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Background. The primary barrier to clinical application of in utero hematopoietic stem cell (HSC) transplantation is limited donor cell engraftment. We hypothesized that limited engraftment was due to competition between host and donor cells for available niches. We reasoned that increased engraftment might be achieved by performing multiple transplants separated by brief intervals to allow time for formation of new niches. To test this we performed multiple transplants in a congenic combination to avoid confounding immunologic effects. Materials and methods. C57Pep3B (H2K b , CD45.1) mice were used as donors of adult bone marrow and C57B1/6 (H2K b , CD45.2) mice were used as 14-day-gestation fetal recipients. All fetuses were injected intraperitoneally with 1 × 10 6 mononuclear cells. Boosted neonates were injected at Days 2, 4, and 7 of life with 5 × 10 6 cells. All animals were analyzed for donor cell engraftment by dual-color flow cytometry using CD45 and CD45.1 antigens. Results are reported as the mean ± SD. Statistical analysis was performed using the two-tailed Student t test with P < 0.05 considered significant. Results. Postnatally boosted animals demonstrated significantly elevated levels of donor cell engraftment (3.30 ± 0.8%; n = 8; P < 0.00001) when compared to the control animals (0.69 ± 0.5%; n = 9) as determined by peripheral blood analysis at 6 weeks of age. This elevated level of engraftment was stable long term. Conclusions. Our results demonstrate a significant increase in donor cell engraftment with postnatal booster injections after in utero transplantation. This supports the hypothesis that a limited number of niches may be a major component of the barrier to engraftment. It also suggests that postnatal booster injections may be a viable therapeutic strategy for improving donor cell engraftment after in utero HSC transplantation.

Background/Purpose: In utero bone marrow transplantation can induce donor-specific tolerance to postnatal solid organ transplantation, although the mechanisms remain poorly defined. In this study, we investigated the role of clonal deletion and clonal anergy in the maintenance of tolerance in a murine model of in utero bone marrow transplantation. Methods: DBA/2 mice (Mls a+ ) were used as donors of adult bone marrow, and 14-day-gestation fetal Balb/c mice (Mls a− ) were used as recipients. Tolerance was defined by donor-specific skin graft survival for more than 8 weeks. Clonal deletion was assessed by flow cytometry for Vβ6 T cell receptor usage. A tolerant animal demonstrating partial deletion of CD4+/Vβ6+ T cells and a nontolerant animal were selected for analysis of clonal anergy by a proliferation assay using plate-bound anti-Vβ6 antibody for stimulation with or without exogenous interleukin-2 (IL2). Results: Vβ6+ splenocytes constituted 6.32% of CD4 + T cells in the tolerant animal compared with 9.19% in the nontolerant animal, demonstrating incomplete clonal deletion in the tolerant animal. Stimulation with plate-bound anti-Vβ6 induced a good proliferative response in the nontolerant animal but a significantly attenuated response in the tolerant animal ( P Conclusions: In this murine model of in utero bone marrow transplantation, the tolerant state is characterized by partial clonal deletion of donor reactive T cells and clonal anergy of nondeleted donor reactive T cells. The anergic state can be abrogated by exogenous IL2, suggesting that the mechanism of anergy is a deficiency of IL2 production.