Anti-thymocyte-globulin (ATG) in the nonmyeloablative conditioning for canine hematopoietic cell transplant (HCT)

Abstract

We tested whether pretransplant immunosuppression with canine-specific rabbit ATG (SangStat), combined with 1 Gy total body irradiation (TBI) and posttransplant mycophenolate mofetil/cyclosporine (MMF/CSP) would assure stable engraftment in our canine HCT model. First, pharmacokinetic studies were done in 4 dogs, with cumulative ATG doses of 2–5 mg/kg, subcutaneously. ATG was most effective in depleting peripheral T cells (CD4+ and CD8+), intermediate on B cells and did not deplete other blood cells. Lymph node biopsies taken after 2 mg/kg ATG showed 50% T-cell depletion. Serum levels of ATG peaked at 6–42 μg/ml (days 4–6), in synchrony with the T-cell nadirs, and became undetectable by day 13. Antibodies to rabbit ATG appeared after 6–7 days, and their titers increased as ATG was cleared from the circulation. We then evaluated the immunosuppressive effects of ATG using allogeneic skin grafts. Median skin graft survival in 5 dogs given ATG was 14 days, compared to 8 days among 28 controls (p = 0.0003). Based on these observations, an HCT protocol was designed; 5 dogs were given ATG (3.5–5 mg/kg) between days –12 and –7 to target a 90–95% depletion of circulating T cells. ATG levels were undetectable by day 0, excluding possible effects on donor T cells. On day 0, dogs were given 1 Gy TBI and marrow from dog leukocyte antigen-identical donors. Median cell doses infused (millions/kg) were: total nucleated cells (TNC) = 263; CD34 = 6.6 and CD3 = 18. Posttransplant immunosuppression was MMF/CSP for 4 and 5 weeks, respectively. All recipients showed initial donor chimerism, with maximal values ranging from 10–75% (median 25%) for granulocytes and 5–40% (median 25%) for mononuclear cells. Four dogs rejected their grafts after a median of 9.5 weeks (range 8–18 weeks), without cytopenias, and they reverted to autologous hematopoiesis. The 5th dog has remained a long-term mixed chimera (>36 weeks). The median times to rejection were 11 weeks (projected) in the study group and 10 weeks in the control group, not given ATG (p = 0.20, log-rank test). Analysis of the impact of cell doses suggested that TNC had the highest Spearman correlation coefficient with the duration of donor chimerism, 0.82 (p = 0.09). We conclude that ATG reliably depleted circulating T cells by 90–95% and lymph node T cells by approximately 50%. Even so, administering ATG before an otherwise inadequate conditioning dose of 1 Gy TBI failed to lead to uniform stable hematopoietic engraftment.