Kidney Transplant Rejection Research Articles

The Most Promising Biomarkers of Allogeneic Kidney Transplant Rejection.

Clinical transplantology is a constantly evolving field of medicine. Kidney transplantation has become standard clinical practice, and it has a significant impact on reducing mortality and improving the quality of life of patients. Allogenic transplantation induces an immune response, which may lead to the rejection of the transplanted organ. The gold standard for evaluating rejection of the transplanted kidney by the recipient's organism is a biopsy of this organ. However, due to the high invasiveness of this procedure, alternative diagnostic methods are being sought. Therefore, the biomarkers may play an essential predictive role in transplant rejection. A review of the most promising biomarkers for early diagnosis and prognosis prediction of allogenic kidney transplant rejection summarizes novel data on neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), C-X-C motif chemokine 10 (CXCL-10), cystatin C (CysC), osteopontin (OPN), and clusterin (CLU) and analyses the dynamics of changes of the biomarkers mentioned above in kidney diseases and the mechanism of rejection of the transplanted kidney.

Effect of regulatory T cells on short-term graft outcome in kidney transplant recipients, a prospective observational, single-center study

BackgroundRegulatory T cells (Tregs) are important for maintaining immune homeostasis, limiting kidney transplant rejection, and promoting transplant tolerance. Tregs are characterized as CD4 + CD25+ T cells and the transcription factor Foxp3; they constitute 5–10% of all peripheral CD4+ T cells in healthy humans. The reduction in Treg cells after transplantation may be a predictive factor in graft rejection. Therefore, in this study, we investigated the association between Tregs and short-term graft outcomes in renal transplant recipients. MethodsThis prospective observational study included 50 patients of both sexes, aged between 18 and 60 years, with end-stage renal disease (ESRD), and who underwent kidney transplantation at the Nizams Institute of Medical Sciences. Flow cytometry was used to measure the percentage of Tregs (CD4 + CD25+) pre-transplant (baseline) and monthly post-transplant in 50 transplant recipients and 20 healthy controls (HC) over six months. The correlation between Treg percentages and graft outcomes was analyzed. ResultsThe percentage of Tregs (Treg%) was significantly lower in ESRD patients before transplantation (baseline) than in the HCs [median 8.5% vs. 14.25%; p < 0.01]. After transplantation, Treg% decreased significantly within a week after grafting compared to the baseline pre-transplant level [median 5.13% vs. 8.5%; p < 0.01]. Moreover, Treg% was lower in the older (> 40 years) than that in the younger age group at one week after transplantation (p > 0.001). Treg% (CD4 + CD25+) was significantly lower in patients with deceased donor renal transplantation (DDRT) than in live renal transplantation (LRT) within the first week of transplantation [median 2.16% vs. 4.6%; p < 0.05] and at the time of graft rejection [median 3.2% vs. 9.2%; p < 0.001]. Induction therapy with anti-IL-2 receptor monoclonal antibody (IL-2RB) lowered Tregs to a greater extent than anti-thymoglobulin (ATG) therapy and no induction therapy [median 3.22% vs. 5.47%, 8.4%, p < 0.05]. In contrast, an increased Treg% was observed in transplant recipients with normal kidney graft function within six months after transplantation compared with the pre-transplant baseline level [median 11.54% vs. 8.5; p < 0.001]. ROC analysis of graft rejection resulted in an area under the curve (AUC) of 0.8 with a p-value <0.05. ConclusionWithin the first week after transplantation, the percentage of pre-transplant Tregs was significantly decreased compared to that in age- and sex-matched HC. The percentage of Tregs was also lower in patients with advanced age, DDRT, and those who received IL-2RB induction therapy. Patients who experienced graft rejection had lower Treg% compared to their pre-transplant levels. In contrast, patients with normal graft function at six months showed increased Treg%. Together, these results suggest that Treg% measurements are correlated with clinical outcomes.

Misidentification of preformed anti-HLA-DP antibodies leads to antibody-mediated kidney transplant rejection: a case report

BackgroundPatients who are HLA-sensitized are at high risk for early antibody-mediated rejection (AMR) and worse outcomes. Therefore, it is crucial to detect the presence of donor-specific antibodies (DSAs) using pretransplant antibody identification and crossmatch assays. An error in antibody identification can lead to disastrous clinical outcomes. We present a case of acute AMR associated with preformed HLA-DPα and HLA-DPβ DSAs that were not identified before transplantation.Case presentationA 27-year-old woman received a second kidney transplant from a deceased donor. Her pretransplant panel-reactive antibody level was 94%. The complement-dependent cytotoxicity crossmatch was negative for T and B cells at the time of transplantation. She experienced early acute AMR proven by a kidney biopsy. Single antigen bead testing of the patient’s serum at the time of rejection as well as the pre-second transplant serum revealed strong antibodies against the DPA1*01:03 and DPB1*02:01 alleles in the second donor. These antibodies were not identified by phenotypic bead assay during the patient’s time on the waiting list. The patient was treated with plasmapheresis and anti-thymocyte globulin. However, she experienced abdominal pain on day 37 post-transplantation. Surgical exploration revealed a laceration on the transplanted kidney, which was then repaired. Subsequently, infected hematoma was suspected and the transplanted kidney was removed.ConclusionThe present case highlights the clinical significance of preformed HLA-DPα and HLA-DPβ DSAs. Accuracy in determination of HLA antibodies before transplantattion is critical for transplant outcome. HLA-DP typing and single antigen bead testing are recommended for a precise antibody interpretation, especially in highly sensitized patients. Careful interpretation of antibody testing results is essential for the success of organ transplantation.

