Masayoshi Okumi

Allosensitization does not increase the risk of xenoreactivity to α1,3-galactosyltransferase gene-knockout miniature swine in patients on transplantation waiting lists

Background. The recent availability of α1,3-galactosyltransferase knockout (GalT-KO) miniature swine has eliminated anti-Gal antibodies as the major barrier to xenotransplantation, potentially bringing this modality closer to clinical application. Highly-allosensitized patients, who have poor prospects of receiving a suitable cross-match negative human organ, might be the first patients to benefit from xenotransplantation of porcine organs. However, concerns exist regarding cross-reactivity of alloreactive anti-human leukocyte antigen (HLA) antibodies against xenogeneic swine leukocyte antigen (SLA) antigens. We have investigated this question using sera from such patients on GalT-KO target cells. Methods. Using flow cytometry and complement-dependent cytotoxicity (CDC) assays, we have tested a panel of 88 human serum samples from patients awaiting cadaveric renal allotransplantation for reactivity against: 1) human; 2) standard miniature swine; and 3) GalT-KO peripheral blood lymphocytes (PBL) and cultured endothelial cells. Results. Anti-swine IgM and IgG antibody binding, as well as CDC, were significantly attenuated on GalT-KO versus standard swine. No correlation was found between the degree of anti-human panel reactive antibodies (PRA) and xenoreactivity against either standard or GalT-KO miniature swine. Treatment of sera with dithiothreitol (DTT) showed that the majority of remaining lymphocytotoxicity against GalT-KO swine was mediated by preformed IgM antibodies. Patients with high alloreactivity but low anti-GalT-KO xenoreactivity were readily identified. Conclusions. Highly allosensitized patients awaiting renal transplants appear to be at no increased risk of xenosensitization over their non-sensitized cohorts, and could therefore be candidates for xenotransplantation using GalT-KO swine donors.

LINKED SUPPRESSION ACROSS AN MHC-MISMATCHED BARRIER IN A MINIATURE SWINE KIDNEY TRANSPLANTATION MODEL

We have demonstrated previously that a 12-day course of FK506 permits the induction of tolerance to fully MHC-mismatched renal transplants in miniature swine. In the present study, we examined the mechanism of this tolerance by assessing the possibility that the survival of one-haplotype mismatched third-party kidneys might be prolonged via linked suppression. Ten SLAd/d miniature swine received fully MHC-mismatched renal allografts from SLAc/c donors with 12 days of FK506. Six animals received second SLAc/c kidneys without immunosuppression to confirm tolerance. Regulatory mechanisms were assessed by mixed lymphocyte reaction (MLR) and cell-mediated lympholysis coculture assays and ELISA for regulatory cytokines. Linked suppression was investigated by transplanting SLAa/c or SLAa/d allografts into long-term tolerant recipients without immunosuppression. All recipients showed donor-specific unresponsiveness in standard cell-mediated lympholysis and MLR assays. Tolerant cells prestimulated with donor Ag and then cocultured with naive recipient MHC-matched cells inhibited antidonor responses, confirming the presence of regulatory cells. ELISA and MLR assays showed that TGF-β2 was involved in mediating the suppression in vitro. SLAa/d renal allografts transplanted into tolerant recipients were rejected by postoperative day 8 (median, 7 days; range, 6–8). In contrast, SLAa/c allografts showed markedly prolonged survival (median, 52 days; range, 28–78; p = 0.0246), suggesting linked suppression. Animals not challenged with a second donor-matched graft did not manifest linked suppression consistent with in vitro data showing that re-exposure to tolerated Ags is important for generation of regulatory cells. To our knowledge, these data represent the first evidence of linked suppression across fully MHC-mismatched barriers in a large animal model.

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.

Carbon monoxide reduces pulmonary ischemia–reperfusion injury in miniature swine

OBJECTIVESnCarbon monoxide is produced endogenously as a by-product of heme catalysis and has been shown to reduce ischemia-reperfusion injury in a variety of organs in murine models. The aims of this translational research were to establish an in situ porcine lung model of warm ischemia-reperfusion injury and to evaluate the cytoprotective effects of low-dose inhaled carbon monoxide in this model.nnnMETHODSnWarm ischemia was induced for 90 minutes by clamping the left pulmonary artery and veins in 8 Clawn miniature swine (Japan Farm CLAWN Institute, Kagoshima, Japan). The left main bronchus was also dissected and reanastomosed just before reperfusion. Four animals were treated with inhaled carbon monoxide at a concentration of approximately 250 ppm throughout the procedure. Lung function and structure were serially accessed via lung biopsy, chest x-ray films, and blood gas analysis.nnnRESULTSnCarbon monoxide inhalation dramatically decreased the lung injury associated with ischemia and reperfusion. Two hours after reperfusion, the arterial oxygen tension of the carbon monoxide-treated group was 454 +/- 34 mm Hg, almost double the arterial oxygen tension of the control group (227 +/- 57 mm Hg). There were fewer pathologic changes seen on chest x-ray films and in biopsy samples from animals in the carbon monoxide-treated group. Animals in the carbon monoxide-treated group also had fewer inflammatory cell infiltrates and a markedly smaller increase in serum concentrations of the proinflammatory cytokines interleukin 1beta, interleukin 6, and high-mobility group box 1 after ischemia-reperfusion injury.nnnCONCLUSIONSnThe perioperative administration of low-dose inhaled carbon monoxide decreases warm ischemia-reperfusion injury in lungs in miniature swine. This protective effect is mediated in part by the downregulation of proinflammatory mediators.

