Application of tolerance induced by transfusion of uvb-irradiated leukocytes for establishments of mixed chimerism

Our earlier study showed that 4 weekly transfusions of UVB-irradiated leukocytes from H-2d BALB/c mice into H-2k (CBA mice can induce immune tolerance to H-2d antigens (Blood 88:4375,1996). To explore whether the induced tolerance can be applied for establishing mixed chimerism, female CBA mice with and without tolerance induction were treated with 100 or 200 cGy γ ray and transplanted with 5×10e6 bone marrow cells from male BALB/c mice. Five hours before transplantation, CBA mice were injected i.p. with or without 0.5 mg MR-1 anti-CD40L monoclonal antibody. A second dose of 0.25mg MR-1 antibody was given 48 hours later. The transplanted mice were followed for GVHD, H-2d positive WBC in blood by flow cytometry, and presence of Y chromosome by PCR. There were 4 mice in each group. Our results demonstrated that increasing numbers of male H-2d positive WBC became detectable only in the blood of tolerant CBA mice treated with MR-1 and 100 or 200 cGy γ ray. On day 25 after BMT, H-2d positive WBC in the blood of 4 tolerant 200-cGy recipient CBA mice were 290/μl(3.4%), 133/μl(2.4%), 99/μl(2%) and 86/μl(1.3%). In tolerant 100-cGy CBA mice, H-2d positive WBC were 23/μl(0.6%), 28/μl(0.5%), 20/μl(0.3%) and 3/μl (0.06%). No male H-2 d positive WBC were detected in the naive control CBA mice. There were no signs of GVHD. Our results show that tolerance induction and anti-CD40L administration are necessary for establishmen mixed chimerism and the dose of γ radiation significantly influences the degree of chimerism.