Histological Evidence Of Rejection Research Articles

Elevated Plasma CXCL8 Concentrations in Significant Fibrosis but Not in Subclinical Rejection After Adult Liver Transplantation

Background. The noninvasive detection of subclinical graft injury including subclinical T cell–mediated rejection (subTCMR) is one of the unresolved challenges after liver transplantation. Recently, serum C-X-C motif chemokine ligand 8 (CXCL8) was proposed as a highly accurate marker of subTCMR in pediatric liver transplant recipients. We aimed to evaluate the accuracy of the quantification of this chemokine for predicting subTCMR in adult liver transplant recipients, as well as its capacity to classify patients who could benefit from immunosuppression reduction. Methods. Plasma CXCL8 concentrations were measured retrospectively in a prospectively collected cohort of adult liver transplant recipients with well-characterized histologic phenotypes. Results. In total, 78 patients were included. Median plasma CXCL8 concentrations did not differ (P = 0.24) between patients without histological evidence of rejection (3.6 [0.4–22.0] pg/mL), subTCMR (11.5 [0.4–41.0] pg/mL), clinical TCMR (9.4 [0.4–40.5] pg/mL), and other etiologies of graft injury (8.7 [0.4–31.2] pg/mL). Likewise, plasma CXCL8 concentrations did not discriminate between patients within and outside histologic criteria for immunosuppression reduction that were proposed by the 2016 Banff Working Group on Liver Allograft Pathology (cutoff: 10.9 pg/mL, sensitivity: 0.48, and specificity: 0.79). Furthermore, weak correlation was found between plasma CXCL8 and alanine aminotransferase and aspartate aminotransferase (Spearman ρ = 0.18 and 0.25). Patients with significant fibrosis (17.8 [0.4–40.5] pg/mL) showed higher plasma CXCL8 concentrations than patients without fibrosis (8.2 [0.4–41.0] pg/mL; P = 0.05). Conclusions. Plasma CXCL8 concentrations are not predictive of subclinical graft injury or of histological criteria for the minimization of immunosuppression in adult liver transplant recipients.

The Problem of Subclinical Antibody-mediated Rejection in Kidney Transplantation.

Defined as histologic evidence of rejection on a protocol biopsy in the absence of kidney dysfunction, subclinical rejection has garnered attention since the 1990s. The major focus of much of this research, however, has been subclinical T cell-mediated rejection (TCMR). Herein, we review the literature on subclinical antibody-mediated rejection (AMR), which may occur with either preexisting donor-specific antibodies (DSA) or upon the development of de novo DSA (dnDSA). In both situations, subsequent kidney function and graft survival are compromised. Thus, we recommend protocol biopsy routinely within the first year with preexisting DSA and at the initial detection of dnDSA. In those with positive biopsies, baseline immunosuppression should be maximized, any associated TCMR treated, and adherence stressed, but it remains uncertain if antibody-reduction treatment should be initiated. Less invasive testing of blood for donor DNA or gene profiling may have a role in follow-up of those with negative initial biopsies. If a protocol biopsy is positive in the absence of detectable HLA-DSA, it also remains to be determined whether non-HLA-DSA should be screened for either in particular or on a genome-wide basis and how these patients should be treated. Randomized controlled trials are clearly needed.

Cutaneous leukocyte lineages in tolerant large animal and immunosuppressed clinical vascularized composite allograft recipients.

Vascularized composite allografts (VCAs) can restore fully functional anatomic units in patients with limb amputations or severe facial tissue loss. However, acute rejection of the skin is frequently observed and underscores the importance of developing tolerance induction protocols. In this study, we have characterized the skin immune system in VCAs. We demonstrate infiltration of recipient leukocytes, regardless of rejection status, and in tolerant mixed hematopoietic chimeras, the co-existence of these cells with donor leukocytes in the absence of rejection. Here we characterize the dermal T cell and epidermal Langerhans cell components of the skin immune system in our porcine model of VCA tolerance, and the kinetics of cutaneous chimerism in both of these populations in VCAs transplanted to tolerant and nontolerant recipients, as well as in host skin. Furthermore, in biopsies from the first patient to receive a hand transplant in our program, we demonstrate the presence of recipient T cells in the skin of the transplanted limb in the absence of clinical or histological evidence of rejection.

