*Purpose: We describe a case of tolerance from our study investigating multilineage chimerism under immunological conditions after intestinal transplantation using a porcine model with defined class I and II MHC loci.

*Methods: The MHC Class Idd/Class IIdd(dd) recipient underwent single-haplotype mismatched orthotopic intestinal transplantation from a Class Icd/Class IIcd(cd) donor. Induction immunosuppression consisted of CD3-immunotoxin and anti-CD8 antibody. Maintenance immunosuppression consisted of tacrolimus and steroids, which were weaned completely off between days 90-139 after transplant. Multilineage chimerism was assessed by flow cytometry. Stoma biopsies were evaluated for rejection. Cellular responses were evaluated by mixed lymphocyte reaction (MLR).

*Results: The recipient has no clinical or histological signs of rejection 260 days after transplantation and >120 days without immunosuppression. Flow cytometry of peripheral blood and bone marrow demonstrates persistent chimerism, primarily in the T cell lineage (Fig. 1). Donor-specific hyporesponsiveness that was attenuated by CD25 depletion was consistently observed by MLR, even after immunosuppression was discontinued (Fig. 2). MLR also demonstrates increased anti-recipient (“self”) proliferative activity that was markedly diminished by CD25 depletion.

*Conclusions: In a model favoring rejection (the recipient’s MHC alleles are all shared by the donor, preventing anti-MHC graft-versus-host reactivity, while the donor expresses an entire haplotype not shared by the recipient), we achieved long-term survival off immunosuppression after intestinal transplantation with in vivo and in vitro evidence of tolerance. Attenuation of donor-specific hyporesponsiveness and anti-recipient responses with CD25 depletion suggests that the mechanism of tolerance involves regulatory T cells and that donor T cells recognizing recipient minor histocompatibility antigens may have been activated in vivo and thereby expressed CD25.

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