Vascularized composite allotransplantation (VCA) has emerged as a viable limb replacement strategy for selected patients with upper limb amputation. However, allograft rejection has been seen in essentially all reported VCA recipients indicating a requirement for substantial immunosuppressive therapy. Calcineurin inhibitors have served as the centerpiece agent in all reported cases, and CNI-associated complications associated with the broad therapeutic effects and side effects of calcineurin inhibitors have been similarly common. Recently, belatacept has been approved as a calcineurin inhibitor replacement in kidney transplantation, but to date, its use in VCA has not been reported. Herein, we report on the case of a hand transplant recipient who developed recurrent acute rejection with alloantibody formation and concomitant calcineurin inhibitor nephrotoxicity, all of which resolved upon conversion from a maintenance regimen of tacrolimus, mycophenolate mofetil and steroids to belatacept and sirolimus. This case indicates that belatacept may be a reasonable maintenance immunosuppressive alternative for use in VCA, providing sufficient prophylaxis from rejection with a reduced side effect profile, the latter being particularly relevant for nonlife threatening conditions typically treated by VCA.

The purpose of the STAR 2019 Working Group was to build on findings from the initial STAR report to further clarify the expectations, limitations, perceptions, and utility of alloimmune assays that are currently in use or in development for risk assessment in the setting of organ transplantation. The goal was to determine the precision and clinical feasibility/utility of such assays in evaluating both memory and primary alloimmune risks. The process included a critical review of biologically driven, state-of-the-art, clinical diagnostics literature by experts in the field and an open public forum in a face-to-face meeting to promote broader engagement of the American Society of Transplantation and American Society of Histocompatibility and Immunogenetics membership. This report summarizes the literature review and the workshop discussions. Specifically, it highlights (1) available assays to evaluate the attributes of HLA antibodies and their utility both as clinical diagnostics and as research tools to evaluate the effector mechanisms driving rejection; (2) potential assays to assess the presence of alloimmune T and B cell memory; and (3) progress in the development of HLA molecular mismatch computational scores as a potential prognostic biomarker for primary alloimmunity and its application in research trial design.

BENEFIT and BENEFIT-EXT were phase III studies of cytotoxic T-cell crossmatch–negative kidney transplant recipients randomized to belatacept more intense (MI)-based, belatacept less intense (LI)-based, or cyclosporine-based immunosuppression. Following study completion, presence/absence of HLA-specific antibodies was determined centrally via solid-phase flow cytometry screening. Stored sera from anti-HLA–positive patients were further tested with a single-antigen bead assay to determine antibody specificities, presence/absence of donor-specific antibodies (DSAs), and mean fluorescent intensity (MFI) of any DSAs present. The effect of belatacept-based and cyclosporine-based immunosuppression on MFI was explored post hoc in patients with preexisting DSAs enrolled to BENEFIT and BENEFIT-EXT. In BENEFIT, preexisting DSAs were detected in 4.6%, 4.9%, and 6.3% of belatacept MI-treated, belatacept LI-treated, and cyclosporine-treated patients, respectively. The corresponding values in BENEFIT-EXT were 6.0%, 5.7%, and 9.2%. In both studies, most preexisting DSAs were of class I specificity. Over the first 24 months posttransplant, a greater proportion of preexisting DSAs in belatacept-treated versus cyclosporine-treated patients exhibited decreases or no change in MFI. MFI decline was more apparent with belatacept MI-based versus belatacept LI-based immunosuppression in both studies and more pronounced in BENEFIT-EXT versus BENEFIT. Although derived post hoc, these data suggest that belatacept-based immunosuppression decreases preexisting DSAs more effectively than cyclosporine-based immunosuppression.

Donor-specific antibodies (DSAs) are associated with an increased risk of antibody-mediated rejection and graft failure. In BENEFIT and BENEFIT-EXT, kidney-transplant recipients were randomized to receive belatacept more intense (MI)–based, belatacept less intense (LI)–based, or cyclosporine-based immunosuppression for up to 7 years (84 months). The presence/absence of HLA-specific antibodies was determined at baseline, at months 6, 12, 24, 36, 48, 60, and 84, and at the time of clinically suspected episodes of acute rejection, using solid-phase flow-cytometry screening. Samples from anti-HLA-positive patients were further tested with a single-antigen bead assay to determine antibody specificities, presence/absence of DSAs, and mean fluorescence intensity (MFI) of any DSAs present. In BENEFIT, de novo DSAs developed in 1.4%, 3.5%, and 12.1% of belatacept MI-treated, belatacept LI-treated, and cyclosporine-treated patients, respectively. The corresponding values in BENEFIT-EXT were 3.8%, 1.1%, and 11.2%. Per Kaplan-Meier analysis, de novo DSA incidence was significantly lower in belatacept-treated vs cyclosporine-treated patients over 7 years in both studies (P <.01). In patients who developed de novo DSAs, belatacept-based immunosuppression was associated with numerically lower MFI vs cyclosporine-based immunosuppression. Although derived post hoc, these data suggest that belatacept-based immunosuppression suppresses de novo DSA development more effectively than cyclosporine-based immunosuppression.

