John C. LaMattina

Induction of transplantation tolerance with a short course of tacrolimus (FK506): I. Rapid and stable tolerance to two-haplotype fully mhc-mismatched kidney allografts in miniature swine.

Background. Inbred miniature swine provide a large animal model inwhich the effects of selective major histocompatibility complex (MHC) matchingcan be reproducibly studied. We have previously demonstrated that although a12-day course of cyclosporine uniformly induces tolerance to classI-mismatched renal allografts, it does not induce tolerance across full MHCbarriers. In this study, we assessed whether and at what dose tacrolimus mightpermit allografts to induce tolerance across different MHCbarriers. Methods. Recipients of MHC disparate renal allografts were treatedwith a 12-day course of tacrolimus by continuous intravenous infusion. Groupswere divided as follows: (1) class I-mismatched kidneys with 0.3 mg/kg/daytacrolimus (n=3); (2) fully MHC-mismatched kidneys with 0.3 mg/kg/daytacrolimus (n=2); and (3) fully MHC-mismatched kidneys with0.12–0.16 mg/kg/day tacrolimus(n=4). Results. In groups 1 and 2, recipients with tacrolimus levels of45–80 ng/ml accepted renal allografts long-term with stable renalfunction. Donor-specific hyporesponsiveness was demonstrated by cell-mediatedlymphocytotoxicity and mixed lymphocyte response, and subsequent donor-matchedgrafts were also accepted, without further immunosuppression (n=4),confirming systemic tolerance. In group 3, recipients that achieved tacrolimuslevels of 35 ng/ml (n=2) accepted their grafts without chronic changes,whereas recipients with levels of 20–26 ng/ml (n=2) developedchronic allograft glomerulopathy, suggesting 35 ng/ml as the threshold bloodlevel for tolerance induction. In vitro assays demonstrated that peripheralblood lymphocytes from tolerant animals produced inhibitory cytokines,suggesting the involvement of regulatorymechanisms. Conclusions. To our knowledge, this study represents the firstdemonstration of the induction of transplant tolerance across a two-haplotypefull MHC barrier with a short course of immunosuppression in a large animalmodel. These studies may also have clinical applicability, because the timecourse required to induce tolerance was sufficiently short that the high druglevels required might be expected to be tolerated clinically with onlytransienttoxicity.

COMPLICATIONS ASSOCIATED WITH LIVER TRANSPLANTATION IN THE OBESE RECIPIENT

The prevalence of the metabolic syndrome with attendant morbid obesity continues to increase nationwide. A concomitant increase in non‐alcoholic steatohepatitis (NASH) and associated end‐stage liver disease requiring transplantation is expected to parallel this trend. Between January 1, 1997 and December 31, 2008, our center performed 813 solitary adult deceased‐donor liver transplants. Patients were divided into groups based on the World Health Organization International Classification of obesity. Patients within each obesity class were compared to normal weight recipients. Preoperative demographics among all groups were similar. NASH was more common in higher BMI groups. Operative time, blood product usage, ICU length of stay, infectious complications, and biliary complications requiring intervention were all higher in obese recipients. Deep venous thrombosis occurred more commonly in patients with Class III obesity. Patients with Class II obesity had lower patient (HR 1.82, CI 1.09–3.01, p = 0.02) and allograft survival (HR 1.62, CI 1.02–2.65, p = 0.04). Obesity class did not reach statistical significance on multivariate analysis. Despite increased technical operative challenges and medical complexities associated with increasing recipient BMI, morbid obesity in and of itself should not be an absolute contraindication to liver transplantation as these patients have reasonable long‐term outcomes.

Xenogeneic thymokidney and thymic tissue transplantation in a pig-to-baboon model: I. Evidence for pig-specific T-cell unresponsiveness.

