Antibody-mediated Allograft Rejection Research Articles

Therapeutic Apheresis in Prevention and Treatment of Antibody-Mediated Rejection of Renal Allografts

Antibody-mediated rejection represents a significant barrier to favourable long-term outcomes after kidney transplantation and is the most common cause of allograft failure. Therapeutic apheresis techniques associated with other treatments, such as immunosuppressive drugs, are commonly used in the prevention and treatment of antibody-mediated injury in pre- and post-transplant protocols. The rationale is related to the removal of donor-specific antibodies as well as other inflammatory mediators, including cytokines, chemokines, complement degradation molecules, and immunomodulatory effects also which determine an increased susceptibility of cell-mediated and humoral immunity to immunosuppressive agents. In this review article, we discuss current knowledge in the use of therapeutic apheresis in the prevention and management of antibody-mediated rejection, in combination with known and other emerging biological immunosuppressive therapies.

Research Highlights

A Peripheral Blood Gene Expression Signature to Diagnose Subclinical Acute Rejection Zhang W, Yi Z, Keung KL, et al. J Am Soc Nephrol. 2019;30:1481–1494. Development and Validation of a Peripheral Blood mRNA Assay for the Assessment of Antibody-mediated Kidney Allograft Rejection: A Multicentre, Prospective Study Van Loon E, Gazut S, Yazdani S, et al. EBioMedicine. 2019;46:463–472. The early and accurate diagnosis of renal allograft rejection is critical to ensuring continued function and survival of the transplanted organ. Regular monitoring of transplant recipients is therefore an integral part of their management post transplantation. The challenge, however, is the delivery of personalized therapy under conditions in which current monitoring techniques are imprecise and, in the case of renal biopsy, subject to sampling error and procedural complications. The current strategy of most centers is to monitor serum creatinine according to Kidney Disease Improving Global Outcomes (KDIGO) guidelines, as well as protein excretion and viral screening. Due to its nonspecific nature, a rise in serum creatinine normally triggers a biopsy, which is then assessed according to the Banff classification of allograft rejection. In many units, surveillance biopsies are also performed which often demonstrate features that may be consistent with rejection in the absence of a rise in serum creatinine. The significance of this “subclinical rejection” is uncertain and indicates that either our current diagnostic tests are not sensitive enough to detect rejection, or that the biopsy findings lack specificity. While the Banff classification offers standardization of rejection criteria, there remains interobserver variability between pathologists. HLA alloantibody screening post transplantation is also part of the armamentarium, yet there are often cases in which there are clinical features of antibody-mediated rejection but no significant serum donor-specific antibody. Overall, the situation results in a degree of clinical uncertainty and a lack of diagnostic accuracy to help guide treatment with precision. There is therefore a need to develop noninvasive assays that can accurately evaluate renal function and alloimmune status. Three broad classes of biomarkers have been defined: prognostic, predictive, and surrogate end point biomarkers.1 There have been a number of incremental steps in the development of such biomarkers, with some tests coming to market, such as AlloSure’s donor-derived cell-free DNA assay, which may assist in the discrimination of rejection from other causes of raised creatinine such as BK nephropathy, thus obviating the need for a biopsy.2 Other examples include the Clinical Trials in Organ Transplantation (CTOT) study, which initially demonstrated an association between urinary CXCL9 levels or IFNγ ELISPOT with acute cellular rejection within the first year (CTOT-01), although the association of these markers with 5-year outcomes was less informative (CTOT-17).3 With the recent advent of accessible advanced transcriptomic analysis including RNA sequencing, a number of groups have examined peripheral blood mRNA to provide further insight into the alloimmune status.4 In the two studies highlighted this month, transcriptomic analyses have been used to help address the challenges in the diagnosis of subclinical and antibody-mediated rejection, producing gene sets with predictive and occasionally prognostic value. In the study from Zhang et al,5 RNA sequencing was performed on 88 patients at the time of a 3-month surveillance biopsy to examine for transcriptomic changes associated with rejection at that time point. Of these, 22 patients had a diagnosis of acute cellular rejection at the time of transcriptomic analysis. A 17-gene set was identified as being associated with acute cellular rejection and then validated on a training set of patients, demonstrating a statistically significant ability to identify patients at higher risk of acute rejection and future graft loss. In the study from Van Loon et al,6 a transcriptomic analysis on peripheral blood was performed in an effort to detect antibody-mediated rejection. Here, the authors used discovery (117 blood samples and 95 biopsies from 117 patients) and derivation (183 patients) cohorts to develop an 8-gene set with the ability to diagnose antibody-mediated rejection on a test cohort of patients. In contrast to the previous study, here RNA was analyzed by microarray rather than sequencing. Importantly, while the gene set correlated with histologic markers of antibody-mediated rejection (such as glomerulitis and peritubular capillaritis), there was no correlation with features of T-cell–mediated rejection in biopsies. Both studies highlight the utility that advanced analysis techniques are bringing to the biomarker discovery field and have successfully produced gene panels with real potential for clinical implementation. The advantage will ultimately be a reduced reliance on biopsies without a loss of diagnostic accuracy, as well as enhanced guidance on immunosuppression management without the troublesome clinical uncertainty.

