GAMMA RADIATION OF BLOOD PRODUCTS PREVENTS REJECTION OF SUBSEQUENT DLA-IDENTICAL MARROW GRAFTS (1996)

Previous studies in dogs unexpectedly showed that treatment of whole blood with gamma irradiation before transfusion was effective in preventing sensitization of marrow graft recipients to minor histocompatibility antigens of donor origin, resulting in almost uniform engraftment of marrow grafts from DLA-identical canine littermates (1). We next determined that gamma irradiation also prevented sensitization and subsequent marrow graft rejection when unrelated blood transfusions were the source of minor histocompatibility transplantation antigens (2). The mechanisms by which gamma irradiation prevents sensitization to non-DLA antigens and, thereby, allays the risk of graft rejection are unknown. At least two possibilities exist. Firstly, irradiation of transfusion products may prevent sensitization by interfering with antigen processing/presentation or with proliferation, maturation, and/or homing of the minor histocompatibility antigen sensitizing cells in recipient dogs. Second, irradiated blood cells may induce functional tolerance or immune deviation by inducing clonal deletion or anergy akin to what has been described in rodents(3, 4) and humans (5, 6). We tested the second possibility in the present study. To this purpose, marrow graft recipients were first given three gamma irradiated blood transfusions from their DLA-identical marrow donors over a period of three weeks. They next received three unirradiated transfusions from the same donors, and, finally, they were given total-body irradiation and marrow grafts. All recipients rejected their grafts, consistent with the notion that tolerance was not induced by the initial treatment of three irradiated transfusions. Research protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the Fred Hutchinson Cancer Research Center, and the research was conducted according to the principles outlined in the Guide for Laboratory Animal Facilities and Care prepared by the National Academy of Sciences, National Research Council. DLA-identical littermate pairs 8-12 months old derived from the breeding colony at the Fred Hutchinson Cancer Research Center were selected as previously described(1, 2, 7-9). Recipients were first given three 50-ml transfusions of 2000 cGy-treated heparinized blood from DLA-identical littermates, a protocol previously shown to abrogate the ability of blood to sensitize (1). Radiation was delivered by a gamma cell 1000137 cesium source(1, 3). The three irradiated transfusions were given on days -48, -41, and -34 before transplant. On days -24, -17, and -10 before transplant, recipients received 50 ml of unirradiated blood from the same donors, a protocol previously shown to result in 100% graft rejection(1, 8). Dogs were prepared for marrow transplantation by 9.2 cGy total-body irradiation (TBI)* at 7 cGy/min from a dual 60Co source (1). Donor marrow at 1 to 4.8×108 cells/kg was infused intravenously within 4 hr of TBI. In addition, marrow donor peripheral blood leukocytes obtained by leukapheresis were administered at 6-20×208 cells/kg intravenously on days 1 and 2 after TBI. Posttransplant immunosuppression was not administered, and postgrafting care has been described in detail elsewhere (1, 7, 8). Daily blood counts were obtained, and complete autopsies and histologic examinations were performed on the animals that died or were euthanized. Failure to engraft was confirmed by the failure of granulocyte and platelet counts to rise following the postirradiation nadir, absence of marrow cellularity at autopsy, and absence of graft-versus-host disease (GVHD)(1, 7, 8). A statistical test comparing proportions of dogs rejecting their grafts was based on Monte Carlo simulation methods (9). Current results are compared with those of previous studies with preceding transfusions in this DLA-identical littermate model (Table 1). Without preceding marrow donor transfusions, 61 of 62 dogs engrafted(1, 2). With three preceding transfusions of 50 ml of untreated whole heparinized donor blood given on days -24, -17, and -10 before transplant, all 27 recipients rejected their DLA-identical littermate marrow grafts (1, 8). However, when the three preceding transfusions were treated with 2000 cGy, only 1 of 10 littermates rejected the graft (1). In the current study, four dogs first received three whole-blood transfusions treated with 2000 cGy (days -48, -41, and -34) followed then by three unirradiated transfusions (days -24, -17, and -10). All four rejected their subsequent DLA-identical marrow grafts (P=0.0014). Had we entered an additional dog into this experiment and had that recipient engrafted, the results (4/5 rejecting) would remain statistically significant(P<0.0056). The uniform graft rejection seen in the present dogs argues against “tolerance” induction to minor MHC antigens as the mechanism by which gamma radiation of blood transfusions allays the risk of marrow graft rejection. Rather, other mechanisms must be operative, including interference with antigen-processing/presentation and with division or homing of the sensitizing cells rather than active immunomodulation of the recipient. Acknowledgments. We wish to thank Jacquelyn S. White, Ana Gervassi, Robert Raff, Eileen Severns, Lori Ausburn, Mimi Arcand, Cindy David, and Jeanine Pazasis for technical support; Robert Burnett, Ph.D., for RFLP analysis of littermates; Bonnie Larson, Harriet Childs, and Julie Sentkowski for preparation of the manuscript; and the technicians of the Hematology, Pathology, and Bacteriology laboratories of the Fred Hutchinson Cancer Research Center.