A New Generation of Cell Therapies Employing Regulatory T Cells (Treg) to Induce Immune Tolerance in Pediatric Transplantation.

Kidney transplantation is the most common solid organ transplant and the preferred treatment for pediatric patients with end-stage renal disease, but it is still not a definitive solution due to immune graft rejection. Regulatory T cells (Treg) and their control over effector T cells is a crucial and intrinsic tolerance mechanism in limiting excessive immune responses. In the case of transplants, Treg are important for the survival of the transplanted organ, and their dysregulation could increase the risk of rejection in transplanted children. Chronic immunosuppression to prevent rejection, for which Treg are especially sensitive, have a detrimental effect on Treg counts, decreasing the Treg/T-effector balance. Cell therapy with Treg cells is a promising approach to restore this imbalance, promoting tolerance and thus increasing graft survival. However, the strategies used to date that employ peripheral blood as a Treg source have shown limited efficacy. Moreover, it is not possible to use this approach in pediatric patients due to the limited volume of blood that can be extracted from children. Here, we outline our innovative strategy that employs the thymus removed during pediatric cardiac surgeries as a source of therapeutic Treg that could make this therapy accessible to transplanted children. The advantageous properties and the massive amount of Treg cells obtained from pediatric thymic tissue (thyTreg) opens a new possibility for Treg therapies to prevent rejection in pediatric kidney transplants. We are recruiting patients in a clinical trial to prevent rejection in heart-transplanted children through the infusion of autologous thyTreg cells (NCT04924491). If its efficacy is confirmed, thyTreg therapy may establish a new paradigm in preventing organ rejection in pediatric transplants, and their allogeneic use would extend its application to other solid organ transplantation.

A Decentralized Kidney Transplant Biopsy Classifier for Transplant Rejection Developed Using Genes of the Banff-Human Organ Transplant Panel.

IntroductionA decentralized and multi-platform-compatible molecular diagnostic tool for kidney transplant biopsies could improve the dissemination and exploitation of this technology, increasing its clinical impact. As a first step towards this molecular diagnostic tool, we developed and validated a classifier using the genes of the Banff-Human Organ Transplant (B-HOT) panel extracted from a historical Molecular Microscope® Diagnostic system microarray dataset. Furthermore, we evaluated the discriminative power of the B-HOT panel in a clinical scenario.Materials and MethodsGene expression data from 1,181 kidney transplant biopsies were used as training data for three random forest models to predict kidney transplant biopsy Banff categories, including non-rejection (NR), antibody-mediated rejection (ABMR), and T-cell-mediated rejection (TCMR). Performance was evaluated using nested cross-validation. The three models used different sets of input features: the first model (B-HOT Model) was trained on only the genes included in the B-HOT panel, the second model (Feature Selection Model) was based on sequential forward feature selection from all available genes, and the third model (B-HOT+ Model) was based on the combination of the two models, i.e. B-HOT panel genes plus highly predictive genes from the sequential forward feature selection. After performance assessment on cross-validation, the best-performing model was validated on an external independent dataset based on a different microarray version.ResultsThe best performances were achieved by the B-HOT+ Model, a multilabel random forest model trained on B-HOT panel genes with the addition of the 6 most predictive genes of the Feature Selection Model (ST7, KLRC4-KLRK1, TRBC1, TRBV6-5, TRBV19, and ZFX), with a mean accuracy of 92.1% during cross-validation. On the validation set, the same model achieved Area Under the ROC Curve (AUC) of 0.965 and 0.982 for NR and ABMR respectively.DiscussionThis kidney transplant biopsy classifier is one step closer to the development of a decentralized kidney transplant biopsy classifier that is effective on data derived from different gene expression platforms. The B-HOT panel proved to be a reliable highly-predictive panel for kidney transplant rejection classification. Furthermore, we propose to include the aforementioned 6 genes in the B-HOT panel for further optimization of this commercially available panel.

