The Banff 2017 Kidney Meeting Report: Revised diagnostic criteria for chronic active T cell-mediated rejection, antibody-mediated rejection, and prospects for integrative endpoints for next-generation clinical trials.

The kidney sessions of the 2017 Banff Conference focused on 2 areas: clinical implications of inflammation in areas of interstitial fibrosis and tubular atrophy (i-IFTA) and its relationship to T cell-mediated rejection (TCMR), and the continued evolution of molecular diagnostics, particularly in the diagnosis of antibody-mediated rejection (ABMR). In confirmation of previous studies, it was independently demonstrated by 2 groups that i-IFTA is associated with reduced graft survival. Furthermore, these groups presented that i-IFTA, particularly when involving >25% of sclerotic cortex in association with tubulitis, is often a sequela of acute TCMR in association with underimmunosuppression. The classification was thus revised to include moderate i-IFTA plus moderate or severe tubulitis as diagnostic of chronic active TCMR. Other studies demonstrated that certain molecular classifiers improve diagnosis of ABMR beyond what is possible with histology, C4d, and detection of donor-specific antibodies (DSAs) and that both C4d and validated molecular assays can serve as potential alternatives and/or complements to DSAs in the diagnosis of ABMR. The Banff ABMR criteria are thus updated to include these alternatives. Finally, the present report paves the way for the Banff scheme to be part of an integrative approach for defining surrogate endpoints in next-generation clinical trials.

Evaluation of C1q Status and Titer of De Novo Donor-Specific Antibodies as Predictors of Allograft Survival.

De novo donor-specific antibodies (dnDSAs) that develop after renal transplantation are independent predictors of allograft loss. However, it is unknown if dnDSA C1q status or titer at the time of first detection can independently predict allograft loss. In a consecutive cohort of 508 renal transplant recipients, 70 developed dnDSAs. Histologic and clinical outcomes were correlated with the C1q assay or dnDSA titer. C1q positivity correlated with dnDSA titer (p < 0.01) and mean fluorescence intensity (p < 0.01) and was more common in class II versus class I dnDSAs (p < 0.01). C1q status correlated with tubulitis (p = 0.02) and C4d status (p = 0.03) in biopsies at the time of dnDSA development, but not T cell-mediated rejection (TCMR) or antibody-mediated rejection (ABMR). De novo DSA titer correlated with Banff g, i, t, ptc, C4d scores, TCMR (p < 0.01) and ABMR (p < 0.01). Post-dnDSA graft loss was observed more frequently in recipients with C1q-positve dnDSA (p < 0.01) or dnDSA titer ≥ 1:1024 (p ≤ 0.01). However, after adjustment for clinical phenotype and nonadherence in multivariate models, neither C1q status nor dnDSA titer were independently associated with allograft loss, questioning the utility of these assays at the time of dnDSA development.

The Synergistic Effect of Class II HLA Epitope-Mismatch and Nonadherence on Acute Rejection and Graft Survival.

Predicting long-term outcomes in renal transplant recipients is essential to optimize medical therapy and determine the frequency of posttransplant histologic and serologic monitoring. Nonadherence and human leukocyte antigen (HLA) mismatch are risk factors that have been associated with poor long-term outcomes and may help individualize care. In the present study, class II HLA mismatches were determined at the HLA epitope level in 195 renal transplant recipients in whom medication adherence was prospectively measured using electronic monitors in medication vial caps. Recipients were grouped by medication adherence and high (≥10 HLA-DR, ≥17 HLA-DQ) or low epitope-mismatch load. We found that the combination of higher epitope mismatch and poor adherence acted synergistically to determine the risk of rejection or graft loss. Nonadherent recipients with HLA-DR epitope mismatch ≥10 had increased graft loss (35% vs. 8%, p < 0.01) compared to adherent recipients with low epitope mismatch. At the HLA-DQ locus nonadherent recipients with HLA-DQ epitope mismatch ≥17 had increased graft loss (33% vs. 10%, p < 0.01) compared to adherent recipients with low epitope mismatch. Subclinical nonadherence early posttransplant combined with HLA class II epitope mismatch may help identify recipients that could benefit from increased clinical, histologic, and serologic monitoring.

cPRA Increases With DQA, DPA, and DPB Unacceptable Antigens in the Canadian cPRA Calculator.