Background. The potential of xenotransplantation for clinical application will require overcoming barriers of humoral and cellular rejection, through strategies using immune suppression or tolerance induction. This laboratory has previously reported the induction of tolerance in the discordant xenogeneic model of pig-to-rodent thymic transplantation. We also have described a miniature swine model of fully mismatched allogeneic composite vascularized thymokidney transplantation that induced transplantation tolerance. We tested a combination of these approaches in a clinically relevant pig-to-primate model of xenotransplantation. Methods. Composite thymokidney grafts were prepared 40 to 80 days before transplantation by the autologous implantation of thymic tissue under the renal capsule of human decay-accelerating factor transgenic swine. Baboons received xenotransplants of both human decay-accelerating factor composite thymokidneys and omental implants of thymic tissue. Recipients were treated with an immunosuppressive-conditioning regimen including thymectomy or thymic irradiation, extracorporeal immunoadsorption of anti-&agr;Gal antibodies and T-cell depletion. Recipients were followed for indicators of xenograft rejection, T-cell depletion and reconstitution, anti-&agr;Gal antibody levels, and mixed lymphocyte responses. Immunologic responses were studied in those animals that survived for more than 3 weeks. Results. Thymokidney xenografts survived for up to 30 days, with evidence of viable thymic epithelium and Hassall’s corpuscles under the renal capsule and in the omental implants, and with evidence of few host lymphocytes. Three animals demonstrated donor-specific unresponsiveness, while maintaining normal alloresponses, in mixed–lymphocyte-response assays performed after immunosuppression had been stopped. Rejected grafts demonstrated humoral damage without evidence of cellular infiltrates. After graftectomy, one animal maintained donor-specific cellular unresponsiveness and stable anti-&agr;Gal antibody levels for more than 2 months. Conclusions. We concluded that composite thymokidney and thymic-tissue xenotransplantation from swine to baboons can induce donor-specific cellular unresponsiveness and stable anti-&agr;Gal antibody levels, suggesting avoidance of sensitization after xenotransplantation. The presence of viable donor-swine thymic epithelium could have a role in the development of donor-specific T-cell tolerance. Further strategies to address humoral rejection could prolong graft survival and result in long-term tolerance to xenografts.

Vascularized thymic lobe transplantation in miniature swine: I. Vascularized thymic lobe allografts support thymopoiesis.

Background. Vascularized thymokidney transplants have previously been shown to induce tolerance across major histocompatibility complex barriers. The ability to perform vascularized thymic lobe transplantation could permit such tolerance to be induced with any cotransplanted solid organ or tissue. For this reason, we have developed a technique for vascularized thymic lobe transplantation in miniature swine. Methods. Thymic vessels (n=2) were anastomosed to the carotid artery and the external jugular vein of naïve minor-mismatched recipients treated with a 12-day course of cyclosporine A (10 mg/kg/day). Graft survival and thymopoiesis were assessed by histology, immunohistochemistry, and fluorescence-activated cell sorting. Allele-specific antibodies 74–12–4 and pig allelic antigen (PAA) were used to distinguish donor and recipient cells. Results. Allografts showed intact cortical and medullary structure posttransplantation, without evidence of rejection or ischemia. Recipient thymocytes repopulated the donor cortical thymus by POD30 and increased in the cortex and medulla by POD60. Conclusions. Our study demonstrates the technical feasibility of vascularized thymic lobe transplantation and the support of thymopoiesis by such transplants in a large animal model. This technique may offer a novel strategy to induce transplant tolerance across allogeneic and xenogeneic barriers, and to support long-term thymopoiesis in immunodeficient hosts.

Thymic transplantation in miniature swine: III. Induction of tolerance by transplantation of composite thymokidneys across fully major histocompatibility complex-mismatched barriers

Background. This study determines whether composite thymokidney (TK) grafts, created by implantation of autologous thymic tissue beneath the donor’s renal capsule before transplantation, could induce allogeneic transplantation tolerance across two-haplotype fully major histocompatibility complex (MHC)- mismatched barriers in juvenile MGH-miniature swine. Methods. TK grafts were prepared by implanting autologous thymic tissue under the renal capsule of donor animals 2 to 3 months before transplantation. Four recipients were treated with a T-cell–depleting immunotoxin and received fully MHC-mismatched TK grafts plus a 12-day course of cyclosporine A (CsA). Control animals were treated with CsA alone or both CsA and immunotoxin, but with a normal kidney or a kidney implanted with autologous lymph node rather than thymus. Renal graft function was assessed by plasma creatinine levels and histologic analyses. Immunologic status was monitored by cell-mediated lympholysis assays. Results. All four recipients of fully MHC-mismatched TK transplants treated with immunotoxin and a 12-day course of CsA accepted their composite renal allografts long-term. All control recipients receiving a TK and CsA alone, a normal kidney or a composite kidney containing lymph node tissue acutely rejected their grafts. Conclusions. To our knowledge, this is the first demonstration that functional vascularized thymic grafts can induce transplantation tolerance across fully MHC-mismatched barriers in a large animal model.

Alemtuzumab as compared to alternative contemporary induction regimens.

