Robust Progenitor T-Cell Production From Human Hematopoietic Progenitor Cell Expanded with Stemregenin-1

T cells are critical mediators of anti-viral and fungal immunity, and are key for the prevention of relapse post hematopoietic stem cell transplant (HSCT). Although HSCT can restore a functional immune system, profound immune deficiency still exists due to impaired recovery of the T cell compartment. Underlying causes are a defective thymic microenvironment due to conditioning regimens and decreased production of T cell progenitors (Tprogs) in the transplanted host. This results in an increased risk of relapse and infection. We have adapted a novel methodology to circumvent this issue. Stem-Reginin 1 (SR-1) has recently been shown by our group to expand CD34+ umbilical cord blood (UCB) stem cells in culture by a median of 330-fold. In a Phase I/II clinical trial, engraftment of SR-1-expanded CD34+ cells successfully expedited neutrophil engraftment (Wagner et al, 2014). In addition, we have shown that murine and human Tprogs generated in vitro in the presence of Notch ligands can accelerate T-cell immunity post-HSCT. In order to maximize the number of Tprogs in culture, we explored the combination of these approaches—SR-1 expansion of a donor cord blood graft to be infused for hematopoietic rescue with the co-infusion of Tprogs generated from the SR-1 expanded cord blood—as a means to overcome immunodeficiency after HSCT. To assess this, CD34+ UCB cells were first expanded in the presence of SR-1 and cytokines for 15 days; a portion of SR-1 expanded cells were then co-cultured with murine OP9-DL1 cells with cytokines for an additional 14 days. We found that SR-1 expanded cord blood used as input for Tprog culture resulted in hundreds of millions of Tprogs due the in large input of cells. We also compared the phenotypes of SR-1 and non-expanded (control) Tprogs throughout the 14-day culture. Interestingly, the phenotype of the SR-1 UCB Tprogs was very different than control Tprogs in regards to CD34 expression. SR-1 UCB Tprogs had significantly less CD34+CD7+ cells compared to control UCB (5% and 25%, respectively, P < .01). To assess immune reconstitution in vivo of SR-1 expanded UCB Tprogs, we utilized a humanized mouse model with neonatal nonobese diabetic/severe combined immunodeficient (NOD/SCID)/γcnull mice (NSG mice). Neonatal mice were injected with 1e6 SR-1 Tprogs that had been sorted as CD34+CD7+ or CD34−CD7+subsets, and thymuses were assessed after 4 weeks. Both control and SR-1 CD34+CD7+ Tprogs showed robust thymic engraftment; however, SR-1 Tprogs CD34−CD7+ cells were uniquely able to engraft the thymus in mice. The thymic cellularity was ~60% lower in mice engrafted with CD34−CD7+ compared to CD34+CD7+ SR-1 Tprogs (Figure 1, P < .05). Taken together, these data support a novel methodology for generating scalable numbers of Tprogs capable of thymic engraftment that can be translated to patients undergoing HSCT as a potential approach to decrease infections and mitigate relapse risk.