First birth from a deceased donor uterus in the United States: from severe graft rejection to successful cesarean delivery

Uterus transplantation is the only known potential treatment for absolute uterine factor infertility. It offers a unique setting for the investigation of immunologic adaptations of pregnancy in the context of the pharmacologic-induced tolerance of solid organ transplants, thus providing valuable insights into the early maternal-fetal interface. Until recently, all live births resulting from uterus transplantation involved living donors, with only 1 prior birth from a deceased donor. The Cleveland Clinic clinical trial of uterus transplantation opened in 2015. In 2017, a 35 year old woman with congenital absence of the uterus was matched to a 24 year old parous deceased brain-dead donor. Transplantation of the uterus was performed with vaginal anastomosis and vascular anastomoses bilaterally from internal iliac vessels of the donor to the external iliac vessels of the recipient. Induction and maintenance immunosuppression were achieved and subsequently modified in anticipation of pregnancy 6 months after transplant. Prior to planned embryo transfer, ectocervical biopsy revealed ulceration and a significant diffuse, plasma cell-rich mixed inflammatory cell infiltrate, with histology interpreted as grade 3 rejection suspicious for an antibody-mediated component. Aggressive immunosuppressive regimen targeting both cellular and humoral rejection was initiated. After 3 months of treatment, there was no histologic evidence of rejection, and after 3 months from complete clearance of rejection, an uneventful embryo transfer was performed and a pregnancy was established. At 21 weeks, central placenta previa with accreta was diagnosed. A healthy neonate was delivered by cesarean hysterectomy at 34 weeks' gestation. In summary, this paper highlights the first live birth in North America resulting from a deceased donor uterus transplant. This achievement underscores the capacity of the transplanted uterus to recover from a severe, prolonged rejection and yet produce a viable neonate. This is the first delivery from our ongoing clinical trial in uterus transplantation, including the first reported incidence of severe mixed cellular/humoral rejection as well as the first reported placenta accreta.

2659. Retrospective Review of Biopsy Proven Acute Graft Pyelonephritis in Renal Transplant Patients

BackgroundThere are few studies of histologic acute graft pyelonephritis (HAGPN) following kidney transplant (KT). The goals of this study are to determine incidence, identify potential risk factors and describe outcomes of HAGPN in a large cohort of KT recipients.MethodsRenal allograft biopsies of all patients undergoing first KT at our medical center between 2008 and 2017 were reviewed. HAGPN was defined as the presence of neutrophils within the interstitium and tubules (casts). Medical charts of patients with HAGPN were reviewed. Episodes of bacteriuria (≥10:5 cfu/mL growth in culture) were classified as urinary tract infection (UTI) or asymptomatic bacteruria (ASB) based upon documented symptoms. An episode of acute rejection was defined as pulse parenteral immunosuppressive therapy for histologic evidence of rejection.ResultsHAGPN was identified in 43 of 1,391 (3.1%) KT recipients at a median of 298 days post-transplant. There were no significant differences between recipient age or gender, donor age or transplant type (deceased, living related, living unrelated) between recipients with and without HAGPN. Urologic malformation was diagnosed in 14 (33%) by day 30 post-transplant. Twenty-five (58%), 17 (40%), and 13 (30%) sustained one or more episodes of acute rejection, UTI and ASB, respectively, prior to HAGPN. At diagnosis of HAGPN, 28 (65%), 7 (16%), and 16 (37%) had histologic evidence of rejection, UTI and ASB, respectively. Twenty-two (51%) and 37 (86%) were treated with pulse immunosuppression and antibiotics, respectively. Median nadir serum creatinine before HAGPN was 1.1 mg/day while median serum creatinine at 6 and 12 months after HAGPN were 1.5 and 1.6. Three patients (7%) developed graft failure within 1 year after HAGPN.ConclusionHAGPN is an infrequent complication of KT. A majority of patients with HAGPN have histologic evidence of rejection and either UTI or ASB at diagnosis, though over 40% have neither UTI nor ASB. When rejection accompanying HAGPN is routinely treated with pulse immunosuppression and antibiotic therapy is administered, graft function is preserved for most patients but a minority (7%) loses graft function within 1 year. Potential risk factors to be assessed in further study include post-transplant urologic dysfunction, acute rejection and UTI.Disclosures All authors: No reported disclosures.

