Atsushi Hirakata

Results of Gal-Knockout porcine thymokidney xenografts

Clinical transplantation for the treatment of end‐stage organ disease is limited by a shortage of donor organs. Successful xenotransplantation could immediately overcome this limitation. The development of homozygous α1,3‐galactosyltransferase knockout (GalT‐KO) pigs removed hyperacute rejection as the major immunologic hurdle to xenotransplantation. Nevertheless, GalT‐KO organs stimulate robust immunologic responses that are not prevented by immunosuppressive drugs. Murine studies show that recipient thymopoiesis in thymic xenografts induces xenotolerance. We transplanted life‐supporting composite thymokidneys (composite thymus and kidneys) prepared in GalT‐KO miniature swine to baboons in an attempt to induce tolerance in a preclinical xenotransplant model. Here, we report the results of seven xenogenic thymokidney transplants using a steroid‐free immunosuppressive regimen that eliminated whole‐body irradiation in all but one recipient. The regimen resulted in average recipient survival of over 50 days. This was associated with donor‐specific unresponsiveness in vitro and early baboon thymopoiesis in the porcine thymus tissue of these grafts, suggesting the development of T‐cell tolerance. The kidney grafts had no signs of cellular infiltration or deposition of IgG, and no grafts were lost due to rejection. These results show that xenogeneic thymus transplantation can support early primate thymopoiesis, which in turn may induce T‐cell tolerance to solid organ xenografts.

Thymic rejuvenation and the induction of tolerance by adult thymic grafts

The thymus, the site of origin of T cell immunity, shapes the repertoire of T cell reactivity through positive selection of developing T cells and prevents autoimmunity through negative selection of autoreactive T cells. Previous studies have demonstrated an important role for the thymus not only in central deletional tolerance, but also in the induction of peripheral tolerance by vascularized renal allografts in juvenile miniature swine recipients. The same protocol did not induce tolerance in thymectomized recipients nor in recipients beyond the age of thymic involution. We subsequently reported that vascularized thymic lobe grafts from juvenile donors were capable of inducing tolerance in thymectomized juvenile hosts. However, the important question remained whether aged, involuted thymus could also induce tolerance if transplanted into thymectomized hosts, which, if true, would imply that thymic involution is not an intrinsic property of thymic tissue but is rather determined by host factors extrinsic to the thymus. We report here that aged, involuted thymus transplanted as a vascularized graft into juvenile recipients leads to rejuvenation of both thymic structure and function, suggesting that factors extrinsic to the thymus are capable of restoring juvenile thymic function to aged recipients. We show furthermore that rejuvenated aged thymus has the ability to induce transplant tolerance across class I MHC barriers. These findings indicate that it may be possible to manipulate thymic function in adults to induce transplantation tolerance after the age of thymic involution.

Role of Persistence of Antigen and Indirect Recognition in the Maintenance of Tolerance to Renal Allografts

Background. We have previously shown that a 12-day treatment with cyclosporine A (CyA) facilitates induction of tolerance to class-I disparate kidneys, as demonstrated by acceptance of second, donor-matched kidneys without immunosuppression. In the present study, we have examined 1) the duration of tolerance in the absence of donor antigen and 2) the pathway of antigen recognition determining maintenance or loss of tolerance. Methods. Seventeen miniature swine received class-I mismatched kidneys with 12 days of CyA, and received second donor-matched kidneys without immunosuppression at 0, 1, 3, or 4 months after nephrectomy of the primary graft. Five were sensitized 6 weeks after nephrectomy of the primary graft, three with donor-matched skin grafts, and two with donor class-I peptides to eliminate direct pathway involvement. In addition, two long-term tolerant animals received class-Ic peptides. Results. Rejection of second grafts required at least a 3 month absence of donor antigen. Although donor-matched skin grafts in animals tolerant to kidneys induced antidonor cytotoxic T lymphocyte responses, second renal transplants revealed no evidence of sensitization. In contrast, immunization of recipients with donor class-I peptides after nephrectomy of the primary graft led to loss of tolerance at both T-cell and B-cell levels, as evidenced by rejection of the second graft in 5 days and development of antidonor immunoglobulin G. Peptide immunization of long-term tolerant in recipients bearing long-term renal grafts did not break tolerance. Conclusions. These data indicate that the renal allograft is required for the indefinite maintenance of tolerance, that indirect antigen presentation is capable of breaking tolerance, and that in tolerant animals, direct antigen presentation may suppress rejection, allowing tolerance to persist.

Occurrence of specific humoral non-responsiveness to swine antigens following administration of GalT-KO bone marrow to baboons.

Griesemer A, Liang F, Hirakata A, Hirsh E, Lo D, Okumi M, Sykes M, Yamada K, Huang CA, Sachs DH. Occurrence of specific humoral non‐responsiveness to swine antigens following administration of GalT‐KO bone marrow to baboons. Xenotransplantation 2010; 17: 300–312.

The Induction of Tolerance of Renal Allografts by Adoptive Transfer in Miniature Swine

Our previous in vitro data have demonstrated that regulatory mechanisms are involved in tolerance of class I‐mismatched renal allografts in miniature swine treated with 12 days of high dose Cyclsporin A. In this study, we attempted to induce tolerance of class I‐mismatched kidneys by adoptive transfer of cells and/or kidneys from long‐term tolerant animals. Fifteen SLAdd miniature swine received 1.5 Gy whole body irradiation and class I‐mismatched (SLAgg) kidneys from naïve pigs with or without cotransplanted kidneys and/or adoptively transferred cells from long‐term tolerant (LTT) SLAdd recipients of SLAgg grafts. In addition, three SLAdd miniature swine received class I mismatched kidney with adoptively transferred cells from LTT SLAdd recipients. Naïve kidneys transplanted without a LTT kidney were rejected within 9 days. All recipients of naive kidneys along with cells and kidney grafts from LTT animals showed markedly prolonged survival of the naive renal grafts (day 28, >150 and >150 days). These studies suggest that (1) tolerated kidneys have potent regulatory effects and (2) cells from LTT animals infused in conjunction with kidney grafts augment these regulatory effects. To our knowledge, these studies represent the first demonstration of successful adoptive transfer of tolerance in large animals.

