Enhanced T Cell Reconstitution by Hematopoietic Progenitors Expanded Ex-Vivo Using the Notch Ligand Delta1.
Authors: Dallas Mari H. , Varnum-Finney Barbara , Martin Paul J. , Bernstein Irwin D. 
Published: April 2020

Abstract
Previously, we demonstrated that activation of Notch receptors by culture of murine lin−Sca-1+c-kit+ (LSK) hematopoietic progenitors with the Notch ligand Delta1ext−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 Delta1ext−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 Delta1ext−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 Delta1ext−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 Delta1ext−IgG-cultured cells. At 3 and 5 wks after HCT, blood samples from recipients of Delta1ext−IgG-cultured cells contained 9- and 3-fold higher numbers of CD3+ cells compared to recipients of LSK cells (p&lt;0.04 and p&lt;0.05). At 1 wk after HCT, thymuses from recipients of Delta1ext−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&lt;0.007 and p&lt;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&lt;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&lt;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 Delta1ext−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.