Durable engraftment of purified allogeneic hematopoietic stem cells following non-myeloablative conditioning

Transplantation of purified allogeneic hematopoietic stem cells (HSC) has the potential to be a curative treatment for autoimmune diseases. Before it becomes a viable therapy, however, the treatment-related mortality and difficulty of achieving engraftment must be addressed. Our research has focused on developing non-myeloablative regimens that lead to donor-derived engraftment of purified HSC in a murine model. Total lymphoid irradiation (TLI) consists of low-dose fractionated irradiation targeted to the thymus, abdomen, and peripheral nodes, while the skull, lungs, and long bones remain shielded. The non-myeloablative conditioning regimen of TLI and anti-thymocyte globulin (ATG) was followed by HSC transplantation. HSC were isolated by the composite phenotype of Thy1.1+, c-kit+, Sca-1+, and lineage− (KTLS). We tested HSC transplantations across three major histocompatibility complex (MHC)-matched strain combinations known through previous studies in our group to have significantly different barriers to engraftment. In all three strain combinations we observed stable mixed chimerism (approximately 50% donor-derived cells by day 90) when high doses (10000/mouse) of HSC were administered. In prior studies from our laboratory in a spontaneously arising autoimmune diabetes model, we demonstrated that mixed allogeneic chimerism alone following non-myeloablative conditioning was sufficient to block autoimmune pathogenesis. To study how TLI/ATG allows engraftment, we have examined the marrow of TLI/ATG conditioned, untransplanted animals. Though TUNEL and caspase-3 assays do not show a significant increase in apoptosis compared to controls, a 71% decrease in the quantitative number of HSCs isolated from these animals was observed. This depletion of HSCs in the marrow could provide a niche for donor HSCs to inhabit. Further histologic studies on lymphoid organs exposed to radiation through TLI are ongoing. The durable mixed chimerism observed following TLI/ATG conditioning and HCT will be applied to mice affected with the rodent form of multiple sclerosis (experimental autoimmune encephalomyelitis) and to tolerance induction of solid-organ grafts. Summary: The combination of TLI/ATG non-myeloablative conditioning and transplantation of allogeneic HSC leads to a durable mixed chimeric state between donor and host and will next be applied to the induction of tolerance to autoantigens and alloantigens.