As shown in Desk 2, human T cells from hu-mice with long-term porcine thymic graft survival (i.e., those treated with BTI-322) exhibited donor-specific non-responsiveness to the porcine thymic donors. treatment for the mind-boggling scarcity of human organ donors that presents a major limiting factor in clinical transplantation, the best available therapy for end-stage organ failure (1). The successful production of viable pigs with homozygous deletion of 1 1,3Gal transferase (2-4) made it possible to avoid both hyperacute rejection (HAR) and acute humoral xenograft rejection (AHXR) (5,6). However, 1,3Gal-deficient porcine xenografts can still be vigorously rejected by T cells, and the use of nonspecific immunosuppressive drugs has not been successful in preventing 1,3Gal-deficient porcine xenograft rejection without severe toxicity in primate recipients (5-7). Thus, tolerance induction is likely to be essential for clinical success of xenotransplantation. We have recently shown that co-transplantation of human fetal thymus and CD34+ cells achieves long-term repopulation with multilineage human lymphohematopoietic cells and formation of secondary lymphoid organs in immunodeficient mice (8-10). Furthermore, these humanized mice (hu-mice) mediate strong antigen-specific immune responses and rejection of porcine skin and islet xenografts. We have previously shown that porcine thymus can generate human T cells that are tolerant of the porcine thymic donor (11). In this study, we used this hu-mouse model to investigate the possibility of porcine thymus transplantation to induce human T cell tolerance in hu-mice with a pre-established human immune system. We observed that brief conditioning with depleting anti-human CD2 mAb results in acceptance of porcine thymic grafts and donor-specific tolerance in human thymic graftectomized hu-mice with a pre-established human immune system. Materials and Methods Animals and human fetal tissues Immunodeficient nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice were purchased from National Malignancy Institute (Bethesda, MD) or The Jackson Laboratory (Bar Harbor, ME), and housed in a specific pathogen-free microisolator environment. Human fetal thymus and liver tissues of gestational age 17 to 20 weeks were obtained from Advanced Bioscience Resource (Alameda, CA). Porcine fetal thymi were harvested from fetuses (60-70 gestational days) of Massachusetts General Hospital inbred miniature swine (kindly provided by Dr. David H. Sachs) (12). Protocols using human tissues and animals in this study were Tetrahydrobiopterin approved by the Massachusetts General Hospital Human Research Committee and Subcommittee of Research Animal Care, and all of the experiments were performed in accordance with the protocols. Hu-mouse preparation Humanized NOD/SCID mice were produced as previously Tetrahydrobiopterin explained (8-10). Briefly, female NOD/SCID mice (7-10 weeks aged) were conditioned with sublethal (2-3Gy) whole body irradiation. Eight to 20 hours later, mice were implanted with human fetal thymus (Thy) and liver (Liv) tissue fragments measuring about 1 Rabbit Polyclonal to NDUFA4 mm3 under the kidney capsule, and injected (i.v.) with 1-5105 CD34+ human fetal liver cells (FLCs). CD34+ FLCs were purified from same donor by the magnetic-activated cell sorter (MACS) separation system using anti-human CD34 microbeads (Miltenyi Biotec, Auburn, CA). Levels of human hematopoietic cells in peripheral blood of the reconstituted mice were determined by circulation cytometric (FCM) analysis using the following mAbs: anti-human CD3, CD4, CD8, CD45, and isotype control mAbs (all purchased from BD Bioscience, San Jose, CA). FACS analysis was performed on a FACScalibur (BD Bioscience, San Jose, CA). The investigators performing this study have been generating consistent results by using this protocol for several years, with approximately 80% success rates in most experiments. The surgical procedure is usually well tolerated in mice and no GVHD has been seen in the reconstituted hu-mice. Hu-mice used in this study were from different cohorts, but all experienced 4% of human CD3+ cells in PBMCs 1 week prior to BTI-322 treatment and porcine thymus transplantation. Porcine thymus transplantation Hu-mice underwent surgical removal of human thymic grafts by nephrectomy 9-15weeks after human Thy/Liv/CD34+ FLC transplantation, which was followed immediately by transplantation of a fetal porcine thymus fragment measuring about 1 mm3 into the capsular space of Tetrahydrobiopterin the contralateral kidney. Hu-mouse recipients were.