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CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo
To present antigens to cognate T cells, dendritic cells (DCs) exploit the chemokine receptor CCR7 to travel from peripheral tissue via afferent lymphatic vessels to directly enter draining lymph nodes through the floor of the subcapsular sinus. Here, we combined unlimited proliferative capacity of c...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120996/ https://www.ncbi.nlm.nih.gov/pubmed/30210501 http://dx.doi.org/10.3389/fimmu.2018.01949 |
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author | Hammerschmidt, Swantje I. Werth, Kathrin Rothe, Michael Galla, Melanie Permanyer, Marc Patzer, Gwendolyn E. Bubke, Anja Frenk, David N. Selich, Anton Lange, Lucas Schambach, Axel Bošnjak, Berislav Förster, Reinhold |
author_facet | Hammerschmidt, Swantje I. Werth, Kathrin Rothe, Michael Galla, Melanie Permanyer, Marc Patzer, Gwendolyn E. Bubke, Anja Frenk, David N. Selich, Anton Lange, Lucas Schambach, Axel Bošnjak, Berislav Förster, Reinhold |
author_sort | Hammerschmidt, Swantje I. |
collection | PubMed |
description | To present antigens to cognate T cells, dendritic cells (DCs) exploit the chemokine receptor CCR7 to travel from peripheral tissue via afferent lymphatic vessels to directly enter draining lymph nodes through the floor of the subcapsular sinus. Here, we combined unlimited proliferative capacity of conditionally Hoxb8-immortalized hematopoietic progenitor cells with CRISPR/Cas9 technology to create a powerful experimental system to investigate DC migration and function. Hematopoietic progenitor cells from the bone marrow of Cas9-transgenic mice were conditionally immortalized by lentiviral transduction introducing a doxycycline-regulated form of the transcription factor Hoxb8 (Cas9-Hoxb8 cells). These cells could be stably cultured for weeks in the presence of doxycycline and puromycin, allowing us to introduce additional genetic modifications applying CRISPR/Cas9 technology. Importantly, modified Cas9-Hoxb8 cells retained their potential to differentiate in vitro into myeloid cells, and GM-CSF-differentiated Cas9-Hoxb8 cells showed the classical phenotype of GM-CSF-differentiated bone marrow-derived dendritic cells. Following intralymphatic delivery Cas9-Hoxb8 DCs entered the lymph node in a CCR7-dependent manner. Finally, we used two-photon microscopy and imaged Cas9-Hoxb8 DCs that expressed the genetic Ca(2+) sensor GCaMP6S to visualize in real-time chemokine-induced Ca(2+) signaling of lymph-derived DCs entering the LN parenchyma. Altogether, our study not only allows mechanistic insights in DC migration in vivo, but also provides a platform for the immunoengineering of DCs that, in combination with two-photon imaging, can be exploited to further dissect DC dynamics in vivo. |
format | Online Article Text |
id | pubmed-6120996 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-61209962018-09-12 CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo Hammerschmidt, Swantje I. Werth, Kathrin Rothe, Michael Galla, Melanie Permanyer, Marc Patzer, Gwendolyn E. Bubke, Anja Frenk, David N. Selich, Anton Lange, Lucas Schambach, Axel Bošnjak, Berislav Förster, Reinhold Front Immunol Immunology To present antigens to cognate T cells, dendritic cells (DCs) exploit the chemokine receptor CCR7 to travel from peripheral tissue via afferent lymphatic vessels to directly enter draining lymph nodes through the floor of the subcapsular sinus. Here, we combined unlimited proliferative capacity of conditionally Hoxb8-immortalized hematopoietic progenitor cells with CRISPR/Cas9 technology to create a powerful experimental system to investigate DC migration and function. Hematopoietic progenitor cells from the bone marrow of Cas9-transgenic mice were conditionally immortalized by lentiviral transduction introducing a doxycycline-regulated form of the transcription factor Hoxb8 (Cas9-Hoxb8 cells). These cells could be stably cultured for weeks in the presence of doxycycline and puromycin, allowing us to introduce additional genetic modifications applying CRISPR/Cas9 technology. Importantly, modified Cas9-Hoxb8 cells retained their potential to differentiate in vitro into myeloid cells, and GM-CSF-differentiated Cas9-Hoxb8 cells showed the classical phenotype of GM-CSF-differentiated bone marrow-derived dendritic cells. Following intralymphatic delivery Cas9-Hoxb8 DCs entered the lymph node in a CCR7-dependent manner. Finally, we used two-photon microscopy and imaged Cas9-Hoxb8 DCs that expressed the genetic Ca(2+) sensor GCaMP6S to visualize in real-time chemokine-induced Ca(2+) signaling of lymph-derived DCs entering the LN parenchyma. Altogether, our study not only allows mechanistic insights in DC migration in vivo, but also provides a platform for the immunoengineering of DCs that, in combination with two-photon imaging, can be exploited to further dissect DC dynamics in vivo. Frontiers Media S.A. 2018-08-28 /pmc/articles/PMC6120996/ /pubmed/30210501 http://dx.doi.org/10.3389/fimmu.2018.01949 Text en Copyright © 2018 Hammerschmidt, Werth, Rothe, Galla, Permanyer, Patzer, Bubke, Frenk, Selich, Lange, Schambach, Bošnjak and Förster. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Immunology Hammerschmidt, Swantje I. Werth, Kathrin Rothe, Michael Galla, Melanie Permanyer, Marc Patzer, Gwendolyn E. Bubke, Anja Frenk, David N. Selich, Anton Lange, Lucas Schambach, Axel Bošnjak, Berislav Förster, Reinhold CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo |
title | CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo |
title_full | CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo |
title_fullStr | CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo |
title_full_unstemmed | CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo |
title_short | CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo |
title_sort | crispr/cas9 immunoengineering of hoxb8-immortalized progenitor cells for revealing ccr7-mediated dendritic cell signaling and migration mechanisms in vivo |
topic | Immunology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120996/ https://www.ncbi.nlm.nih.gov/pubmed/30210501 http://dx.doi.org/10.3389/fimmu.2018.01949 |
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