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 SLA^dd miniature swine received 1.5 Gy whole body irradiation and class I‐mismatched (SLA^gg) kidneys from naïve pigs with or without cotransplanted kidneys and/or adoptively transferred cells from long‐term tolerant (LTT) SLA^dd recipients of SLA^gg grafts. In addition, three SLA^dd miniature swine received class I mismatched kidney with adoptively transferred cells from LTT SLA^dd 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.     

The Role of Regulatory Cells in Miniature Swine Rendered Tolerant to Cardiac Allografts by Donor Kidney Cotransplantation

To determine the mechanism by which cotransplantation of a kidney allograft induces tolerance to a donor heart in miniature swine, we examined the role of CD25⁺ cells in heart/kidney recipients. Tolerance was induced to class‐I MHC mismatched hearts by cotransplanting a donor‐specific kidney with a 12‐day course of cyclosporine. Peripheral blood leukocytes (PBL) were isolated from tolerant heart/kidney recipients and used in cell‐mediated lympholysis (CML) coculture assays as either unmodified PBL, PBL enriched for CD25⁺ cells or PBL depleted of CD25⁺ cells to assess their ability to suppress CML responses of naïve recipient‐matched leukocytes against mismatched target cells. Primed PBL from tolerant heart/kidney recipients completely suppressed lysis by naïve cells. Complete suppression of the response of naïve recipient‐matched leukocytes against donor‐matched target cells was lost following the depletion of CD25⁺ cells from tolerant heart/kidney animal PBL, but it was reestablished by incubation of naïve cells with small populations of CD25⁺ cells from tolerant heart/kidney animals. These data suggest that peripheral blood from tolerant heart/kidney recipients contains regulatory cells that, upon priming, can suppress the response of naïve‐matched PBL in coculture CML assays, and that suppression appears to be dependent on cells expressing CD25.     

Effects of Lung Cotransplantation on Cardiac Allograft Tolerance Across a Full Major Histocompatibility Complex Barrier in Miniature Swine

A 12‐day course of high‐dose tacrolimus induces tolerance of major histocompatibility complex–mismatched lung allografts in miniature swine but does not induce tolerance of heart allografts unless a kidney is cotransplanted. To determine whether lungs share with kidneys the ability to induce cardiac allograft tolerance, we investigated heart–lung cotransplantation using the same induction protocol. Hearts (n = 3), heart–kidneys (n = 3), lungs (n = 6), and hearts–lungs (n = 3) were transplanted into fully major histocompatibility complex–mismatched recipients treated with high‐dose tacrolimus for 12 days. Serial biopsy samples were used to evaluate rejection, and in vitro assays were used to detect donor responsiveness. All heart–kidney recipients and five of six lung recipients demonstrated long‐term graft survival for longer than 272 days, while all heart recipients rejected their allografts within 35 days. Tolerant recipients remained free of alloantibody and showed persistent donor‐specific unresponsiveness by cell‐mediated lympholysis/mixed‐lymphocyte reaction. In contrast, heart–lung recipients demonstrated rejection of both allografts (days 47, 55, and 202) and antidonor responsiveness in vitro. In contrast to kidneys, lung cotransplantation leads to rejection of both heart and lung allografts, indicating that lungs do not have the same tolerogenic capacity as kidneys. We conclude that cells or cell products present in kidney, but not heart or lung allografts, have a unique capacity to confer unresponsiveness on cotransplanted organs, most likely by amplifying host regulatory mechanisms.     

Characteristics of Cardiac Allograft Vasculopathy Induced by Immunomodulation in the Miniature Swine

Purpose: We aimed to develop swine cardiac transplantation model for study of cardiac allograft vasculopathy (CAV) and to characterize the mechanisms of its formation.Methods: Heterotropic cardiac transplantation was performed in swine leukocyte antigen mismatched miniature swine, and CAV was induced by immunomodulation by cyclosporine A (CyA). Histology and immunohistochemistry were performed to identify cellular components of CAV. Fluorescence in situ hybridization (FISH) was developed for detection of 1 and Y-chromosome for identification of cell origin in the female donor to the male recipient heart transplantation model.Results: CAV was successfully developed by immunomodulation of CyA. Severity of CAV revealed more prominent in the distal epicardial coronary arteries than proximal coronary arteries. Phenotype of the SMCs proliferated in the intimal thickening of CAV were mostly embryonal/secretory type. Our new chromosome specific probes for FISH method were useful for discrimination of sex of each cell, and proliferated SMCs were revealed to be mainly donor origin.Conclusion: CAV mimicking human heart transplantation can be developed by appropriate immunomodulation in the swine. In swine CAV, proliferated SMCs seen in the intimal thickening were demonstrated to be from the donor origin.     

