Association of BKV viremia and nephropathy with adverse alloimmune outcomes in kidney transplant recipients.

BACKGROUND: Immunosuppression reduction for BK polyoma virus (BKV) must be balanced against risk of adverse alloimmune outcomes. We sought to characterize risk of alloimmune events after BKV within context of HLA-DR/DQ molecular mismatch (mMM) risk score. METHODS: This single-center study evaluated 460 kidney transplant patients on tacrolimus-mycophenolate-prednisone from 2010-2021. BKV status was classified at 6-months post-transplant as "BKV" or "no BKV" in landmark analysis. Primary outcome was T-cell mediated rejection (TCMR). Secondary outcomes included all-cause graft failure (ACGF), death-censored graft failure (DCGF), de novo donor specific antibody (dnDSA), and antibody-mediated rejection (ABMR). Predictors of outcomes were assessed in Cox proportional hazards models including BKV status and alloimmune risk defined by recipient age and molecular mismatch (RAMM) groups. RESULTS: At 6-months post-transplant, 72 patients had BKV and 388 had no BKV. TCMR occurred in 86 recipients, including 27.8% with BKV and 17% with no BKV (p = .05). TCMR risk was increased in recipients with BKV (HR 1.90, (95% CI 1.14, 3.17); p = .01) and high vs. low-risk RAMM group risk (HR 2.26 (95% CI 1.02, 4.98); p = .02) in multivariable analyses; but not HLA serological MM in sensitivity analysis. Recipients with BKV experienced increased dnDSA in univariable analysis, and there was no association with ABMR, DCGF, or ACGF. CONCLUSIONS: Recipients with BKV had increased risk of TCMR independent of induction immunosuppression and conventional alloimmune risk measures. Recipients with high-risk RAMM experienced increased TCMR risk. Future studies on optimizing immunosuppression for BKV should explore nuanced risk stratification and may consider novel measures of alloimmune risk.

Safety and efficacy of a reduced frequency viral monitoring strategy for Epstein-Barr virus, cytomegalovirus, and BK polyomavirus post-kidney transplant: A quality assurance initiative.

BACKGROUND: There is variability in recommended viral monitoring protocols after kidney transplant. In response to increased demand for laboratory testing during the COVID-19 pandemic, the Transplant Manitoba Adult Kidney Program updated its monitoring protocols for cytomegalovirus (CMV), Epstein-Barr virus (EBV), and BK polyomavirus (BKV) to a reduced frequency. METHODS: This single-center nested case-control study evaluated 252 adult kidney transplant recipients transplanted from 2015 to 2021, with the updated protocols effective on March 19th 2020. Cases included recipients transplanted after the protocol update who developed CMV, EBV, and BKV DNAemia and were matched to controls with DNAemia transplanted prior to the protocol update. The primary outcome was the difference in maximum DNA load titers between cases and matched controls. Secondary outcomes included time to initial DNAemia detection and DNAemia clearance. Safety outcomes of tissue-invasive viral disease were described. RESULTS: There were 216 recipients transplanted preupdate and 36 recipients postupdate. There was no difference between cases and controls in maximum or first DNA load titers for EBV, CMV, or BKV. Cases experienced earlier EBV DNAemia detection (26 (IQR 8, 32) vs. 434 (IQR 96, 1184) days, p = .005). Median follow-up was significantly longer for recipients transplanted preupdate (4.3 vs. 1.3 years, p < .0001). After adjusting for follow-up time, there was no difference in DNAemia clearance or tissue-invasive viral disease. CONCLUSION: Our findings suggest that reduced frequency viral monitoring protocols may be safe and cost-effective. This quality assurance initiative should be extended to detect longer-term and tissue-invasive disease outcomes.

Identification of Antibodies to DQß:DRα Interisotypic Heterodimers in Human Sera.