A calculated panel reactive antibody (cPRA) estimates the percentage of donors with unacceptable antigens (UA) for a recipient. cPRA may be underestimated in transplant candidates with UA to DQA, DPA, and DPB if these are not included in the calculation program. To serve the National Canadian Transplant Programs, a cPRA calculator was developed with complete molecular typing for all donors at HLA-A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1, and DPB1, all resolved to serologic equivalents. The prevalence of UA at DQA, DPA and DPB was evaluated in a sensitized regional population. The impact of adding these additional UA to cPRA was calculated alone and in combination, and compared to the baseline cPRA for UA at A, B, C, DR, DR51/52/53, and DQ. Of 740 sensitized transplant candidates, 18% of total and 32% with cPRA≥95% had DQA UA. Twenty-seven percent of total and 54% with cPRA≥95% had DPB UA. Of 280/740 subjects with these UA, 36/280 (13%) had cPRA increase of >20% when they were included, 7% increased cPRA to ≥80% and 6% to ≥95%. Inclusion of DQA, DPA, and DPB UA in Canadian cPRA calculations improves the accuracy of cPRA where these are relevant in allocation.

Interferon Gamma ELISPOT Testing as a Risk-Stratifying Biomarker for Kidney Transplant Injury: Results From the CTOT-01 Multicenter Study.

Previous studies suggest that quantifying donor-reactive memory T cells prior to kidney transplantation by interferon gamma enzyme-linked immunosorbent spot assay (IFNγELISPOT) can assist in assessing risk of posttransplant allograft injury. Herein, we report an analysis of IFNγELISPOT results from the multicenter, Clinical Trials in Organ Transplantation-01 observational study of primary kidney transplant recipients treated with heterogeneous immunosuppression. Within the subset of 176 subjects with available IFNγELISPOT results, pretransplant IFNγELISPOT positivity surprisingly did not correlate with either the incidence of acute rejection (AR) or estimated glomerular filtration rate (eGFR) at 6- or 12-month. These unanticipated results prompted us to examine potential effect modifiers, including the use of T cell-depleting, rabbit anti-thymocyte globulin (ATG). Within the no-ATG subset, IFNγELISPOT(neg) subjects had higher 6- and 12-month eGFRs than IFNγELISPOT(pos) subjects, independent of biopsy-proven AR, peak PRA, human leukocyte antigen mismatches, African-American race, donor source, and recipient age or gender. In contrast, IFNγELISPOT status did not correlate with posttransplant eGFR in subjects given ATG. Our data confirm an association between pretransplant IFNγELISPOT positivity and lower posttransplant eGFR, but only in patients who do not receive ATG induction. Controlled studies are needed to test the hypothesis that ATG induction is preferentially beneficial to transplant candidates with high frequencies of donor-reactive memory T cells.

Rates and determinants of progression to graft failure in kidney allograft recipients with de novo donor-specific antibody.

Understanding rates and determinants of clinical pathologic progression for recipients with de novo donor-specific antibody (dnDSA), especially subclinical dnDSA, may identify surrogate endpoints and inform clinical trial design. A consecutive cohort of 508 renal transplant recipients (n = 64 with dnDSA) was studied. Recipients (n = 388) without dnDSA or dysfunction had an eGFR decline of -0.65 mL/min/1.73 m(2) /year. In recipients with dnDSA, the rate eGFR decline was significantly increased prior to dnDSA onset (-2.89 vs. -0.65 mL/min/1.73 m(2) /year, p < 0.0001) and accelerated post-dnDSA (-3.63 vs. -2.89 mL/min/1.73 m(2) /year, p < 0.0001), suggesting that dnDSA is both a marker and contributor to ongoing alloimmunity. Time to 50% post-dnDSA graft loss was longer in recipients with subclinical versus a clinical dnDSA phenotype (8.3 vs. 3.3 years, p < 0.0001). Analysis of 1091 allograft biopsies found that dnDSA and time independently predicted chronic glomerulopathy (cg), but not interstitial fibrosis and tubular atrophy (IFTA). Early T cell-mediated rejection, nonadherence, and time were multivariate predictors of IFTA. Independent risk factors for post-dnDSA graft survival available prior to, or at the time of, dnDSA detection were delayed graft function, nonadherence, dnDSA mean fluorescence intensity sum score, tubulitis, and cg. Ultimately, dnDSA is part of a continuum of mixed alloimmune-mediated injury, which requires solutions targeting T and B cells.

Should HLA mismatch acceptability for sensitized transplant candidates be determined at the high-resolution rather than the antigen level?

