Qiuheng Zhang

Phosphorylated S6 Ribosomal Protein: A Novel Biomarker of Antibody‐Mediated Rejection in Heart Allografts

We tested the hypothesis that phosphorylation of S6 ribosomal protein (S6RP), a downstream target of the PI3K/Akt/mTOR pathway, is a biomarker of antibody‐mediated rejection (AMR) in heart allografts. Primary cultures of human aortic and microvascular endothelial cells (EC) were treated with anti‐HLA class I and class II antibodies (Ab) and cell lysates were studied for phosphorylation of S6 ribosmal protein at Serine235/236 (p‐S6RP). Treatment of cultured EC with anti‐class I and class II Ab stimulated S6RP phosphorylation. Immunohistochemical techniques were used to detect the level of p‐S6RP in endomyocardial biopsies (n = 131) from 46 heart transplant recipients and the results were correlated with histopathological diagnosis of rejection, C4d staining, production of posttransplant anti‐HLA Ab and clinical outcome. Increased phosphorylation of S6RP in endomyocardial biopsies was significantly associated with the diagnosis of AMR (p < 0.0001). No significant association between acute cellular rejection (ACR) and p‐S6RP was observed. C4d staining was positively associated with both AMR and p‐S6RP. Posttransplant anti‐HLA class II Ab production was also significantly associated with a positive p‐S6RP status in cardiac biopsies. These results indicate that p‐S6RP is a useful biomarker for the diagnosis of AMR.

HLA and MICA: targets of antibody-mediated rejection in heart transplantation.

Background. The goal of this study was to determine whether antidonor antibodies directed against human leukocyte antigen (HLA) or endothelial cells (ECs) expressed antigens, including major histocompatibility complex class I chain-related antigens A (MICA) are associated with the diagnosis of antibody-mediated rejection (AMR) in heart transplant recipients. Methods. We studied posttransplant antidonor HLA antibodies in 168 heart allograft recipients transplanted from October 2001 to December 2005. Among them, there were 37 AMR+ patients and 131 age- and sex- matched AMR− controls. Sera were collected at the time of protocol biopsies and tested for the presence of HLA antibodies. Seventy-two of the 168 patients were genotyped for donor and recipient MICA alleles and were tested for the presence of anti-MICA antibodies. Thirty-one patients who never developed antibodies to HLA or MICA were further tested for anti-EC antibodies. Results and Conclusions. Of 37 AMR+ patients, 22 (60%) developed donor-specific antibodies (DSA) to HLA compared with 6 of 131(4%) AMR− patients (P<0.0001). Of the remaining 15 AMR+ patients, 5 had anti-HLA antibodies that were not donor specific and 10 did not show any HLA antibodies. In the subgroup of 72 patients, all 19 AMR+ patients had clearly demonstrable antibodies reactive with donor HLA, MICA or with nondonor-derived ECs, with 30% of them showed antibodies directed to non-HLA antigens. The incidence of transplant coronary artery disease was significantly higher in patients who had DSA to HLA and MICA compared with patients without DSA.

Non-MHC antigenic targets of the humoral immune response in transplantation.

There is a growing body of data supporting a role for non-HLA antibodies in acute and chronic rejection of solid organ transplants. While many of these non-HLA antigens remain poorly defined, the principal antigenic targets are expressed on cells of the allograft including endothelium and epithelium. These non-HLA antigens are classified as either alloantigens, such as the major histocompatibility complex class I chain-related gene A (MICA) or MICB, or tissue-specific autoantigens such as vimentin, cardiac myosin (CM), collagen V (Col V), agrin, and angiotensin II receptor type I (AT1). Herein we provide an overview of the non-MHC antigenic targets that have been implicated in graft rejection and discuss the interplay between alloimmunity and autoreactivity in graft rejection.

