FRACTIONATED DOSING OF CYCLOPHOSPHAMIDE FOR ESTABLISHING LONG-LASTINGSKIN ALLOGRAFT SURVIVAL, STABLE MIXED CHIMERISM, AND INTRATHYMIC CLONAL DELETION IN MICE PRIMED WITH ALLOGENEIC SPLEEN-CELLS

Background., Injection of allo-spleen cells (SC) followed by a single dose of cyclophosphamide (CP) can induce tolerance of tumor and/or ski n allografts in mice. To minimize the damage caused by CP, fractionati on of CP that can establish long-lasting skin graft survival, stable m ixed chimerism, and intrathymic clonal deletion in the host was invest igated in the present study. Methods. Allo-SC (10(8)) were given intra venously on day 0. CP at 200 mg/kg was given intraperitoneally on day 2 in a single dose (CP 200x1 group). CP at 100, 66, 50, 40, and 33 mg/ kg was given daily from day 1 through days 2, 3, 4, 5 and 6, respectiv ely, in the fractionated doses (CP 100x2, 66x3, 50x4, 40x5, and 33x6 g roups; total dose=200 mg/kg). Allografting was performed on day 14. Re sults. In a fully allogeneic combination of C57BL/6 (H2(b))-->AKR (H2( k), Mls-1(a)), an EL-4 tumor (H2(b)) was specifically accepted to kill the AKR mice in all of the SC+CP 200x1, 100x2, 66x3, 50x4, 40x5, and 33x6 groups (n=6), but C57BL/6 skin graft survival was not prolonged i n any of the tumor-tolerant groups. In an H2-identical combination of AKR-->C3H (H2(k), Mls-1(b)), AKR skin graft survival was prolonged rem arkably (80-90 days) in the SC+CP 200x1, 100x2, and 66x3 groups (n=5-1 1), but was prolonged moderately (20-60 days) in the SC+CP 50x4 and 40 x5 groups. In both of the SC+CP 200x1 and 66x3 groups in the AKR-->C3H combination, mixed chimerism was maintained for as long as 100 days a fter tolerance induction in both the spleen and thymus, associated wit h intrathymic clonal deletion of V beta 6(+) T cells. The decreases in leukocyte count, hemoglobin level, spleen weight, SC count, and body weight were significantly smaller in the SC+CP 66x3 group than in the SC+CP 200x1 group. Conclusions. Fractionated CP is effective in amelio rating the compromised state induced by a single dose of CP. To induce a long-lasting skin allograft survival associated with stable mixed c himerism and intrathymic clonal deletion in an H2-identical combinatio n, 200 mg/kg of CP can be divided into three or fewer fractions.