Enhanced T Cell Reconstitution by Cord Blood Progenitors Expanded Ex-Vivo Using the Notch Ligand Delta1
Authors: Dallas Mari , Delaney Colleen , Bernstein Irwin D. 
Published: October 2019

Abstract
Previously, we demonstrated that activation of Notch receptors by culture of CD34+CD38− cord blood (CB) 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 CB cells cultured with Delta1ext-IgG compared to recipients of control IgG-cultured or non-cultured CB cells. Furthermore, data suggest that addition of Delta1ext-IgG-cultured CB cells to a non-cultured CB graft facilitated engraftment of the non-cultured CB cells. CD34+CD38− CB cells cultured on immobilized Delta1 in serum free media with Il3, Il6, Flt3l, SCF and TPO for 16 days generated a107-fold increase in total number of cells as well as a 105-fold increase in the number of T-lymphoid biased (CD34+CD7+CD45RA+) cells. Sub-lethally irradiated (275cGy) NOD/SCIDγc−/− mice received 103 non-cultured CB cells or the progeny of 103 Delta1ext-IgG-cultured or IgG-cultured cells. At 6 and 8 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 non-cultured CB cells (p<0.03 and p<0.04). At 4 wks after HCT, thymuses from recipients of Delta1ext-IgG-cultured cells showed a 9-fold increase in the number of donor CD4+ and/or CD8+ cells compared to recipients of non-cultured cells (p<0.05). These data suggest that Delta1ext-IgG-cultured CB cells have the potential for enhancing early T cell recovery after HCT compared to the noncultured CB cells. Next, we transplanted equal numbers of cells by injecting 106 Delta1ext-IgG-cultured or 106 IgG-cultured CB cells into sub-lethally irradiated NOD/SCIDγc−/− mice. Human engraftment was not observed in mice up to 20 wks after HCT in recipients of IgG-cultured CB cells. Recipients of Delta1ext-IgG-cultured CB cells had detectable CD3+ cells 4 wks after HCT. Despite injecting the equivalent number of cells, the IgG-cultured CB cells did not engraft while the Delta1ext-IgG-cultured cells were able to provide early T cell reconstitution. Lastly, we determined the effect of T cell reconstitution by augmenting a non-cultured CB graft with Delta1ext-IgG-cultured CB. The relative contribution of T cells by the two different CB cells was determined using two HLA disparate CB units. Sub-lethally irradiated NOD/SCIDγc−/− mice received 106 Delta1ext-IgG-cultured cells along with 103 non-cultured CB cells or 106 IgG-cultured CB cells along with 103 non-cultured CB cells or 103 non-cultured CB cells alone. At 6 and 8 wks after HCT, blood samples from the recipient of the non-cultured CB cells augmented with Delta1ext-IgG-cultured had 5-fold higher number of CD3+ cells compared to the other two control groups (p<0.04). Furthermore, CD3+ cells derived from the non-cultured CB cells was 2-fold higher in the mice that received the addition of Delta1ext-IgG-cultured cells compared to mice that received non-cultured cells alone (p<0.05), suggesting a facilitating effect of the Delta1ext-IgG-cultured cells. Augmentation of engraftment was not observed in mice that received IgG-cultured cells. In summary, although both non-cultured and Delta1ext-IgG-cultured CB cells were able to reconstitute the T cell lineage, the Delta1ext-IgG-cultured CB cells had the potential to enhance immune reconstitution early after HCT. We also found that the addition of Delta1ext-IgG-cutlured cells to non-cultured CB cells facilitated the lymphoid engraftment of non-cultured CB. This report describes a novel and clinically feasible exvivo culture system using Delta1 for the generation of CB cell progenitors as a means to accelerate initial T-cell recovery after HCT.