Engineering an inducible leukemia-associated fusion protein enables large-scale ex vivo production of functional human phagocytes

Windisch, Roland ; Soliman, Sarah; Hoffmann, Adrian; Chen-Wichmann, Linping ; Danese, Anna; Vosberg, Sebastian ; Bravo, Jimena; Lutz, Sebastian; Kellner, Christian ; Fischer, Alexander ; Gebhard, Claudia ; Redondo Monte, Enric; Hartmann, Luise; Schneider, Stephanie; Beier, Fabian ; Strobl, Carolin Dorothea ; Weigert, Oliver ; Peipp, Matthias; Schündeln, Michael ; Stricker, Stefan H. ; Rehli, Michael ; Bernhagen, Jürgen ; Humpe, Andreas; Klump, Hannes ; Brendel, Christian ; Krause, Daniela S. ; Greif, Philipp A. und Wichmann, Christian (2024): Engineering an inducible leukemia-associated fusion protein enables large-scale ex vivo production of functional human phagocytes. In: Proceedings of the National Academy of Sciences, Bd. 121, Nr. 25, e2312499121

Abstract

Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells remains a challenge due to rapid differentiation after detachment from the bone marrow niche. In this study, we assessed the capacity of an inducible fusion protein to enable sustained ex vivo proliferation of hematopoietic precursors and their capacity to differentiate into functional phagocytes. We fused the coding sequences of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the myeloid/lymphoid lineage leukemia/eleven nineteen leukemia (MLL-ENL) fusion gene to generate the fusion protein DD-MLL-ENL and retrovirally expressed the protein switch in human CD34+ progenitors. Using Shield1, a chemical inhibitor of DD fusion protein degradation, we established large-scale and long-term expansion of late monocytic precursors. Upon Shield1 removal, the cells lost self-renewal capacity and spontaneously differentiated, even after 2.5 y of continuous ex vivo expansion. In the absence of Shield1, stimulation with IFN-γ, LPS, and GM-CSF triggered terminal differentiation. Gene expression analysis of the obtained phagocytes revealed marked similarity with naïve monocytes. In functional assays, the novel phagocytes migrated toward CCL2, attached to VCAM-1 under shear stress, produced reactive oxygen species, and engulfed bacterial particles, cellular particles, and apoptotic cells. Finally, we demonstrated Fcγ receptor recognition and phagocytosis of opsonized lymphoma cells in an antibody-dependent manner. Overall, we have established an engineered protein that, as a single factor, is useful for large-scale ex vivo production of human phagocytes. Such adjustable proteins have the potential to be applied as molecular tools to produce functional immune cells for experimental cell-based approaches.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Medizin > Physiologisches Institut ; Medizin > Munich Cluster for Systems Neurology (SyNergy) ; Medizin > Institut für Schlaganfall- und Demenzforschung (ISD) ; Medizin > Klinikum der LMU München ; Medizin > Klinikum der LMU München > Klinik für Anaesthesiologie ; Medizin > Klinikum der LMU München > Medizinische Klinik und Poliklinik III (Onkologie)
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• ISSN: 0027-8424
• Sprache: Englisch
• DFG: Gefördert durch die Deutsche Forschungsgemeinschaft (DFG) - 390857198