MO994: Impact of Hla-A, B DR and DQ on renal transplant outcomes in the Tacrolimus/Mmf Era: An Artificial Intelligence Approach

Abstract BACKGROUND AND AIMS One of the biological barriers that can increase risk of acute rejection in kidney transplantation is HLA mismatch. Several immunotherapy protocols have been implemented to reduce the effect of HLA-mismatch and improve outcomes. The aim of our study is to assess the effect of HLA mismatches on acute rejection rates in the Tacrolimus era. METHOD All kidney transplant patients registered in UNOS database between 1 January 2005 and 1 December 2019 were retrospectively reviewed. Inclusion criteria: deceased donor transplants that were discharged on Tacrolimus/Mycophenolate Mofetil. Exclusion criteria: multiple organ transplants, previous kidney transplants, recipient age &amp;lt;18 years old, living donor transplants, patients not discharged on Tacrolimus/Mycophenolate Mofetil immunotherapy, missing HLA mismatch or ABO incompatible transplant. We used double-selection lasso (‘least absolute shrinkage and selection operator) logistic regression model to assess for the effect of HLA-A, B, DR and DQ on acute rejection rates at one-year post-transplant. We used square-root lassos for the variables of interest. Variables of interest were HLA-A, B, DR and DQ mismatch. Variables Lasso selected from were: recipient characteristics (age, sex, BMI, ethnicity, diabetes, recipient/donor CMV status, time on dialysis), donor characteristics (KDPI score) and transplant characteristics (type of induction therapy, steroid therapy at time of discharge, cold ischemia time, delayed graft function, PRA). For survival analysis, we fit a penalized Cox model to the entire set of data and obtained the estimated set of alphas. The variables included in the penalized cox model donor, recipient and transplant factors in addition to the HLA mismatches. Then we determined the set of alpha for evaluation using optimized cross-validated grid-search. Moreover, we visualized how the coefficients changed for varying α using ridge regression model. RESULTS About 73 910 were included in our study. Worse acute rejection rates at one-year post-transplant were noted with incremental increase in HLA-DQ (Two HLA-DQ: OR = 1.56, P = 0.005, 95%CI: 1.04–1.27; One HLA DQ: OR = 1.15, P = 0.01, 95%CI: 1.03–1.24), HLA-DR mismatches (two HLA-DR: HR = 1.46, P &amp;lt; 0.01, 95%CI: 1.31–1.62; One HLA-DR: OR = 1.29, P &amp;lt; 0.01, 95%CI: 1.16–1.43), and Two HLA-A (OR = 1.12, P = 0.049, 95%CI: 1.0006–1.26). However, the effect of different HLA-mismatches on death-censored graft survival were minimal (HLA-DQ coefficient: 0.003, HLA-DR coefficient: 0.05, HLA-A coefficient: 0.008 and HLA-B coefficient: 0.007). Mean follow-up time was 3.79 years. Figure 1 shows the visualisation of coefficients among different alphas for the ridge penalised cox regression model. CONCLUSION HLA-DQ, DR and A mismatches still play a vital role in the occurrence of acute rejection in the Tacrolimus/MMF era. However, the effect of HLA mismatches on the graft survival is minimal.

FC 117: Clinical Validation of Automated Urinary Chemokine Assays for Non-Invasive Detection of Kidney Transplant Rejection: A Large Prospective Cohort Study

Abstract BACKGROUND AND AIMS The urinary chemokines CXCL9 and CXCL10 have been extensively studied as biomarkers for kidney transplant rejection. However, final validation in a sufficiently large, real-world prospective cohort is needed to determine the relation to rejection subtypes and clinical confounders, the context of use and the added benefit for clinical practice. METHOD In this single-centre prospective cohort study, we analysed 1559 biopsy-paired urine samples at both indication and protocol time points from 622 kidney transplants performed between April 2013 and July 2019. We quantified urinary CXCL9 and CXCL10 using automated immunoassays and normalized the values to urinary creatinine. In addition to evaluating diagnostic performance for Banff-grade rejection, we also considered rejection as assessed by the molecular microscope. RESULTS The urinary chemokines strongly associated with the inflammation severity in the biopsy, when considering classical semi-quantitative histological scores, but also when considering molecular rejection scores. The diagnostic performance of urinary chemokines alone was moderate for Banff acute rejection, and increased when considering treated rejection or molecular rejection instead (Figure 1). Taking into account confounding factors rather than excluding them, an integrated model of urinary chemokines with eGFR, donor-specific antibodies, time post-transplant and polyoma viremia had a high diagnostic value for acute rejection (N = 150) [ROCAUC 82.6%, 95% confidence interval (95% CI) 78.9–86.2] and added value on top of clinical standard-of-care. The integrated score would help to safely avoid 59 protocol biopsies per 100 patients when the predicted rejection risk is less than 10%. CONCLUSION The integration of urinary chemokines and clinical markers allows for personalized noninvasive monitoring of rejection probability and enables to importantly and safely reduce the number of unnecessary biopsies. We expect this study to pave the way for the final implementation of urinary chemokine measurements as noninvasive markers of rejection in routine clinical follow-up after kidney transplantation.

