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Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting

BACKGROUND: On encountering a susceptible target, natural killer (NK) cells mediate cytotoxicity through highly regulated steps of directed degranulation. Cytotoxic granules converge at the microtubule organizing center and are polarized toward the immunological synapse (IS), followed by granule exo...

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Autores principales: Eitler, Jiri, Wotschel, Natalie, Miller, Nicole, Boissel, Laurent, Klingemann, Hans G, Wels, Winfried, Tonn, Torsten
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BMJ Publishing Group 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817806/
https://www.ncbi.nlm.nih.gov/pubmed/33468562
http://dx.doi.org/10.1136/jitc-2020-001334
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author Eitler, Jiri
Wotschel, Natalie
Miller, Nicole
Boissel, Laurent
Klingemann, Hans G
Wels, Winfried
Tonn, Torsten
author_facet Eitler, Jiri
Wotschel, Natalie
Miller, Nicole
Boissel, Laurent
Klingemann, Hans G
Wels, Winfried
Tonn, Torsten
author_sort Eitler, Jiri
collection PubMed
description BACKGROUND: On encountering a susceptible target, natural killer (NK) cells mediate cytotoxicity through highly regulated steps of directed degranulation. Cytotoxic granules converge at the microtubule organizing center and are polarized toward the immunological synapse (IS), followed by granule exocytosis. NK cell retargeting by chimeric antigen receptors (CARs) or mAbs represents a promising strategy for overcoming tumor cell resistance. However, little is known about the lytic granule dynamics of such retargeted NK cells toward NK-cell-resistant tumors. METHODS: Here, we used spinning disk confocal microscopy for live-cell imaging to analyze granule-mediated NK cell cytotoxicity in ErbB2-targeted CAR-expressing NK-92 cells (NK-92/5.28.z) and high-affinity FcR transgenic NK-92 cells plus Herceptin toward ErbB2-positive breast cancer cells (MDA-MB-453), which are resistant to parental NK-92. RESULTS: Unmodified NK-92 cells cocultured with resistant cancer cells showed stable conjugate formation and granule clustering, but failed to polarize granules to the IS. In contrast, retargeting by CAR or FcR+Herceptin toward the MDA-MB-453 cells enabled granule polarization to the IS, resulting in highly effective cytotoxicity. We found that in NK-92 the phosphoinositide 3-kinase pathway was activated after contact with resistant MDA-MB-453, while phospholipase C-γ (PLCγ) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) were not activated. In contrast, retargeting by CAR or antibody-dependent cell-mediated cytotoxicity (ADCC) provided the missing PLCγ and MEK/ERK signals. CONCLUSIONS: These observations suggest that NK cells can create conjugates with resistant cancer cells and respond by granule clustering, but the activation signals are insufficient to induce granule polarization and consequent release of lytic enzymes. Retargeting by CAR and/or the FcR/mAb (ADCC) axis provide the necessary signals, leading to granule polarization and thereby overcoming tumor cell resistance. Keywords: NK cells, NK-92, haNK, ADCC, Chimeric Antigen Receptor (CAR), breast cancer, cancer immunotherapy, live-cell imaging, granule polarization
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spelling pubmed-78178062021-01-28 Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting Eitler, Jiri Wotschel, Natalie Miller, Nicole Boissel, Laurent Klingemann, Hans G Wels, Winfried Tonn, Torsten J Immunother Cancer Basic Tumor Immunology BACKGROUND: On encountering a susceptible target, natural killer (NK) cells mediate cytotoxicity through highly regulated steps of directed degranulation. Cytotoxic granules converge at the microtubule organizing center and are polarized toward the immunological synapse (IS), followed by granule exocytosis. NK cell retargeting by chimeric antigen receptors (CARs) or mAbs represents a promising strategy for overcoming tumor cell resistance. However, little is known about the lytic granule dynamics of such retargeted NK cells toward NK-cell-resistant tumors. METHODS: Here, we used spinning disk confocal microscopy for live-cell imaging to analyze granule-mediated NK cell cytotoxicity in ErbB2-targeted CAR-expressing NK-92 cells (NK-92/5.28.z) and high-affinity FcR transgenic NK-92 cells plus Herceptin toward ErbB2-positive breast cancer cells (MDA-MB-453), which are resistant to parental NK-92. RESULTS: Unmodified NK-92 cells cocultured with resistant cancer cells showed stable conjugate formation and granule clustering, but failed to polarize granules to the IS. In contrast, retargeting by CAR or FcR+Herceptin toward the MDA-MB-453 cells enabled granule polarization to the IS, resulting in highly effective cytotoxicity. We found that in NK-92 the phosphoinositide 3-kinase pathway was activated after contact with resistant MDA-MB-453, while phospholipase C-γ (PLCγ) and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) were not activated. In contrast, retargeting by CAR or antibody-dependent cell-mediated cytotoxicity (ADCC) provided the missing PLCγ and MEK/ERK signals. CONCLUSIONS: These observations suggest that NK cells can create conjugates with resistant cancer cells and respond by granule clustering, but the activation signals are insufficient to induce granule polarization and consequent release of lytic enzymes. Retargeting by CAR and/or the FcR/mAb (ADCC) axis provide the necessary signals, leading to granule polarization and thereby overcoming tumor cell resistance. Keywords: NK cells, NK-92, haNK, ADCC, Chimeric Antigen Receptor (CAR), breast cancer, cancer immunotherapy, live-cell imaging, granule polarization BMJ Publishing Group 2021-01-18 /pmc/articles/PMC7817806/ /pubmed/33468562 http://dx.doi.org/10.1136/jitc-2020-001334 Text en © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. https://creativecommons.org/licenses/by-nc/4.0/This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/ (https://creativecommons.org/licenses/by-nc/4.0/) .
spellingShingle Basic Tumor Immunology
Eitler, Jiri
Wotschel, Natalie
Miller, Nicole
Boissel, Laurent
Klingemann, Hans G
Wels, Winfried
Tonn, Torsten
Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting
title Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting
title_full Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting
title_fullStr Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting
title_full_unstemmed Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting
title_short Inability of granule polarization by NK cells defines tumor resistance and can be overcome by CAR or ADCC mediated targeting
title_sort inability of granule polarization by nk cells defines tumor resistance and can be overcome by car or adcc mediated targeting
topic Basic Tumor Immunology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7817806/
https://www.ncbi.nlm.nih.gov/pubmed/33468562
http://dx.doi.org/10.1136/jitc-2020-001334
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