montpellier Universit\u00e9 d\u2019Excellence","award":["ANR-10-LABX-53"],"award-info":[{"award-number":["ANR-10-LABX-53"]}]},{"name":"\u201cInvestissements d\u2019avenir\u201d Grant LabEx MAbImprove","award":["2021-014"],"award-info":[{"award-number":["2021-014"]}]},{"name":"\u201cInvestissements d\u2019avenir\u201d Grant LabEx MAbImprove","award":["ANR-10-LABX-53"],"award-info":[{"award-number":["ANR-10-LABX-53"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cancers"],"abstract":"<jats:p>CD20 monoclonal antibodies (mAbs) eliminate B cells in several clinical contexts. At least two of these Abs, obinutuzumab (OBI) and rituximab (RTX), induce quick elimination of targets and put cancer patients at risk of tumor lysis syndrome (TLS) within 12\u201324 h of the first dose. The mechanisms of killing can require the recruiting of effector mechanisms from the patient\u2019s immune system, but they can induce direct killing as well. This can be more rapid than recruiting cellular effectors and\/or complement. We showed here that OBI and RTX induce quick (&lt;1 h) and high (up to 60% for OBI) killing of two different B cell lines. This was unveiled by using two different techniques that circumvent cell centrifugation steps: a Muse\u00ae Cell Analyzer-based approach and a direct examination of the cells\u2019 physical properties by using forward scatter (FS) area and side scatter (SS) area by flow cytometry. These results excluded the presence of aggregates and were also confirmed by developing a normalized survival ratio based on the co-incubation of RTX- and OBI-sensitive cells with MOLM-13, an insensitive cell line. Finally, this normalized survival ratio protocol confirmed the RTX- and OBI-direct killing on primary tumor B cells from B cell chronic lymphocytic leukemia (B-CLL) and Non-Hodgkin\u2019s lymphoma (NHL) patients. Moreover, we unveiled that direct killing is higher than previously expected and absent in patients\u2019 samples at relapse. We also observed that these mAbs, prior to increasing intracellular calcium levels, decrease calcium entry, although manipulating calcium levels did not affect their cytotoxicity. Altogether, our results show that direct killing is a major mechanism to induce cell death by RTX and OBI mAbs.<\/jats:p>","DOI":"10.3390\/cancers15041109","type":"journal-article","created":{"date-parts":[[2023,2,10]],"date-time":"2023-02-10T01:35:29Z","timestamp":1675992929000},"page":"1109","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Direct Cell Death Induced by CD20 Monoclonal Antibodies on B Cell Lymphoma Cells Revealed by New Protocols of Analysis"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3507-9161","authenticated-orcid":false,"given":"Michael","family":"Constantinides","sequence":"first","affiliation":[{"name":"IRMB, University of Montpellier, INSERM, CHRU de Montpellier, F-34090 Montpellier, France"},{"name":"D\u00e9partement d\u2019H\u00e9matologie Clinique, CHRU de Montpellier, F-34090 Montpellier, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexis","family":"Fayd\u2019herbe De Maudave","sequence":"additional","affiliation":[{"name":"IRMB, University of Montpellier, INSERM, CHRU de Montpellier, F-34090 Montpellier, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1361-3397","authenticated-orcid":false,"given":"Marie","family":"Potier-Cartereau","sequence":"additional","affiliation":[{"name":"Inserm UMR 1069, Nutrition Croissance Cancer, Facult\u00e9 de M\u00e9decine, Universit\u00e9 de Tours, F-37032 Tours, France; R\u00e9seau 3MC \u201cMol\u00e9cules Marines, M\u00e9tabolisme et Cancer\u201d and R\u00e9seau CASTOR \u201cCancers des Tissus Hormono-D\u00e9pendants\u201d Canc\u00e9rop\u00f4le Grand Ouest, F-34090 Montpellier, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mauricio","family":"Campos-Mora","sequence":"additional","affiliation":[{"name":"IRMB, University of Montpellier, INSERM, CHRU de Montpellier, F-34090 Montpellier, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guillaume","family":"Cartron","sequence":"additional","affiliation":[{"name":"D\u00e9partement d\u2019H\u00e9matologie Clinique, CHRU de Montpellier, F-34090 Montpellier, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4385-4888","authenticated-orcid":false,"given":"Martin","family":"Villalba","sequence":"additional","affiliation":[{"name":"IRMB, University of Montpellier, INSERM, CHRU de Montpellier, F-34090 Montpellier, France"},{"name":"IRMB, University of Montpellier, INSERM, CNRS, CHRU de Montpellier, F-34090 Montpellier, France"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,2,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"163","DOI":"10.3389\/fonc.2018.00163","article-title":"Past, Present, and Future of Rituximab-The World\u2019s First Oncology Monoclonal Antibody Therapy","volume":"8","author":"Pierpont","year":"2018","journal-title":"Front. 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Oncol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"477","DOI":"10.1182\/bloodadvances.2019001148","article-title":"Rituximab leads to early elimination of circulating CD20+ T and B lymphocytes in patients with iTTP despite ongoing TPEx","volume":"4","author":"Carden","year":"2020","journal-title":"Blood Adv."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"3372","DOI":"10.1182\/blood-2010-06-289736","article-title":"Possible misinterpretation of the mode of action of therapeutic antibodies in vitro: Homotypic adhesion and flow cytometry result in artefactual direct cell death","volume":"116","author":"Golay","year":"2010","journal-title":"Blood"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1039","DOI":"10.1158\/1078-0432.CCR-11-1429","article-title":"Regulation of CD20 in Rituxi-mab-Resistant Cell Lines and B-cell Non-Hodgkin Lymphoma","volume":"18","author":"Tsai","year":"2012","journal-title":"Clin. 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