Paired Immunoglobulin-Like Receptors Impact The Differentiation of Reparative Macrophages Following Allogeneic Challenge

Abstract Monocytes differentiate into CD206+CD301+ reparative macrophages (MΦs) that orchestrate tissue repair in response to damage signals and cytokines. Yet, transplanting allogeneic materials produces a novel situation where innate allorecognition of mismatched MHCI may disrupt normal MΦ responses. B6 monocyte paired immunoglobulin-like receptor (PIR)-A3 recognizes BALB/c H2-Dd to generate pro-inflammatory dendritic cells that promote kidney and heart transplant rejection. Conversely, monocyte PIR-B binding to self MHCI negatively regulates PIR-A signals. How PIRs shape MΦ differentiation after Tx was not understood, so we tested the hypothesis that recognition of self-versus-allogeneic MHCI by PIRs divergently modulates MΦ differentiation. Monocyte-derived MΦ differentiation in wild type (WT), Rag2−/−γc−/−, Pira−/− and Pirb−/− B6 mice was assessed by flow cytometry 3 days after exposure to 20×106 radiation-damaged BALB/c allogeneic (allo) or B6 syngeneic (syn) splenocytes administered intraperitoneally. Syn materials predominantly stimulated monocyte differentiation into phenotypically reparative MΦs. Allo cells instead transformed monocytes exclusively into pro-inflammatory Ly6chiCD86hi MΦs. Rag2−/−γc−/− mice behaved similarly. IL-33 released from injured cells metabolically reprograms infiltrating monocytes and MΦs to support their differentiation into CD206+CD301+MΦs. Interestingly, Pirb−/− mice display greatly reduced C206+CD301+MΦs, which correlated with their decreased expression of the IL-33 receptor. Our data provide important insights into how innate allorecognition by PIR-A disrupts the typical generation of reparative MΦs in response to injured self that is supported by PIR-B. Supported by 5T32AI074490-14

Histologic and Molecular Patterns in Responders and Non-responders With Chronic-Active Antibody-Mediated Rejection in Kidney Transplants.

IntroductionThere is no proven therapy for chronic-active antibody-mediated rejection (caABMR), the major cause of late kidney allograft failure. Histological and molecular patterns associated with possible therapy responsiveness are not known.MethodsBased on rigorous selection criteria this single center, retrospective study identified 16 out of 1027 consecutive kidney transplant biopsies taken between 2008 and 2016 with pure, unquestionable caABMR, without other pathologic features. The change in estimated GFR pre- and post-biopsy/treatment were utilized to differentiate subjects into responders and non-responders. Gene sets reflecting active immune processes of caABMR were defined a priori, including endothelial, inflammatory, cellular, interferon gamma (IFNg) and calcineurin inhibitor (CNI) related-genes based on the literature. Transcript measurements were performed in RNA extracted from stored, formalin-fixed, paraffin-embedded (FFPE) samples using NanoString™ technology. Histology and gene expression patterns of responders and non-responders were compared.ResultsA reductionist approach applying very tight criteria to identify caABMR and treatment response excluded the vast majority of clinical ABMR cases. Only 16 out of 139 cases with a written diagnosis of chronic rejection fulfilled the caABMR criteria. Histological associations with therapy response included a lower peritubular capillaritis score (p = 0.028) along with less glomerulitis. In contrast, no single gene discriminated responders from non-responders. Activated genes associated with NK cells and endothelial cells suggested lack of treatment response.ConclusionIn caABMR active microvascular injury, in particular peritubular capillaritis, differentiates treatment responders from non-responders. Transcriptome changes in NK cell and endothelial cell associated genes may further help to identify treatment response. Future prospective studies will be needed which include more subjects, who receive standardized treatment protocols to identify biomarkers for treatment response.Clinical Trial Registration[ClinicalTrials.gov], identifier [NCT03430414].

Tocilizumab and Active Antibody-Mediated Rejection in Kidney Transplantation: A Literature Review.

