Microchimerism and tolerance after in utero bone marrow transplantation in mice

Background. Donor-specific tolerance has been induced after both fetal and neonatal hematopoietic stem cell (HSC) transplantation in mice. However, the relationship between hematopoietic microchimerism and tolerance in these models has not been defined due to the insensitivity of donor cell detection methodology. To address this problem we developed a semi-quantitative polymerase chain reaction (PCR)-based assay for detection of microchimerism after major histocompatibility (MHC) class I disparate HSC transplantation. This assay was used to examine the relationship between microchimerism and tolerance after fetal and neonatal transplantation of fully allogeneic bone marrow cells. Materials and methods. C57BL/6 mice (H2-Kb) were used as adult bone marrow donors and Balb/c mice (H2-Kd) were used as fetal or newborn recipients. A dose of 10¹⁰ BM cells/kg was injected intraperitoneally into recipient animals. Peripheral blood of animals which survived beyond 3 weeks of age was analyzed by PCR for the presence of donor MHC class I DNA. Tolerance was tested by placement of donor-specific skin grafts after determination of chimerism status. Results. Our assay was found to be specific for H2Kb donor cells in an H2-Kd background with a sensitivity of <0.0001%. Of 49 animals injected in utero 19 (38%) had donor DNA present in peripheral blood at low levels (<0.1%) whereas only 1 of 18 neonatally injected animals had detectable donor cells (P < 0.01). Tolerance to donor- specific skin grafts was found in 6 of 9 animals which were chimeric after in utero HSC transplantation whereas none of the 18 neonatally injected animals including the chimeric animal were tolerant. Conclusions. Our results indicate the following. (1) Hematopoietic microchimerism can be detected by PCR in peripheral blood after in utero injection of fully allogeneic HSCs. (2) Fetal injections yield a higher incidence of microchimerism than newborn injections. (3) Tolerance can be induced across a fully allogeneic barrier by in utero HSC transplantation and this is associated with the presence of peripheral blood microchimerism.