Dog Leukocyte Antigen (DLA)-Identical Sibling Cord Blood Transplantation (CBT) Following Myeloablative Total Body Irradiation (TBI).
Authors: Lesnikov Vladimir , Baran Szczepan , Abrams Kraig , Nikitine Alla , Hwang Billie , Zellmer Eustacia , Heimfeld Shelly , Lesnikova Marina , Georges George E. 
Published: October 2019

Abstract
Cord blood (CB) is increasingly used for hematopoietic cell transplantation (HCT) due to its rapid availability and less stringent HLA matching requirements. However, low cell dose of available CB units and delayed engraftment remain significant obstacles for increased use of CBT in adults. Recent clinical results with 2-unit CBT may help overcome these problems. We sought to develop a large animal model of CBT to improve the understanding of engraftment across histocompatibility barriers and cell dose limitations of multiple units of CBT. Since the outbred dog is a very well-established preclinical model of HCT, our first aim was to establish feasibility of canine CBT. We harvested and cryopreserved individual units of canine CB obtained from litters following Caesarian section at day 56 to 60 of gestation. We asked if multiple units of previously cryopreserved, DLA-identical sibling CB could engraft in DLA-identical recipient dogs after myeloablative total body irradiation (TBI). Five adult dogs (8–18 months of age) received 920 cGy TBI (7 cGy/min) followed by intravenous infusion of 2 to 4 units of thawed CB with a combined total nucleated cell dose range 0.4x107 − 2.6x107/kg. Transplanted total CD34+/kg cell dose range was 0.5−2.5x105/kg. Postgrafting immunosuppression was combined cyclosporine and mycophenolate mofetil for 35 and 28 days, respectively. G-CSF was given until recovery of neutrophil counts, and dogs received blood product transfusion support and intravenous and oral antibiotics. Four dogs engrafted and survived; they are currently 35 to 212 days after CBT. One dog died on day 13 due to neutropenic sepsis. All 4 surviving dogs engrafted with sustained neutrophil recovery >1000/mL between 29 – 35 days after CBT. Bi-weekly chimerism analysis was assessed by PCR using informative microsatellite markers. For each of the 4 surviving recipients all transplanted donor CB units were detected after engraftment. However, among the 3 dogs currently at 3 to 7 months after CBT, only 1 of the multiple transplanted CB units dominated hematopoiesis with 75–95% donor chimerism. For 2 of the 3 dogs, the CB unit with the highest CD34+ dose was the dominant donor. There was no evidence of acute or chronic graft-versus-host disease (GVHD). Among the 3 dogs at 3 to 7 months after CBT, immune reconstitution studies included normal T cell proliferation stimulation index (mixed lymphocyte reaction) and gradual recovery of the absolute number of CD4 and CD8 T-cell subsets. In summary, cryopreserved DLA-matched CB successfully engrafted and provided durable hematopoietic recovery in lethally irradiated dogs. The cell dose of transplanted CB units was comparable with CB units for adult human transplantation. These results establish that multiple units of canine CB with limited cell dose can engraft and initiate immune reconstitution without GVHD in DLA-identical dogs transplanted after marrow-lethal irradiation.