350.6: The impact of circulating DSA in visceral transplant recipients without histological evidence of rejection: How should we treat or not treat?

Backgrounds: There has been accumulating evidence that donor-specific antibody (DSA) has a negative impact on the outcome of visceral allografts. Prospective DSA monitoring allows detection of circulating DSA without histological evidence of acute rejection. However, its association with acute rejection and natural course without treatment are yet to be determined. Methods: Between 2012 and 2019, 94 visceral transplantation with 25 liver-contained grafts were performed at a single institution. Induction therapy for preformed DSA included rituximab or bortezomib. DSA were monitored at 1 and 2weeks, followed by 1, 3, and 6 months, and yearly after transplant. De-novo DSA was not treated without histologically proved rejection. The characteristics and impact of DSA in terms of predictability of rejection, and its correlation of severity of acute rejection were analyzed. Results: One-year cumulative rejection rate in the entire cohort was 67%. With the protocol of anti-B cell therapy, preformed DSA did not increase the risk of rejection, regardless of the status of liver-free allografts, high titer (>4000 MFI) or persistence of DSA. Moreover, there was no difference in severity of rejection with the presence of preformed DSA (n=12) compared to that without DSA (n=36). However, 3-years graft survival was inferior in patients with preformed DSA to those without (52% vs. 64%, p=0.069), due to increased infectious complications. De-novo DSA was detected in 32 patients (35%) with an HLA-ABDR mismatch as the only predictor (Odds ratio 1.7, p=0.036). De-novo DSAs were more likely to be class II with high inclusion of anti-DQ DSA (69%). Prospective monitoring detected de-novo DSA in 25 occasions without ongoing rejection. Accordingly, it turned negative without rejection episode in 19 (76%) (Silent DSA), while subsequent rejection occurred in 6 occasions (24%) (DSA before rejection). In 10 occasions, de-novo DSA turned positive during the rejection episodes (DSA during rejection). The rejection episodes that induced DSA circulation was with a more severe grade. Conclusions: Increased immunosuppressant induction to treat preformed DSA may compensate the rejection risk, but can negatively impact the overall graft survival. With prospective monitoring and no preemptive treatment, high proportion of de-novo DSA was self-limiting, and rarely precipitated rejection episodes particularly with a severe grade.

Metabolomic Characterization of Human Model of Liver Rejection Identifies Aberrancies Linked to Cyclooxygenase (COX) and Nitric Oxide Synthase (NOS).

BackgroundAcute liver rejection (ALR), a significant complication of liver transplantation, burdens patients, healthcare payers, and the healthcare providers due to an increase in morbidity, cost, and resources. Despite clinical resolution, ALR is associated with an increased risk of graft loss. A unique protocol of delayed immunosuppression used in our institute provided a model to characterize metabolomic profiles in human ALR.Material/MethodsTwenty liver allograft biopsies obtained 48 hours after liver transplantation in the absence of immunosuppression were studied. Hepatic metabolites were quantitated in these biopsies by liquid chromatography and mass spectroscopy (LC/MS). Metabolite profiles were compared among: 1) biopsies with reperfusion injury but no histological evidence of rejection (n=7), 2) biopsies with histological evidence of moderate or severe rejection (n=5), and 3) biopsies with histological evidence of mild rejection (n=8).ResultsThere were 133 metabolites consistently detected by LC/MS and these were prioritized using variable importance to projection (VIP) analysis, comparing moderate or severe rejection vs. no rejection or mild rejection using partial least squares discriminant statistical analysis (PLS-DA). Twenty metabolites were identified as progressively different. Further PLS-DA using these metabolites identified 3 metabolites (linoleic acid, γ-linolenic acid, and citrulline) which are associated with either cyclooxygenase or nitric oxide synthase functionality.ConclusionsHepatic metabolic aberrancies associated with cyclooxygenase and nitric oxide synthase function occur contemporaneous with ALR. Additional studies are required to better characterize the role of these metabolic pathways to enhance utility of the metabolomics approach in diagnosis and outcomes of ALR.

Desensitization and Prevention of Antibody-Mediated Rejection in Vascularized Composite Allotransplantation by Syngeneic Hematopoietic Stem Cell Transplantation.

