In Vitro Exposure to DL-4 Increases the in Vitro and In Vivo T-Lymphopoietic Potential of CB Derived CD34+ Progenitor Cells

Abstract 2980

Notchligand-based culture systems such as OP9-DL1 cells induce HSC to engage towards the T-cell developmental program and allow generation of T-lymphoid progenitors in vitro. In vitro generated murine T-lymphoid progenitors accelerated T-cell reconstitution in vivo. In consistency, human T-lymphoid progenitors generated in co-culture with OP9-DL1 cells enhanced thymic repopulation when injected into NOD/SCID/gc^−/− mice (NSG). However, positive effects of human T-lymphoid progenitors on peripheral T-cell reconstitution have not been reported yet. Besides, Notchligand-based culture systems, consisting of genetically modified murine cells might raise safety concern for clinical use. It has been described that exposure of CD34⁺ cells to immobilized DL4 induces the T-cell developmental program even in absence of stromal cell support. Recently, we have made use of this system to generate T-lymphoid progenitors in vitro. In the present study we have further characterized their T-lymphoid potential in vitro and in vivo.

Exposure of human CB-derived CD34⁺ cells to immobilized DL4 allowed generation of CD34⁺CD7⁺ and CD34⁻CD7⁺⁺CD5⁺ progenitors displaying a similar phenotype as early thymic progenitors (ETP) and the prethymocytes (pre-T). Within the DL-4 derived ETP- and preT-like progenitors we observed subsequent up regulation of genes involved in T-cell development and silencing of genes implied in B-cell and myeloid differentiation. T-cell commitment of DL-4 progenitors could be further confirmed by early and intermediate rearrangement events within the TCR d/g/b genes. The pattern of gene expression profile and TCR-rearrangement events displayed a pattern similar to what we observed in corresponding intrathymic developmental stages.

DL4-progenitors obtained after 7 days of culture displayed a 30-fold increased in vitro T-lymphoid potential as compared to untreated CD34⁺ CB progenitors. DL4 ETP-like and preT-like progenitors further completed T-cell differentiation in vitro (in OP9DL1 co-culture) faster than native CD34⁺ CB progenitors. When transferred into NSG, DL4 progenitors obtained after 7 days of culture were able to repopulate the recipients' thymus and to give rise to mature, polyclonal intrathymic and peripheral T-cells. Two months after transfer recipients of DL4 progenitors displayed advanced intrathymic T-cell development as compared to recipients of CD34⁺ CB cells. Furthermore, peripheral T-cells could be observed in a number of DL-4 progenitor recipients but not in control mice. Our experiments provide further evidence that DL4 allows in vitro induction of T-cell development and generation of early T-lymphoid progenitors in a system devoid of stromal cell support. These progenitors feature phenotypical and molecular characteristics of immature thymic developmental stages. Moreover, they are able to accelerate T-cell development in vitro and when transferred into NSG.

This work provides further evidence of the ability of in vitro -generated human T-cell progenitors to accelerate T-cell reconstitution and simultaneously introduces a culture technique that could be rapidly transferred into a clinical setting.