Ex vivo-generated lymphoid progenitors encompass both T cell and innate lymphoid cell fates

Juliette Paillet^1,2*Pierre GAUDEAUX^1,2Monah Abou Alezz^3,4Ranjita Devi Moirangthem^1,2Sara Cascione^3,4Marta Martin Corredera^1,2Anne-Catherine Dolens⁵Katrien De Mulder⁵Imke Velghe⁵Marieke Lavaert⁵Bart Vandekerckhove⁵Robil Noémie⁶Aurélien Corneau^7,8Hanem SADEK¹Pauline Rault¹Akshay Joshi²Pierre De La Grange⁶Frank Staal^10,9Tom Taghon⁵NEGRE Olivier¹Andrea Ditadi^3,4Isabelle André²Tayebeh-Shabi Soheili^1*

• ¹Smart Immune, Paris, France
• ²Laboratory of Human Lymphohematopoiesis, Institut Imagine, Paris, France
• ³San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), Milan, Lombardy, Italy
• ⁴San Raffaele Scientific Institute (IRCCS), Milan, Lombardy, Italy
• ⁵Cancer Research Institute, Ghent University, Ghent, East Flanders, Belgium
• ⁶GenoSplice technology SARL, Paris, France
• ⁷INSERM U938 Centre de Recherche Saint Antoine (CRSA), Paris, Île-de-France, France
• ⁸Cytometry Core Facility CyPS, Sorbonne Université, Paris, France
• ⁹Department of Immunology, Leiden University Medical Center (LUMC), Leiden, Netherlands
• ¹⁰Willem-Alexander Children's Hospital, Leiden University Medical Center (LUMC), Leiden, Netherlands

We previously established a feeder-free cell therapy platform for the ex vivo generation of lymphoid-primed progenitors using immobilized Delta-like ligand 4 (DLL4). In vivo studies demonstrated that adoptive transfer of these progenitors accelerates T cell reconstitution following thymic engraftment. To further explore the full therapeutic potential of this cell product, we performed a comprehensive molecular and phenotypic characterization using single-cell RNA sequencing and mass cytometry analysis. Our analysis revealed the presence of distinct cell subsets within the cellular product characterized mainly by commitment to lymphoid lineages. Using integrated transcriptomic analyses to compare these ex vivogenerated progenitors to in vivo human thymocytes, we revealed strong similarities with early stages of T cell development, underscoring the physiological relevance of our system.We also delineated two distinct developmental trajectories within the CD7 + progenitor population: a T cell-oriented path, marked by CD5 upregulation, and an innate lymphoid cell (ILC)-oriented branch, identified by CD161 expression and an ILC-like gene signature. Despite these lineage predispositions, both subsets demonstrated plasticity, retaining the ability to differentiate into both T cells and natural killer (NK) cells in vitro. Additionally, in our experimental setting, we observed that BCL11B, a transcription factor essential for T cell commitment, regulates negatively myeloid cell differentiation while preserving the potential for NK cell development.These findings underscore the versatility of DLL4-based lymphoid progenitors in generating either T cells or ILCs in response to environmental cues. This research paves the way for innovative cell therapy approaches to treat immune deficiencies and cancer-and age-related immune dysfunctions.