Update on C1 Esterase Inhibitor in Human Solid Organ Transplantation.

Complement plays important roles in both ischemia-reperfusion injury (IRI) and antibody-mediated rejection (AMR) of solid organ allografts. One approach to possibly improve outcomes after transplantation is the use of C1 inhibitor (C1-INH), which blocks the first step in both the classical and lectin pathways of complement activation and also inhibits the contact, coagulation, and kinin systems. C1-INH can also directly block leukocyte-endothelial cell adhesion. C1-INH contrasts with eculizumab and other distal inhibitors, which do not affect C4b or C3b deposition or noncomplement pathways. Authors of reports on trials in kidney transplant recipients have suggested that C1-INH treatment may reduce IRI and delayed graft function, based on decreased requirements for dialysis in the first month after transplantation. This effect was particularly marked with grafts with Kidney Disease Profile Index ≥ 85. Other clinical studies and models suggest that C1-INH may decrease sensitization and donor-specific antibody production and might improve outcomes in AMR, including in patients who are refractory to other modalities. However, the studies have been small and often only single-center. This article reviews clinical data and ongoing trials with C1-INH in transplant recipients, compares the results with those of other complement inhibitors, and summarizes potentially productive directions for future research.

Development and validation of a peripheral blood mRNA assay for the assessment of antibody-mediated kidney allograft rejection: A multicentre, prospective study

BackgroundAntibody-mediated rejection, a leading cause of renal allograft graft failure, is diagnosed by histological assessment of invasive allograft biopsies. Accurate non-invasive biomarkers are not available. MethodsIn the multicentre, prospective BIOMARGIN study, blood samples were prospectively collected at time of renal allograft biopsies between June 2011 and August 2016 and analyzed in three phases. The discovery and derivation phases of the study (N = 117 and N = 183 respectively) followed a case-control design and included whole genome transcriptomics and targeted mRNA expression analysis to construct and lock a multigene model. The primary end point was the diagnostic accuracy of the locked multigene assay for antibody-mediated rejection in a third validation cohort of serially collected blood samples (N = 387). This trial is registered with ClinicalTrials.gov, number NCT02832661. FindingsWe identified and locked an 8-gene assay (CXCL10, FCGR1A, FCGR1B, GBP1, GBP4, IL15, KLRC1, TIMP1) in blood samples from the discovery and derivation phases for discrimination between cases with (N = 49) and without (N = 134) antibody-mediated rejection. In the validation cohort, this 8-gene assay discriminated between cases with (N = 41) and without antibody-mediated rejection (N = 346) with good diagnostic accuracy (ROC AUC 79·9%; 95% CI 72·6 to 87·2, p < 0·0001). The diagnostic accuracy of the 8-gene assay was retained both at time of stable graft function and of graft dysfunction, within the first year and also later after transplantation. The 8-gene assay is correlated with microvascular inflammation and transplant glomerulopathy, but not with the histological lesions of T-cell mediated rejection. InterpretationWe identified and validated a novel 8-gene expression assay that can be used for non-invasive diagnosis of antibody-mediated rejection. FundingThe Seventh Framework Programme (FP7) of the European Commission.

2013 Banff Criteria for Acute Antibody-Mediated Rejection Are Superior to 2007 Banff Criteria in the Diagnosis and Assessment of Renal Allograft Outcomes