Kidney‐Induced Cardiac Allograft Tolerance in Miniature Swine is Dependent on MHC‐Matching of Donor Cardiac and Renal Parenchyma

Kidney allografts possess the ability to enable a short course of immunosuppression to induce tolerance of themselves and of cardiac allografts across a full‐MHC barrier in miniature swine. However, the renal element(s) responsible for kidney‐induced cardiac allograft tolerance (KICAT) are unknown. Here we investigated whether MHC disparities between parenchyma versus hematopoietic‐derived “passenger” cells of the heart and kidney allografts affected KICAT. Heart and kidney allografts were co‐transplanted into MHC‐mismatched recipients treated with high‐dose tacrolimus for 12 days. Group 1 animals (n = 3) received kidney and heart allografts fully MHC‐mismatched to each other and to the recipient. Group 2 animals (n = 3) received kidney and heart allografts MHC‐matched to each other but MHC‐mismatched to the recipient. Group 3 animals (n = 3) received chimeric kidney allografts whose parenchyma was MHC‐mismatched to the donor heart. Group 4 animals (n = 3) received chimeric kidney allografts whose passenger leukocytes were MHC‐mismatched to the donor heart. Five of six heart allografts in Groups 1 and 3 rejected <40 days. In contrast, heart allografts in Groups 2 and 4 survived >150 days without rejection (p < 0.05). These data demonstrate that KICAT requires MHC‐matching between kidney allograft parenchyma and heart allografts, suggesting that cells intrinsic to the kidney enable cardiac allograft tolerance.     

Donor Brain Death Inhibits Tolerance Induction in Miniature Swine Recipients of Fully MHC‐Disparate Pulmonary Allografts

We have previously shown that a short course of high‐dose tacrolimus induces long‐term tolerance to fully mismatched lung allografts procured from healthy MHC‐inbred miniature swine. Here, we investigate whether donor brain death affects tolerance induction. Four recipient swine were transplanted with fully mismatched lung grafts from donors that were rendered brain dead and mechanically ventilated for 4 h before procurement (Group 1). These recipients were compared to two control groups (Group 2: 4 h of donor ventilation without brain death [n = 5]; and Group 3: no donor brain death with <1 h of ventilation [n = 6]). All recipients were treated with a 12‐day course of tacrolimus. In contrast to both groups of control animals, the swine transplanted with lung allografts from brain dead donors all rejected their grafts by postoperative day 45 and showed persistent responsiveness to donor antigen by MLR. Several additional swine underwent brain death induction and/or mechanical ventilation alone to determine the effects of these procedures on the expression of proinflammatory molecules. Significant increases in serum concentrations of IL‐1, TNF‐α and IL‐10 were seen after brain death. Upregulation of IL‐1 and IL‐6 gene expression was also observed.     

Composite ''Thymoheart'' Transplantation Improves Cardiac Allograft Survival

We have generated a novel composite organ, the thymoheart, which facilitates the contemporaneous transfer of fully vascularized and functional donor thymic tissue to the host at the time of cardiac transplantation. Composite thymoheart allografts were prepared in MHC-inbred miniature swine by implanting autologous thymic tissue into donor hearts 60-90 days before organ procurement. Thymoheart allografts and unmanipulated control hearts were then transplanted into three groups, each treated with the same 12 days of cyclosporine. MHC-matched thymohearts transplanted into euthymic recipients had a minimum survival ranging between 72 and 194 days vs. 42-64 days for unmanipulated control hearts (p = 0.02). MHC class I-disparate thymohearts transplanted into euthymic recipients had a minimum survival ranging between 64 and 191 days vs. 30-55 days for unmanipulated control hearts (p = 0.01). MHC class I-disparate thymohearts transplanted into thymectomized recipients survived between 41 and 70 days vs. 8-27 days for unmanipulated control hearts (p = 0.01). Cellular and humoral functional assays, and skin grafting, confirmed the presence of donor-specific hyporesponsiveness in long-term thymoheart allografts recipients. The transfer of vascularized, functional donor thymic tissue to the host at the time of cardiac transplantation may provide a novel approach to the induction of tolerance in human heart transplant recipients.     