Between 1 January 2002 and 31 December 2007, our center performed 1687 adult renal transplants. A retrospective analysis was performed to compare outcomes between patients receiving alemtuzumab (n = 632) and those receiving either basiliximab (n = 690) or thymoglobulin (n = 125). Patients receiving alemtuzumab were younger (49 vs. 51 years, P = 0.02), had fewer HLA matches (1.7 vs. 2.0, P < 0.0001), were more likely to have a cytomegalovirus (CMV) donor(+)/recipient(−) transplant (22% vs. 17%, P = 0.03) and were less likely to receive a living donor allograft (32% vs. 37%, P = 0.04). Alemtuzumab recipients were less likely to receive tacrolimus (35% vs. 47%, P < 0.0001). The 1‐, 3‐, and 5‐year cumulative incidence of antibody‐mediated rejection (AMR) in alemtuzumab‐treated patients was 19%, 24%, and 27%, vs. 11%, 15%, and 18% for the other group (P < 0.0001). The 1‐, 3‐, and 5‐year allograft survival in the alemtuzumab group was 88%, 75%, and 67%, vs. 91%, 82%, and 74% for the other group (P < 0.0001). Patient survival was equivalent. Alemtuzumab was an independent risk factor for living donor allograft loss (HR 2.0, P = 0.004), opportunistic infections (HR 1.3, P = 0.01), CMV infections (HR 1.6, P = 0.001), and AMR (HR 1.5, P = 0.002). The significantly worse graft survival in the alemtuzumab cohort may be due to the increased rates of AMR and infectious complications.

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.

Shorter waitlist times and improved graft survivals are observed in patients who accept hepatitis C virus+ renal allografts

Background There is a paucity of data regarding long-term renal graft survival in hepatitis C virus positive (HCV+) patients. We analyzed our institution’s experience with HCV+ renal transplantation and factors contributing to subsequent renal graft failure. Methods We analyzed 1,679 adult, deceased donor, single-organ renal transplants occurring between 2000 and 2012. Recipient and donor demographics, HCV serostatus, and graft outcome and function were evaluated. Results Of 1,679 patients, 195 HCV+ recipients (R+) received renal transplants from HCV+ donors (D+), in contrast to 1,418 HCV negative (HCV−) recipients (R−) who received grafts from HCV− donors (D−), and 66 R+ patients who received D− kidneys. Death-censored graft survival in the R+/D+ population was better than graft survival for R+/D− patients, despite R+/D+ patients having higher rates of hypertension and African Americans. Waitlist times for patients accepting HCV+ grafts was 318 days (for R+/D+ patients) versus 613 days (R−/D−) or 570 days (R+/D−). On multivariate analysis, waitlist times were independently predictive of graft failure. Conclusion R+/D+ patients spent less time on the transplant waitlist, which contributed to improved death censored graft survival when compared with R+/D− patients.

Safety of belatacept bridging immunosuppression in hepatitis C-positive liver transplant recipients with renal dysfunction.

Background Perioperative renal dysfunction in liver transplant recipients complicates maintenance immunosuppressive therapy, particularly in patients with hepatitis C. Calcineurin inhibitors exacerbate renal dysfunction and mammalian target-of-rapamycin inhibitors are generally avoided because of perceived perioperative risks. The authors’ experience with seven liver transplant patients who received belatacept and mycophenolic acid maintenance immunosuppression is reported. Methods A retrospective review of adult liver transplant recipients with hepatitis C receiving belatacept was conducted under Institutional Review Board approval. All patients were Epstein-Barr virus IgG seropositive. The primary endpoint was patient and graft survival, with secondary endpoints including the incidence of acute rejection, degree of renal function recovery, and occurrence of major side effects. Results Between December 19, 2011 and January 25, 2013, seven liver transplant recipients with hepatitis C received belatacept immunosuppression in the perioperative period. The primary indication for belatacept was perioperative renal dysfunction. Belatacept was initiated between 2 and 90 days posttransplant and the duration of belatacept therapy ranged from 19 to 89 days. Patients were transitioned onto calcineurin inhibitor therapy when they reached chronic kidney disease stage 2 or better. Six-month patient and graft survival was 86%. There was one episode of graft rejection on belatacept therapy in a patient who had also had early rejection before initiation of belatacept. Conclusions The results in this initial group of patients suggest that belatacept with mycophenolic acid may be a safe maintenance immunosuppression regimen in hepatitis C–positive liver transplant recipients with renal dysfunction, and that this regimen can serve as an effective bridge to calcineurin inhibitor therapy.

Native kidney function following liver transplantation using calcineurin inhibitors: single-center analysis with 20 years of follow-up

The incidence of chronic kidney disease (CKD) in liver transplant recipients has been estimated to be from 18% to 28% at 10 yr after transplantation. As outcomes from liver transplantation continue to improve, long‐term native kidney function in these recipients becomes more critical to patient survival.