BackgroundThe 2013 Banff meeting updated the requirements for the diagnosis of acute/active antibody-mediated rejection (AAMR) in kidney allografts. There has been speculation that the changes lower the threshold for diagnosing AAMR, and may lead to possible unnecessary and expensive treatment. MethodsWe compared the 2013 Banff classification for AAMR to the previous 2007 Banff to determine if there was an increase in the number of patients receiving a diagnosis of AAMR and if the diagnosis affected allograft survival and post-biopsy 3-month and 6-month creatinine and eGFR values. ResultsA total of 212 renal allograft biopsies were compared to both 2007 and 2013 Banff classification requirements for AAMR. Ten patients (11 biopsies) met the 2007 criteria. An additional 15 patients (20 biopsies) met the 2013 criteria. These 2 groups showed no statistically significant demographic differences. By applying the 2013 Banff classification, we observed a 2.5-fold increase in the number of AAMR cases. One-year post-transplant allograft survival was higher in the 2013 group (.85 vs .55) and the 3-month and 6-month post-biopsy creatinine values were significantly lower for the 2013 group (1.6 ± .6 vs 3.3 ± 2.2, P value .01, and 1.7 ± .6 vs 3.4 ± 2.8, P value .03). The 3-month and 6-month eGFR values were higher in the 2013 group, although not statistically significant. ConclusionsThese results suggest that use of Banff 2013 criteria in place of Banff 2007 may result in diagnosing milder and earlier cases of AAMR with the possibility of initiating earlier treatment and improving graft outcomes.

Kidney Allograft Recipient Absence of Myeloperoxidase Decreases Donor-Specific Antibody Titers and Attenuates Antibody-Mediated Allograft Rejection

Abstract The incidence of antibody-mediated rejection (AMR) in clinical kidney transplants is increasingly observed. We have reported that strong donor-specific antibody (DSA) responses are induced in B6.CCR5−/−mice transplanted with complete MHC mismatched A/J kidney allografts and that NK cells play a critical role in acute injury and graft failure. Since this acute AMR is accompanied by intense macrophage infiltration into the allograft, we tested the role of recipient-derived myeloperoxidase (MPO) production on kidney allograft survival. B6.CCR5−/−and B6.CCR5−/−MPO−/−mice were transplanted with complete MHC mismatched A/J kidney grafts. Allografts were rejected between days 18 and 25 post-transplant in B6.CCR5−/−recipients but not until days 46–54 in B6.CCR5−/−MPO−/− recipients. DSA titers in B6.CCR5−/−MPO−/−recipients were 4–5 fold lower than those induced in the B6.CCR5−/− recipients. There was also a 60% decrease in the number of donor-reactive T cells producing IFN-g in the spleens of the B6.CCR5−/−MPO−/− vs. B6.CCR5−/−recipients on day 7 post-transplant. Despite the extended survival, qPCR analyses indicated slight increases in mRNA encoding TNFa, IL-6 and FasL in kidney allografts on day 14 post-transplant in B6.CCR5−/−MPO−/− vs. B6.CCR5−/−recipients as well as the pro-fibrogenic factors P-selectin and connective tissue growth factor on day 50 post-transplant. These results suggest MPO regulates the magnitude of the DSA and T cell response in kidney transplant recipients and that the decreased DSA titers attenuate acute AMR but induce the indolent development of interstitial fibrosis and glomerular injury that will eventually lead to graft dysfunction and failure at later times.

C5b9 Deposition in Glomerular Capillaries Is Associated With Poor Kidney Allograft Survival in Antibody-Mediated Rejection.

C4d deposition in peritubular capillaries (PTC) reflects complement activation in antibody-mediated rejection (ABMR) of kidney allograft. However, its association with allograft survival is controversial. We hypothesized that capillary deposition of C5b9—indicative of complement-mediated injury—is a severity marker of ABMR. This pilot study aimed to determine the frequency, location and prognostic impact of these deposits in ABMR. We retrospectively selected patients diagnosed with ABMR in two French transplantation centers from January 2005 to December 2014 and performed C4d and C5b9 staining by immunohistochemistry. Fifty-four patients were included. Median follow-up was 52.5 (34.25–73.5) months. Thirteen patients (24%) had C5b9 deposits along glomerular capillaries (GC). Among these, seven (54%) had a global and diffuse staining pattern. Twelve of the C5b9+ patients also had deposition of C4d in GC and PTC. C4d deposits along GC and PTC were not associated with death-censored allograft survival (p = 0.42 and 0.69, respectively). However, death-censored allograft survival was significantly lower in patients with global and diffuse deposition of C5b9 in GC than those with a segmental pattern or no deposition (median survival after ABMR diagnosis, 6 months, 40.5 months and 44 months, respectively; p = 0.015). Double contour of glomerular basement membrane was diagnosed earlier after transplantation in C5b9+ ABMR than in C5b9– ABMR (median time after transplantation, 28 vs. 85 months; p = 0.058). In conclusion, we identified a new pattern of C5b9+ ABMR, associated with early onset of glomerular basement membrane duplication and poor allograft survival. Complement inhibitors might be a therapeutic option for this subgroup of patients.

