Matthew J. Vitalone

A Peripheral Blood Diagnostic Test for Acute Rejection in Renal Transplantation

Monitoring of renal graft status through peripheral blood (PB) rather than invasive biopsy is important as it will lessen the risk of infection and other stresses, while reducing the costs of rejection diagnosis. Blood gene biomarker panels were discovered by microarrays at a single center and subsequently validated and cross‐validated by QPCR in the NIH SNSO1 randomized study from 12 US pediatric transplant programs. A total of 367 unique human PB samples, each paired with a graft biopsy for centralized, blinded phenotype classification, were analyzed (115 acute rejection (AR), 180 stable and 72 other causes of graft injury). Of the differentially expressed genes by microarray, Q‐PCR analysis of a five gene‐set (DUSP1, PBEF1, PSEN1, MAPK9 and NKTR) classified AR with high accuracy. A logistic regression model was built on independent training‐set (n = 47) and validated on independent test‐set (n = 198)samples, discriminating AR from STA with 91% sensitivity and 94% specificity and AR from all other non‐AR phenotypes with 91% sensitivity and 90% specificity. The 5‐gene set can diagnose AR potentially avoiding the need for invasive renal biopsy. These data support the conduct of a prospective study to validate the clinical predictive utility of this diagnostic tool.

Biomarkers in solid organ transplantation: establishing personalized transplantation medicine

Technological advances in molecular and in silico research have enabled significant progress towards personalized transplantation medicine. It is now possible to conduct comprehensive biomarker development studies of transplant organ pathologies, correlating genomic, transcriptomic and proteomic information from donor and recipient with clinical and histological phenotypes. Translation of these advances to the clinical setting will allow assessment of an individual patients risk of allograft damage or accommodation. Transplantation biomarkers are needed for active monitoring of immunosuppression, to reduce patient morbidity, and to improve long-term allograft function and life expectancy. Here, we highlight recent pre- and post-transplantation biomarkers of acute and chronic allograft damage or adaptation, focusing on peripheral blood-based methodologies for non-invasive application. We then critically discuss current findings with respect to their future application in routine clinical transplantation medicine. Complement-system-associated SNPs present potential biomarkers that may be used to indicate the baseline risk for allograft damage prior to transplantation. The detection of antibodies against novel, non-HLA, MICA antigens, and the expression of cytokine genes and proteins and cytotoxicity-related genes have been correlated with allograft damage and are potential post-transplantation biomarkers indicating allograft damage at the molecular level, although these do not have clinical relevance yet. Several multi-gene expression-based biomarker panels have been identified that accurately predicted graft accommodation in liver transplant recipients and may be developed into a predictive biomarker assay.

Progressive histological damage in renal allografts is associated with expression of innate and adaptive immunity genes.

The degree of progressive chronic histological damage is associated with long-term renal allograft survival. In order to identify promising molecular targets for timely intervention, we examined renal allograft protocol and indication biopsies from 120 low-risk pediatric and adolescent recipients by whole-genome microarray expression profiling. In data-driven analysis, we found a highly regulated pattern of adaptive and innate immune gene expression that correlated with established or ongoing histological chronic injury, and also with development of future chronic histological damage, even in histologically pristine kidneys. Hence, histologically unrecognized immunological injury at a molecular level sets the stage for the development of chronic tissue injury, while the same molecular response is accentuated during established and worsening chronic allograft damage. Irrespective of the hypothesized immune or nonimmune trigger for chronic allograft injury, a highly orchestrated regulation of innate and adaptive immune responses was found in the graft at the molecular level. This occurred months before histologic lesions appear, and quantitatively below the diagnostic threshold of classic T-cell or antibody-mediated rejection. Thus, measurement of specific immune gene expression in protocol biopsies may be warranted to predict the development of subsequent chronic injury in histologically quiescent grafts and as a means to titrate immunosuppressive therapy.

A rapid noninvasive assay for the detection of renal transplant injury.