BACKGROUND: HLA class II antigens, DR, DQ, and DP, comprised an α and ß chains, which typically combine, within the same isotype, to form the major histocompatibility complex:peptide complex. Interisotypic pairing is not commonly observed. Although reports of DQß:DRα heterodimers exist, the pairing was reported to be unstable and, therefore, not studied to any extent. METHODS: DQß:DRα single antigens were produced through transfectant cell lines and used to identify and characterize positive reactive human sera by a multiplex bead-based assay. RESULTS: Stable DQß:DRα transfectants were constructed. Cell surface staining with class II-specific monoclonal antibodies revealed that some DQB1 alleles appear to be more efficient in expressing DQß:DRα heterodimers. Interestingly, alleles within the same serological group varied in their efficiency of forming dimers on the cell surface. For example, DQß0601:DRα had the highest transfection and cell membrane expression efficiency among 16 common DQB1 alleles tested. In contrast, DQß0603:DRα-positive transfectants demonstrated minimal surface expression. Assembly of DQß0601:DRα was not affected by the presence of a DQα chain. DQß0601:DRα and DQß0603:DRα single-antigen beads were used to screen human sera. Positive sera were identified that reacted to the unique epitopes of DQß0601:DRα protein on the cell surface of the transfectants. CONCLUSIONS: Our studies have demonstrated that unique DQß:DRα heterodimers can be formed and are stably expressed on the cell surface. Such antigenic combinations, presented on single-antigen beads, demonstrated that patient sera can react with such heterodimers. Investigations on the potential clinical roles of antibodies against such interisotypic heterodimers are now possible.

HLA class I peptide polymorphisms contribute to class II DQß0603:DQα0103 antibody specificity.

Antibodies reactive to human leukocyte antigens (HLA) represent a barrier for patients awaiting transplantation. Based on reactivity patterns in single-antigen bead (SAB) assays, various epitope matching algorithms have been proposed to improve transplant outcomes. However, some antibody reactivities cannot be explained by amino acid motifs, leading to uncertainty about their clinical relevance. Antibodies against the HLA class II molecule, DQß0603:DQα0103, present in some candidates, represent one such example. Here, we show that peptides derived from amino acids 119-148 of the HLA class I heavy chain are bound to DQß0603:DQα0103 proteins and contribute to antibody reactivity through an HLA-DM-dependent process. Moreover, antibody reactivity is impacted by the specific amino acid sequence presented. In summary, we demonstrate that polymorphic HLA class I peptides, bound to HLA class II proteins, can directly or indirectly be part of the antibody binding epitope. Our findings have potential important implications for the field of transplant immunology and for our understanding of adaptive immunity.

A rational approach to guide cost-effective de novo donor-specific antibody surveillance with tacrolimus immunosuppression.

De novo donor-specific antibody (dnDSA) after renal transplantation has been shown to correlate with antibody-mediated rejection and allograft loss. However, the lack of proven interventions and the time and cost associated with annual screening for dnDSA are difficult to justify for all recipients. We studied a well-characterized consecutive cohort (n = 949) with over 15 years of prospective dnDSA surveillance to identify risk factors that would help institute a resource-responsible surveillance strategy. Younger recipient age and HLA-DR/DQ molecular mismatch were independent predictors of dnDSA development. Combining both risk factors into recipient age molecular mismatch categories, we found that 52% of recipients could be categorized as low-risk for dnDSA development (median subclinical dnDSA-free survival at 5 and 10 years, 98% and 97%, respectively). After adjustment, multivariate correlates of dnDSA development included tacrolimus versus cyclosporin maintenance immunosuppression (hazard ratio [HR], 0.37; 95% CI, 0.2-0.6; P < .0001) and recipient age molecular mismatch category: intermediate versus low (HR, 2.48; 95% CI, 1.5-4.2; P = .0007), high versus intermediate (HR, 2.56; 95% CI, 1.6-4.2; P = .0002), and high versus low (HR, 6.36; 95% CI, 3.7-10.8; P < .00001). When combined, recipient age and HLA-DR/DQ molecular mismatch provide a novel data-driven approach to reduce testing by >50% while selecting those most likely to benefit from dnDSA surveillance.

Rationale for the IMAGINE study for chronic active antibody-mediated rejection (caAMR) in kidney transplantation.