Relative Frequencies of Alloantigen-Specific Helper CD4 T Cells and B Cells Determine Mode of Antibody-Mediated Allograft Rejection.

Humoral alloimmunity is now recognized as a major determinant of transplant outcome. MHC glycoprotein is considered a typical T-dependent antigen, but the nature of the T cell alloresponse that underpins alloantibody generation remains poorly understood. Here, we examine how the relative frequencies of alloantigen-specific B cells and helper CD4 T cells influence the humoral alloimmune response and how this relates to antibody-mediated rejection (AMR). An MHC-mismatched murine model of cardiac AMR was developed, in which T cell help for alloantibody responses in T cell deficient (Tcrbd−/−) C57BL/6 recipients against donor H-2Kd MHC class I alloantigen was provided by adoptively transferred “TCR75” CD4 T cells that recognize processed H-2Kd allopeptide via the indirect-pathway. Transfer of large numbers (5 × 105) of TCR75 CD4 T cells was associated with rapid development of robust class-switched anti-H-2Kd humoral alloimmunity and BALB/c heart grafts were rejected promptly (MST 9 days). Grafts were not rejected in T and B cell deficient Rag2−/− recipients that were reconstituted with TCR75 CD4 T cells or in control (non-reconstituted) Tcrbd−/− recipients, suggesting that the transferred TCR75 CD4 T cells were mediating graft rejection principally by providing help for effector alloantibody responses. In support, acutely rejecting BALB/c heart grafts exhibited hallmark features of acute AMR, with widespread complement C4d deposition, whereas cellular rejection was not evident. In addition, passive transfer of immune serum from rejecting mice to Rag2−/− recipients resulted in eventual BALB/c heart allograft rejection (MST 20 days). Despite being long-lived, the alloantibody responses observed at rejection of the BALB/c heart grafts were predominantly generated by extrafollicular foci: splenic germinal center (GC) activity had not yet developed; IgG secreting cells were confined to the splenic red pulp and bridging channels; and, most convincingly, rapid graft rejection still occurred when recipients were reconstituted with similar numbers of Sh2d1a−/− TCR75 CD4 T cells that are genetically incapable of providing T follicular helper cell function for generating GC alloimmunity. Similarly, alloantibody responses generated in Tcrbd−/− recipients reconstituted with smaller number of wild-type TCR75 CD4 T cells (103), although long-lasting, did not have a discernible extrafollicular component, and grafts were rejected much more slowly (MST 50 days). By modeling antibody responses to Hen Egg Lysozyme protein, we confirm that a high ratio of antigen-specific helper T cells to B cells favors development of the extrafollicular response, whereas GC activity is favored by a relatively high ratio of B cells. In summary, a relative abundance of helper CD4 T cells favors development of strong extrafollicular alloantibody responses that mediate acute humoral rejection, without requirement for GC activity.This work is composed of two parts, of which this is Part I. Please read also Part II: Chhabra et al., 2019.

Abstract 17377: The Effect of Mechanical Circulatory Support on HLA Antibodies and Antibody-Mediated Rejection in Cardiac Allografts

Introduction: Due to the shortage of donor organs, mechanical circulatory support systems (MCS) are now widely used as a treatment option to bridge the failing heart to transplant. It has been observed that MCS patients show a higher frequency of antibody-mediated rejection (AMR) episodes. The latter is thought to represent an immune reaction directed against the device in terms of allosensitation. Hypothesis: Capillary C4d and/or C3d depositions are required to diagnose AMR in cardiac allografts. We aimed to analyze the effect of MCS on pre- and post-HTx HLA antibodies and their correlation to cardiac AMR with due regards to capillary C3d and C4d depositions. Methods: We evaluated all consecutive right ventricular protocol endomyocardial biopsies (EMBs) of cardiac allografts which fulfilled the ISHLT- criteria (254 patients) during two consecutive calendar years. With regards to pre- transplant MCS treatment patients were divided into two groups (MCS group n=82 patients, non MCS group n= 138 patients). Conventional histology and immunohistochemical stains were used on formalin-fixed paraffin-embedded tissue sections (FFPE) to evaluate the EMBs regarding AMR (complements C3d and C4d). Permutation test (x 2 ) was performed to analyze the relationship between C4d and C3d and pre-and post-transplant HLA-antibodies, measured using Luminex assay, in all patient subgroups. Cochran-Mantel-Haenzsel was performed to assure the significance of the difference of interactions in both tables. Results: The statistical analysis showed in the MCS group a highly significant positive correlation between C3d and Pre-Tx-HLA (p=0.012, Pre-Tx-HLA Class I and II p= 0.042, respectively vs p=0.884 and p=0.349 in non-MCS group), which levelled out following HTx (p= 0.867 and 0.697 for HLA Class I and II, vs p=0.212 and p=0.682 in non-MCS group, respectively). Statistical significance was not reached between C4d and pre-or post Tx HLA-antibodies in any group (p=0.349 vs. p=0.694 for entire study population). Conclusions: We demonstrated that C3d correlates solely in the MCS group with pre-Tx HLA antibodies and could therefore add to the diagnosis of AMR if the pre-transplant treatment is being considered in the diagnostic setting.