IntroductionChronic kidney disease (CKD) is a major public-health problem that increases the risk of end-stage kidney disease (ESKD), cardiovascular diseases, and other complications. Kidney transplantation is a renal-replacement therapy that offers better survival compared to dialysis. Antibody-mediated rejection (ABMR) is a significant complication following kidney transplantation: it contributes to both short- and long-term injury. The standard-of-care (SOC) therapy combines plasmapheresis and Intravenous Immunoglobulins (IVIg) with or without steroids, with or without rituximab: however, despite this combined treatment, ABMR remains the main cause of graft loss. IL-6 is a key cytokine: it regulates inflammation, and the development, maturation, and activation of T cells, B cells, and plasma cells. Tocilizumab (TCZ) is the main humanized monoclonal aimed at IL-6R and appears to be a safe and possible strategy to manage ABMR in sensitized recipients. We conducted a literature review to assess the place of the anti-IL-6R monoclonal antibody TCZ within ABMR protocols.Materials and MethodsWe systematically reviewed the PubMed literature and reviewed six studies that included 117 patients and collected data on the utilization of TCZ to treat ABMR.ResultsMost studies report a significant reduction in levels of Donor Specific Antibodies (DSAs) and reduced inflammation and microvascular lesions (as found in biopsies). Stabilization of the renal function was observed. Adverse events were light to moderate, and mortality was not linked with TCZ treatment. The main side effect noted was infection, but infections did not occur more frequently in patients receiving TCZ as compared to those receiving SOC therapy.ConclusionTCZ may be an alternative to SOC for ABMR kidney-transplant patients, either as a first-line treatment or after failure of SOC. Further randomized and controlled studies are needed to support these results.

The Inhibition of CD40/CD154 Costimulatory Signaling in the Prevention of Renal Transplant Rejection in Nonhuman Primates: A Systematic Review and Meta Analysis.

The prevention of allograft transplant rejection by inhibition of the CD40/CD40L costimulatory pathway has been described in several species. We searched pubmed for studies reporting the prevention of kidney transplant rejection in nonhuman primates utilizing either anti CD40 or anti CD40L (CD154) treatment. Inclusion of data required treatment with anti CD40 or anti CD154 as monotherapy treatment arms, full text available, studies conducted in nonhuman primate species, the transplant was renal transplantation, sufficient duration of treatment to assess long term rejection, and the reporting of individual graft survival or survival duration. Eleven publications were included in the study. Rejection free survival was calculated using the Kaplan-Meier (KM) life test methods to estimate the survival functions. The 95% CI for the medians was also calculated. A log-rank test was used to test the equality of the survival curves between control and treatment arms (CD40 and CD154). The hazard ratio for CD154 compared to CD40 and 95% CI was calculated using a Cox proportional-hazards model including treatment as the covariate to assess the magnitude of the treatment effect. Both anti CD40 and anti CD154 treatments prevented acute and long term graft rejection. The median (95% CI) rejection free survival was 131 days (84,169 days) in the anti CD40 treated animals and 352 days (173,710 days) in the anti CD154 treated animals. Median survival in the untreated animals was 6 days. The inhibition of transplant rejection was more durable in the anti CD154 group compared to the anti CD40 group after cessation of treatment. The median (95% CI) rejection free survival after cessation of treatment was 60 days (21,80 days) in the anti CD40 treated animals and 230 days (84,552 days) in the anti CD154 treated animals.

The role of the NGAL biomarker in the assessment of early and prediction of late graft function in kidney transplantation from a living related donor

The aim. To study the role of NGAL in assessing the recovery of early graft function (GF) and in predicting GF in the first year after kidney transplantation (KT) from a living-related donor (LRD). Materials and methods. For this aim, a total of 60 kidney recipients who underwent KT from LRDs were examined. To study the early GF, NGAL concentrations in urine samples were measured on days 1, 2, 3, 7 after KT. To study the late GF, the glomerular filtration rate (GFR) was calculated at 3, 6, 12 months after KT. To study the prognosis of GF during the first year after KT, the relationship between u-NGAL and GFR levels at 3, 6, 12 months after KT was analyzed. The incidence of acute kidney transplant rejection (AKTR), delay of graft function (DGF), primary non-function was recorded. Results. DGF was observed in 5 (8.33 %) recipients with statistically significantly increased mean NGAL levels (P &lt; 0.05) on days 1, 2, 3, 7 as compared to those in normal GF. AKTR was detected in 10 (16.67 %) recipients with statistically significantly increased mean NGAL levels (P &lt; 0.05) on days 1, 2, 3, 7 compared to those in normal GF. Primary allograft non-function was diagnosed in 8 (13.33 %) recipients with statistically significantly increased mean NGAL levels (P &lt; 0.05) on days 1, 2, 3, 7 compared to those in normal GF. Assessing the relationship between NGAL levels on days 1, 2, 3, 7 and GFR at 3, 6, 12 months after transplantation, it was found that GFR was higher in recipients with NGAL levels on days 1, 2, 3, 7 after transplantation within the reference values (&lt;131.7 ng/ml) than that in recipients with NGAL levels on days 1, 2, 3, 7 after transplantation above 131.7 ng/ml. Conclusions. Evaluation of NGAL after KT informs about the restoration of renal function (rapid decrease in NGAL) or the development of complications (DGF, primary allograft non-function, AKTR) with slow decrease or increase in NGAL levels. Thus, u-NGAL is an early, non-invasive and accurate predictor of the need for dialysis in the first week after KT and the restoration of GF within 12 months.

SLAMF8 Participates in Acute Renal Transplant Rejection via TLR4 Pathway on Pro-Inflammatory Macrophages.