Indirect Recognition of MHC Class I Allopeptides Accelerates Lung Allograft Rejection in Miniature Swine

The role of indirect allorecognition in graft rejection is examined in two experiments using a swine lung transplantation model. First, two swine received class I mismatched grafts without immunosuppression; another two recipients were treated postoperatively with cyclosporine (CsA). These swine exhibited acute and chronic rejection, respectively. All four recipients developed T-cell reactivity to donor-derived class I major histocompatibility complex (MHC) peptides. Second, six swine were immunized with synthetic donor-derived class I allopeptides prior to transplantation. Control groups consisted of nonimmunized recipients (n = 6) and recipients immunized with an irrelevant peptide (n = 3). These recipients all received a 12-day course of post-operative CsA. Swine immunized with allopeptides exhibited accelerated graft rejection, as compared to both control groups (p < 0.01 and p = 0.03, respectively). Within the experimental group, the dominant histologic finding was acute rejection (AR). Obliterative bronchiolitis (OB) was seen in the graft with the longest survival. Both control groups showed a lesser degree of AR, with four out of six nonimmunized swine ultimately developing OB. These studies suggest that indirect allorecognition is operative during lung allograft rejection, and that pre-transplant sensitization to donor-derived MHC allopeptides can accelerate graft rejection.     

The Indirect Alloresponse Impairs the Induction but Not Maintenance of Tolerance to MHC Class I-Disparate Allografts

We studied the effects of indirect allorecognition on the induction and maintenance phases of tolerance in miniature swine cotransplanted with heart and kidney allografts. MHC class I-mismatched heart and kidney grafts were cotransplanted in recipients receiving CyA for 12 days. Recipients were unimmunized or immunized with a set of donor-derived or control third-party MHC class I peptides either 21 days prior to transplantation or over 100 days after transplantation. T-cell proliferation, delayed type hypersensitivity reaction (DTH) and antibody production were assessed. All animals injected with donor MHC class I peptides developed potent indirect alloresponses specific to the immunizing peptides. While untreated recipients developed stable tolerance, all animals preimmunized with donor allopeptides rejected kidney–heart transplants acutely. In contrast, when peptide immunization was delayed until over 100 days after kidney–heart transplantation, no effects were observed on graft function or in vitro measures of alloimmunity. Donor peptide immunization prevented tolerance when administered to recipients pre transplantation but did not abrogate tolerance when administered to long-term survivors post transplantation. This suggests that the presence of T cells activated via indirect allorecognition represent a barrier to the induction but not the maintenance of tolerance.     

Depletion of CD8 Memory T Cells for Induction of Tolerance of a Previously Transplanted Kidney Allograft

Heterologous immunologic memory has been considered a potent barrier to tolerance induction in primates. Induction of such tolerance for a previously transplanted organ may be more difficult, because specific memory cells can be induced and activated by a transplanted organ. In the current study, we attempted to induce tolerance to a previously transplanted kidney allograft in nonhuman primates. The conditioning regimen consisted of low dose total body irradiation, thymic irradiation, antithymocyte globulin, and anti‐CD154 antibody followed by a brief course of a calcineurin inhibitor. This regimen had been shown to induce mixed chimerism and allograft tolerance when kidney transplantation (KTx) and donor bone marrow transplantation (DBMT) were simultaneously performed. However, the same regimen failed to induce mixed chimerism when delayed DBMT was performed after KTx. We found that significant levels of memory T cells remained after conditioning, despite effective depletion of naïve T cells. By adding humanized anti‐CD8 monoclonal antibody (cM‐T807), CD8 memory T cells were effectively depleted and these recipients successfully achieved mixed chimerism and tolerance. The current studies provide ‘proof of principle’ that the mixed chimerism approach can induce renal allograft tolerance, even late after organ transplantation if memory T‐cell function is adequately controlled.     