Chronic active antibody-mediated rejection (caAMR) in kidney transplantation is a major cause of late graft loss and despite all efforts to date, there is no proven effective therapy. Indeed, the Transplant Society (TTS) consensus opinion called for a conservative approach optimizing baseline immunosuppression and supportive care focused on blood pressure, blood glucose, and lipid control. This review provides the rationale and early evidence in kidney transplant recipients with caAMR that supported the design of the IMAGINE study whose goal is to evaluate the potential impact of targeting the IL6/IL6R pathway.

Clazakizumab for the treatment of chronic active antibody-mediated rejection (AMR) in kidney transplant recipients: Phase 3 IMAGINE study rationale and design.

BACKGROUND: Chronic active antibody-mediated rejection (AMR) is a major cause of graft loss with no approved drugs for its treatment. Currently, off-label regimens are used, reflecting the high unmet need for effective therapies based on well-controlled trials. Clazakizumab is a high-affinity, humanized monoclonal antibody that binds interleukin-6 and decreases donor-specific antibody (DSA) production and inflammation. Phase 2 pilot studies of clazakizumab in kidney transplant recipients with chronic active AMR suggest modulation of DSA, stabilization of glomerular filtration rate (GFR), and a manageable safety profile. We report the design of the Phase 3 IMAGINE study (NCT03744910) to evaluate the safety and efficacy of clazakizumab for the treatment of chronic active AMR. METHODS: IMAGINE is a multicenter, double-blind trial of approximately 350 kidney transplant recipients with chronic active AMR (Banff chronic glomerulopathy [cg] >0 with concurrent positive human leukocyte antigen DSA) randomized 1:1 to receive clazakizumab or placebo (12.5 mg subcutaneous once every 4 weeks). The event-driven trial design will follow patients until 221 occurrences of all-cause graft loss are observed, defined as return to dialysis, graft nephrectomy, re-transplantation, estimated GFR (eGFR) <15 mL/min/1.73m2, or death from any cause. A surrogate for graft loss (eGFR slope) will be assessed at 1 year based on prior modeling validation. Secondary endpoints will include measures of pharmacokinetics/pharmacodynamics. Recruitment is ongoing across North America, Europe, Asia, and Australia. DISCUSSION: IMAGINE represents the first Phase 3 clinical trial investigating the safety and efficacy of clazakizumab in kidney transplant recipients with chronic active AMR, and the largest placebo-controlled trial in this patient population. This trial includes prognostic biomarker enrichment and uniquely utilizes the eGFR slope at 1 year as a surrogate endpoint for graft loss, which may accelerate the approval of a novel therapy for patients at risk of graft loss. The findings of this study will be fundamental in helping to address the unmet need for novel therapies for chronic active AMR. TRIAL REGISTRATION: ClinicalTrials.gov NCT03744910 . Registered on November 19, 2018.

Premortem anticoagulation timing and dose in donation after circulatory death: multicentre study of associations with graft function.

BACKGROUND: In controlled donation after circulatory determination of death (DCD), it is common to administer premortem heparin to potential donors. This practice remains controversial because there is limited evidence for it and there is the possibility of inducing hemorrhage. To our knowledge, no previous studies have assessed the effects of heparin timing and dose on graft function. METHODS: We performed a multicentre cohort study of consecutive DCD donors and the recipients of their organs. Anticoagulation administration was considered early if given near the time of withdrawal of life-sustaining measures and late if delayed until the onset of donor hypoxemia (oxygen saturation < 70%) or hypotension (systolic blood pressure < 60 mm Hg or mean blood pressure < 50 mm Hg). The anticoagulation dose was considered high if it was 300 units/kg or greater. RESULTS: Donor anticoagulation data were available for 301 kidney, 75 liver and 46 lung recipients. Heparin was administered in 92% of cases and was most commonly withheld in donors with cerebrovascular causes of death (p = 0.01). Administration was late in 59% and the dose was low in 27%. Among kidney recipients, there were no significant differences in need for dialysis, glomerular filtration rate over the first year after transplantation or graft survival on the basis of whether or not the donor received heparin, the timing of heparin administration or the dose of heparin. Among liver recipients, alkaline phosphatase concentrations over the first year were significantly higher among recipients who received organs from donors to whom lower doses of heparin had been administered. CONCLUSION: Premortem heparin is widely used in DCD cases, but there is variability in timing and dose, which was not associated with kidney outcomes in this study. Donor anticoagulation may have a greater impact in preventing biliary complications following liver transplantation.

