Enhanced T Cell Reconstitution by Cord Blood Progenitors Expanded Ex-Vivo Using the Notch Ligand Delta1

Previously, we demonstrated that activation of Notch receptors by culture of CD34⁺CD38⁻ cord blood (CB) 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 CB cells cultured with Delta1^ext-IgG compared to recipients of control IgG-cultured or non-cultured CB cells. Furthermore, data suggest that addition of Delta1^ext-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/SCIDγ_c^−/− mice received 10³ non-cultured CB cells or the progeny of 10³ Delta1^ext-IgG-cultured or IgG-cultured cells. At 6 and 8 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 non-cultured CB cells (p<0.03 and p<0.04). At 4 wks after HCT, thymuses from recipients of Delta1^ext-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 Delta1^ext-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 Delta1^ext-IgG-cultured or 10⁶ IgG-cultured CB cells into sub-lethally irradiated NOD/SCIDγ_c^−/− mice. Human engraftment was not observed in mice up to 20 wks after HCT in recipients of IgG-cultured CB cells. Recipients of Delta1^ext-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 Delta1^ext-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 Delta1^ext-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/SCIDγ_c^−/− mice received 10⁶ Delta1^ext-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 Delta1^ext-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 Delta1^ext-IgG-cultured cells compared to mice that received non-cultured cells alone (p<0.05), suggesting a facilitating effect of the Delta1^ext-IgG-cultured cells. Augmentation of engraftment was not observed in mice that received IgG-cultured cells. In summary, although both non-cultured and Delta1^ext-IgG-cultured CB cells were able to reconstitute the T cell lineage, the Delta1^ext-IgG-cultured CB cells had the potential to enhance immune reconstitution early after HCT. We also found that the addition of Delta1^ext-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.