Stable Mixed Hematopoietic Chimerism in DLA-Identical Littermate Dogs Given Sublethal Total Body Irradiation Before and Pharmacological Immunosuppression After Marrow Transplantation

Abstract

Postgrafting cyclosporine (CSP) given for 35 days resulted in establishment of stable marrow grafts from DLA-identical canine littermates after otherwise suboptimal, but nevertheless, lethal conditioning with 450 cGy of total body irradiation (TBI). We now asked whether sustained allografts could be achieved after sublethal TBI or without TBI. Five groups of recipients were studied. Dogs in group 1 were given 200 cGy TBI and postgrafting CSP, 15 mg/kg twice daily by mouth on days −1 to 35 posttransplant. Dogs in group 2 were given 200 cGy TBI and methotrexate (MTX), 0.4 mg/kg intravenously (IV) on days 1, 3, 6, and 11 along with CSP. Dogs in group 3 were given 200 cGy TBI and CSP along with mycophenolate mofetil (MMF ), 10mg/kg twice daily subcutaneously (SC) on days 0 to 27 after transplant, a novel immunosuppressive combination. Dogs in group 4 were given 100 cGy TBI and MMF/CSP. Dogs in group 5 were not given TBI and they received MMF/CSP posttransplant. Allografts were assessed by (Ca)n dinucleotide repeat polymorphism studies in cells from peripheral blood, lymph nodes, and marrow. Dogs in group 1 had transient mixed donor-host hematopoietic chimerism for no more than 4 weeks. Three of six dogs in group 2 had transient mixed chimerism for 3 to 11 weeks, and three have remained stable mixed chimeras for up to 60 weeks now. Four of five dogs in group 3 have remained stable mixed chimeras for 54 to 57 weeks now, while one lost the allograft after 12 weeks. All dogs in groups 4 and 5 rejected their allografts after 2 to 12 weeks. In summary, the establishment of stable mixed hematopoietic chimerism following nonmyelosuppressive and nontoxic conditioning programs has remained a difficult goal. Here we present evidence in a large random-bred animal species that this goal may be achievable with pharmacological immunosuppression postgrafting, capable of inhibiting both host-versus-graft (HVG) and graft-versus-host (GVH) reactions in the setting of DLA-identical grafts.

What Role Is There for Antithymocyte Globulin in Allogeneic Nonmyeloablative Canine Hematopoietic Cell Transplantation?

“…The fifth dog has remained a mixed chimera at Ͼ36 weeks of follow-up, with donor contributions of 1% for granulocytes and 25% for MNCs. The median durations of engraftment in the 5 dogs (11 weeks) were similar to those among 6 historical controls [1] not given ATG (median, 10 weeks; P ϭ .20; log-rank test).…”

“…The median duration of mixed chimerism was estimated with the Kaplan-Meier method. The duration of mixed chimerism was compared with that of 6 historical controls [1] by using the log-rank test. The association between cell doses in the grafts and the mixed chimerism duration was evaluated with the Spearman rank correlation coefficient.…”

“…Chimerism was also assessed at least once in unfractionated marrow samples and among circulating T cells, sorted by fluorescence-activated cell sorting. PCR amplification with informative radioactively labeled VNTR primers was performed [1]. PCR products were analyzed by gel electrophoresis, and percentages of donor chimerism in the recipients were determined by visual approximation after autoradiography.…”

“…We have developed a nonmyeloablative regimen for hematopoietic cell transplantation (HCT) between dog leukocyte antigen (DLA)-identical canine littermates. It consists of conditioning with 2 Gy of total body irradiation (TBI) and a short course of postgrafting immunosuppression with mycophenolate mofetil (MMF) and cyclosporine (CSP) [1]. This regimen has been successfully translated into clinical trials for patients thought to be at high risk for toxicities from conventional myeloablative regimens [2][3][4].…”

“…When the TBI conditioning dose was decreased to 1 Gy, initial uniform allogeneic engraftment was seen, but all dogs eventually lost their grafts, despite MMF and CSP; this shows a delicate immunologic balance between host and donor [1]. Attempts at establishing stable allografts after 1 Gy of TBI have met with mixed success.…”