Candidates for vascularized composite allotransplantation (VCA) are frequently sensitized, putting them at risk for antibody-mediated rejection. Current desensitization strategies are imperfect and require a living-donor setting. Here we investigated the impact of sensitization on and the efficacy of a desensitization protocol utilizing syngeneic hematopoietic stem cell transplantation (HSCT) to prevent antibody-mediated rejection in VCA. Skin transplants from Dark Agouti to Lewis rats were performed for sensitization. Orthotopic hind limb transplants from Dark Agouti donors were performed to sensitized and nonsensitized recipients, and the animals were treated with either daily tacrolimus or no immunosuppression. A desensitization protocol consisting of total body irradiation, fludarabine, and syngeneic HSCT was applied to sensitized animals. Graft rejection was monitored by clinical assessment and histological analysis. Serum levels of donor-specific antibodies (DSA IgG) were measured using flow cytometry. Sensitized recipients exhibited accelerated rejection by 5.5 ± 1.2 days without immunosuppression and 10.2 ± 3.6 days with daily tacrolimus compared with 8.7 ± 1.2 days and longer than 30 days in nonsensitized recipients, respectively. Serum levels of DSA IgG were markedly elevated (37.3 ± 3.34-fold from baseline) in sensitized recipients after VCA and correlated with histologic evidence of rejection and C4d deposition. Desensitization significantly reduced DSA compared with sensitized controls (2.6 ± 0.5-fold vs 6.0 ± 1.2-fold, P < 0.01) and along with daily tacrolimus led to improved VCA survival longer than 30 days without evidence of C4d deposition (n = 6). In summary, sensitization leads to accelerated rejection of VCA, and syngeneic HSCT combined with conventional immunosuppression effectively reduces DSA and improves allograft survival in sensitized rats.

The effects of brain death and ischemia on tolerance induction are organ-specific.

We have previously shown that 12days of high-dose calcineurin inhibition induced tolerance in MHC inbred miniature swine receiving MHC-mismatched lung, kidney, or co-transplanted heart/kidney allografts. However, if lung grafts were procured from donation after brain death (DBD), and transplanted alone, they were rejected within 19-45days. Here, we investigated whether donor brain death with or without allograft ischemia would also prevent tolerance induction in kidney or heart/kidney recipients. Four kidney recipients treated with 12days of calcineurin inhibition received organs from donors rendered brain dead for 4hours. Six heart/kidney recipients also treated with calcineurin inhibition received organs from donors rendered brain dead for 4hours, 8hours, or 4hours with 4 additional hours of cold storage. In contrast to lung allograft recipients, all isolated kidney or heart/kidney recipients that received organs from DBD donors achieved long-term survival (>100days) without histologic evidence of rejection. Proinflammatory cytokine gene expression was upregulated in lungs and hearts, but not kidney allografts, after brain death. These data suggest that the deleterious effects of brain death and ischemia on tolerance induction are organ-specific, which has implications for the application of tolerance to clinical transplantation.

Immune Desensitization Allows Pediatric Blood Group Incompatible Kidney Transplantation.

Blood group incompatible transplantation (ABOi) in children is rare as pretransplant conditioning remains challenging and concerns persist about the potential increased risk of rejection. We describe the results of 11 ABOi pediatric renal transplant recipients in the 2 largest centers in the United Kingdom, sharing the same tailored desensitization protocol. Patients with pretransplant titers of 1 or more in 8 received rituximab 1 month before transplant; tacrolimus and mycophenolate mofetil were started 1 week before surgery. Antibody removal was performed to reduce titers to 1 or less in 8 on the day of the operation. No routine postoperative antibody removal was performed. Death-censored graft survival at last follow-up was 100% in the ABOi and 98% in 50 compatible pediatric transplants. One patient developed grade 2A rejection successfully treated with antithymocyte globulin. Another patient had a titer rise of 2 dilutions treated with 1 immunoadsorption session. There was no histological evidence of rejection in the other 9 patients. One patient developed cytomegalovirus and BK and 2 others EBV and BK viremia. Tailored desensitization in pediatric blood group incompatible kidney transplantation results in excellent outcomes with graft survival and rejection rates comparable with compatible transplants.

Use of Quantitative Real Time Polymerase Chain Reaction to Assess Gene Transcripts Associated With Antibody-Mediated Rejection of Kidney Transplants.