Defining HLA mismatch acceptability of organ transplant donors for sensitized recipients has traditionally been based on serologically defined HLA antigens. Now, however, it is well accepted that HLA antibodies specifically recognize a wide range of epitopes present on HLA antigens and that molecularly defined high resolution alleles corresponding to the same low resolution antigen can possess different epitope repertoires. Hence, determination of HLA compatibility at the allele level represents a more accurate approach to identify suitable donors for sensitized patients. This approach would offer opportunities for increased transplant rates and improved long term graft survivals.

Precision diagnostics in transplantation: from bench to bedside.

The Canadian and American Societies of Transplantation held a symposium on February 22, 2012 in Quebec City focused on discovery, validation and translation of new diagnostic tools into clinical transplantation. The symposium focused on antibody testing, transplantation pathology, molecular diagnostics and laboratory support for the incompatible patient. There is an unmet need for more precise diagnostic approaches in transplantation. Significant potential for increasing the diagnostic precision in transplantation was recognized through the integration of conventional histopathology, molecular technologies and sensitive antibody testing into one enhanced diagnostic system.

Comprehensive assessment and standardization of solid phase multiplex-bead arrays for the detection of antibodies to HLA.

Solid phase multiplex-bead arrays for the detection and characterization of HLA antibodies provide increased sensitivity and specificity compared to conventional lymphocyte-based assays. Assay variability due to inconsistencies in commercial kits and differences in standard operating procedures (SOP) hamper comparison of results between laboratories. The Clinical Trials in Organ Transplantation Antibody Core Laboratories investigated sources of assay variation and determined if reproducibility improved through utilization of SOP, common reagents and normalization algorithms. Ten commercial kits from two manufacturers were assessed in each of seven laboratories using 20 HLA reference sera. Implementation of a standardized (vs. a nonstandardized) operating procedure greatly reduced MFI variation from 62% to 25%. Although laboratory agreements exceeded 90% (R(2) ), small systematic differences were observed suggesting center specific factors still contribute to variation. MFI varied according to manufacturer, kit, bead type and lot. ROC analyses showed excellent consistency in antibody assignments between manufacturers (AUC > 0.9) and suggested optimal cutoffs from 1000 to 1500 MFI. Global normalization further reduced MFI variation to levels near 20%. Standardization and normalization of solid phase HLA antibody tests will enable comparison of data across laboratories for clinical trials and diagnostic testing.

Multicenter validation of urinary CXCL9 as a risk-stratifying biomarker for kidney transplant injury.

Noninvasive biomarkers are needed to assess immune risk and ultimately guide therapeutic decision-making following kidney transplantation. A requisite step toward these goals is validation of markers that diagnose and/or predict relevant transplant endpoints. The Clinical Trials in Organ Transplantation-01 protocol is a multicenter observational study of biomarkers in 280 adult and pediatric first kidney transplant recipients. We compared and validated urinary mRNAs and proteins as biomarkers to diagnose biopsy-proven acute rejection (AR) and stratify patients into groups based on risk for developing AR or progressive renal dysfunction. Among markers tested for diagnosing AR, urinary CXCL9 mRNA (odds ratio [OR] 2.77, positive predictive value [PPV] 61.5%, negative predictive value [NPV] 83%) and CXCL9 protein (OR 3.40, PPV 67.6%, NPV 92%) were the most robust. Low urinary CXCL9 protein in 6-month posttransplant urines obtained from stable allograft recipients classified individuals least likely to develop future AR or a decrement in estimated glomerular filtration rate between 6 and 24 months (92.5-99.3% NPV). Our results support using urinary CXCL9 for clinical decision-making following kidney transplantation. In the context of acute dysfunction, low values can rule out infectious/immunological causes of injury. Absent urinary CXCL9 at 6 months posttransplant defines a subgroup at low risk for incipient immune injury.

Class II HLA epitope matching-A strategy to minimize de novo donor-specific antibody development and improve outcomes.