Noninvasive and quantitative measurement of C4d deposition for the diagnosis of antibody-mediated cardiac allograft rejection.

BackgroundC4d is a specific biomarker for the diagnosis of antibody-mediated rejection (AMR) after cardiac transplantation. Although strongly recommended, routine C4d surveillance is hindered by the invasive nature of endomyocardial biopsy. Targeted ultrasound (US) has high sensitivity, and C4d is abundantly expressed within the graft of patients experiencing AMR, which makes it possible to visualize C4d deposition in vivo using targeted US.MethodsWe designed a serial dilution of C4d-targeted microbubbles (MBC4d) using a streptavidin-biotin conjugation system. A rat model of AMR with C4d deposition was established by pre-sensitization with skin transplantation before cardiac transplantation. MBC4d were injected into recipients and then qualitatively and quantitatively analyzed using the destruction-replenishment method with a clinical US imaging system and analyzed by software.FindingsWe successfully obtained qualitative images of C4d deposition in a wide cardiac allograft section, which, for the first time, reflected real-time C4d distribution. Moreover, normal intensity difference was used for quantitative analysis and exhibited an almost nearly linear correlation with the grade of C4d deposition according to the pathologic evidence. In addition, MBC4d injection did not affect the survival and aggravate injury, which demonstrates its safety.InterpretationThis study demonstrates a noninvasive, quantitative and safe evaluation method for C4d. As contrast-enhanced US has been widely used in clinical settings, this technology is expected to be applied quickly to clinical practice.FundNational Natural Science Foundation of China and Guangdong Province, Leading Scientific Talents of Guangdong special support program, the Science and Technology Project of Guangdong Province and Guangzhou City.

A method to reduce variability in scoring antibody-mediated rejection in renal allografts: implications for clinical trials - a retrospective study.

Poor reproducibility in scoring antibody-mediated rejection (ABMR) using the Banff criteria might limit the use of histology in clinical trials. We evaluated the reproducibility of Banff scoring of 67 biopsies by six renal pathologists at three institutions. Agreement by any two pathologists was poor: 44.8-65.7% for glomerulitis, 44.8-67.2% for peritubular capillaritis, and 53.7-80.6% for chronic glomerulopathy (cg). All pathologists agreed on cg0 (n = 20) and cg3 (n = 9) cases, however, many disagreed on scores of cg1 or cg2. The range for the incidence of composite diagnoses by individual pathologists was: 16.4-22.4% for no ABMR; 17.9-47.8% for active ABMR; and 35.8-59.7% for chronic, active antibody-mediated rejection (cABMR). A "majority rules" approach was then tested in which the scores of three pathologists were used to reach an agreement. This increased consensus both for individual scores (ex. 67.2-77.6% for cg) and for composite diagnoses (ex. 74.6-86.6% cABMR). Modeling using these results showed that differences in individual scoring could affect the outcome assessment in a mock study of cABMR. We conclude that the Banff schema has high variability and a majority rules approach could be used to adjudicate differences between pathologists and reduce variability in scoring in clinical trials.

Histological picture of antibody-mediated rejection without donor-specific anti-HLA antibodies: Clinical presentation and implications for outcome.