Microarray studies have shown elevated transcript levels of endothelial and natural killer (NK) cell-associated genes during antibody-mediated rejection (AMR) of the renal allograft. This study aimed to assess the use of quantitative real-time polymerase chain reaction as an alternative to microarray analysis on a subset of these elevated genes. Thirty-nine renal transplant biopsies from patients with de novo donor-specific antibodies and eighteen 1-year surveillance biopsies with no histological evidence of rejection were analyzed for expression of 11 genes previously identified as elevated in AMR. Expression levels of natural killer markers were correlated to microcirculation inflammation and graft outcomes to a greater extent than endothelial markers. Creating a predictive model reduced the number of gene transcripts to be assessed to 2, SH2D1b and MYBL1, resulting in 66.7% sensitivity and 89.7% specificity for graft loss. This work demonstrates that elevated gene expression levels, proposed to be associated with AMR, can be detected by established quantitative real-time polymerase chain reaction technology, making transition to the clinical setting feasible. Transcript analysis provides additional diagnostic information to the classification schema for AMR diagnosis but it remains to be determined whether significant numbers of centres will validate transcript analysis in their laboratories and put such analysis into clinical use.

G473 Outcomes of abo incompatible kidney transplantation in children

Objective ABO blood group incompatible transplantation (ABOi) has become increasingly common over the last decade, in part due to a shortage of suitable deceased donor allografts. Whilst encouraging data is emerging on short and medium term graft outcomes in adults, ABOi in children is rare; pre-transplant conditioning in paediatric patients remains challenging and concerns persist about an increased risk of rejection. Encouraged by good results in a large number of adult ABOi transplants, we extended our programme to paediatric recipients, and here report the largest European cohort. Methods A retrospective analysis of all ABOi paediatric renal transplant recipients in the 2 largest centres in the UK sharing the same tailored desensitisation protocol. Patients with pre-transplant titres 1 in 8 or above received rituximab one month prior to transplant; tacrolimus and mycophenolate mofetil were started one week pre-op. Antibody removal was performed to reduce titres to 1 in 8 or less at surgery. No routine post-op removal was performed. Results Ten children (age 2–14 years) underwent an ABOi kidney transplant (Figures 1 and 2 and Table 1). Graft and patient survival was 100%. Baseline titres, tailored desensitisation and graft outcomes are shown in table. One patient developed grade IIa rejection after 2 weeks successfully treated with anti-thymocyte globulin;no histological evidence of rejection in other 9 patients. Another patient had a rise in titre of 2 dilutions at week one treated with 2 immunoadsorption sessions. Nine patients had good graft function (eGFR 30–130 mls/min/1.73m2) at last follow up (range 1–36 months); one patient had eGFR 22 ml/min/1.73m2. One patient developed CMV and BK; another EBV and BK. Conclusion ABOi transplantation in children appears to have an optimal outcome with good graft survival, low risk of rejection and infectious complications.

A case of chronic antibody-mediated rejection in the making

A kidney transplant recipient developed chronic antibody-mediated rejection (ABMR) with clinically significant transplant glomerulopathy while under careful clinical monitoring. The patient developed a de novo donor-specific antibody (DSA) posttransplantation, and a protocol renal biopsy showed C4d deposition with no histological evidence of rejection. Subsequently he developed peritubular capillary basement membrane multilayering, with negative C4d and DSA. Finally, he developed proteinuria and transplant glomerulopathy, with reappearance of DSA and C4d. Despite having a de novo antibody and progressive antibody-mediated damage, this patient under close histological and serological surveillance did not fulfill Banff criteria for acute or chronic ABMR until his disease was advanced. This case illustrates the limitations of current Banff criteria in this setting, due to the fluctuating nature of DSA and C4d staining.

Use of Surveillance Biopsies in Pediatric Renal Transplant Recipients

Copyright: © 2013 Winnicki ED, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The term ‘surveillance biopsy’, also often described as ‘protocol’ biopsy, refers to biopsies that are performed at pre-determined time points after renal transplantation in patients with stable allograft function in order to detect subclinical insults to the allograft, with the intent of early intervention so as to prolong allograft survival. Primarily, surveillance biopsies have been used to detect Sub Clinical Rejection (SCR), defined as histologic evidence of rejection in the absence of any clinical or biochemical signs of graft dysfunction [1]. Other potential benefits include early identification of acute humoral rejection in patients with known high donor specific antibodies, calcineurininhibitor toxicity and other post-transplant complications such as BK virus nephropathy and recurrent disease, all of which may be amenable to intervention. Perhaps due to concerns related to the potential risks associated with the biopsy procedure, especially in the setting of a wellfunctioning allograft, surveillance biopsies have not been universally accepted as a standard of care. Here, we briefly review the literature and attempt to answer the question: should surveillance biopsies be part of routine post-transplant care in pediatric renal transplant recipients?