De novo donor-specific antibody (dnDSA) develops in 15-25% of renal transplant recipients within 5 years of transplantation and is associated with 40% lower graft survival at 10 years. HLA epitope matching is a novel strategy that may minimize dnDSA development. HLAMatchmaker software was used to characterize epitope mismatches at 395 potential HLA-DR/DQ/DP conformational epitopes for 286 donor-recipient pairs. Epitope specificities were assigned using single antigen HLA bead analysis and correlated with known monoclonal alloantibody epitope targets. Locus-specific epitope mismatches were more numerous in patients who developed HLA-DR dnDSA alone (21.4 vs. 13.2, p < 0.02) or HLA-DQ dnDSA alone (27.5 vs. 17.3, p < 0.001). An optimal threshold for epitope mismatches (10 for HLA-DR, 17 for HLA-DQ) was defined that was associated with minimal development of Class II dnDSA. Applying these thresholds, zero and 2.7% of patients developed dnDSA against HLA-DR and HLA-DQ, respectively, after a median of 6.9 years. Epitope specificity analysis revealed that 3 HLA-DR and 3 HLA-DQ epitopes were independent multivariate predictors of Class II dnDSA. HLA-DR and DQ epitope matching outperforms traditional low-resolution antigen-based matching and has the potential to minimize the risk of de novo Class II DSA development, thereby improving long-term graft outcome.

Dynamic challenges inhibiting optimal adoption of kidney paired donation: findings of a consensus conference.

While kidney paired donation (KPD) enables the utilization of living donor kidneys from healthy and willing donors incompatible with their intended recipients, the strategy poses complex challenges that have limited its adoption in United States and Canada. A consensus conference was convened March 29-30, 2012 to address the dynamic challenges and complexities of KPD that inhibit optimal implementation. Stakeholders considered donor evaluation and care, histocompatibility testing, allocation algorithms, financing, geographic challenges and implementation strategies with the goal to safely maximize KPD at every transplant center. Best practices, knowledge gaps and research goals were identified and summarized in this document.

Evolution and clinical pathologic correlations of de novo donor-specific HLA antibody post kidney transplant.

The natural history for patients with de novo donor-specific antibodies (dnDSA) and the risk factors for its development have not been well defined. Furthermore, clinical and histologic correlation with serologic data is limited. We studied 315 consecutive renal transplants without pretransplant DSA, with a mean follow-up of 6.2 ± 2.9 years. Protocol (n = 215) and for cause (n = 163) biopsies were analyzed. Solid phase assays were used to screen for dnDSA posttransplant. A total of 47 out of 315 (15%) patients developed dnDSA at a mean of 4.6 ± 3.0 years posttransplant. Independent predictors of dnDSA were HLA-DRß1 MM > 0 (OR 5.66, p < 0.006); and nonadherence (OR 8.75, p < 0.001); with a strong trend toward clinical rejection episodes preceding dnDSA (OR 1.57 per rejection episode, p = 0.061). The median 10-year graft survival for those with dnDSA was lower than the No dnDSA group (57% vs. 96%, p < 0.0001). Pathology consistent with antibody-mediated injury can occur and progress in patients with dnDSA in the absence of graft dysfunction and furthermore, nonadherence and cellular rejection contribute to dnDSA development and progression to graft loss.

Detection of clinical and subclinical tubulo-interstitial inflammation by the urinary CXCL10 chemokine in a real-life setting.

Urinary CXCL10 is a promising noninvasive biomarker for tubulo-interstitial allograft inflammation, but its diagnostic characteristics have not been assessed in a real-life setting. We investigated urinary CXCL10 in 213 consecutive renal allograft recipients having 362 surveillance biopsies at 3/6 months and 80 indication biopsies within the first year posttransplant. Allograft histology results were classified as (i) acute Banff score zero, (ii) interstitial infiltrates only, (iii) tubulitis t1, (iv) tubulitis t2-3 and (v) isolated vascular compartment inflammation. For clinical and subclinical pathologies, urinary CXCL10 correlated well with the extent of tubulo-interstitial inflammation. To determine diagnostic characteristics of urinary CXCL10, histological groups were separated into two categories: no relevant inflammation (i.e. acute Banff score zero and interstitial infiltrates only) versus all other pathologies (i.e. tubulitis t1-3 and isolated vascular compartment inflammation). For subclinical pathologies, AUC was 0.69 (sensitivity 61%, specificity 72%); for clinical pathologies, AUC was 0.74 (sensitivity 63%, specificity 80%). A urinary CXCL10-guided biopsy strategy would have reduced performance of surveillance and indication biopsies by 61% and 64%, respectively. Missed (sub)clinical pathologies were mostly tubulitis t1 and isolated vascular compartment lesions. In real life, urinary CXCL10 had clinically useful diagnostic properties making it a candidate biomarker to guide allograft biopsies.

Lifetime probabilities of needing an organ transplant versus donating an organ after death.

