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Antibody (Ab) mediated rejection of transplanted organs presents a formidable barrier to solid organ transplantation. We have previously shown that intravenous (i.v.) exposure to MHC-mismatched donor peripheral blood cells using a novel, mild conditioning protocol results in B-cell tolerance with transient T-cell unresponsiveness. Here we show that B-cell tolerance is stable and allo-Ab cannot be induced even after loss of chimerism and repeated exposure to donor cells without immunosuppression.MGH miniature swine conditioned with CD3-immunotoxin, low-dose (100cGy) total body irradiation (TBI), and a short, 45 day course of cyclosporine A, received cells i.v. from a haplo- or fully MHC-mismatched donor. Weekly immunophenotyping of peripheral blood was performed. Hematopoietic cells from donor and third-party MHC-mismatched pigs were injected subcutaneously (s.c.) +/- complete Freund’s adjuvant (CFA) monthly, starting 60 days following conditioning. Control animals with and without conditioning, but without donor HCT, were challenged in the same manner. The draining lymph nodes were analyzed by Histology and bulk RNAseq. Allo-Ab was measured by flow cytometry and complement dependent Ab mediated cellular cytotoxicity assay.Phenotypic analysis of recipient cells during conditioning revealed that most cell populations, including CD4+ Foxp3+ Treg cells, were only partially depleted and quickly recovered to baseline. Allo-Ab responses were not detected even after repeated s.c. injections of donor cells. The addition of CFA provoked a transient, weak IgM response suggestive of an extrafollicular B-cell response with no germinal center development. Control animals consistently developed Allo-Ab responses against donor cells. Histologic analysis of draining lymph nodes shows similar morphology and presence of active Germinal centers in both groups. However, preliminary analysis reveals an abundance of FoxP3+ cells in tolerant animals. This suggests that donor cells trigger a germinal center reaction in tolerant animals that may be controlled by memory T-follicular regulatory cells. Further histologic and transcriptome analysis of draining LN cells are currently underway and may yield additional insights into the mechanical underpinnings.Stable B-cell tolerance to donor antigens across fully MHC-mismatched barriers can be induced by a novel, mild conditioning protocol that does not result in prolonged T-cell depletion. We are currently investigating the mechanism of this phenomenon. Understanding immunomodulatory mechanisms involved in controlling B-cell responses to transplantation antigens could lead to significant advances in improving transplantation outcomes.