Hyperacute kidney rejection is unusual in crossmatch positive recipients of simultaneous liver-kidney transplants (SLKT). However, recent data suggest that these patients remain at risk for antibody-mediated kidney rejection. To further investigate the risk associated with donor-specific alloantibodies (DSA) in SLKT, we studied 86 consecutive SLKT patients with an available pre-SLKT serum sample. Serum samples were analyzed in a blinded fashion for HLA DSA using single antigen beads (median florescence intensity ≥ 2,000 = positive). Post-SLKT samples were analyzed when available (76%). Thirty patients had preformed DSA, and nine developed de novo DSA. Preformed class I DSA did not change the risk of rejection, patient or allograft survival. In contrast, preformed class II DSA was associated with a markedly increased risk of renal antibody mediated rejection (AMR) (p = 0.006), liver allograft rejection (p = 0.002), patient death (p = 0.02), liver allograft loss (p = 0.02) and renal allograft loss (p = 0.045). Multivariable modeling showed class II DSA (preformed or de novo) to be an independent predictor of patient death (HR = 2.2; p = 0.043) and liver allograft loss (HR = 2.2; p = 0.044). These data warrant reconsideration of the approach to DSA in SLKT.

Apolipoprotein L1 gene (APOL1) nephropathy variants in African American deceased kidney donors were associated with shorter renal allograft survival in a prior single-center report. APOL1 G1 and G2 variants were genotyped in newly accrued DNA samples from African American deceased donors of kidneys recovered and/or transplanted in Alabama and North Carolina. APOL1 genotypes and allograft outcomes in subsequent transplants from 55 U.S. centers were linked, adjusting for age, sex and race/ethnicity of recipients, HLA match, cold ischemia time, panel reactive antibody levels, and donor type. For 221 transplantations from kidneys recovered in Alabama, there was a statistical trend toward shorter allograft survival in recipients of two-APOL1-nephropathy-variant kidneys (hazard ratio [HR] 2.71; p = 0.06). For all 675 kidneys transplanted from donors at both centers, APOL1 genotype (HR 2.26; p = 0.001) and African American recipient race/ethnicity (HR 1.60; p = 0.03) were associated with allograft failure. Kidneys from African American deceased donors with two APOL1 nephropathy variants reproducibly associate with higher risk for allograft failure after transplantation. These findings warrant consideration of rapidly genotyping deceased African American kidney donors for APOL1 risk variants at organ recovery and incorporation of results into allocation and informed-consent processes.

Objective The first US Food and Drug Administration–approved clinical trial to treat amyotrophic lateral sclerosis (ALS) with neural stem cell–based therapy is in progress. The goal of the current study was to identify and assess the survival of human spinal cord–derived neural stem cells (HSSCs) transplanted into the spinal cord in patients with ALS. Methods Spinal cords transplanted with HSSCs were examined from six autopsy cases. Homogenized tissues were interrogated for the presence of donor versus recipient DNA using real-time PCR methods (qPCR). Fluorescence in situ hybridization (FISH) was performed using DNA probes for XY chromosomes to identify male donor HSSCs in one female case, and immunohistochemistry (IHC) was used to characterize the identified donor cells. Results Genomic DNA from donor HSSCs was identified in all cases, comprising 0.67–5.4% of total tissue DNA in patients surviving 196 to 921 days after transplantation. In the one female patient a “nest” of cells identified on H&E staining were XY-positive by FISH, confirming donor origin. A subset of XY-positive cells labeled for the neuronal marker NeuN and stem cell marker SOX2. Interpretation This is the first study to identify human neural stem cells transplanted into a human spinal cord. Transplanted HSSCs survived up to 2.5 years posttransplant. Some cells differentiated into neurons, while others maintained their stem cell phenotype. This work is a proof of concept of the survival and differentiation of human stems cell transplanted into the spinal cord of ALS patients.

