CD3 Antibody Treatment Prevents Gvhd and Allows For Rapid, Long Term, Multilineage Engraftment Of Unfractionated UCB Into Immunodeficient Mice

Abstract

NSG mice are currently the best hosts for human hematopoietic stem cell xenografts, due in large part to their ability to initiate robust grafts comprised of diverse human blood cell lineages. Unfortunately, NSG mice are susceptible to GVHD induced by contaminating T cells. Therefore, stem and progenitor cells must be selected with a high degree of purity. This not only complicates the procedure, but also prevents modeling of whole umbilical cord blood (WUCB) and bone marrow transplantation. Here, we have explored both in vivo and in vitro methods to prevent xenogenic GVHD utilizing the anti-T cell antibodies ATG and OKT3. IP injection of either antibody within the first 72 hours of transplant proved effective at preventing T cell expansion. Alternatively, a simple short-term incubation of the cell suspension with antibody was equally effective. ATG, but not OKT3, had significant non-specific activity resulting in reduction and even prevention of the non-T cell portion of the graft, including SCID repopulating cells, using either the in vivo or in vitro approach. However, using the OKT3 in vitro incubation protocol, we were able to initiate stable, long term, multi-lineage engraftment from whole, unselected UCB without GVHD. Additionally, WUCB transplanted mice engrafted more readily, with a wider array of human cell types in the peripheral blood as early as 2 weeks, relative to CD34+ engrafted mice. Stem cell engraftment was confirmed by successful transplant of the human graft to secondary recipients. Limiting dilution experiments showed that significant human grafts could be generated with as few as 1 million total WBCs per mouse, indicating that very large cohorts of mice can be generated with a single UCB sample. This protocol not only has the potential to significantly streamline hematopoietic xenograft studies while greatly reducing the cost and time commitments involved, but also to allow for whole cord blood transplantation modeling and generation of unique subsets of human cells in vivo. Disclosures:No relevant conflicts of interest to declare.