Production of cloned NIBS (Nippon Institute for Biological Science) and α‐1, 3‐galactosyltransferase knockout MGH miniature pigs by somatic cell nuclear transfer using the NIBS breed as surrogates

Nuclear transfer (NT) technologies offer a means for producing the genetically modified pigs necessary to develop swine models for mechanistic studies of disease processes as well as to serve as organ donors for xenotransplantation. Most previous studies have used commercial pigs as surrogates.

Reversal of age-related thymic involution by an LHRH agonist in miniature swine.

UNLABELLED BACKGROUND AND AIMS OF STUDY: We have previously demonstrated a requirement for the presence of a juvenile thymus for the induction of transplantation tolerance to renal allografts by a short-course of calcineurin inhibition in miniature swine. We have also shown that aged, involuted thymi can be rejuvenated when transplanted as vascularized thymic lobes into juvenile swine recipients. The present studies were aimed at elucidating the extrinsic factors facilitating this restoration of function in the aged thymus. In particular, we tested the impact of sex steroid blockade by Luteinizing Hormone-Releasing Hormone (LHRH). MATERIALS AND METHODS 30 naive animals (25 males and 5 females) were used for measurement of serum testosterone levels. 3 mature male pigs (aged at 22, 22 and 29 months old) were used to test the effects of Lupron (LHRH analog) injection at 45 mg (per 70-80 kg body weight) as a 3-month depot on testosterone levels and thymic rejuvenation. Thymic rejuvenation was assessed by histology, flow cytometric analysis, morphometric analysis and TREC assays. RESULTS Hormonal alterations were induced by Lupron and resulted in macroscopic and histologic regeneration of the thymus of aged animals within 2 months, as evidenced by restoration of juvenile thymus architecture and increased cellularity. Two animals that were evaluated for TREC both showed increased levels in the periphery following Lupron treatment. CONCLUSION Treatment of aged animals with Lupron leads to thymic rejuventaion in adult miniature swine. This result could expand the applicability of thymus-dependent tolerance-inducing regimens to adult recipients.

Porcine CFSE mixed lymphocyte reaction and PKH-26 cell-mediated lympholysis assays

UNLABELLED Mixed lymphocyte reaction (MLR) and cell-mediated lympholysis (CML) are widely used to assess T cell responses. A major limitation of the traditional MLR and CML assays is that they require radioisotope labeling with (3)H for MLR and (51)Cr for CML, thereby limiting their use to laboratories with the capabilities to deal safely with these materials. Recently, flow cytometry with CFSE labeling has been used to detect cell division in rodent and human assays, and flow cytometry with PKH-26 labeling has been used to study cytotoxicity in murine models. Partially inbred miniature swine provide a unique large animal preclinical model for experimental transplantation, helping to bridge the gap between rodent and clinical studies. In this study, we modified the reported CFSE and PKH-26 labeling procedures for use with porcine cells, and established that these radioactive-free MLR and CML assays are comparable to traditional radioactive CML and MLR assays for assessing immunologic responses in miniature swine. To our knowledge, this is the first report that has directly compared the traditional CML/MLR with radiation-free CML/MLR in MHC-defined swine models. OBJECTIVE The aim of this study is to establish non-radiolabeled CSFE and PKH-26 labeling procedures for flow cytometry based CML/MLR assays that are comparable to radioactive CML/MLR assays in preclinical large animals.

Tolerance and Long-Lasting Peripheral Chimerism After Allogeneic Intestinal Transplantation in MGH Miniature Swine

Background and Objective. Clinical intestinal transplantation (Int-Tx) is limited by high rates of rejection, infection, and graft versus host disease. To improve clinical outcomes and eliminate the comorbidities associated with chronic immunosuppression, the induction of donor-specific tolerance to intestinal grafts is desirable, especially in the pediatric population. This study determined the ability of intestinal grafts to facilitate tolerance induction in major histocompatibility complex (MHC)–inbred miniature swine. Methods. Seven MGH-miniature swine received heterotopic intestinal grafts, two across MHC-matched, minor-antigen disparities, three across a class I MHC disparity with 12 days of cyclosporine A, and two across a class I MHC disparity without an immunosuppressant. Chimerism was assessed by FACS analysis and immunohistochemistry. Cell-mediated lympholysis assays were used to assess antidonor responses. Results. Two animals receiving intestinal grafts without an immunosuppressant developed antidonor IgG in 14 days and rejected these completely. All other grafts were accepted with 12 days of cyclosporine A across both MHC-matched and MHC class I barriers. Cell-mediated lympholysis assays showed donor-specific unresponsiveness by day 30 across MHC class I barriers. Greater than 15% peripheral donor cell chimerism persisted for more than 60 days after MHC-matched Int-Tx. Although less than 1.5% peripheral donor cell chimerism was seen during the maintenance period after class I-mismatched Int-Tx, 5% to 10% myeloid chimerism was found in the peripheral blood 14 to 90 days after Int-Tx. FACS analysis demonstrated that 1% to 2% of lymphocytes in the graft mesenteric lymph nodes were CD4+/CD25high+/Foxp3+ cells. Conclusion. To our knowledge, this is the first demonstration of tolerance induction and persistence of chimerism in a large animal intestinal transplant model.

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.