BackgroundAcute rejection (AR) in kidney transplantation is an established risk factor that reduces the survival rate of allografts. Despite standard immunosuppression, molecules with regulatory control in the immune pathway of AR can be used as important targets for therapeutic operations to prevent rejection.MethodsWe downloaded the microarray data of 15 AR patients and 37 non-acute rejection (NAR) patients from Gene Expression Omnibus (GEO). Gene network was constructed, and genes were classified into different modules using weighted gene co-expression network analysis (WGCNA). Kyoto Encyclopedia of Genes and Genomes (KEGG) and Cytoscape were applied for the hub genes in the most related module to AR. Different cell types were explored by xCell online database and single-cell RNA sequencing. We also validated the SLAMF8 and TLR4 levels in Raw264.7 and human kidney tissues of TCMR.ResultsA total of 1,561 differentially expressed genes were filtered. WGCNA was constructed, and genes were classified into 12 modules. Among them, the green module was most closely associated with AR. These genes were significantly enriched in 20 pathway terms, such as cytokine–cytokine receptor interaction, chemokine signaling pathway, and other important regulatory processes. Intersection with GS > 0.4, MM > 0.9, the top 10 MCC values and DEGs in the green module, and six hub genes (DOCK2, NCKAP1L, IL2RG, SLAMF8, CD180, and PTPRE) were identified. Their expression levels were all confirmed to be significantly elevated in AR patients in GEO, Nephroseq, and quantitative real-time PCR (qRT-PCR). Single-cell RNA sequencing showed that AR patient had a higher percentage of native T, CD1C+_B DC, NKT, NK, and monocytes in peripheral blood mononuclear cells (PBMCs). Xcell enrichment scores of 20 cell types were significantly different (p<0.01), mostly immune cells, such as B cells, CD4+ Tem, CD8+ T cells, CD8+ Tcm, macrophages, M1, and monocytes. GSEA suggests that highly expressed six hub genes are correlated with allograft rejection, interferon γ response, interferon α response, and inflammatory response. In addition, SLAMF8 is highly expressed in human kidney tissues of TCMR and in M1 phenotype macrophages of Raw264.7 cell line WGCNA accompanied by high expression of TLR4.ConclusionThis study demonstrates six hub genes and functionally enriched pathways related to AR. SLAMF8 is involved in the M1 macrophages via TLR4, which contributed to AR process.

Can Heart Allografts Protect Simultaneous Kidney Allografts from Antibody Injury?

<h3>Purpose</h3> Pre-formed donor specific antibodies (DSA) are associated with antibody mediated rejection (AMR) and graft loss in kidney transplants. However, simultaneous liver/kidney transplants show a lower risk for DSA mediated AMR in kidney allograft, likely due to absorbance of DSAs by the liver allograft. The goal of this study is to determine if heart allografts can protect subsequent kidney grafts from antibody mediated injury. <h3>Methods</h3> 93 patients were transplanted with combined heart and kidney allografts between 2016 and 2020 at our center. The median time (interquartile range [IQR]) of post-transplant follow up is 883 days (459-1322). Treatment for patients transplanted across DSA included intravenous immune globulin, obinutuzumab, and eculizumab. <h3>Results</h3> 12 of 93 recipients had pre-formed DSA, with 3 recipients having strong pre-formed DSA (MFI>10,000); 5 recipients having moderate DSA (MFI >5,000); 4 recipients having weak DSA (MFI>2,500). Survival was not different between recipients with preformed DSA and recipients without preformed DSA (p=0.436) (Fig. 1). Of these 12 patients with pre-formed DSA, 4 patients had positive flow crossmatches and were treated with intravenous immune globulin. Notably, one of these 4 patients also had a positive CDC crossmatch with pre-formed C1q-binding DSA and was treated with obinutuzumab and eculizumab in addition to intravenous immune globulin. All 4 patients had stable renal and cardiac function at the end of the follow up. DSA in 3 of these 4 patients including the patient with positive CDC crossmatch, became negative during the follow up (Fig. 2). <h3>Conclusion</h3> Levels of DSA can decrease post heart/kidney transplant, suggesting the heart/kidney transplant may modulate antibody response. Our study indicates that the kidney can be transplanted across strong DSA in heart/kidney combined transplant with acceptable outcomes.

Can Gene Expression Analysis in Zero-Time Biopsies Predict Kidney Transplant Rejection?