Background The copy number of donor-derived cell-free DNA (dd-cfDNA) in blood correlates with acute rejection (AR) in heart transplantation. We analyzed urinary dd-cfDNA as a surrogate marker of kidney transplant injury. Methods Sixty-three biopsy-matched urine samples (41 stable and 22 allograft injury) were analyzed from female recipients of male donors for chromosome Y (donor)–specific dd-cfDNA. All biopsies were semiquantitatively scored by a single pathologist. Standard statistical measures of correlation and significance were used. Results There was baseline scatter for urinary dd-cfDNA/&mgr;g urine creatinine across different patients, even at the time of stable graft (STA) function (undetected to 12.26 copies). The mean urinary dd-cfDNA in AR (20.5±13.9) was significantly greater compared with STA (2.4±3.3; P<0.0001) or those with chronic allograft injury (CAI; 2.4±2.4; P=0.001) but no different from BK virus nephropathy (BKVN; 20.3±15.7; P=0.98). In AR and BKVN, the intrapatient drift was highly significant versus STA or CAI patients (10.3±7.4 in AR; 12.3±8.4 in BKVN vs. −0.5±3.5 in STA and 2.3±2.6 in CAI; P<0.05). Urinary dd-cfDNA correlated with protein/creatinine ratio (r=0.48; P<0.014) and calculated glomerular filtration rate (r=−0.52; P<0.007) but was most sensitive for acute allograft injury (area under the curve=0.80; P<0.0006; 95% confidence interval, 0.67–0.93). Conclusion Urinary dd-cfDNA after renal transplantation has patient specific thresholds, reflecting the apoptotic injury load of the donor organ. Serial monitoring of urinary dd-cfDNA can be a surrogate sensitive biomarker of acute injury in the donor organ but lacks the specificity to distinguish between AR and BKVN injury.

The dual role of epithelial-to-mesenchymal transition in chronic allograft injury in pediatric renal transplantation.

Background. Tubulointerstitial damage (TID) is a key feature of chronic allograft injury (CAI) and loss. One proposed mechanism attributing to TID is epithelial-to-mesenchymal transition (EMT); however, it has recently been shown to be unrelated to early TID in adult renal allografts. This has yet to be studied in late TID or in pediatric renal transplantation; both questions were investigated. Methods. By using 83 unique pediatric renal transplant recipients, 126 protocol, serial, posttransplant renal biopsies were examined by centralized, blinded Banff grading for CAI and transcriptional profiling (AffyU133+2.0) at 3 (n=20), 6 (n=45), 12 (n=19), and 24 months (n=42). Two hundred forty-three EMT-associated genes, identified from the literature, were interrogated for their differential expression in biopsies with and without CAI, using standard bioinformatic algorithms. Results. Early (3–6 months) enrichment of EMT (P≤0.05) related gene expression was noted, correlating with inflammation in the graft (total i scores), with upregulation of hepatocyte growth factor at 24 months, indicating a time-dependent mechanism of action. We observed a strong correlation of EMT-related gene expression with early interstitial fibrosis (r<0.45) for size-mismatched allograft recipients. Throughout 24 months posttransplant, EMT signaling and epithelial-mesenchymal-epithelial cycling were associated with progressive CAI injury, with the greatest risk factors being ischemia, immune burden, and the calcineurin inhibitor toxicity score. Conclusions. EMT has a role in the evolution of CAI in pediatric transplantation. We postulate that EMT dysregulation plays a dual role in fibrosis/injury repair and healing. The evolution of this chronic injury response stems from size- mismatched transplant ischemia, calcineurin inhibitor nephrotoxicity, and inflammatory response within the allograft.

Differential immunogenicity and clinical relevance of kidney compartment specific antigens after renal transplantation.

To evaluate the pathogenic role of non-HLA antibodies after organ transplantation, 81 unique serum samples from renal transplant patients were analyzed by protein array technology on integrative genomics approach (Li, L.; et al. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (11), 4148-53; Higgins, J. P.; et al. Mol. Biol. Cell 2004, 15 (2), 649-56), validated by ELISA, and the results correlated with clinical relevance with time post-transplantation or post-transplant graft function. There was a significant association of de novo non-HLA antibodies with time post-transplantation (n = 1,785) and decline in graft function over the subsequent year (n = 105). There was an enrichment of immunogenic antigens in the renal cortex (p = 0.01) with post-transplant time, and for glomerular specific targets (p = 0.02) with decline in graft function. Two targets with very strong correlation in each category (AGT and SPDYA) were validated by customized ELISA assays in independent patient sera and their localization confirmed by immunohistochemistry. In conclusion, defined profiles of these non-HLA antibodies to renal cortical proteins develop with increasing length of engraftment, and may reflect the increasing recognition of altered localization or exposure of renal tubular and interstitial proteins, affected by advancing chronic nonimmune graft injury. The panel of non-HLA antibodies to glomerular targets is most interesting, as these corresponding antigenic targets may be important pathways in functional graft injury and could provide novel targets for drug design.

Transcriptional Perturbations in Graft Rejection.

