*Purpose: Clinical data suggest allogeneic pregnancy is a sensitizing event; however, data from mouse models suggests it induces fetal-specific regulatory T cell (Treg) expansion and differentiation into memory Tregs that mediate systemic, fetal-specific immune regulation post-partum (PP). In this study, we sought to define the immunological consequences of allogeneic pregnancy for the induction of allograft tolerance in mice.

*Methods: Wild-type B6 (WT) mice were mated with B/c-2W-OVA transgenic mice. Fetus-specific antibodies were monitored using B/c targets and flow cytometry. Fetus-specific responses were tracked using K^d+IE^d+L^d tetramers for donor-specific B cells, and 2W:I-A^b and OVA:K^b tetramers for donor-specific CD4⁺ and CD8⁺ T cells, respectively. F1-2W-OVA heart transplants (HTx) were performed and PP-WT recipients were treated with costimulatory blockade [CoB, anti-CD154 plus donor-specific splenocytes] to induce allograft tolerance. Graft rejection was determined by direct palpation. We also used female PP-sIgKO and -µKO B6 mice that are unable to express functional antibodies and are deficient in B cells, respectively.

*Results: Fetus-specific antibodies increased during and post-pregnancy to a peak 9-fold increase by PP day 21. Prior donor-matched pregnancy, but not syngeneic pregnancy, prevented subsequent CoB-mediated induction of HTx tolerance in 60% of recipients, despite the animals bearing a significant increase in percentage of fetal-specific FOXP3⁺ cells as well as a modest reduction in number of fetal-specific conventional CD4⁺ T cells (Tconvs) at PP day 21-189, compared to PP-WT females who were not transplanted. In contrast, there was an ongoing donor-specific B cell response, with increased percentages acquiring a germinal center phenotype. To test the hypothesis that pregnancy-triggered donor-specific B cell responses and/or antibodies were the cause of this resistance to transplant tolerance induction, we performed experiments in PP-µKO and PP-sIgKO recipients. PP-µKO recipients achieved long-term graft acceptance when treated with CoB but PP-sIgKO mice did not, while both PP-µKO and PP-sIgKO mice spontaneously accepted B/c allograft without CoB but the non-PP counterparts did not.

*Conclusions: Despite the sustained expansion of maternal FoxP3⁺ Tregs with fetal/allograft-specificity, allogeneic pregnancy results in a resistance to transplant tolerance that is due to the presence of fetal/transplant-specific alloantibodies, whereas the presence of B cells incapable of producing antibodies are not sufficient. Strikingly, pregnancy in the absence of B cells induces T cell tolerance to F1 heart allografts. These data suggest that pregnancy-sensitized B cells and antibodies are a barrier to pregnancy-induced and CoB-induced tolerance to paternal/fetus matched allografts.

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