Antibody-mediated Renal Allograft Rejection Research Articles

Integrative metagenomic and metabolomic analyses reveal the role of gut microbiota in antibody-mediated renal allograft rejection

BackgroundAntibody-mediated rejection (AMR) remains one of the major barriers for graft survival after kidney transplantation. Our previous study suggested a gut microbiota dysbiosis in kidney transplantation recipients with AMR. However, alternations in gut microbial function and structure at species level have not been identified. In the present study, we investigated the metagenomic and metabolic patterns of gut microbiota in AMR patients to provide a comprehensive and in-depth understanding of gut microbiota dysbiosis in AMR.MethodsWe enrolled 60 kidney transplantation recipients, 28 showed AMR and 32 were non-AMR controls with stable post-transplant renal functions. Shotgun sequencing and untargeted LC/MS metabolomic profiling of fecal samples were performed in kidney transplantation recipients with AMR and controls.ResultsTotally, we identified 311 down-regulated and 27 up-regulated gut microbial species associated with AMR after kidney transplantation, resulting in the altered expression levels of 437 genes enriched in 22 pathways, of which 13 were related to metabolism. Moreover, 32 differential fecal metabolites were found in recipients with AMR. Among them, alterations in 3b-hydroxy-5-cholenoic acid, l-pipecolic acid, taurocholate, and 6k-PGF1alpha-d4 directly correlated with changes in gut microbial species and functions. Specific differential fecal species and metabolites were strongly associated with clinical indexes (Cr, BUN, etc.), and could distinguish the recipients with AMR from controls as potential biomarkers.ConclusionsAltogether, our findings provided a comprehensive and in-depth understanding of the correlation between AMR and gut microbiota, which is important for the etiological and diagnostic study of AMR after kidney transplantation.

Safety, tolerability, and efficacy of monoclonal CD38 antibody felzartamab in late antibody-mediated renal allograft rejection: study protocol for a phase 2 trial

BackgroundAntibody-mediated rejection (ABMR) is a cardinal cause of renal allograft loss. This rejection type, which may occur at any time after transplantation, commonly presents as a continuum of microvascular inflammation (MVI) culminating in chronic tissue injury. While the clinical relevance of ABMR is well recognized, its treatment, particularly a long time after transplantation, has remained a big challenge. A promising strategy to counteract ABMR may be the use of CD38-directed treatment to deplete alloantibody-producing plasma cells (PC) and natural killer (NK) cells.MethodsThis investigator-initiated trial is planned as a randomized, placebo-controlled, double-blind, parallel-group, multi-center phase 2 trial designed to assess the safety and tolerability (primary endpoint), pharmacokinetics, immunogenicity, and efficacy of the fully human CD38 monoclonal antibody felzartamab (MOR202) in late ABMR. The trial will include 20 anti-HLA donor-specific antibody (DSA)-positive renal allograft recipients diagnosed with active or chronic active ABMR ≥ 180 days post-transplantation. Subjects will be randomized 1:1 to receive felzartamab (16 mg/kg per infusion) or placebo for a period of 6 months (intravenous administration on day 0, and after 1, 2, 3, 4, 8, 12, 16, and 20 weeks). Two follow-up allograft biopsies will be performed at weeks 24 and 52. Secondary endpoints (preliminary assessment) will include morphologic and molecular rejection activity in renal biopsies, immunologic biomarkers in the blood and urine, and surrogate parameters predicting the progression to allograft failure (slope of renal function; iBOX prediction score).DiscussionBased on the hypothesis that felzartamab is able to halt the progression of ABMR via targeting antibody-producing PC and NK cells, we believe that our trial could potentially provide the first proof of concept of a new treatment in ABMR based on a prospective randomized clinical trial.Trial registrationEU Clinical Trials Register (EudraCT) 2021-000545-40. Registered on 23 June 2021. ClinicalTrials.gov NCT05021484. Registered on 25 August 2021

Deletion of the Natural Killer Cell Receptor NKG2C Encoding KLR2C Gene and Kidney Transplant Outcome.

