Further Characterization of T-Cellular Precursors Generated From CD34+ Progenitors by Exposure to Immobilized Notch Ligand Delta-Like 4 In Vitro.
Authors: Reimann Christian , Schiavo Andrea , Rouiller Julien , Vidal Elodie , Beldjord Kheira , Dal-Cortivo Liliane , Cavazzana-Calvo Marina , Andre-Schmutz Isabelle 
Published: October 2019

Abstract
Abstract 3712
Injection of donor derived T-cellular precursors has been proposed as a novel strategy to shorten delayed reconstitution of the T-lymphoid compartment following HSCT. In the past years, several research groups have successfully generated murine and human T-cellular precursors in vitro using Notchligand-based coculture systems such as OP9-DL1 or Tst-DL4. Murine T-cellular precursors generated in vitro, promoted reconstitution of the T-cellular compartment when applied in murine HSCT-models. In consistency, transfer of human T-cellular precursors, generated in vitro in coculture with OP9-DL1 or Tst-DL4 resulted in enhanced thymic repopulation in NOD/SCID/gc−/− mice. Yet, positive effects on peripheral T-cell reconstitution have not been reported. Moreover, clinical application of OP9-DL1 or Tst-DL4 coculture systems is limited, since they consist of murine stromal cells transduced with either DL1 or DL4. It has been described that exposure of CD34+ cells to immobilized DL4 induces T-cell differentiation in vitro and allows expansion human T-cellular precursors even in absence of stromal cell support. However, the hypothesis that DL4 alone can drive hematopoietic progenitors towards a T-cell fate in vitro, requires more evidence. Here, we further characterized the in vitro and in vivo potential of T-cellular precursors generated by single exposure to DL4.
We exposed human CD34+ progenitors to immobilized DL4 in the presence of different cytokine combinations implicated in human haematopoiesis. Within 7 days, CD34+CD7+ and CD34−CD7++ T-cellular precursors emerged in the presence of DL4, but not under control conditions. After 7 days the CD34+CD7+ population subsequently declined while the CD34−CD7++ population further expanded. Two distinct progenitor subsets, CD5+ and CD5-, emerged within the CD34−CD7++ population. The CD34−CD7++CD5+ subset partially acquired CD1a, corresponding to a developmental stage between the early thymic progenitor (ETP) and the prethymocyte (pre-T) stage. Conversely to what observed in the OP9-DL1 system, T-cell development did not progress beyond the pre-T-stage. Indeed, we neither observed more advanced stages of T-cell development, such as immature single positive CD4+ cells, nor complete TCR-rearrangements.
7-day exposure to immobilized DL4 induced a 90-fold increase of T-precursor frequency in CD34+ progenitors (1/8800 before culture vs. 1/90 after culture) as confirmed by limiting dilution assays on OP9-DL1. All T-cellular precursor activity was restricted to cells expressing CD34, CD7 or both (frequency: 1/9). In particular, elevated T-cellular precursor levels were found in the subsets expressing CD7 (CD34+/CD7+ and CD34−/CD7+), while the T-cellular precursor frequency in the CD34+/CD7− subset was equal to that seen in non-cultured CD34+ progenitors. In consistency the CD34−CD7− population did not contain any detectable T-cellular precursors.
After 7 day exposure to DL4, cells phenotypically corresponding to T-cellular precursors were transferred into NOD/SCID/gc−/− mice. Within 2 months following HSCT, cells exposed to DL4 were able to reconstitute the recipients' thymus and partially gave rise to peripheral T-cells. When injecting non-cultured CD34+ progenitors, thymic reconstitution was likewise seen 2 months after HSCT. However, intrathymic T-cell development was less advanced and peripheral T-cells were absent. In contrast, cells cultured in presence of a control peptide did not retain any potential to repopulate the recipients' thymus.
Our experiments provide further evidence that exposure DL4 induces early human T-cell development and allows generation of large numbers of T-cellular precursors in vitro. These precursors feature phenotypical and molecular properties corresponding to early precursors found in the human thymus. Furthermore, they have an increased potential to further differentiate into mature T-cells in vitro and when transferred into immunodeficient mice. Our preliminary data suggest, that injection of T-cellular precursors accelerates T-cell reconstitution after HSCT and provides further evidence for the feasibility of this novel strategy of immunotherapy.

Disclosures:
No relevant conflicts of interest to declare.