Radiation Dose Determines Degree of Donor Chimerism after Nonmyeloablative Hematopoietic Cell Transplantation.

Efforts to replace total body irradiation (TBI) used for transplant conditioning with agents that have less acute and long-term toxicities require a better understanding of the biological effects of low dose TBI. We therefore retrospectively analyzed the role of radiation dose, stem cell source, and type of immunosuppression on both the stability and degree of donor chimerism in canine recipients of matched littermate hematopoietic cell transplants. Recipients were prepared with 200 cGy (n=26), 100 cGy (n=76) or 50 cGy (n=19) total body irradiation (TBI) at 7 cGy/min. Stem cell sources included bone marrow (BM) alone (n=58), BM plus G-CSF mobilized peripheral blood mononuclear cells (G-PBMC) (n=42), BM and CD14-depleted G-PBMC (n=13), or BM and T-cell-depleted G-PBMC (n=8). Posttransplant immunosuppression consisted of cyclosporin (CSP) only (n=53), CSP plus mycophenolate mofetil (MMF) (n=23), CSP and rapamycin (n=12), CSP, MMF and rapamycin (n=5); or CSP and MMF in combination with pretransplant immunosuppression (n=28). The percentage of donor granulocytes in the peripheral blood, as determined by PCR amplification of variable numbers of tandem repeats (VNTR), served as a marker for engraftment. TBI dose and stem cell source were both significantly associated with long-term (>26 weeks) stable engraftment in multivariate analysis (p=0.0001 and p=0.004, respectively). Among the 39 dogs with stable engraftment, however, TBI dose was the only factor examined that was associated with the degree of donor chimerism (mean % of donor granulocytes after 200 cGy, 100 cGy and 50 cGy of TBI: 65%, 52%, and 24%, respectively; p=0.008). To determine whether low-dose irradiation directly affected recipient stem/progenitor cell numbers and thereby conferred a competitive disadvantage to donor cells, CD34+ cells were isolated from two normal human donors. One preparation of CD34 cells was ex vivo irradiated (=200 cGy) and then injected into NOD/SCID beta2m-/- mice in combination with an equal number of unirradiated CD34 cells from the second donor. The contributions of each donor to human engraftment were assessed at 10 weeks by VNTR. After 200 cGy, the irradiated population contributed 74% less than expected, 24% less after 100 cGy, but only 6% less after 50 cGy. Flow analysis of Caspase-3 activation indicated that a significant percentage of cells irradiated with 200 cGy were apoptotic, and that this was associated with the loss of L-selectin and P-selectin glycoprotein ligand-1. In conclusion, our findings suggest that TBI, in addition to its well-characterized immunosuppressive effects, determines the degree of donor cell engraftment by directly compromising recipient stem cells, thereby providing a competitive advantage to donor stem cells.