Zero-time biopsies are taken to determine the quality of the donor organ at the time of transplantation. Histological analyses alone have so far not been able to identify parameters that allow the prediction of subsequent rejection episodes or graft survival. This study investigated whether gene expression analyses of zero-time biopsies might support this prediction. Using a well-characterized cohort of 26 zero-time biopsies from renal transplant patients that include 4 living donor (LD) and 22 deceased donor (DD) biopsies that later developed no rejection (Ctrl, n = 7), delayed graft function (DGF, n = 4), cellular (T-cell mediated rejection; TCMR, n = 8), or antibody-mediated rejection (ABMR, n = 7), we analyzed gene expression profiles for different types of subsequent renal transplant complication. To this end, RNA was isolated from formalin-fixed, paraffin-embedded (FFPE) sections and gene expression profiles were quantified. Results were correlated with transplant data and B-cell, and plasma cell infiltration was assessed by immunofluorescence microscopy. Both principal component analysis and clustering analysis of gene expression data revealed marked separation between LDs and DDs. Differential expression analysis identified 185 significant differentially expressed genes (adjusted p < 0.05). The expression of 68% of these genes significantly correlated with cold ischemia time (CIT). Furthermore, immunoglobulins were differentially expressed in zero-time biopsies from transplants later developing rejection (TCMR + ABMR) compared to non-rejected (Ctrl + DGF) transplants. In addition, immunoglobulin expression did not correlate with CIT but was increased in transplants with previous acute renal failure (ARF). In conclusion, gene expression profiles in zero-time biopsies derived from LDs are markedly different from those of DDs. Pre-transplant ARF increased immunoglobulin expression, which might be involved in triggering later rejection events. However, these findings must be confirmed in larger cohorts and the role of early immunoglobulin upregulation in zero-biopsies needs further clarification.

US payer budget impact of a microarray assay with machine learning to evaluate kidney transplant rejection in for-cause biopsies

Aim This study evaluates the economic impact to US commercial payers of MMDx-Kidney used in conjunction with histologic evaluation of for-cause kidney transplant biopsies. Materials and methods An Excel-based model was developed to assess the cost impact of histology plus MMDx-Kidney versus histology alone for the evaluation of potential rejection in kidney transplant patients who receive a for-cause biopsy. Different model time periods were assessed, ranging from 1 to 5 years post-biopsy. A targeted literature review was used to identify parameter estimates, validated by two external clinicians with expertise in managing kidney transplant rejection. A sensitivity analysis was conducted to evaluate the relative impact of key clinical and cost parameters. In particular, the model identified the magnitude of MMDx-Kidney’s impact on graft failure from rejection that would be required for MMDx-Kidney to be cost-neutral. Results By more accurately characterizing rejection, MMDx-Kidney is estimated to increase antirejection treatment costs by $1,126 per test. Nevertheless, a break-even analysis shows that the costs of MMDx-Kidney and anti-rejection medication, as well as the costs associated with an increase in the number of patients with functioning transplants, may be offset by reductions in costs associated with graft failure (i.e. costs of hospitalizations, dialysis, and repeat transplants) over 5 years, assuming MMDx-Kidney reduces annual graft failure from rejection by at least 5%. For the base case, with a 25% relative reduction in annual rate of graft failures from rejection, MMDx-Kidney increases overall costs incurred in the first year of the model but starts generating savings by the second year of the model. Conclusions Compared with histologic evaluation of for-cause kidney transplant biopsies alone, the use of MMDx-Kidney in conjunction with histologic evaluation improves the diagnoses of graft dysfunction and may have the potential to generate overall savings from reductions in rejection-related graft failure.

Use of a donor-derived cell-free DNA assay to monitor treatment response in pediatric renal transplant recipients with allograft rejection.

Detection of donor-derived cell-free DNA (dd-cfDNA) reliably identifies allograft rejection in pediatric and adult kidney transplant (KT) recipients. Here, we evaluate the utility of dd-cfDNA for monitoring response to treatment among pediatric renal transplant recipients suffering graft rejection. 58 pediatric transplant recipients were enrolled between April 2018 and March 2020 and underwent initial dd-cfDNA testing to monitor for rejection. Allograft biopsy was performed for dd-cfDNA scores >1.0%. Patients with histologically proven rejection formed the study cohort and underwent appropriate treatment. Results of dd-cfDNA, serum creatinine (SCr), biopsy findings, and treatment outcomes were evaluated. Standard statistical analyses were applied. Nineteen of 58 (31%) patients had dd-cfDNA score >1.0%, of which 18 (94.7%) had biopsy-proven rejection. Median dd-cfDNA value was 1.90% (interquartile range 1.43%-3.23%), and biopsy results showed 11 patients (61.1%) with antibody-mediated rejection (AMR), 2 patients (11.1%) with T-cell mediated rejection (TCMR), and 5 patients (27.7%) with mixed AMR/TCMR. SCr at time of biopsy was 1.28±1.09mg/dl. Following treatment, dd-cfDNA scores decreased for all types of rejection but still remained >1.0% in both AMR (1.50% [0.90%-3.10%]) and mixed (1.40% [0.95%-4.15%]) groups. Repeat dd-cfDNA values were <1.0% for patients with TCMR (0.20%-0.28%). SCr showed minimal change from pre-treatment levels regardless of rejection subtype. Patients with TCMR may be reliably followed by dd-cfDNA; however, it remains unclear whether persistently elevated dd-cfDNA levels in AMR is a reflection of ongoing subclinical rejection or an inherent limitation of the assay's utility.

