The rejuvenating effects of leuprolide acetate on the aged baboon's thymus.

BACKGROUND: We have previously demonstrated that the juvenile thymus plays an essential role in tolerance induced by both renal transplantation and a short course of calcineurin inhibitors. Aged thymi have a decreased ability to induce tolerance. Luteinizing hormone-releasing hormone (LHRH) is known to pharmacologically rejuvenate the thymus in rodents. In order to develop a clinically applicable regimen of transplantation tolerance in adults, we sought to determine if thymic rejuvenation would occur with LHRH agonism in non-human primates. METHODS AND RESULTS: Thymic rejuvenation was evaluated by magnetic resonance imaging (MRI), histology, as well as in-vitro cellular and molecular tests. Four aged male hamadryas baboons underwent subcutaneous injection of a 3-month depot of Lupron (11.25mg; LI) and were followed for 3 months. Thymi increased volumetrically by MRI. After LI, thymic cellularity markedly increased within the cortical and medullary thymus. Additionally, a significant increase in the CD4(+)/CD45RA(hi+) population in the peripheral blood occurred for 50 days after LI, and flow cytometry of thymic tissue revealed a large increase in the percentage of CD4(+)/CD8(+) cells. TREC assay corroborated enhancement in thymic function. CONCLUSION: These data indicate that LI is associated with thymic rejuvenation in baboons, and further confirm that extrinsic factors play an important role in thymic rejuvenation in a non-human primate model.

Development of antidonor antibody directed toward non-major histocompatibility complex antigens in tolerant animals.

BACKGROUND: The clinical significance of antibodies directed against antigens other than major histocompatibility complex (MHC) antigens is poorly understood, and there are few large animal models in which such antibodies can be examined. We studied, both retrospectively and prospectively, the development of antibodies to non-MHC antigens in tolerant miniature swine. METHODS: Our database was assessed for cases of antidonor antibody formation in tolerant animals over the last 20 years. Flow cytometry, absorption assays, and familial analyses for inheritance pattern of the gene(s) potentially responsible for the antibody reactivities were carried out, and an animal determined to be negative for this reactivity was immunized by a skin graft and subcutaneous injections of peripheral blood monocyte cells from an antigen-positive donor. RESULTS: Sixteen of 469 tolerant animals tested were found to have developed antidonor antibodies. These antibodies were found to be specific for the same, presumably single, non-MHC antigen. Familial analyses indicated that the gene encoding this antigen was expressed in an autosomal-dominant manner in approximately 95% of the herd. In a prospective study, antidonor antibodies with the same specificity as those observed retrospectively were successfully induced in an antigen-negative animal after immunization with peripheral blood monocyte cells. CONCLUSION: To our knowledge, this is the first report of the development of antibodies to a highly prevalent, non-MHC antigen present on peripheral blood mononuclear cells and developing in tolerant animals without signs of graft dysfunction. Considering the concern often raised by the appearance of antidonor antibodies in transplant recipients, these data could have important implications for clinical transplantation.

Vascularized composite allograft transplant survival in miniature swine: is MHC tolerance sufficient for acceptance of epidermis?

BACKGROUND: We have previously reported that Massachusetts General Hospital miniature swine, which had accepted class I-mismatched kidneys long-term after 12 days of high-dose cyclosporine A, uniformly accepted donor-major histocompatibility complex (MHC)-matched kidneys without immunosuppression but rejected donor MHC-matched split-thickness skin grafts by day 25, without changes in renal graft function or antidonor in vitro responses. We have now tested whether this "split tolerance" would also be observed for the primarily vascularized skin of vascularized composite allografts (VCAs). METHODS: Group 1 animals (n=3) received donor MHC-matched VCAs less than 70 days after primary kidney transplant (KTx). Group 2 animals (n=3) received a second donor-matched kidney transplant followed by a donor-matched VCA more than 200 days after primary KTx. RESULTS: Animals in Group 1 lost the epidermis on days 28, 30, and 40, with all other components of the VCAs remaining viable. Histology showed cellular infiltration localized to dermal-epidermal junction. One of three recipients of VCAs in Group 2, accepted all components of the VCA, including epidermis (>200 days). The other two recipients lost only the epidermis on days 45 and 85, with survival of the remainder of the VCA long-term. CONCLUSIONS: All tissues of a VCA are accepted long-term on animals tolerant of class I-mismatched kidneys, with the exception of epidermis, the survival of which is markedly prolonged compared with split-thickness skin grafts but not indefinite. Exposure of tolerant animals to second donor-matched kidneys before VCA increases the longevity of the VCA epidermis, suggesting an increase in the immunomodulatory mechanisms associated with tolerance of the kidney.