Renal Allograft Thrombosis in the Early Post Transplant Period

Renal allograft thrombosis involving either the renal artery or the veins is a well known entity in clinical practice. This complication of the renal transplant surgery is more common in the early part of the post transplant period and it is usually associated with acute antibody mediated rejection. This more commonly occurs in the paediatric renal transplant and also seems to have some relation to the duration of peritoneal dialysis pretransplant. However, the occurrence of graft thrombosis in isolation without clinical or histological graft rejection is not rare. We encountered a patient in whom the renal allograft thrombosis occurred after 6 weeks of commercial renal transplantation without any histological evidence of rejection.

Chemokines and Their Receptors in Human Renal Allotransplantation

Chemokines and their receptors play a critical role in leukocyte trafficking, and inhibition of select chemokines has been shown to attenuate kidney disease and allograft rejection in animal models. Therefore, we evaluated chemokine and chemokine receptor transcripts in human renal allograft biopsies, correlating transcript levels with clinical course and immunohistochemical analysis to relate chemokine expression to relevant clinical human disease phenotypes. Renal biopsies were grouped as postreperfusion (n=10), stable function (n=10), subclinical (n=10) or acute rejection (n=17), or calcineurin inhibitor nephrotoxicity (n=9) based on clinical presentation and histopathologic assessment. Using quantitative real-time polymerase chain reaction analysis, chemokine transcripts were assessed relative to transcript levels in preprocurement biopsies from live donor kidneys (n=15). Transcripts from several inflammatory chemokines (CCL3, CCL5, CXCL9, CXCL10, and CXCL11) and chemokine receptors (CCR5, CCR7, and CXCR3) were significantly increased in allografts with subclinical and clinical acute rejection, indicating a strong polarization toward a T-helper 1 effector phenotype during rejection. These transcripts also distinguished acutely rejecting allografts from allografts with nonrejection causes of renal dysfunction. Biopsies from patients with stable function without histologic evidence of rejection had increased chemokine transcript levels that were qualitatively similar but quantitatively reduced compared with rejecting allografts. This comprehensive evaluation of chemokines and their receptors in human renal transplantation defines associations between chemokine expression and clinical phenotypes, may have diagnostic utility, and highlights relevant pathways for therapeutic intervention.

Temporal Pattern of Transfusion and Its Relation to Rejection After Lung Transplantation

Blood transfusion has been shown to impact rejection after renal and cardiac transplantation, but it has not been studied after lung transplantation (LTx). In this study we assess: (1) patterns of transfusion, and (2) temporal interrelationships with histologic evidence of rejection. From July 1998 to January 2006, 326 of 331 patients undergoing LTx had available for study both time-related post-operative blood transfusion data and their series of transbronchial biopsy evaluations of perivascular rejection grade (Grades A0 to A4). Longitudinal temporal decomposition for ordinal variables was used to characterize prevalence of rejection grade and simultaneously assess the influence of (a) red blood cell (RBC), (b) platelet and (c) plasma administration. Although peri-operative transfusion was common, transfusions continued at a low, steady rate throughout the life of LTx patients; patients received a total of 2,841 RBC units through follow-up. Immediately after LTx, the prevalence of Grade A0 rejection was 51%, and this increased to 84% by 6 months. RBC transfusion between biopsies was associated with lower histologic grade of rejection (70%, 73% and 77% with Grade A0 for 0, 1 and 12 units, respectively; p = 0.009), and this was particularly evident early after LTx. Histologic grade was not influenced by platelets or plasma. Transfusion requirements are high and continue throughout life after LTx; causes and effective treatment of persistent anemia should be sought. RBC transfusion appears to have an immunosuppressive effect, particularly early after transplant.

Chimerism and Tolerance after Combined Human Kidney and Hematopoietic Cell Transplantation Using Conditioning with Total Lymphoid Irradiation and Anti-Thymocyte Globulin.