How Should Acute T-cell Mediated Rejection of Kidney Transplants Be Treated: Importance of Follow-up Biopsy.

Background.Limited published data exist to guide patient monitoring after the treatment of T-cell mediated rejection (TCMR) of kidney allografts.Methods.We reviewed the kidney function and histological outcomes after treatment of 163 first episodes of biopsy-proven TCMR between January 1‚ 2015‚ and July 31‚ 2020.Results.Of the 146 patients treated with steroid pulse alone, complete histological response was seen in 83% of patients with borderline rejection, 82.5% with grade 1A, 67% with grade 1B, and 50% with grade IIA. Of the 17 patients treated with steroids plus antithymocyte globulin, the complete histological response rate was 100% with grade 1A, 75% with grade 1B, 100% with grade IIA, and 57% with grade IIB. Among the patients with complete response as assessed by kidney function, 14% only had a partial or no response histologically. Among patients with no kidney function response, 68% had a complete response histologically.Conclusion.We thus find that responses based on kidney function alone do not correlate well with histological responses. If further treatment had been based solely on changes in estimated glomerular filtration rate, a significant number of patients would have been subsequently undertreated or overtreated. These results support the use of protocol follow-up biopsies after the treatment of TCMR.

A Novel Method of CD31-Combined ABO Carbohydrate Antigen Microarray Predicts Acute Antibody-Mediated Rejection in ABO-Incompatible Kidney Transplantation.

Isohemagglutinin assays employing red blood cells (RBCs) are the most common assays used to measure antibody titer in ABO-incompatible kidney transplantation (ABOi KTx). However, ABO antigens expressed on RBCs are not identical to those of kidney and antibody titers do not always correlate with clinical outcome. We previously reported that CD31 was the main protein linked to ABO antigens on kidney endothelial cells (KECs), which was different from those on RBCs. We developed a new method to measure antibody titer using a microarray of recombinant CD31 (rCD31) linked to ABO antigens (CD31-ABO microarray). Mass spectrometry analysis suggested that rCD31 and native CD31 purified from human kidney had similar ABO glycan. To confirm clinical use of CD31-ABO microarray, a total of 252 plasma samples including volunteers, hemodialysis patients, and transplant recipients were examined. In transplant recipients, any initial IgG or IgM antibody intensity >30,000 against the donor blood type in the CD31-ABO microarray showed higher sensitivity, specificity, positive predictive value, and negative predictive value of AABMR, compared to isohemagglutinin assays. Use of a CD31-ABO microarray to determine antibody titer specifically against ABO antigens expressed on KECs will contribute to precisely predicting AABMR or preventing over immunosuppression following ABOi KTx.

The Pathological and Clinical Diversity of Acute Vascular Rejection in Kidney Transplantation.

Vascular rejection (VR) is characterized by arteritis, steroid resistance, and increased graft loss but is poorly described using modern diagnostics. We screened 3715 consecutive biopsies and retrospectively evaluated clinical and histological phenotypes of VR (n = 100) against rejection without arteritis (v0REJ, n = 540) and normal controls (n = 1108). Biopsy sample size affected the likelihood of arterial sampling, VR diagnosis, and final Banff v scores ( P < 0.001). Local v and cv scores were greatest in larger arteries (n = 258). VR comprised 15.6% of all rejection episodes, presented earlier (median 1.0 mo, interquartile range, 0.4-8 mo) with higher serum creatinine levels and inferior graft survival, versus v0REJ ( P < 0.001). Early VR (≤1 mo) was common (54%) and predicted by sensitization, delayed function, and prior corticosteroid use, with associated acute dysfunction and optimal therapeutic response, independent of Banff v score. Late VR followed under-immunosuppression in 71.4% (noncompliance 38.8%, iatrogenic 32.6%), and was associated with chronic interstitial fibrosis, incomplete renal functional recovery and persistent inflammation using sequential histopathology. The etiology was "pure" antibody-mediated VR (n = 21), mixed VR (n = 36), and "pure" T cell-mediated VR (n = 43). Isolated VR (n = 34, Banff i < 1 without tubulitis) comprised 24 T cell-mediated VR and 10 antibody-mediated VR, presenting with mild renal dysfunction, minimal Banff acute scores, and better graft survival compared with inflamed VR. Interstitial inflammation influenced acute renal dysfunction and early treatment response, whereas chronic tubulointerstitial damage determined long-term graft loss. VR is a heterogenous entity influenced by time-of-onset, pathophysiology, accompanying interstitial inflammation and fibrosis. Adequate histological sampling is essential for its accurate diagnostic classification and treatment.