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. H owever, the relationship between hematopoietic microchimerism and tole rance in these models has not been defined due to the insensitivity of donor cell detection methodology. To address this problem we develope d a semiquantitative polymerase chain reaction (PCR)-based assay for d etection of microchimerism after major histocompatibility (MHC) class I disparate HSC transplantation. This assay was used to examine the re lationship between microchimerism and tolerance after fetal and neonat al transplantation of fully allogeneic bone marrow cells. Materials an d 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 do se of 10(10) 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. To lerance was tested by placement of donor-specific skin grafts after de termination of chimerism status. Results. Our assay was found to be sp ecific for H2-Kb donor cells in an HS-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 1 8 neonatally injected animals had detectable donor cells (P < 0.01), T olerance to donor-specific skin grafts was found in 6 of 9 animals whi ch were chimeric after in utero HSC transplantation whereas none of th e 18 neonatally injected animals including the chimeric animal were to lerant. Conclusions. Our results indicate the following. (1) Hematopoi etic microchimerism can be detected by PCR in peripheral blood after i n utero injection of fully allogeneic HSCs. (2) Fetal injections yield a higher incidence of microchimerism than newborn injections. (3) Tol erance can be induced across a fully allogeneic barrier by in utero HS C transplantation and this is associated with the presence of peripher al blood microchimerism. (C) 1998 Academic Press.