Although transplantation tolerance to organ allografts has been achieved using a wide variety of immunologic interventions in laboratory animals, few tolerance induction protocols with complete immunosuppressive drug withdrawal have been tested in humans. In preclinical rodent models our group showed that long-term tolerance to vascularized organ allografts and donor specific unresponsiveness was achieved when hosts developed persistent mixed hematopoietic chimerism following conditioning with nonmyeloablative fractionated total lymphoid irradiation (TLI) combined with depletive anti-T cell antibodies (ATG) and infusions of donor bone marrow cells. We now report the translation of the preclinical model to human transplantation and have studied 8 patients enrolled in a tolerance induction protocol with a goal of complete immunosuppressive drug withdrawal after combined HLA- matched kidney and hematopoietic cell transplantation. Donors received a 5 day course of G-CSF 4-6 weeks before kidney transplant surgery, and purified CD34+ hematopoietic progenitor cells from the apheresis products were mixed with 1x10^6 donor CD3+ T cells/kg recipient weight (n = 7) or in the case of the patient with life-threatening lupus 10 x10^6 donor CD3+ T cells/kg before cryopreservation. After kidney transplantation surgery, recipients were conditioned with 10 equal doses of TLI administered once daily to a total dose of 800 (n=3) or 1200 (n=5) cGy combined with 5 intravenous infusions of rabbit anti-thymocyte globulin (ATG) at 1.5mg/kg/ dose, total dose of 7.5 mg/kg. Immediately after conditioning (day 10 after kidney transplantation), recipients were given an intravenous injection of the thawed donor cells in the outpatient clinic. Mycophenolate mofetil was given until day 30, and a tapering course of cyclosporine until day 180. Follow up for the first enrolled patient is 1230 days and the last enrolled patient is 105 days. The mean duration of hospitalization for the transplant was 5 days. The mean nadir WBC was 1100 cells/mm3 and no patient developed significant thrombocytopenia. Of the 8 patients transplanted, five have developed persistent chimerism; four of the five have persistent mixed chimerism and the one patient with lupus has complete chimerism. Among the patients with persistent chimerism two have had complete immunosuppressive drug withdrawal and three are in the midst of taper; none of these five patients have had clinical rejection episodes or have histologic evidence of rejection on protocol kidney biopsies. The three patients with transient hematopoietic cell chimerism are on maintenance low doses of immune suppression medications. All patients have excellent graft function with serum creatinine levels between 0.8 to 1.3 mg/dL. None of the patients developed acute or chronic GVHD. Two patients developed 1–2 dermatomal zoster and one patient had CMV reactivation in the blood; all were treated to resolution without hospitalization. Analysis of the gene array profiles of PBMC from the two patients off immune suppressive drugs showed the development of a “ tolerance” pattern after transplantation, and donor specific unresponsivesness in the MLR after drug withdrawal. In conclusion, using a CD34 selected G-CSF mobilized graft with a defined T cell dose and 1200 cGy of TLI we can establish persistent mixed hematopoietic cell chimerism for tolerance induction in patients undergoing kidney transplantation. Tolerance does not develop as soon as chimerism is established, and drug withdrawal without allograft rejection was feasible several months after persistent chimerism. The results suggest that tolerance induction in this protocol is an active and dynamic process.

Randomized Controlled Trial of Total Immunosuppression Withdrawal in Liver Transplant Recipients: Role of Ursodeoxycholic Acid