Natural killer (NK) cells may contribute to antibody-mediated rejection (ABMR) of renal allografts. The role of distinct NK cell subsets in this specific context, such as NK cells expressing the activating receptor NKG2C, is unknown. Our aim was to investigate whether KLRC2 gene deletion variants which determine NKG2C expression affect the pathogenicity of donor-specific antibodies (DSA) and, if so, influence long-term graft survival. We genotyped the KLRC2 wt/del variants for two distinct kidney transplant cohorts, (i) a cross-sectional cohort of 86 recipients who, on the basis of a positive post-transplant DSA result, all underwent allograft biopsies, and (ii) 1,860 recipients of a deceased donor renal allograft randomly selected from the Collaborative Transplant Study (CTS) database. In the DSA+ patient cohort, KLRC2 wt/wt (80%) was associated with antibody-mediated rejection (ABMR; 65% versus 29% among KLRC2 wt/del subjects; P=0.012), microvascular inflammation [MVI; median g+ptc score: 2 (interquartile range: 0-4) versus 0 (0-1), P=0.002], a molecular classifier of ABMR [0.41 (0.14-0.72) versus 0.10 (0.07-0.27), P=0.001], and elevated NK cell-related transcripts (P=0.017). In combined analyses of KLRC2 variants and a functional polymorphism in the Fc gamma receptor IIIA gene (FCGR3A-V/F158), ABMR rates and activity gradually increased with the number of risk genotypes. In DSA+ and CTS cohorts, however, the KLRC2 wt/wt variant did not impact long-term death-censored graft survival, also when combined with the FCGR3A-V158 risk variant. KLRC2 wt/wt may be associated with DSA-triggered MVI and ABMR-associated gene expression patterns, but the findings observed in a highly selected cohort of DSA+ patients did not translate into meaningful graft survival differences in a large multicenter kidney transplant cohort not selected for HLA sensitization.

Functional Fc Gamma Receptor Gene Polymorphisms and Long-Term Kidney Allograft Survival

The functional Fc gamma receptor (FcγR) IIIA polymorphism FCGR3A-V/F158 was earlier suggested to determine the potential of donor-specific HLA antibodies to trigger microcirculation inflammation, a key lesion of antibody-mediated renal allograft rejection. Associations with long-term transplant outcomes, however, have not been evaluated to date. To clarify the impact of FCGR3A-V/F158 polymorphism on kidney transplant survival, we genotyped a cohort of 1,940 recipient/donor pairs. Analyzing 10-year death-censored allograft survival, we found no significant differences in relation to FCGR3A-V/F158. There was also no independent survival effect in a multivariable Cox model. Similarly, functional polymorphisms in two other activating FcγR, FCGR2A-H/R131 (FcγRIIA) and FCGR3B-NA1/NA2 (FcγRIIIB), were not associated with outcome. There were also no significant survival differences among patient subgroups at increased risk of rejection-related injury, such as pre-sensitized recipients (> 0% panel reactivity; n = 438) or recipients treated for rejection within the first year after transplantation (n = 229). Our study results suggest that the earlier shown association of FcγR polymorphism with microcirculation inflammation may not be strong enough to exert a meaningful effect on graft survival.

Clinicopathological Analysis of Acute/Active Antibody-Mediated Rejection in Renal Allografts According to the Banff 2013 Classification

Aim: This study evaluated the clinicopathological findings of acute/active antibody-mediated rejection (AABMR) according to the Banff 2013 classification. Methods: We analyzed 345 biopsies of 269 kidney transplant recipients. Pathological AABMR (PAABMR) was defined as histological evidence of acute tissue injury and endothelial injury by light microscopy regardless of donor-specific antibodies (DSAs). Results: Among the 345 biopsies, 29 (8.4%) were diagnosed as PAABMR. The mean g score was 1.17 ± 0.60, the mean ptc score was 1.97 ± 1.32, and DSA positivity was found in 69% of PAABMR. The mean duration after transplantation was 22.9 ± 26.7 months. Among 3 groups (DSA-high, mean fluorescence intensity (MFI) ≥ 5,000; DSA-low, MFI < 5,000 to ≥1,000; below cutoff), ABO incompatibility in DSA-high was significantly lower and second transplantation in DSA-high was significantly higher. We found 83% of PAABMR by the protocol biopsy (subclinical AABMR [SAABMR]). The short-term clinical and light microscopical changes in 8 cases of SAABMR did not show worsening during follow-up period (9–24 months). However, ultrastructural finding, including glomerular endothelial swelling, subendothelial electron-lucent widening, and early glomerular basement duplication, were found by electron microscopy (EM) in the first biopsies, and half of the SAABMR cases developed de novo circular peritubular capillary multilayering in the follow-up biopsies. Conclusion: PAABMR was mainly found by the protocol biopsy. The short-term follow-up of SAABMR patients did not show worsening clinically and light microscopically, but ultrastructural examination by EM was useful to detect early lesions of endothelial injury and progression of glomerular and peritubular capillary basement membrane alterations.