The negative impact of T cell-mediated rejection on renal allograft survival in the modern era.

The prevalence and long-term impact of T cell-mediated rejection (TCMR) is poorly defined in the modern era of tacrolimus/mycophenolate-based maintenance therapy. This observational study evaluated 775 kidney transplant recipients with serial histology and correlated TCMR events with the risk of graft loss. After a ~30% incidence of a first Banff Borderline or greater TCMR detected on for-cause (17%) or surveillance (13%) biopsies, persistent (37.4%) or subsequent (26.3%) TCMR occurred in 64% of recipients on follow-up biopsies. Alloimmune risk categories based on the HLA-DR/DQ single molecule eplet molecular mismatch correlated with the number of TCMR events (p = .002) and Banff TCMR grade (p = .007). Both a first and second TCMR event correlated with death-censored and all-cause graft loss when adjusted for baseline covariates and other significant time-dependent covariates such as DGF and ABMR. Therefore, a substantial portion of kidney transplant recipients, especially those with intermediate and high HLA-DR/DQ molecular mismatch scores, remain under-immunosuppressed, which in turn identifies the need for novel agents that can more effectively prevent or treat TCMR.

Role of HLA molecular mismatch in clinical practice.

To date, traditional pre-transplant risk factors have failed to provide accurate risk stratification in transplantation. As a result, the practice of precision medicine remains elusive, resulting in a one-size-fits-all therapeutic approach for most patients. However, recent advancements in the understanding of HLA molecules at the molecular level have revitalized interest in HLA mismatch assessment. This review discusses HLA molecular mismatch as a potential prognostic and predictive biomarker available at the time of transplantation and answers some of the common questions and critiques of this approach. We highlight the retrospective data that supports single molecule risk categorization and explore the next steps required to evaluate its potential in clinical practice.

A noninferiority design for a delayed calcineurin inhibitor substitution trial in kidney transplantation.

Improving long-term kidney transplant outcomes requires novel treatment strategies, including delayed calcineurin inhibitor (CNI) substitution, tested using informative trial designs. An alternative approach to the usual superiority-based trial is a noninferiority trial design that tests whether an investigational agent is not unacceptably worse than standard of care. An informative noninferiority design, with biopsy-proven acute rejection (BPAR) as the endpoint, requires determination of a prespecified, evidence-based noninferiority margin for BPAR. No such information is available for delayed CNI substitution in kidney transplantation. Herein we analyzed data from recent kidney transplant trials of CNI withdrawal and "real world" CNI- based standard of care, containing subjects with well-documented evidence of immune quiescence at 6 months posttransplant-ideal candidates for delayed CNI substitution. Our analysis indicates an evidence-based noninferiority margin of 13.8% for the United States Food and Drug Administration's composite definition of BPAR between 6 and 24 months posttransplant. Sample size estimation determined that ~225 randomized subjects would be required to evaluate noninferiority for this primary clinical efficacy endpoint, and superiority for a renal function safety endpoint. Our findings provide the basis for future delayed CNI substitution noninferiority trials, thereby increasing the likelihood they will provide clinically implementable results and achieve regulatory approval.

More precise donor-recipient matching: the role of eplet matching.

PURPOSE OF REVIEW: A precise understanding of the alloimmune risk faced by individual recipients at the time of transplant is an unmet need in transplantation. Although conventional HLA donor-recipient mismatch is too imprecise to fulfil this need, HLA molecular mismatch increases the precision in alloimmune risk assessment by quantifying the difference between donors and recipients at the molecular level. RECENT FINDINGS: Within each conventional HLA mismatch the number, type, and position of mismatched amino acids create a wide range of HLA molecular mismatches between recipients and donors. Multiple different solid organ transplant groups from across the world have correlated HLA molecular mismatch with transplant outcomes including de novo donor-specific antibody development, antibody-mediated rejection, T-cell-mediated rejection, and allograft survival. SUMMARY: All alloimmunity is driven by differences between donors and recipients at the molecular level. HLA molecular mismatch may represent an advancement compared to traditional HLA antigen mismatch as a fast, reproducible, cost-effective way to improve alloimmune risk assessment at the time of transplantation to move the field towards precision medicine.