In this cohort study (n=935 transplantations), we investigated the phenotype and risk of graft failure in patients with histological criteria for antibody-mediated rejection (ABMR) in the absence of circulating donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA), and compared this to patients with definite ABMR and HLA-DSA-positivity. The histological picture did not differ between HLA-DSA-positive (n=85) and HLA-DSA-negative (n=123) cases of ABMR histology, apart from increased complement split product 4d (C4d) deposition in the peritubular capillaries in HLA-DSA-positive cases. Histology of ABMR without HLA-DSA was more transient than DSA-positive ABMR, and patients with ABMR histology without HLA-DSA had graft survival superior to that of HLA-DSA-positive patients, independent of concomitant T cell-mediated rejection (38.2%) or borderline changes (17.9%). Multivariate analysis showed that the risk of graft failure was not higher in patients with histological picture of ABMR (ABMRh ) in the absence of HLA-DSA, compared to patients without ABMRh . Despite an association between C4d deposition and HLA-DSA-positivity, using C4d deposition as alternative for the DSA criterion in the diagnosis of ABMR, as proposed in Banff 2017, did not contribute to the prognosis of graft function and graft failure. We concluded that biopsies with ABMRh but without detectable HLA-DSA represent a distinct, often transient phenotype with superior allograft survival.

The relationship between pathologic lesions of active and chronic antibody-mediated rejection in renal allografts.

The Banff classification of renal allograft pathology defines specific morphologic lesions that are used in the diagnosis of active (glomerulitis, peritubular capillaritis, endarteritis) and chronic (transplant glomerulopathy, peritubular capillary basement membrane multilayering, transplant arteriopathy) antibody-mediated rejection (ABMR). However, none of these individual lesions are specific for ABMR, and for this reason Banff requires 1 or more additional findings, including C4d deposition in peritubular capillaries, presence of circulating donor-specific antibodies (DSAs), and/or expression in the tissue of transcripts strongly associated with ABMR, for a definitive diagnosis of ABMR to be made. In addition, while animal studies examining serial biopsies have established the progression of morphologic lesions of active to chronic ABMR as well as intermediate forms (chronic active ABMR) exhibiting features of both, clear documentation that lesions of chronic ABMR require the earlier presence of corresponding active and intermediate lesions is less well established in human renal allografts. This review examines temporal relationships between key morphologic lesions of active and chronic ABMR in biopsies of human grafts, likely intermediate forms, and findings for and possibly against direct and potentially interruptible progression from active to chronic lesions.

Graft dysfunction in simultaneous pancreas kidney transplantation (SPK): Results of concurrent kidney and pancreas allograft biopsies.

Simultaneous pancreas and kidney transplants offer significant therapeutic advantages but present a diagnostic approach dilemma in the diagnosis of rejection. Because both organs are from the same donor, the kidney has been treated traditionally as the "sentinel" organ to biopsy, presumably representing the status of both allografts. Truly concurrent biopsy studies, however, are needed to confirm this hypothesis. We examined 101 concurrent biopsies from 70 patients with dysfunction in either or both organs. Results showed concurrent rejection in 23 of 57 (40%) of cases with rejection; 19 of 57 (33.5%) and 15 of 57 (26.5%) showed kidney or pancreas only rejection, respectively. The degree and type of rejection differed in the majority (13 of 23, 56.5%) of cases with concurrent rejection, with the pancreas more often showing higher rejection grade. Taking into account pancreas dysfunction, a positive kidney biopsy should correctly predict pancreas rejection in 86% of the instances. However, the lack of complete concordance between the 2 organs, the discrepancies in grade and type of rejection, and the tendency for higher rejection grades in concurrent or pancreas only rejections, all support the rationale for pancreas biopsies. The latter provide additional data on the overall status of the organ, as well as information on nonrejection-related pathologies.

Transfusion of leukoreduced blood products and risk of antibody-mediated rejection of renal allografts.

Antibody-mediated rejection (AMR) is a major barrier to the long-term function of renal allografts. White blood cells, which may be present in red blood cell (RBC) units, and platelets (PLTs) express HLA antigens that may increase the risk of AMR by inducing or increasing humoral sensitization to HLA. A retrospective cohort study of HLA-incompatible (HLAi) renal transplant recipients between 2004 and 2015 was conducted. Data on apheresis PLT and leukoreduced RBC transfusions within 4 weeks of transplantation, demographic information, and biopsy-proven AMR were collected from medical records and the Scientific Registry of Transplant Recipients. Patients were evaluated until they showed evidence of AMR or until 1 year posttransplant, whichever came first. Multivariable analysis with Cox modeling was performed. Of 244 individuals, 182 (74.6%) received RBCs and 20 (8.2%) of those also received PLTs. During the first year posttransplant, 97 (39.8%) had AMR. RBC-alone or RBC and PLT transfusions were not associated with increased risk of AMR after adjustment for panel-reactive antibody, years on dialysis, HLA antibody strength, and number of therapeutic plasma exchange treatments (adjusted hazard ratio [adjHR] 1.00, 95% confidence interval [95% CI] 0.59-1.69; and adjHR 0.68, 95% CI 0.28-1.68, respectively). For each 1-unit increase in RBC transfusions, there was no association with AMR (adjHR 0.94, 95% CI 0.85-1.05). Only HLA antibody strength before transplantation was associated with AMR (adjHR 2.23, 95% CI 1.10-4.52; cytotoxic crossmatch compared to crossmatch negative but detectable donor-specific HLA antibodies). Patients who receive an HLAi transplant who are transfused with leukoreduced RBCs or PLTs in the peritransplant period are at no higher risk of AMR than nontransfused patients.