Induction of Tolerance by Exosomes and Short-Term Immunosuppression in a Fully MHC-Mismatched Rat Cardiac Allograft Model

Exosomes are MHC‐bearing vesicles secreted by a wide array of cells. We have previously shown that donor‐haplotype exosomes from bone marrow dendritic cells (DCs) injected before transplantation significantly prolong heart allograft survival in congenic and fully MHC‐mismatched Lewis rats. Here we show that donor exosomes administered after transplantation are similarly able to prolong allograft survival, however, without inducing tolerance. We therefore tested the effect of exosomes combined with short‐term LF 15‐0195 (LF) treatment, which blocks the maturation of DCs, so that donor‐MHC antigens from exosomes could be presented in a more tolerogenic environment. LF treatment does not preclude the development of a strong antidonor cellular response, and while LF, but not exosome, treatment inhibits the antidonor humoral response and decreases leukocyte graft infiltration, allografts from LF‐treated recipients were either acutely or strongly chronically rejected. Interestingly, when combined with LF treatment, exosomes induced a donor‐specific allograft tolerance characterized by a strong inhibition of the antidonor proliferative response. This donor‐specific tolerance was transferable to naïve allograft recipients. Moreover, exosomes/LF treatment prevented or considerably delayed the appearance of chronic rejection. These results suggest that under LF treatment, presentation of donor‐MHC antigens (from exosomes) can induce regulatory responses that are able to modulate allograft rejection and to induce donor‐specific allograft tolerance.     

Humoral and Cellular Responses to Influenza Vaccination in Human Recipients Naturally Tolerant to a Kidney Allograft

Rare kidney allograft recipients enjoy unaltered graft function years after interruption of their immunosuppressive treatment. To assess the extent to which this state of 'operational tolerance' (TOL) is specific to the graft and not the result of a global immunodeficiency, we analyzed the response of such patients following influenza vaccination. Hemagglutination inhibition titers and frequency of IFNgamma-secreting T cells were measured before 1 and 3 months after vaccination. The proportion of healthy volunteers (HV) responding to vaccine was significantly higher than that of immunosuppressed (IS) patients. Three 'TOL' patients presented a humoral response similar to that of HV, whereas the two others had a poor response, like the IS recipients. Although the small number of patients does not allow for definitive conclusions to be made, these data suggest that the status of tolerance may be heterogeneous, with some patients with a global immunodeficiency and others with an adapted response to vaccination.     

Effects of Donor Age and Cell Senescence on Kidney Allograft Survival

The biological processes responsible for somatic cell senescence contribute to organ aging and progression of chronic diseases, and this may contribute to kidney transplant outcomes. We examined the effect of pre-existing donor aging on the performance of kidney transplants, comparing mouse kidney isografts and allografts from old versus young donors. Before transplantation, old kidneys were histologically normal, but displayed an increased expression of senescence marker p16 ^INK4a . Old allografts at day 7 showed a more rapid emergence of epithelial changes and a further increase in the expression of p16 ^INK4a . Similar but much milder changes occurred in old isografts. These changes were absent in young allografts at day 7, but emerged by day 21. The expression of p16 ^INK4a remained low in young kidney allografts at day 7, but increased with severe rejection at day 21. Isografts from young donors showed no epithelial changes and no increase in p16 ^INK4a . The measurements of the alloimmune response—infiltrate, cytology, expression of perforin, granzyme B, IFN-γ and MHC—were not increased in old allografts. Thus, old donor kidneys display abnormal parenchymal susceptibility to transplant stresses and enhanced induction of senescence marker p16 ^INK4a , but were not more immunogenic. These data are compatible with a key role of somatic cell senescence mechanisms in kidney transplant outcomes by contributing to donor aging, being accelerated by transplant stresses, and imposing limits on the capacity of the tissue to proliferate.     

Expanding the Donor Kidney Pool: Utility of Renal Allografts Procured in a Setting of Uncontrolled Cardiac Death

The chronic shortage of deceased kidney donors has led to increased utilization of donation after cardiac death (DCD) kidneys, the majority of which are procured in a controlled setting. The objective of this study is to evaluate transplantation outcomes from uncontrolled DCD (uDCD) donors and evaluate their utility as a source of donor kidneys. From January 1995 to December 2004, 75,865 kidney-alone transplants from donation after brain death (DBD) donors and 2136 transplants from DCD donors were reported to the United Network for Organ Sharing. Among the DCD transplants, 1814 were from controlled and 216 from uncontrolled DCD donors. The log-rank test was used to compare survival curves. The incidence of delayed graft function in controlled DCD (cDCD) was 42% and in uDCD kidneys was 51%, compared to only 24% in kidneys from DBD donors (p < 0.001). The overall graft and patient survival of DCD donors was similar to that of DBD donor kidneys (p = 0.66; p = 0.88). Despite longer donor warm and cold ischemic times, overall graft and patient survival of uDCD donors was comparable to that of cDCD donors (p = 0.65, p = 0.99). Concerted efforts should be focused on procurement of uDCD donors, which can provide another source of quality deceased donor kidneys.