Tolerogenicity of donor major histocompatibility complex-matched skin grafts in previously tolerant Massachusetts general hospital miniature swine.

BACKGROUND: Long-term tolerance of class I disparate renal allografts in miniature swine can be induced by a short course of cyclosporine and persists for 3 to 4 months after grafts are removed. Donor class I peptide immunization 6 weeks after graftectomy of tolerated kidneys leads to sensitization, but donor skin grafts do not. Here, we tested the hypothesis that skin grafts prevent rejection after simultaneous peptide administration and skin grafting. METHODS: Miniature swine underwent bilateral nephrectomy and class I-mismatched renal transplantation with a 12-day course of cyclosporine A to induce long-term tolerance. Tolerated allografts were then replaced with recipient-matched kidneys, and animals were challenged with simultaneous donor-type skin grafts and peptide. Six weeks later, second donor-matched kidneys were transplanted without immunosuppression, and immune responses were characterized. RESULTS: Animals treated only with peptide (n=2) rejected subsequent renal transplants in 3 to 5 days with strong in vitro antidonor responses. Of five recipients of skin-plus-peptide regimen, two accepted kidneys long term, one demonstrated a modestly prolonged survival (11 days), and two rejected rapidly (5-7 days). The two long-term acceptors maintained donor-specific hyporesponsiveness in vitro. CONCLUSIONS: Sensitization by class I peptide in previously tolerant swine could be prevented by simultaneous class I skin grafts. These data suggest that skin grafts may actually augment rather than abrogate downregulation in some cases. A mechanistic hypothesis for this surprising result is that recognition of class I antigens through the direct rather than the indirect pathway of antigen presentation promotes tolerance by expanding regulatory T cells.

First experience with the use of a recombinant CD3 immunotoxin as induction therapy in pig-to-primate xenotransplantation: the effect of T-cell depletion on outcome.

BACKGROUND: We have previously reported life-supporting kidney xenograft-survival greater than 80 days using a steroid-free antithymocyte globulin (ATG)-based induction regimen (ATG regimen) in a GalT-KO pig-to-baboon thymokidney (TK) model. We evaluated two induction regimens, a newly developed anti-monkey CD3 recombinant immunotoxin (anti-CD3 rIT) and an anti-human CD2 antibody (LoCD2), by assessing T-cell depletion (TCD) and graft survival. METHODS: Four baboons received anti-CD3 rIT; the time course of TCD was studied in two animals and the other two received GalT-KO TK transplants. Two additional baboons underwent GalT-KO TK transplantation after treatment with LoCD2. All other treatments were identical to previous TCD studies with ATG. TCD was assessed by flow-cytometry; renal function was evaluated by serum creatinine and histology. RESULTS: Baboons that received the anti-CD3 rIT died from pneumonia or cardiac failure on days 15 and 23. Both animals in the rIT group died with functioning grafts. Thymokidney grafts from baboons treated with the LoCD2 regimen were rejected by day 14. TCD levels in baboons receiving the anti-CD3 rIT regimen were 150 to 250 cells/µL for at least 14 days, whereas baboons receiving the LoCD2 recovered to more than 300 cells/µL by day 7. CONCLUSIONS: The newly developed anti-CD3 rIT could be a useful TCD agent in baboons. However, optimal dosage, treatment duration, and bioactivity must be studied to avoid side effects. A LoCD2-based regimen was not effective for preventing xenogeneic rejection. Optimal TCD levels less than 250/µL during the induction period seem to be important for success of xeno-thymokidney transplantation.