Background & Aims: A substantial proportion of pediatric liver transplant recipients develop subclinical chronic allograft injury. We studied whether there are distinct patterns of injury based on histopathologic features and identified associated immunologic profiles. Methods: We conducted a cross-sectional study of 157 stable, long-term pediatric recipients of transplanted livers (70 boys; > 6 years old at time of transplantation; mean, 8.9 ± 3.46 years after liver transplantation) who underwent liver biopsy analysis from August 13, 2012, through May 1, 2014. Participants had received livers from a living or deceased donor and had consistently normal results from liver tests. Liver biopsy specimens were scored by a central pathologist; an unsupervised hierarchical cluster analysis of histologic features was used to sort biopsy samples into 3 clusters. We conducted transcriptional and cytometric analyses of liver tissue samples and performed a systems biology analysis that incorporated clinical, serologic, histologic, and transcriptional data. Results: The mean level of alanine aminotransferase in participants was 27.6 ± 14.57 U/L, and the mean level of γ-glutamyl transferase was 17.4 ± 7.93 U/L. Cluster 1 was characterized by interface activity (n = 34), cluster 2 was characterized by periportal or perivenular fibrosis without interface activity (n = 45), and cluster 3 had neither feature (n = 78). We identified a module of genes whose expression correlated with levels of alanine aminotransferase, class II donor-specific antibody, portal inflammation, interface activity, perivenular inflammation, portal and perivenular fibrosis, and cluster assignment. The module was enriched in genes that regulate T-cell–mediated rejection (TCMR) of liver and other transplanted organs. Functional pathway analysis showed overrepresentation of TCMR gene sets for cluster 1 but not clusters 2 or 3. Conclusion: In an analysis of biopsies from an apparently homogeneous group of stable, long-term pediatric liver transplant recipients with consistently normal liver test results, we found evidence of chronic graft injury (inflammation and/or fibrosis). Biopsy samples with interface activity had a gene expression pattern associated with TCMR.

Background. Liver allografts protect renal allografts from the same donor from some, but not all, preformed donor specific alloantibodies (DSA). However, the precise mechanisms of protection and the potential for more subtle alterations/injuries within the grafts resulting from DSA interactions require further study. Methods. We reevaluated allograft biopsies from simultaneous liver-kidney transplant recipients who had both allografts biopsied within 60 d of one another and within 30 d of DSA being positive in serum (positive: mean florescence intensity ≥5000). Routine histology, C4d staining, and specialized immunohistochemistry for Kupffer cells (KCs; CD163) and a C4d receptor immunoglobulin-like transcript-4 were carried out in 4 patients with 6 paired biopsies. Results. Overt antibody-mediated rejection was found in 3 of 4 renal and liver allografts. One patient had biopsy-confirmed renal and liver allograft antibody-mediated rejection despite serum clearance of DSA. All biopsies showed KC hypertrophy (minimal: 1; mild: 2; moderate: 1; severe: 2) and cytoplasmic C4d KC staining was easily detected in 2 biopsies from 2 patients; minimal and negative in 2 biopsies each. Implications of which are discussed. Control 1-y protocol liver allograft biopsies from DSA- recipients showed neither KC hypertrophy nor KC C4d staining (n = 6). Conclusions. Partial renal allograft protection by a liver allograft from the same donor may be partially mediated by phagocytosis/elimination of antibody and complement split products by KCs, as shown decades ago in controlled sensitized experimental animal experiments.

Solid phase multiplex-bead arrays for the detection and characterization of HLA antibodies provide increased sensitivity and specificity compared to conventional lymphocyte-based assays. Assay variability due to inconsistencies in commercial kits and differences in standard operating procedures (SOP) hamper comparison of results between laboratories. The Clinical Trials in Organ Transplantation Antibody Core Laboratories investigated sources of assay variation and determined if reproducibility improved through utilization of SOP, common reagents and normalization algorithms. Ten commercial kits from two manufacturers were assessed in each of seven laboratories using 20 HLA reference sera. Implementation of a standardized (vs. a nonstandardized) operating procedure greatly reduced MFI variation from 62% to 25%. Although laboratory agreements exceeded 90% (R2), small systematic differences were observed suggesting center specific factors still contribute to variation. MFI varied according to manufacturer, kit, bead type and lot. ROC analyses showed excellent consistency in antibody assignments between manufacturers (AUC > 0.9) and suggested optimal cutoffs from 1000 to 1500 MFI. Global normalization further reduced MFI variation to levels near 20%. Standardization and normalization of solid phase HLA antibody tests will enable comparison of data across laboratories for clinical trials and diagnostic testing.