The importance of non-HLA antibodies in transplantation

The development of post-transplantation antibodies against non-HLA autoantigens is associated with rejection and decreased long-term graft survival. Although our knowledge of non-HLA antibodies is incomplete, compelling experimental and clinical findings demonstrate that antibodies directed against autoantigens such as angiotensin type 1 receptor, perlecan and collagen, contribute to the process of antibody-mediated acute and chronic rejection. The mechanisms that underlie the production of autoantibodies in the setting of organ transplantation is an important area of ongoing investigation. Ischaemia–reperfusion injury, surgical trauma and/or alloimmune responses can result in the release of organ-derived autoantigens (such as soluble antigens, extracellular vesicles or apoptotic bodies) that are presented to B cells in the context of the transplant recipients antigen presenting cells and stimulate autoantibody production. Type 17 T helper cells orchestrate autoantibody production by supporting the proliferation and maturation of autoreactive B cells within ectopic tertiary lymphoid tissue. Conversely, autoantibody-mediated graft damage can trigger alloimmunity and the development of donor-specific HLA antibodies that can act in synergy to promote allograft rejection. Identification of the immunologic phenotypes of transplant recipients at risk of non-HLA antibody-mediated rejection, and the development of targeted therapies to treat such rejection, are sorely needed to improve both graft and patient survival.

Impact of three Illumina library construction methods on GC bias and HLA genotype calling.

Next-generation sequencing (NGS) is increasingly recognized for its ability to overcome allele ambiguity and deliver high-resolution typing in the HLA system. Using this technology, non-uniform read distribution can impede the reliability of variant detection, which renders high-confidence genotype calling particularly difficult to achieve in the polymorphic HLA complex. Recently, library construction has been implicated as the dominant factor in instigating coverage bias. To study the impact of this phenomenon on HLA genotyping, we performed long-range PCR on 12 samples to amplify HLA-A, -B, -C, -DRB1, and -DQB1, and compared the relative contribution of three Illumina library construction methods (TruSeq Nano, Nextera, Nextera XT) in generating downstream bias. Here, we show high GC% to be a good predictor of low sequencing depth. Compared to standard TruSeq Nano, GC bias was more prominent in transposase-based protocols, particularly Nextera XT, likely through a combination of transposase insertion bias being coupled with a high number of PCR enrichment cycles. Importantly, our findings demonstrate non-uniform read depth can have a direct and negative impact on the robustness of HLA genotyping, which has clinical implications for users when choosing a library construction strategy that aims to balance cost and throughput with data quality.

Acute Rejection Associated with Donor-Specific Anti-MICA Antibody in a Highly Sensitized Pediatric Renal Transplant Recipient

Narayan S, Tsai EW, Zhang Q, Wallace WD, Reed EF, Ettenger RB. Acute rejection associated with donor‐specific anti‐MICA antibody in a highly sensitized pediatric renal transplant recipient.
Pediatr Transplantation 2011: 15:E1–E7.

Phosphorylated S6 kinase and S6 ribosomal protein are diagnostic markers of antibody-mediated rejection in heart allografts.

BACKGROUND Anti-MHC Class I alloantibodies have been implicated in the processes of acute and chronic rejection. These antibodies (Ab) bind to endothelial cells (EC) and transduce signals leading to the activation of cell survival and proliferation pathways, including Src, FAK and mTOR, as well as downstream targets ERK, S6 kinase (S6K) and S6 ribosomal protein (S6RP). We tested the hypothesis that phosphorylation of S6K, S6RP and ERK in capillary endothelium may serve as an adjunct diagnostic tool for antibody-mediated rejection (AMR) in heart allografts. METHODS Diagnosis of AMR was based on histology or immunoperoxidase staining of paraffin-embedded tissue, consistent with 2013 ISHLT criteria. Diagnosis of acute cellular rejection (ACR) was based on ISHLT criteria. Endomyocardial biopsies from 67 heart transplant recipients diagnosed with acute rejection [33 with pAMR, 18 with ACR (15 with Grade 1R, 3 with Grade ≥2R), 16 with pAMR and ACR (13 with 1R and 3 with ≥2R)] and 40 age- and gender-matched recipients without rejection were tested for the presence of phosphorylated forms of ERK, S6RP and S6K by immunohistochemistry. RESULTS Immunostaining of endomyocardial biopsies with evidence of pAMR showed a significant increase in expression of p-S6K and p-S6RP in capillary EC compared with controls. A weaker association was observed between pAMR and p-ERK. CONCLUSIONS Biopsies diagnosed with pAMR often showed phosphorylation of S6K and S6RP, indicating that staining for p-S6K and p-S6RP is useful for the diagnosis of AMR. Our findings support a role for antibody-mediated HLA signaling in the process of graft injury.

