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

## TL;DR

This study shows that lab-generated lymphoid progenitors can become both T cells and innate lymphoid cells, offering potential for new cell therapies.

## Contribution

The discovery of dual developmental plasticity in DLL4-based lymphoid progenitors is novel.

## Key findings

- Ex vivo-generated progenitors show strong similarities to early T cell development stages.
- Two distinct developmental paths were identified: T cell-oriented and ILC-oriented.
- BCL11B regulates myeloid differentiation while preserving NK cell potential.

## Abstract

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 vivo-generated 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.

Flowchart illustrating the analysis of ProTCells, highlighting their differentiation pathways into T cell or ILC/NK lineages. The scRNAseq and mass cytometry analyses reveal gene expression patterns, including CD7 and CD161 markers, with emphasis on BCL11b expression. The chart details cell trajectory analysis, developmental plasticity between lineages, and experiments including injection into NSG mice for thymus colonization assessment.