Regulatory T Cells Prevent Donor Cell Rejection in a Mouse Model of Delayed Gestational Age in Utero Hematopoietic Cell Transplantation

Abstract Background: In utero hematopoietic cell transplantation (IUHCT) results in long-term, multilineage chimerism without myeloablation/immunosuppression. It has the potential to treat a number of congenital hematologic disorders, including as Sickle cell disease. Central tolerance, through which donor-reactive host T cells and host-reactive donor T cells are deleted, is instrumental to the induction of donor-specific tolerance (DST) and the prevention of graft-versus-host-disease (GVHD). The window of central tolerance induction in humans occurs at a gestational age that will pose significant technical challenges and exclude from treatment all fetuses evaluated after 14 weeks gestation. We have previously demonstrated that IUHCT results in the peripheral induction of traditional and non-traditional regulatory T cells that contribute to the suppression of GVHD (Riley et al. ASH 2016 Abstract #4540). We hypothesized that regulatory T cells also contribute to DST and, furthermore, may be used to expand the window of central tolerance induction to a later point in gestation. Methods: A mouse model of delayed gestational age IUHCT was developed in which 9.7x10^6 C57/Bl6 (B6) GFP T cell-depleted (TCD) bone marrow (BM) cells were injected intravenously into day of life 0 (P0) naive Balb/c pups via the facial vein. The ability of IUHCT-induced regulatory T cells to prevent donor cell rejection was assessed by injecting 9.7x10^6 B6 GFP TCD BM cells plus 5x10^6 CD4+ splenocytes harvested from 2-4 week old chimeric mice. Fetuses injected with 9.7x10^6 B6 GFP TCD BM cells at gestational day 14 (E14) served as positive controls for tolerance and engraftment. Peripheral blood (PB) was analyzed at 4, 8, 12, and 24 weeks of age for donor cell chimerism (%CD45+H2kb+GFP+/CD45+H2kd+) and for engraftment (defined as chimerism >1% at 4 weeks). Weight gain and histology of the skin, liver, lungs, and small bowel were analyzed to confirm the absence of GVHD. PB was analyzed at 6 months of age to confirm multilineage engraftment. An in vivo mixed lymphocyte reaction (MLR) was performed at 6 months of age to confirm long-term DST. B6-sensitized Balb/c mice served as positive controls for alloreactivity. Comparison of continuous data was performed using Student's t test. Binary data were compared using Fischer's exact test. All tests were two-sided with an alpha level set at 0.05 for significance. Results: Our model of delayed gestational age IUHCT was associated with donor cell rejection in 52 of 56 (92.9%) pups injected with TCD BM only. Co-injection of CD4+ chimeric splenocytes significantly improved engraftment (100% vs. 7.1%, p Conclusion: The induction of tolerance following IUHCT is important for maintaining successful long-term engraftment and requires injection at an early gestational age to avoid donor cell rejection by the maturing fetal immune system. This study demonstrates an important role for regulatory T cell populations in the induction of DST following IUHCT. The adoptive transfer of regulatory T cells from a chimeric mouse to a naive P0 pup inhibits donor cell rejection in our model of delayed gestational age IUHCT, allowing central tolerance induction and long-term engraftment to occur despite the increased maturity of the recipient immune system. These findings represent proof-of-principle that augmentation of regulatory T cell-dependent peripheral tolerance is one approach by which IUHCT could be delayed to a later point in gestation, decreasing the technical difficulty of the procedure and allowing fetuses evaluated after 14 weeks to be eligible for treatment. Download : Download high-res image (87KB) Download : Download full-size image Figure 1 . Disclosures No relevant conflicts of interest to declare.