Total immunosuppression withdrawal (TIW) without causing rejection has been reported in stable liver recipients. The role of ursodeoxycholic acid (UDCA) and patient characteristics that predict the success of this tolerance are unclear. There are two goals, to determine: 1) whether TIW is frequently associated with rejection; and 2) whether UDCA decreases the risk of liver disease (both rejection and recurrence) after TIW. Twenty-six liver recipients who had been free of rejection while on immunosuppressive agents for a minimum of 2 years were randomized to receive either (15 mg/kg) of UDCA (n=14) or identical placebo (n=12) followed by sequential withdrawal of their immunosuppressive regimen over several months. Endpoints were defined as biochemical and histological evidence of rejection, graft dysfunction without rejection, recurrence of pretransplant disease, or 6 months without immunosuppression and no rejection or dysfunction on repeat liver biopsy. Rejection occurred in 6 of 14 (43%) of the UDCA group and 9 of 12 (75%) of those receiving placebo (P=0.09). Degree of rejection was mild, moderate, and severe in 73%, 20%, and 7% of patients respectively. All responded to rescue therapy and none developed chronic rejection. Nine of the remaining 11 patients (eight of the UDCA recipients and three of controls) who did not develop rejection developed graft dysfunction which responded to reintroduction of immunosuppressive agents in each case. Disease recurrence was most common in patients with underlying immune-mediated disorders of the liver. One year after withdrawal only two patients were free of immunosuppression, 80% were able to discontinue prednisone therapy (steroid free), and 50% were able to reduce their dose of cyclosporine. Age, underlying cause of liver disease, and regimen of immunosuppression were favorable predictors. The results of this study suggest that TIW: 1) is frequently associated with subsequent rejection, 2) increases the risk of underlying disease recurrence, and 3) is not facilitated by UDCA use and responds properly to the reintroduction of immunosuppressive therapy.

Use of Skin Grafting to Demonstrate Tolerance Before Kidney Transplantation Without Immunosuppression in the Recipient of a Previous Bone Marrow Transplant

Chronic renal insufficiency is a rare complication of bone marrow transplantation (BMT), occurring in 0.8% to 9.5% (1, 2) of patients and may be of sufficient severity to require renal replacement therapy. When BMT has been used as treatment for hematologic malignancy, an immune-mediated graft-versus-leukemia effect may be needed to maintain remission because systemic immunosuppression after cadaveric renal transplantation could cause risk of relapse of leukemia. The ideal kidney donor would be the same individual who donated the bone marrow. The recipient’s immune system could be identical or chimeric with that of the donor. It is difficult to be certain that 100% of the recipient immune system has been eradicated, and a small residual could proliferate to reject a donated kidney. Ethically, to submit a live donor to nephrectomy, a high level of assurance of success is needed. We present a case in which tolerance using a skin graft was demonstrated before successful kidney transplantation without immunosuppression in a BMT recipient. A 64-year-old male was referred for assessment of chronic renal insufficiency in January 2001. Four years previously, he had had chronic myeloid leukemia (CML) treated successfully with an allogenic BMT from his human leukocyte antigen (HLA)-identical (HLA-A, B, and DR only tested) sister. Two episodes of graft-versus-host-disease (GvHD) were treated with short courses of steroids and cyclosporine. In April 2001, relapse of CML was diagnosed on the basis of break-point cluster region Ableson leukemia transcripts found by polymerase chain reaction in peripheral blood and by 11 of 200 cells being of recipient (male) origin on bone-marrow examination. The relapse was successfully treated with donor lymphocyte infusion. PreBMT he had long-standing hypertension controlled by a single agent and normal kidney function. At referral, his serum creatinine had been raised for more than 24 months. An ultrasound examination revealed an absent left kidney and 9 cm right kidney. Renal insufficiency was progressive, and he was begun on peritoneal dialysis. The sister who had donated the bone marrow offered to donate a kidney. A full-thickness skin graft from the sister was performed in July 2002. One month after skin grafting, the graft had been accepted with no clinical or histologic evidence of rejection (Fig. 1). Kidney transplantation then took place without any induction or maintenance immunosuppression. Two years posttransplant, the patient remains well without episodes of transplant kidney rejection or recurrence of leukemia, with a serum creatinine of 1 mg/dL (89 μmol/L).FIGURE 1. Histology of full-thickness skin graft 1 month postgrafting showing no features of rejection.In a patient with prior documented GvHD, skin grafting provided a simple, practical, in vivo immunologic challenge to reassure us that live-donor renal allografting could be successful without immunosuppression. To our knowledge, there have been only two similar cases reported previously (3, 4). Even though the paradigm of solid-organ transplantation from the same donor in previous BMT recipients is not applicable to the vast majority of organ recipients, it offers a unique opportunity to realize the benefits of tolerance and avoid deleterious effects of immunosuppressive medication, including the risk of relapse of the primary hematologic malignancy. Rommel Ravanan Christopher R. K. Dudley Richard M. Smith Christopher J. Burton Richard Bright Renal Unit Southmead Hospital Bristol, UK Paul A. Lear Department of Surgery Southmead Hospital Bristol, UK David J. Unsworth Department of Immunology Southmead Hospital Bristol, UK