Recipient LAG3 deficiency results in antibody-mediated rejection of mouse renal allografts

Abstract The functions of coinhibitory receptor lymphocyte activation gene-3 (LAG3) in T cells are well studied, however its role in humoral immune responses remains poorly characterized. The goal of this study was to test the role of recipient LAG3 in a mouse model of renal allograft rejection Compared to WT animals, naïve B6.LAG3−/− mice have increased splenic cellularity and higher frequencies of CD44hi memory T cells, CXCR5hi follicular T cells, and B220+CD138+plasma cells yet do not develop spontaneous autoimmunity. Furthermore, naïve B6.LAG3−/−(H-2Db) mice have increased frequencies of memory T cells against H-2Dd, H-2Ds, H-2Dq and H-2Dk alloantigens. C3H (H-2Dk) kidney allografts were transplanted into B6.WT or B6.LAG3−/− recipients after bilateral nephrectomy. Whereas 4/4 WT recipients accepted C3H allografts for longer than 60 d, recipient LAG3 deficiency led to rapid allograft rejection (MST of 14 d, n=5) with serum creatinine levels of 0.1 and 1.35 mg/dl respectively at d14 posttransplant. Graft histology at rejection revealed minimal T cell infiltration, diffuse C4d staining, atrophic peritubular capillaries, endothelial swelling and edema characteristic of antibody mediated rejection (AMR). Compared to WT, LAG3−/− recipients had elevated frequencies of anti-donor IFNg producing T cells and increased levels of anti-donor MHC-II antibodies. Recipient CD8 T cell depletion did not alter the rejection kinetics in LAG3−/− recipients (MST of 16 d), while B cell depletion significantly extended C3H kidney allograft survival (MST of &amp;gt;30 d). These results suggest the predominant role of alloantibody rather than T cells in renal allograft injury and identify LAG3 is a potential therapeutic target for AMR prevention and treatment.

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.

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.

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.

Donor Specific Antibody Shows Different IgG Subtype in Early versus Late Antibody Mediated Renal Allograft Rejection

Background Early and late antibody mediated rejection (EAMR/LAMR) are immunologically distinct entities and represent major risk factors for allograft survival in renal transplantation. The aim of this study was to describe the nature of donor-specific anti-HLA antibodies (DSA) that mediate EAMR and LAMR, and to correlate these data with DSA mean fluorescence intensity (MFI) response to proteasome-inhibitor (PI) therapy. Methods DSA levels (Day 0 and Day 50 therapy) were determined in 35 (11 EAMR + 24 LAMR) patients who received PI-based AMR therapy (1 or two bortezomib cycles +/- a single rituximab dose (375mg/m2)). All patients had at least 9 months of follow-up after AMR therapy. EAMR was defined as occurring within 6 months after transplantation. All donors and recipients had HLA-A,-B,-C,-DRB1, DRB3/4/5,-DQB1, DQA1 and DPB1 typing by molecular methods, and all cases were transplanted after negative prospective flow T- and B-cell crossmatches. AMR was diagnosed using Antibody Working Group and Banff criteria. Anti-HLA donor-specific antibody were identified by Luminex single-antigen beads (One Lambda) using both the generic IgG and IgG1-4 subtype-specific mAb (PE-conjugated, Southern Biotech). Epitope fingerprints were used to assess the therapeutic response (figures). Results 33 DSA were detected in EAMR (11 class I, 22 class II) and 52 DSA in LAMR (11 class I, 41 class II). DSA Class distribution was 43 anti-HLA class I, 27 anti-HLA DR, 50 anti-HLA DQ and 4 anti-HLA DP. Anti-HLA DQ DSAs were found in 3/11 EAMR vs 18/24 LAMR (p=0.01). IgG1 was detected in all patients, IgG2 was detected in 3/11 EAMR and 5/24 LAMR, IgG3 was present in 15/24 LAMR versus 0/11 EAMR (p=0.007), and IgG4 was detected in 2/11 EAMR vs 6/24 LAMR. There were 4/11 EAMR versus 16/24 LAMR patients with multiple IgG subtypes. The DSA MFI reduction at 50 days after PI therapy was 79±22% in EAMR versus 30±26% in LAMR (p=0.00001).. Summary Late AMR demonstrated: 1) a greater number of de novo DSAs; 2) a greater proportion of anti-HLA class II DSA, especially anti-HLA DQ; 3) a higher proportion of IgG3, the subtype with the highest affinity for complement (C1q) binding; 4) a higher degree of multiple IgG subtypes. 5) lesser response in DSA reduction with PI therapy.

