Induction of mixed chimerism in mice by employing different conditioning protocols and bone marrow cell transplantation.

Mixed chimerism has been suggested to produce allograft tolerance. Since this phenomenon is not fully understood, the aim of our study was to evaluate various protocols for chimerism induction in a mouse model. B6.SJL-Ptprc(a)Pep3(b) mice were injected with 20 to 30 x 10(6) bone marrow cells from Balb C mice. Conditioning consisted of total body gamma irradiation with 9.5, 5, and 3 Gy on "-1 day" of the experiment, with 200 mg/kg cyclophosphamide (CP) ("+2 day"). Additionally, one group of mice received blocking antibody against CD40L on days 0, 1, 4, and 7. The presence of mixed chimerism in peripheral blood was assessed at 1, 2, 3, 4, 6, and 8 weeks using flow cytometry to detect CD45.1 or CD45.2 antigen expression. Moreover, the chimerism was examined in CD4, CD8, CD45/B220, Mac-1alpha subpopulations in peripheral blood and bone marrow cells at 8 weeks. We also compared chimerism in peripheral blood, bone marrow, and spleen leukocyte populations. We observed that the most effective conditioning method with relatively low toxicity was based on concomitant use of 5 Gy total body irradiation and CP. The percentage of donor cells differed among peripheral blood subpopulations and bone marrow cells, but was similar in leukocyte populations derived from various sources. Our experiments sought to optimize the induction of stable mixed chimerism.