The lifetime probabilities of becoming a deceased organ donor and requiring or receiving an organ transplant are unknown. An actuarial analysis was performed in a representative Canadian sample. Using Canadian organ donation data 1999-2007, provincial waiting list and population census data, actuarial rates were produced that provide the probabilities, by age band and gender, of (1) becoming a deceased organ donor, (2) needing an organ transplant and (3) receiving all organs needed. Regardless of age, the lifetime probability of needing a transplant for males is approximately twice that of females. Depending on age, Canadians are five to six times more likely to need an organ transplant than to become a deceased organ donor. The lifetime probabilities of not receiving a required organ transplant, expressed as a percentage of individuals on the waiting list, ranges from approximately 30% at birth, 20 years and 40 years to approximately 40% at 60 years. Across provinces and genders, Canadians at all ages are much more likely to need an organ transplant than to become an organ donor. Approximately one-third of those in need of a transplant will never receive one. How this information may influence organ donation decisions is currently under study.

Effects of epidermal growth factor and erythropoietin on Müller glial activation and phenotypic plasticity in the adult mammalian retina.

Retinal Müller glia have received considerable attention with regard to their potential to function as quiescent retinal precursors. Various activation strategies induce characteristic features of retinal progenitor cells in Müller glia; however, these are often accompanied by hallmark features of reactive gliosis. We investigated the effects of an intravitreal injection of epidermal growth factor (EGF), a known mitogen, and erythropoietin (EPO) on activation and expression of developmental phenotypes within the adult retina. Using thymidine-analogue labeling as well as immunocytochemical and confocal analyses, we assayed the responses of retinal cells exposed to intravitreal administration of either EGF or EPO. We report that adult Müller glia incorporate bromodeoxyuridine (BrdU) and undergo a process of nuclear translocation to ectopic retinal layers following exposure to EGF. These cells survive within the retina for at least 23 days and express the developmental markers Pax6 and Chx10 as well as nestin and glial fibrillary acidic protein. Furthermore, we demonstrate that cotreatment with EGF and EPO suppresses aspects of EGF-induced glial reactivity, alters the retinal distribution of BrdU-positive nuclei, and serves to regulate the expression of developmental phenotypes seen in these cells. These data further our understanding of Müller cell responsiveness to intravitral, combinatorial growth factor treatments.

Changes in Musashi-1 subcellular localization correlate with cell cycle exit during postnatal retinal development.

RNA-binding proteins, and in particular, the Musashi genes, function as essential regulators of progenitor functioning in both the developing and adult organism. In this report, we characterize the differential subcellular distribution of Musashi-1 in cells engaged in either proliferating or differentiating contexts in the developing mouse retina, and in cultured Müller glia. During retinal cell differentiation, Musashi-1 immunoreactivity shifts from exclusively cytoplasmic in retinal progenitor cells, to predominantly nuclear localization in differentiating neurons. This nuclear shift is transient, with localization in the adult retina becoming predominantly perinuclear and cytoplasmic in Müller glia and photoreceptors. A correlation between cell cycle progression and subcellular distribution of Musashi-1 is observed in passageable, adult Müller glial cells in vitro. Furthermore, treatment of Müller cultures with neuron-promoting differentiation media induces asymmetric cytoplasmic Musashi-1 immunoreactivity in dividing daughter cells. The observed shifts in subcellular Musashi-1 localization are consistent with contrasting roles for Musashi-1 during cell proliferation and differentiation. These data provide evidence that nuclear, and cytoplasmic sequestering of Musashi-1 in retinal cells is context-specific, and may contribute to downstream functioning of Musashi-1.

Urinary CXCL9 and CXCL10 levels correlate with the extent of subclinical tubulitis.

Subclinical tubulitis has been associated with the later development of interstitial fibrosis and tubular atrophy (IF/TA), leading to diminished allograft survival. The aim of this study was to investigate how concentrations of urinary CXC-receptor 3 (CXCR3) chemokines (i.e. CXCL4/9/10/11) and CCL2 relate to the extent of subclinical tubulitis. Using ELISA, urinary CXCR3 chemokines, CCL2 and tubular injury markers (i.e. urinary NGAL and alpha1-microglobulin [alpha1 m]) were measured in patients with stable estimated GFR >or=40 mL/min exhibiting normal tubular histology (n = 24), subclinical borderline tubulitis (n = 18) or subclinical tubulitis Ia/Ib (n = 22), as well as in patients with clinical tubulitis Ia/Ib (n = 17) or IF/TA (n = 10). CXCL9 and CXCL10 were significantly higher in subclinical tubulitis Ia/Ib than in subclinical borderline tubulitis (p