Enhanced T Cell Reconstitution by Hematopoietic Progenitors Expanded Ex-Vivo Using the Notch Ligand Delta1.

Previously, we demonstrated that activation of Notch receptors by culture of murine lin⁻Sca-1⁺c-kit⁺ (LSK) hematopoietic progenitors with the Notch ligand Delta1^ext−IgG, consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG, promoted early T cell differentiation and increased the number of progenitors capable of short-term lymphoid and myeloid reconstitution. Here we show accelerated thymic engraftment and T cell recovery after hematopoietic cell transplantation (HCT) in recipients of LSK cells cultured with Delta1^ext−IgG compared to recipients of non-cultured LSK cells. Furthermore, data suggest that accelerated T cell recovery resulted from progenitor cells induced towards T cell differentiation. 10³ LSK cells cultured with Delta1^ext−IgG generated an 10⁸-fold increase in total number of cells compared to cutlure with control IgG. Using limiting dilution analysis we estimated the number of progenitors with T cell potential in 10³ LSK cells was 2.3; after culture with Delta1^ext−IgG, the number in the 10¹¹ expanded product was 1.9×10⁹, representing a 5×10⁸-fold increase. Lethally irradiated mice received 10⁵ BM cells along with 10³ LSK cells or 10⁶ Delta1^ext−IgG-cultured cells. At 3 and 5 wks after HCT, blood samples from recipients of Delta1^ext−IgG-cultured cells contained 9- and 3-fold higher numbers of CD3⁺ cells compared to recipients of LSK cells (p<0.04 and p<0.05). At 1 wk after HCT, thymuses from recipients of Delta1^ext−IgG-cultured cells showed a 10-and 9-fold increase, respectively, in the percentage of donor CD4⁺ and CD8⁺ cells compared to recipients of LSK cells (p<0.007 and p<0.007). Cultured cells were sorted into fractions containing either Sca-1⁺c-kit⁺ cells or Sca-1⁺c-kit⁺ depleted cells. Both fractions had T cell potential but at 7 wks after HCT, the Sca-1⁺c-kit⁺-enriched fraction maintained 3-fold higher numbers of CD3⁺ cells compared to the other fraction (p<0.04). These data suggest that the Sca-1⁺c-kit⁺ fraction had the potential for more sustained T cell production and contained a more immature precursor compared to the other subsets. Most of the Sca-1⁺c-kit⁺ cells (98%) expressed the lymphoid-associated antigens B220, IL7Rα, CD25 and/or Thy1 (SK-lymph⁺). The SK-lymph⁻ cells were rare, representing 1 in 100 cells in culture. We transplanted proportionate numbers of each population into irradiated mice with 10⁵ BM cells. Recipients of 10⁶ sorted SK-lymph⁺ cells had 20-fold higher numbers of CD3⁺ cells compared to recipients of 10⁴ sorted SK-lymph⁻ cells (p<0.002). Despite injecting 100-fold fewer cells, the SK-lymph⁻ cells generated similar number of B and myeloid reconstitution as SK-lymph⁺ cells. The SK-lymph⁺cells are potent T cell progenitors and serve as more committed progenitors that preferentially reconstitute T cells, since B and myeloid reconstitution was not enhanced. Although, both undifferentiated Sca-1⁺c-kit⁺ cells and cells differentiated toward the lymphoid lineage (SK-lymph⁺) were able to reconstitute the T cell lineage, the SK-lymph⁻ was more efficient on a per cell basis. However, the SK-lymph⁺ population was responsible for the majority of the repopulation as it was the largest population generated following culture with Delta1^ext−IgG. This report describes a novel and clinically feasible ex-vivo culture system using Delta1 for the generation of T cell progenitors as a means to accelerate initial T-cell recovery after HCT.