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.

Clinical Significance of Macrophage Polarization in Antibody-Mediated Rejection of Renal Allograft

BackgroundThe significance of proinflammatory M1 (classically activated) and profibrotic M2 (alternatively activated) macrophages in antibody-mediated rejection (ABMR) after kidney transplantation has not been investigated. MethodsFifty-five biopsy-confirmed ABMR samples were stained with MRP 8/14 (a marker of M1 macrophages) and CD163 (a marker of M2 macrophages), and positive cells were counted in glomeruli and the tubulointerstitium, respectively. Patients were classified into M1 and M2 polarization groups according to the glomerular and tubulointerstitial M1:M2 ratio, and the results were compared with Banff scores, serum creatinine level, estimated glomerular filtration rate (eGFR), and graft survival. ResultsThe glomerular M2 polarization group showed significantly higher chronic glomerulopathy scores, serum creatinine levels, and lower eGFR at the time of biopsy (P = .019 and P = .015, respectively) and 3-month postbiopsy (P = .016 and P = .032, respectively) than the M1 polarization group. The tubulointerstitial M2 polarization group had significantly lower glomerulitis, arteritis, peritubular capillaritis, and glomerulitis + peritubular capillaritis scores than the M1 polarization group, but there was no significant difference in renal function. Long-term graft survival was not associated with macrophage polarization. ConclusionGlomerular M2 polarization in ABMR biopsy samples is associated with chronic glomerular injury and poorer graft function, but without graft survival.

Decreased Expression of MicroRNA-107 in B Lymphocytes of Patients with Antibody-Mediated Renal Allograft Rejection.

MicroRNAs (miRNAs) are small noncoding RNA molecules that participate in normal B cell lineage development through posttranscriptional gene regulation. Antibody-mediated renal allograft rejection (ABMR) is emerging as one of the most common serious threats to renal transplant patients. In this study, we explored the role of miRNAs in the pathogenesis of ABMR. The differentially expressed miRNAs were identified by Affymetrix miRNA microarray analysis using B lymphocytes from 5 recipients and 5 volunteers. Based on quantitative RT-PCR, the expression levels of miR-107 were lower in the B lymphocytes from recipients than in those from volunteers. Computational analysis predicted that 3'-untranslated region of the autophagy-related protein 12 (ATG12) mRNA was targeted by miR-107, and we identified ATG12 as a target of miR-107 by Luciferase assay. Importantly, the expression levels of ATG12 in B lymphocytes of recipients were higher than those in the volunteer group, and miR-107 mimic significantly decreased ATG12 expression and formation of autolysosomes in B lymphocytes of recipients. Furthermore, we observed that levels of autophagy in B lymphocytes of transplant recipients were higher than those in B cells from volunteers. These findings suggest that miR-107 may contribute to the regulation of autophagy via targeting ATG12. Lastly, treatment with an miR-107 mimic caused the decrease in the secretion of IgG and IgM antibodies from B lymphocytes of transplant recipients, indicating that deregulated miR-107 could be involved in the pathogenesis of ABMR. Taken together, we propose that decreased miR-107 expression is associated with autophagy activation in B lymphocytes from patients with ABMR.

Accelerated humoral renal allograft rejection due to HLA-C14 mediated allosensitization to HLA-Bw6

ObjectivesTo investigate immunological mechanisms underlying accelerated antibody-mediated rejection (AMR) of a living-related renal allograft in a patient with no detectable antibodies to donor human leukocyte antigens (HLA) in pre-transplant sera. MethodsPre- and post-transplant HLA antibody specificities were determined by single-antigen bead assay, and crossmatching was performed by flow cytometry- and complement-dependent cytotoxicity-based methods. Intermediate- and high-resolution HLA typing were performed by molecular methods. ResultsPre-transplant patient serum reacted weakly against Bw6-positive beads; cytotoxicity and flow crossmatches were negative. The patient was mismatched for the donor antigens B62 and C10 (Bw6-positive). Following transplantation, strong antibody responses against B62, C10, and all Bw6-positive beads were detected. This reactivity was initially masked by complement interference, but became apparent at 1:20 dilution. High-resolution typing suggested that the anti-C16 antibody reactivity detected was an allele-specific response to donor C∗16:01 (Bw6-positive) but not recipient C∗16:02 (Bw6-negative). Alloimmunization likely occurred during pregnancy, during which HLA-C14 (Bw6-positive) was the only mismatched paternal HLA Class I allele. ConclusionsSensitization to HLA-Bw6 via exposure to paternal HLA-C14 during pregnancy likely predisposed this patient to AMR. The case demonstrates the immunogenicity of HLA-C14-associated Bw6 epitopes in vivo and the clinical significance of low-level antibodies to HLA-Bw6.