Application of High-Throughput Next-Generation Sequencing for HLA Typing on Buccal Extracted DNA: Results from over 10,000 Donor Recruitment Samples

Background Unambiguous HLA typing is important in hematopoietic stem cell transplantation (HSCT), HLA disease association studies, and solid organ transplantation. However, current molecular typing methods only interrogate the antigen recognition site (ARS) of HLA genes, resulting in many cis-trans ambiguities that require additional typing methods to resolve. Here we report high-resolution HLA typing of 10,063 National Marrow Donor Program (NMDP) registry donors using long-range PCR by next generation sequencing (NGS) approach on buccal swab DNA. Methods Multiplex long-range PCR primers amplified the full-length of HLA class I genes (A, B, C) from promotor to 3’ UTR. Class II genes (DRB1, DQB1) were amplified from exon 2 through part of exon 4. PCR amplicons were pooled and sheared using Covaris fragmentation. Library preparation was performed using the Illumina TruSeq Nano kit on the Beckman FX automated platform. Each sample was tagged with a unique barcode, followed by 2×250 bp paired-end sequencing on the Illumina MiSeq. HLA typing was assigned using Omixon Twin software that combines two independent computational algorithms to ensure high confidence in allele calling. Consensus sequence and typing results were reported in Histoimmunogenetics Markup Language (HML) format. All homozygous alleles were confirmed by Luminex SSO typing and exon novelties were confirmed by Sanger sequencing. Results Using this automated workflow, over 10,063 NMDP registry donors were successfully typed under high-resolution by NGS. Despite known challenges of nucleic acid degradation and low DNA concentration commonly associated with buccal-based specimens, 97.8% of samples were successfully amplified using long-range PCR. Among these, 98.2% were successfully reported by NGS, with an accuracy rate of 99.84% in an independent blind Quality Control audit performed by the NDMP. In this study, NGS-HLA typing identified 23 null alleles (0.023%), 92 rare alleles (0.091%) and 42 exon novelties (0.042%). Conclusion Long-range, unambiguous HLA genotyping is achievable on clinical buccal swab-extracted DNA. Importantly, full-length gene sequencing and the ability to curate full sequence data will permit future interrogation of the impact of introns, expanded exons, and other gene regulatory sequences on clinical outcomes in transplantation.

Accelerated rejection, thrombosis, and graft failure with angiotensin II type 1 receptor antibodies

BackgroundAngiotensin II type 1 receptor antibodies (AT1R-Abs) have been implicated in renal transplant rejection and failure; however, the mechanism of allograft damage, patterns of clinical presentation, and response to desensitization of AT1R-Abs have not been clearly established.Case diagnosis/treatmentWe present the case of a 7-year-old boy with preformed AT1R-Abs who developed accelerated vascular and cellular rejection and renal allograft thrombosis despite desensitization and treatment with angiotensin receptor blockade. Although an association between AT1R-Abs and microvascular occlusion has been previously described, we are the first to describe an association between AT1R-Abs and renal artery thrombosis, leading to devastating early allograft failure.ConclusionsThis case highlights the risk of allograft thrombosis associated with AT1R-Abs and illustrates that previous treatments utilized for AT1R-Abs may not always be effective. Further studies are needed to better characterize the mechanisms of AT1R-Ab pathogenesis and to establish safe levels of AT1R-Abs both pre- and post-transplantation. Given the outcome of this patient and the evidence of pro-coagulatory effects of AT1R-Abs, we suggest that the presence of AT1R-Ab may be a risk factor for thrombosis. The role of treatment with anti-coagulation and novel immunomodulatory agents such as tocilizumab and bortezomib require further investigation.

Array-based methods for diagnosis and prevention of transplant rejection

DNA microarray is a microhybridization-based assay that is used to simultaneously study the expression of thousands of genes, thus providing a global view of gene expression in a tissue sample. This powerful technique has been adopted by many biomedical disciplines and will likely have a profound impact on the diagnosis, treatment and prognosis of human diseases. This review article presents an overview of the application of microarray technology to the field of solid-organ transplantation.