Adoptive immunotherapy to increase the level of donor hematopoietic chimerism after nonmyeloablative marrow transplantation for severe canine hereditary hemolytic anemia

Donor chimerism levels in the granulocyte and mononuclear fractions of the peripheral blood, hematocrit, and reticulocyte counts across time after transplantation. Early after nonmyeloablative stem cell transplantation in 3 dogs with pyruvate kinase deficiency and hemolytic anemia, the levels of donor chimerism in the myeloid compartment (granulocyte fraction) decreased in 2 dogs (E854 and E920) with a recurrence of hemolytic anemia. All dogs had 2 infusions of donor lymphocytes (↓), and both dogs with the recurrence of hemolytic anemia had an increase in donor chimerism in the myeloid compartment and resolution of hemolytic anemia. In (A), (B), and (C), the hematocrit, reticulocyte count, and levels of donor chimerism in the peripheral blood mononuclear cell (PBMC) and granulocyte fractions are shown over time for dogs E854, E919, and E920, respectively. • indicates hematocrit; ■, reticulocytes; ○, donor chimerism in the granulocyte fraction; ☆, donor chimerism in the PBMC fraction.

A representative sample of microsatellite marker studies of donor and recipient (E854) cells before transplantation and recipient cells after transplantation. After second donor lymphocyte infusion (DLI), there was a rapid conversion to complete donor chimerism (as noted in Figure 1A). G indicates peripheral blood granulocyte fraction; M, peripheral blood mononuclear cell fraction.

Marrow histology from a pyruvate kinase-deficient dog (E920) at baseline (A) and 3 years after nonmyeloablative transplantation and DLI (B). A, The marrow is hypercellular with a reversed M:E ratio. B, The marrow is normocellular with a normal M:E ratio and maturation of all 3 major cell lines. The vacuoles seen in B are fat cells that are observed in normocellular marrows from dogs (stain, periodic acid-Schiff; magnification, 300×).

Normal liver histology in a pyruvate kinase-deficient dog (E920). Three years after nonmyeloablative transplantation, there were normal hepatocytes (large arrow) and bile ducts (small arrow). Iron deposition was normal. There was no development of significant liver disease from iron overload, which is normally associated with the natural history of pyruvate kinase deficiency in affected dogs.