Background Understanding the regulatory interplay of relevant microRNAs (miRNAs) and messenger RNAs (mRNAs) in the rejecting allograft will result in a better understanding of the molecular pathophysiology of alloimmune injury. Methods One hundred sixty-seven allograft biopsies, with (n = 47) and without (n = 120) central histology for Banff scored acute rejection (AR), were transcriptionally profiled for mRNA and miRNA by whole genome microarrays and multiplexed microfluidic quantitative polymerase chain reaction, respectively. A customized database was curated (GO-Elite) and used to identify AR-specific dysregulated mRNAs and the role of perturbations of their relevant miRNAs targets during AR. Results The AR-specific changes in 1035 specific mRNAs were mirrored by AR-specific perturbations in 9 relevant miRNAs as predicted by Go-Elite and were regulated specifically by p53 and forkhead box P3. Infiltrating lymphocytes and the renal tubules drove the miRNA tissue pertubations in rejection, involving message degradation and transcriptional/translational activation. The expression of many of these miRNAs significantly associated with the intensity of the Banff-scored interstitial inflammation and tubulitis. Conclusions There is a highly regulated interplay between specific mRNA/miRNAs in allograft rejection that drive both immune-mediated injury and tissue repair during AR.

Transcriptional Profiling of Belatacept and Calcineurin Inhibitor Therapy in Renal Allograft Recipients

Calcineurin inhibitor (CNI) use may lead to allograft injury and compromised renal function. Gene expression profiles of 12‐month kidney biopsies from a Phase 3 study of belatacept and a CNI comparator, cyclosporine (CsA), were compared with expression profiles of a set of historical, demographically matched, preimplantation control biopsies. Gene set enrichment analysis was used to test each set of differentially expressed genes (DEGs) for the enrichment of an in vitro‐derived CNI toxicity (CNIT) gene set and published gene sets associated with chronic allograft injury (CAI), immune modulation and tissue remodeling. The unique set of genes differentially expressed in CNI biopsies compared with preimplantation controls was enriched for genes associated with fibrosis, early tubulointerstitial damage and in vitro CNIT. The DEGs from belatacept biopsies were not enriched for the CNIT genes but, instead, exhibited enrichment for gene sets associated with immune response and tissue remodeling. A combined analysis of DEGs across both treatment groups identified select solute transporter and cellular differentiation genes whose expression at 12 months correlated with renal function at 36 months. These results provide mechanistic insights into the reduced CAI and higher renal function observed in belatacept‐ versus CsA‐treated patients.

Liver microRNA Profile of Induced Allograft Tolerance.

Background Although the liver is less immunogenic than other solid organs, most liver transplant recipients receive lifelong immunosuppression. In both experimental models and clinical transplantation, total lymphoid irradiation (TLI) has been shown to induce allograft tolerance. Our goal was to identify the microRNAs (miRNAs) expressed in tolerant liver allograft recipients in an experimental model of TLI-induced tolerance. Methods To identify the miRNAs associated with TLI-induced tolerance, we examined syngeneic recipients (Lewis→Lewis) and allogeneic recipients (Dark Agouti→Lewis) of orthotropic liver transplants that received posttransplant TLI, allogeneic recipients that were not treated posttransplantation and experienced acute rejection, and native Dark Agouti livers. Quantitative-polymerase chain reaction miRNA array cards were used to profile liver grafts. Results We identified 12 miRNAs that were specifically and significantly increased during acute rejection. In early tolerance, 33 miRNAs were altered compared with syngeneic livers, with 80% of the miRNAs increased. In established tolerance, 42 miRNAs were altered. In addition, miR-142-5p and miR-181a demonstrated increased expression in tolerant livers (both early and established tolerance) as compared with syngeneic livers. A principal component analysis of all miRNAs assayed demonstrated a profile in established tolerance that was closely related to that seen in syngeneic livers. Conclusions The miRNA profile of established tolerant allografts is very similar to syngeneic grafts, suggesting tolerance may be a return to an immunological state of quiescence.

Mass cytometry reveals a distinct immunoprofile of operational tolerance in pediatric liver transplantation

Long‐term IS in transplant patients has significant morbidity, poorer quality of life, and substantial economic costs. TOL, defined as graft acceptance without functional impairment in the absence of IS, has been achieved in some pediatric LT recipients. Using mass cytometry, peripheral blood immunotyping was performed to characterize differences between tolerant patients and patients who are stable on single‐agent IS. Single‐cell mass cytometry was performed using blood samples from a single‐center pediatric LT population of operationally tolerant patients to comprehensively characterize the immune cell populations in the tolerant state compared with patients on chronic low‐dose IS. Specific T‐cell populations of interest were confirmed by flow cytometry. This high‐dimensional phenotypic analysis revealed distinct immunoprofiles between transplant populations as well as a CD4+ TOT (CD4+CD5+CD25+CD38−/loCD45RA) that correlates with tolerance in pediatric LT recipients. In TOL patients, the TOT was significantly increased as compared to patients stable on low levels of IS. This TOT cell was confirmed by flow cytometry and is distinct from classic Treg cells. These results demonstrate the power of mass cytometry to discover significant immune cell signatures that have diagnostic potential.