Treatment of Acute Antibody-Mediated Renal Allograft Rejection With Cyclophosphamide.

Antibody-mediated rejection (AMR) is a major risk for renal allograft survival. Throughout decades, cyclophosphamide treatment has been proven to be effective in patients with antibody-associated autoimmune diseases. We investigated whether cyclophosphamide combined with plasmapheresis and intravenous immunoglobulins is an option for patients with AMR. Between March 2013 and November 2015, we initiated treatment of 13 consecutive patients with biopsy-proven acute AMR with intravenous cyclophosphamide pulses (15 mg/kg adapted to age and renal function) at 3-week intervals, PPH (6×), and high-dose intravenous immunoglobulin (1.5 g/kg). Treatment was completed after 6 cyclophosphamide pulses or in case of return to baseline serum creatinine together with reduction of donor-specific HLA antibodies (DSA) below 500 mean fluorescence intensity. Eleven of 13 patients completed treatment. Median follow-up was 18 (12-44) months. At the end of follow-up, graft survival was 77% (10/13). The 3 graft losses were caused at least in part by nonadherence and premature termination of treatment. Serum creatinine increased from 1.7±0.4 mg/dL at 3 months before diagnosis to 3.7±2.4 mg/dL at diagnosis (P = 0.01), and decreased to 2.1 ± 0.7 mg/dL at 3 months after diagnosis (P = 0.01). In 7 (64%) of 11 patients, who completed treatment, DSA decreased, in 4 (36%) of 11 DSA were below 500 mean fluorescence intensity after treatment. Dose reductions had to be performed in 3 of 13 patients for leukopenia. We observed 14 hospitalizations in 9 of 13 patients. To our knowledge, this is the first systematic report on cyclophosphamide-based treatment of acute AMR based on modern diagnostics. Treatment was effective and relatively safe. Future studies will show, whether cyclophosphamide proves to be a valuable alternative for the treatment of AMR.

OR16 Donor-specific antibodies (DSA) detected by C1Q and C3D assays predict antibody-mediated rejection (AMR) in renal transplant (TX) patients, but specific detection of IGG2, IGG3, IGG4, IGA, and IGM DSA, and ANTI-AT1R and anti-etar antibodies do not

Aim To investigate the specific roles for Class I and Class II DSA, complement binding and activation, and non-HLA antibodies, in renal tx patients with AMR. Methods One Lambda®: LABScreen™ (routine total IgG and modified to detect IgG1, IgG2, IgG3, IgG4, IgA, IgM); C1qScreen™; anti-AT1R; anti-ETAR. Immucor®: LIFECODES C3d. Assays were used to detect the presence of antibodies and DSA. The quantity of each antibody and capacity to bind and activate complement were correlated with the histopathologic features found in biopsy specimens. Among 480 renal transplants performed between 2009 and 2014, we enrolled 17 (∼4%) patients with clinical and histopathologic diagnosis of AMR, and compared with matched-controls who had anti-HLA antibodies and apparent DSA, but had not exhibited AMR. We assessed antibodies and DSA MFI values prior to transplantation and at the time of AMR. Results Detection of anti-AT1R, anti-ETAR, and Class I DSA did not correlate with AMR. Class II IgG2, IgG3, IgG4, IgA, and IgM DSA did not correlate with AMR. Class II DSA >500 MFI detected by LABScreen™ + C1q + IgG1 + C3d identified 82% of patients and only 12% of controls, p = 0.00009 (88% PPV, 17% NPV), whereas, C1q + C3d identified 71% of patients and no controls, p = 0.00002 (100% PPV, 23% NPV). Notable is that anti-HLA Class II DSA detected by C1q and C3d correlates with rejection, and the injury found on biopsies was primarily tubulointerstitial, consistent with the region where constitutive expression of HLA Class II occurs in renal tissue. Conclusions Clinically “reversible” AMR appears to be due to Class II anti-HLA DSA, which can bind and activate complement. Detection of anti-HLA DSA by the C1q and C3d assays correlate with histological characteristics (tubulointerstitial injury) of AMR in renal allografts, and indicates that monitoring for anti-HLA DSA, which can bind and activate complement, may be useful to detect patients at risk for AMR; thus allowing for earlier detection and treatment prior to graft injury or graft loss.