Mouse Model Established by Early Renal Transplantation After Skin Allograft Sensitization Mimics Clinical Antibody-Mediated Rejection.

Antibody-mediated rejection (AMR) is the main barrier to renal graft survival, and mouse renal AMR models are important to study this process. Current mouse models are established by priming the recipient to donor skin for over 7 days before kidney transplantation. The robustness of AMR in these cases is too strong to mimic clinical AMR and it is unclear why altering the priming times ranging from 7 to 91 days fails to reduce the AMR potency in these models. In the present study, we found that the donor-recipient combination and skin graft size were determinants of donor-specific antibody (DSA) development patterns after skin transplantation. DSA-IgG was sustained for over 100 days after skin challenge, accounting for an identical AMR robustness upon different skin priming times over 7 days. However, decreasing the skin priming time within 7 days attenuated the robustness of subsequent renal allograft AMR in C3H to Balb/c mice. Four-day skin priming guaranteed that recipients develop acute renal AMR mixed with a high ratio of graft-infiltrating macrophages, renal grafts survived for a mean of 6.4 ± 2.1 days, characterized by typical AMR histological changes, such as glomerulitis, peritubular capillary (PTC) dilation, and capillaritis, deposition of IgG and C3d in PTCs, but less prevalence of microthrombus, whereas the cellular rejection histological change of tubulitis was absent to mild. With this scheme, we also found that the renal AMR model can be developed using common mouse strains such as C57BL/6 and Balb/c, with mean prolonged renal graft survival times of 14.4 ± 5.0 days. Finally, we proved that donor-matched skin challenge after kidney transplantation did not strongly affect DSA development and kidney graft outcome. These findings may facilitate an understanding and establishment of mouse renal allograft AMR models and promote AMR-associated studies.

Pattern of Glomerular Staining for C4d Immunohistochemistry is Useful for the Diagnosis of Immune Complex Mediated Glomerulonephritis in Renal Allograft Biopsies

Introduction Performance of the C4d stain is required for the adequate evaluation of kidney transplant biopsies (KTBx). Positive C4d staining in the renal peritubular capillaries is very useful for the diagnosis of antibody mediated allograft rejection (AMR) and correlates with poorer graft outcomes. Glomerular C4d staining is common in the chronic phases of AMR but this is not diagnostically useful due to inconsistent distribution and intensity of staining. Deposition of the C4d complement component in immune complex mediated (ICM) glomerulonephritis (GN) is expected but this has not been systematically studied in KTBx. Materials and Methods C4d stain (immunohistochemical method) was applied to 2432 consecutive, paraffin embedded KTBx, over a period of 5 years. In KTBx due to abrupt increase in proteinuria or history of GN in the native kidney, immunofluorescence and electron microscopy studies for standard evaluation of GN were also used for diagnosis. Results and Discussion In this unselected biopsy cohort, 33 KTBx from 27 patients showed recurrent or primary GN: 12 membranous glomerulopathy (MGN), 4 lupus nephritis (SLE), 9 IgA nephropathy and 8 focal segmental glomerulosclerosis (FSGS), Distinctive and consistent glomerular C4d stainining was noted in patients with MGN with a pattern paralleling the IF and EM findings. SImilarly, in recurrnet SLE, glomerular C4d staining corresponded to the histological lupus Class determined by light microscopy, IF and EM. The C4d staining pattern in these GNs was clearly different from that seen in transplant glomerulopathy. C4d staining in IgA nephropathy was inconsistent, mostly mesangial and did not correlate clearly with the strength of IF IgA staining. Only the segmental sclerotic lesions in FSGS were highlighted by the C4d staining in a manner that appeared nonspecific. Conclusions C4d staining is routinely evaluated in transplant biopsies and this stain consistently highlights immune complex deposits in MGN and SLE. Recognition of this distinctive staining pattern is helpful in the diagnosis of these processes and should prompt complete evaluation with IF and EM if these studies were not initially available. The findings in this study are based on C4d stain evaluation of paraffin embedded tissue sections. Since C4d staining corresponds to the classical pathway of complement activation, similar findings are expected with the IF staining method in fresh frozen tissue sections.