What have we learned about how to prevent and treat antibody-mediated rejection in kidney transplantation?

Antibody-mediated rejection (ABMR) in kidney transplantation is a major cause of late graft loss, and despite all efforts to date the "standard of care" remains plasmapheresis, IVIg, and steroids, which itself is based on low quality evidence. This review focuses on the risk factors leading to memory and de novo donor-specific antibody (DSA)-associated ABMR, the optimal prevention strategies for ABMR, and advances in  adjunctive and emerging therapies for ABMR. Because new agents require regulatory approval via a Phase 3 randomized control trial (RCT), an overview of progress in innovative trial design for ABMR is provided. Finally, based on the insights gained in the biology of ABMR, current knowledge gaps are identified for future research that could significantly affect our understanding of how to optimally treat ABMR.

Evidence for the alloimmune basis and prognostic significance of Borderline T cell-mediated rejection.

Prognostic biomarkers of T cell-mediated rejection (TCMR) have not been adequately studied in the modern era. We evaluated 803 renal transplant recipients and correlated HLA-DR/DQ molecular mismatch alloimmune risk categories (low, intermediate, high) with the severity, frequency, and persistence of TCMR. Allograft survival was reduced in recipients with Banff Borderline (hazard ratio [HR] 2.4, P = .003) and Banff ≥ IA TCMR (HR 4.3, P < .0001) including a subset who never developed de novo donor-specific antibodies (P = .002). HLA-DR/DQ molecular mismatch alloimmune risk categories were multivariate correlates of Banff Borderline and Banff ≥ IA TCMR and correlated with the severity and frequency of rejection episodes. Recipient age, HLA-DR/DQ molecular mismatch category, and cyclosporin vs tacrolimus immunosuppression were independent correlates of Banff Borderline and Banff ≥ IA TCMR. In the subset treated with tacrolimus (720/803) recipient age, HLA-DR/DQ molecular mismatch category, and tacrolimus coefficient of variation were independent correlates of TCMR. The correlation of HLA-DR/DQ molecular mismatch category with TCMR, including Borderline, provides evidence for their alloimmune basis. HLA-DR/DQ molecular mismatch may represent a precise prognostic biomarker that can be applied to tailor immunosuppression or design clinical trials based on individual patient risk.

Human leukocyte antigen molecular mismatch to risk stratify kidney transplant recipients.

PURPOSE OF REVIEW: Stalled drug development and the lack of improvement in long-term graft survival reflect the unmet need for prognostic and predictive biomarkers in transplantation. Although conventional human leukocyte antigen (HLA) mismatch is too imprecise to fulfill this need, HLA molecular mismatch increases the precision in alloimmune risk assessment by quantifying the difference between donors and recipients at the molecular level. RECENT FINDINGS: Within each conventional HLA mismatch, recipients exhibit a wide range of HLA molecular mismatches with their donors. Quantifying HLA molecular mismatch improves the precision of alloimmune risk assessment for de novo donor-specific antibody development (dnDSA). Alloimmune risk categories developed analyzing dnDSA development were also found to correlate with T-cell-mediated rejection, antibody-mediated rejection, and all cause graft loss in adjusted and unadjusted models. SUMMARY: All alloimmunity is driven by differences between donors and recipients at the molecular level. HLA molecular mismatch may represent a fast, reproducible, cost-effective, way to improve alloimmune risk assessment at the time of transplantation to move the field towards precision medicine.

Early Antibody-Mediated Kidney Transplant Rejection Associated With Anti-Vimentin Antibodies: A Case Report.