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

UNLABELLEDnBACKGROUND 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).nnnMATERIALS AND METHODSn30 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.nnnRESULTSnHormonal 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.nnnCONCLUSIONnTreatment 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.

Abrogation of Renal Allograft Tolerance in MGH Miniature Swine: The Role of Intra-Graft and Peripheral Factors in Long-Term Tolerance

We have previously demonstrated that long‐term tolerance (LTT) of an MHC class‐I mismatched renal allograft can be achieved with a short course of cyclosporine. In order to examine regulatory mechanisms underlying tolerance in this model, we assessed the contributions of factors within the graft and in the peripheral blood for their relative roles in the maintenance of stable tolerance. Twelve LTT recipients of MHC class‐I mismatched primary kidneys were subjected to a treatment consisting of donor‐specific transfusion followed by leukapheresis, in order to remove peripheral leukocytes, including putative regulatory T cells (Tregs). Following treatment, 2 controls were followed clinically and 10 animals had the primary graft removed and received a second, donor‐MHC‐matched kidney. Neither control animal showed evidence of rejection, while 8 of 10 retransplanted animals developed either rejection crisis or full rejection of the second transplant. In vitro assays confirmed that the removed leukocytes were suppressive and that CD4+Foxp3+ Treg reconstitution in blood and kidney grafts correlated with return to normal renal function in animals experiencing transient rejection crises. These data indicate that components of accepted kidney grafts as well as peripheral regulatory components both contribute to the tolerogenic environment required for tolerance of MHC class‐I mismatched allotransplants.

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

UNLABELLEDnMixed 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.nnnOBJECTIVEnThe 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.

BREAKING MHC-CLASS I TOLERANCE WITH DST FOLLOWED BY LEUKAPHERESIS: 1429

Introduction: Understanding the cellular mechanisms underlying the development and loss of tolerance may lead to clinical protocols of transplant tolerance, obviating the need for immunosuppression. In this study, we attempted to break tolerance in an established renal tolerance model in MHC inbred MGH miniature swine. Methods: Tolerance to class I mismatched kidney allografts was induced by a 12-day course of high dose Cyclosporine in 6 MGH miniature swine. Long-Term Tolerance (LTT) was defined as stable renal function for 100 days following primary transplant and in-vitro evidence of donor specific unresponsiveness by both CML and MLR. LTT animals underwent DST (10ml/kg donor type whole blood) followed by leukapheresis (8 days after DST) of 1.6-3.15x109cells/kg. One day after leukapheresis, 4 animals underwent nephrectomy and retransplant of a donor-type kidney without immunosuppression. Two animals, serving as controls, did not receive a second graft. Renal function was evaluated by serum creatinine and open biopsies. Characterization of cell populations, including CD4+/FoxP3+, cells was assessed by FACS at multiple time points before and after DST, leukapheresis, and Ktxp. Presence of antibody was evaluated using flow cytometry. Results: All 6 recipients became tolerant. Leukapheresis decreased lymphocyte count peripherally by > 50% in all cases. 2 controls (no retransplant) showed no increase in creatinine over baseline (1.0-1.3mg/dL) > 30days after DST and leukapheresis. Of 4 animals retransplanted, 3 had a rejection crisis between POD4 and POD8 (Peak creatinine 3.7mg/dL-11.0mg/dL). 1 of these 3 animals proceeded to develop chronic renal failure and reject completely by 3 months. The remaining animal did not have a rejection crisis following DST+leukapheresis. This animal had only 1.6x109cells/kg removed with leukapheresis. Decrement in the absolute number of CD4+/FoxP3+ was observed following DST and leukapheresis. Absolute CD8+ count increased following DST. All cell populations decreased with leukapheresis. Immunohistochemistry of the explanted grafts from tolerant animals showed bright staining of FoxP3+ cells, whereas biopsies of the retransplanted grafts did not. Animals did not develop antibody (IgM or IgG) before or after DST, leukapheresis, or transplantation. Conclusions: DST + Leukapheresis does not break tolerance in a LTT animal unless the primary graft is removed and replaced with a donor-matched kidney. Further, if leukapheresis was not extensive (i.e. <1.6x109cells/kg) rejection crisis did not occur. These data suggest that both graft infiltrating/protecting cells in tolerant kidneys and peripheral T-regs may be involved in the maintenance of tolerance. Lack of antibody development following retransplant suggests that rejection crises were due to a T-cell mediated response. As animals developed increased CD8+ cells, but decreased T-regs following DST, the loss of tolerance may be associated with a change in the balance between alloreactive T-cells and T-regs (CD4+/ FoxP3+).