Urinary C-X-C Motif Chemokines 13 is a Noninvasive Biomarker of Antibody-Mediated Renal Allograft Rejection

Background Since acute rejection remains one of the major complications which necessitate periodic surveillance, noninvasive diagnostic/prognostic methods are preferred by renal transplant recipients. Here we explored whether urinary C-X-C motif chemokines 13(CXCL13) could be apotential candidate to reflect ongoing immune processes within the renal graft. Methods We investigated urinary CXCL13 levels by a cross-sectional analysisof 146 renal allograft recipients and 40 healthy controls. Besides, a subset of patients (n = 57) were followed-up for kinetic monitoring of immune status. Results Urinary CXCL13/Cr was lower in normal transplants compared to those withacute tubular necrosis (ATN, P = 0.001), chronic allograft nephropathy (CAN, P = 0.01) andacute rejection (AR, P < 0.0001), which yielded a good diagnosis performance of urinary CXCL13 for AR (AUC = 0.818, P< 0.0001). Interestingly, urinary CXCL13 furtherdistinguished acute antibody mediated rejection (ABMR)from acute cellular rejection, with an AUC of 0.856. Besides,patients with steroid-resistant acute rejection had distinctly greater urinary CXCL13/Cr levels than patients with reversible acute rejection, P =0.001. Follow-up data revealed that urinary CXCL13/Cr varied in line with the occurrence of ABMR. Furthermore, elevated levels of urinary CXCL13/Cr within the first monthwas predictive of graft function at 3, 6 months, P=0.044 and 0.4. Conclusion This study demonstrated that monitoring of urinary CXCL13/Cr might be a valuable noninvasive approach for the detection of AR, especially ABMR. Additionally, high urinary CXCL13/Cr levels related to the poor response to steroid treatment and predicted a compromised graft function after AR. Key Words:C-X-C motif chemokines 13; kidney transplantation; rejection; urine Key Laboratory of Multiple Organ Transplantation, Ministry ofHealth.

B Cell Activating Factor, Renal Allograft Antibody-Mediated Rejection, and Long-Term Outcome.

Antibody-mediated rejection (ABMR) of renal allograft lacks typical phenotypes and clinical manifestations, always resulting in delayed diagnosis and treatment. It has been considered to be an elemental factor influencing the improvement of the long-term outcome of renal allograft. The B cell activating factor (BAFF) signal plays a fundamental function in the process of antibody-mediated immune response. Data from recipients and the nonhuman primate ABMR model suggest that the BAFF signal participates in the ABMR of renal allograft, while there are objections. The challenges in the diagnosis of ABMR, different study population, and details of research may explain the discrepancy. Large quantities of dynamic, credible data of BAFF ligands and their association with renal allograft pathological characteristics would constitute a direct proof of the role of BAFF in the progression of renal allograft ABMR.

Evolving criteria for the diagnosis of antibody-mediated rejection in renal allografts.

To review changes in the Banff schema for antibody-mediated renal allograft rejection over the past decade, including key revisions agreed upon during and immediately subsequent to the 2017 Banff Conference on Allograft Pathology. The original Banff schema for diagnosis of acute and chronic active antibody-mediated rejection (ABMR) in renal allografts was formulated at the 2001 and 2007 Banff Conferences, and required histologic (primarily microvascular inflammation and transplant glomerulopathy), immunohistologic (C4d in peritubular capillaries), and serologic [circulating donor-specific antibodies (DSA)] evidence for a definitive diagnosis of ABMR. This schema was updated at the 2013 Banff Conference, recognizing C4d-negative ABMR, intimal arteritis as a potential manifestation of ABMR, and revising definitions and thresholds for glomerulitis and transplant glomerulopathy to improve interobserver agreement and correlation with clinical, molecular, and serologic data. Compared with the 2007 criteria, Banff 2013 improved the sensitivity of the classification for diagnosing ABMR and the correlation of ABMR diagnosis with graft outcomes. At the 2017 Banff Conference, new modifications to the classification were discussed and have subsequently been agreed upon, accepting C4d and thoroughly validated molecular classifiers as surrogate markers for DSA. From a consensus reached at the 2017 Banff Conference, updated criteria for diagnosis of active and chronic active ABMR have been developed that recognize C4d and molecular classifiers as surrogate markers for DSA. In addition, specific recommendations for the use of molecular diagnostics in the diagnosis of ABMR were developed.