Improving precision in predicting alloreactivity is an important unmet need and may require individualized consideration of non-HLA antibodies. We report a 21-year-old man with kidney failure from immunoglobulin A nephropathy who met all traditional criteria for a "low-risk" transplant for immune memory. He was unsensitized and received a haplotype-matched living donor kidney transplant from his mother. There were no anti-HLA donor-specific antibodies and flow cross-match was negative. After immediate function, he developed delayed graft function on postoperative day 2. The transplant biopsy specimen was suggestive of antibody-mediated rejection and acute tubular injury with increased vimentin proximal tubular expression compared to the implantation biopsy specimen. He had a history of juvenile idiopathic arthritis, and non-HLA antibody screening demonstrated preformed anti-vimentin antibody. He was successfully treated with plasmapheresis, intravenous immunoglobulin, antithymocyte globulin, and methylprednisolone, with renal recovery. The follow-up biopsy specimen demonstrated decreased vimentin expression with decreased alloinflammation, and graft function remains stable at 1 year posttransplantation (estimated glomerular filtration rate, 62mL/min/1.73m2). We postulate that preformed anti-vimentin autoantibodies bound to vimentin expressed on apoptotic tubular epithelial cells induced by ischemia-reperfusion injury and to constitutively expressed vimentin on peritubular capillaries and podocytes. Our case is suggestive of the involvement of anti-vimentin antibody, for which the pathogenic epitopes may be exposed during ischemia-reperfusion injury.

Activity-based Protein Profiling Approaches for Transplantation.

Enzyme activity may be more pathophysiologically relevant than enzyme quantity and is regulated by changes in conformational status that are undetectable by traditional proteomic approaches. Further, enzyme activity may provide insights into rapid physiological responses to inflammation/injury that are not dependent on de novo protein transcription. Activity-based protein profiling (ABPP) is a chemical proteomic approach designed to characterize and identify active enzymes within complex biological samples. Activity probes have been developed to interrogate multiple enzyme families with broad applicability, including but not limited to serine hydrolases, cysteine proteases, matrix metalloproteases, nitrilases, caspases, and histone deacetylases. The goal of this overview is to describe the overall rationale, approach, methods, challenges, and potential applications of ABPP to transplantation research. To do so, we present a case example of urine serine hydrolase ABPP in kidney transplant rejection to illustrate the utility and workflow of this analytical approach. Ultimately, developing novel transplant therapeutics is critically dependent on understanding the pathophysiological processes that result in loss of transplant function. ABPP offers a new dimension for characterizing dynamic changes in clinical samples. The capacity to identify and measure relevant enzyme activities provides fresh opportunities for understanding these processes and may help identify markers of disease activity for the development of novel diagnostics and real-time monitoring of patients. Finally, these insights into enzyme activity may also help to identify new transplant therapeutics, such as enzyme-specific inhibitors.

HLA-DR/DQ molecular mismatch: A prognostic biomarker for primary alloimmunity.

Alloimmune risk stratification in renal transplantation has lacked the necessary prognostic biomarkers to personalize recipient care or optimize clinical trials. HLA molecular mismatch improves precision compared to traditional antigen mismatch but has not been studied in detail at the individual molecule level. This study evaluated 664 renal transplant recipients and correlated HLA-DR/DQ single molecule eplet mismatch with serologic, histologic, and clinical outcomes. Compared to traditional HLA-DR/DQ whole antigen mismatch, HLA-DR/DQ single molecule eplet mismatch improved the correlation with de novo donor-specific antibody development (area under the curve 0.54 vs 0.84) and allowed recipients to be stratified into low, intermediate, and high alloimmune risk categories. These risk categories were significantly correlated with primary alloimmune events including Banff ≥1A T cell-mediated rejection (P = .0006), HLA-DR/DQ de novo donor-specific antibody development (P < .0001), antibody-mediated rejection (P < .0001), as well as all-cause graft loss (P = .0012) and each of these correlations persisted in multivariate models. Thus, HLA-DR/DQ single molecule eplet mismatch may represent a precise, reproducible, and widely available prognostic biomarker that can be applied to tailor immunosuppression or design clinical trials based on individual patient risk.