Introduction: Clinical data suggest allogeneic pregnancy is a sensitizing event; yet, recent data from mouse models indicates it induces fetal-specific regulatory T cell (Treg) expansion that mediates systemic, fetal-specific immune regulation post-partum (PP). In this study, we aimed to define the mechanism for acquired resistance to allograft tolerance after allogeneic pregnancy.
Methods: Virgin female wild-type (WT) B/6, µKO, or anti-HEL BCR-tg (MD4) mice were mated with male B/c-2W-OVA transgenic mice.  Fetus-specific IgG were monitored by flow cytometry, while donor-specific cellular responses were tracked using K^d+IE^d+L^d tetramers for B cells, and 2W:I-A^b and OVA:K^b tetramers for CD4⁺ and CD8⁺ T cells, respectively.  At PP day 30–45, F1 (B/6 x B/c)-2W-OVA heart transplants (HTx) were performed and anti-CD154 (POD 0, 7, 14) and donor splenocytes (DST, POD 0) were used to induce tolerance.
Results: Fetus-specific IgG increased during and post-pregnancy to a 9-fold peak by PP day 21.  Prior donor-matched pregnancy, but not syngeneic pregnancy, prevented the induction of tolerance in 60% of PP-WT B/6 recipients, despite a significant increase in percentage of fetal-specific FOXP3⁺ cells and a modest reduction in number of fetal-specific conventional CD4⁺ T cells (Tconvs) examined at POD 21–189, compared to PP-WT B/6 untransplanted controls.  In contrast, ongoing donor-specific germinal center B cell responses were detected suggesting a B cell-mediated resistance to transplant tolerance. Strikingly, anti-CD154/DST successfully achieved long-term graft acceptance in PP-µKO B/6 and PP-MD4 (normal B cell numbers but ≤5% donor-specific B cells compared to WT) recipients.
Conclusion: Despite the sustained expansion of maternal FoxP3⁺ Tregs with fetal/allograft-specificity, allogeneic pregnancy results in subsequent resistance to transplant tolerance induced with anti-CD154/DST.  These studies provide new insights into the mechanism of pregnancy-induced sensitization by demonstrating a necessity for B cells in preventing subsequent induction of fetal/allograft-specific transplantation tolerance.