C4d-positive Antibody-mediated Rejection Research Articles

Preformed Angiotensin II Type-1 Receptor Antibodies Are Associated With Rejection After Kidney Transplantation: A Single-Center, Cohort Study

Antibodies against angiotensin II type-1 receptors (AT1R) have been increasingly recognized in association with rejection and poor allograft outcomes. Our goal was to define the prevalence of preformed antibodies against AT1R and evaluate the association with renal allograft outcomes in a consecutive cohort of 150 transplant recipients. IgG antibodies against AT1R were measured by enzyme-linked immunosorbent assay using cryopreserved serum samples obtained for HLA testing at the time of transplantation. Results were categorized as negative if <10 U/mL (44%), intermediate from 10 to 17 U/mL (38%), or strongly positive if >17 U/mL (18%). The presence of AT1R antibodies was inversely associated with age, dialysis status, and diabetes. We found a strong association between the presence of AT1R antibodies and acute cellular rejection using multivariate analyses, odds ratio 3.86 (95% CI, 1.03–14.47) for intermediate titers and 9.99 (95% CI, 2.6–38.4) for strongly positive titers. There was no association with HLA sensitization or C4d-positive antibody-mediated rejection. We did not observe a significant association with graft failure, allograft function, or proteinuria. Preformed AT1R antibodies are prevalent and highly associated with acute cellular rejection early after transplant, independent of anti-HLA antibodies. The presence of AT1R antibodies correlates with recipient characteristics that may denote stronger immune responses. Future studies are needed to evaluate the mechanism and causative effect of AT1R antibodies.

OR20 Characterization of de novo donor-specific HLA ANTIBODIES AND correlation with C4d+ antibody-mediated rejection of kidney transplants

Characteristics of de novo donor-specific HLA antibodies (dnDSA) that are associated with C4d positive antibody-mediated rejection (C4d+ AMR) in kidney transplants have not been well defined. We determined the nature of dnDSA, their capacity to bind complement components (C1q and C3d), and their correlation with biopsy proven C4d+ AMR in kidney transplant recipients. Sera from 120 kidney transplant recipients (68 males; 14 re-Tx; 43 LD), who had dnDSA (by One Lambda single antigen beads) at the time of a protocol or for-cause kidney biopsy (median 3.8 yrs post-tx), were tested for C1q (One Lambda) and C3d (Immucor) binding dnDSA. Results were assessed using two-tailed Fisher’s exact probabilities. Most of the recipients had a single DSA (53.3%) while 23.3% had 2 DSAs and 23.3% had >2 DSAs. Most of the DSA were directed against HLA class II (103/120; 86%). The strongest DSAs in most patients were directed to DQ antigens (80/120; 67%). Fifty-seven patients (48%) had C3d-binding dnDSA, and sixty-seven patients (56%) had C1q-binding DSA. Most of the C3d and C1q-binding dnDSA had high MFI (13932 ± 5278 and 14144 ± 5363, respectively) compared with the C3d/C1q-negative dnDSA (5970 ± 3347). Sixty-three patients (52.5%) were diagnosed with C4d+ AMR (Table). Most of the C4d+ AMR patients had C3d (63.5%) and C1q (66.7%) binding dnDSA compared to the AMR negative group, and the differences were statistically significant. Furthermore, the patients with C3d and C1q-binding dnDSA had higher C4d scores on their biopsy. Most dnDSA in kidney transplant are directed against HLA-DQ molecules. There is a strong correlation between dnDSA with MFI > 10000 and C3d/C1q-binding. The presence of C3d/C1q-binding MFI > 10000 dnDSA is associated with a greater incidence of AMR. In addition, kidney recipients with high MFI/C3d/C1q-binding dnDSA had higher score of C4d deposition on the biopsy.Download : Download high-res image (55KB)Download : Download full-size image

(446) - Metabolic Profile of Broncho-Alveolar Lavage Fluid in Lung Transplant Recipients Suggests Potential Early Biomarkers of Bronchiolitis Obliterans Syndrome

Purpose: The definition of antibody-mediated rejection (AMR) after lung transplantation (LT) has undergone revision as recognition of this entity has increased. Although considered a key criterion for the diagnosis of AMR, C4d deposition has been an inconsistent finding. We compared outcomes of LT recipients who developed C4d-negative AMR to those who developed C4d-positive AMR. Methods: We performed a retrospective cohort study of patients transplanted between 1/2006 and 7/2015, and identified those who developed AMR defined as acute allograft dysfunction, DSA, and a lung injury pathology. We excluded those who did not have C4d staining at the time of diagnosis (n= 20). We grouped those who developed AMR into a C4d-positive cohort and a C4d-negative cohort and compared outcomes of the 2 groups using Kaplan-Meier method and the log rank test. Results: 37 patients developed AMR during the study period; 15 developed C4d-positive AMR and 22 developed C4d-negative AMR. All patients with AMR were treated with IVIG. Of these, 36 were treated with rituximab, 7 with bortezomib, and 9 with plasma exchange (PLEX) (Table 1). Survival and CLAD-free survival after the diagnosis of AMR were poor in both groups, and there was no significant difference in either survival or CLAD-free survival between the 2 groups (Figure 1). Conclusion: A large proportion of patients with AMR in this cohort did not have C4d deposition. However, there was no significant difference in outcome between those who had C4d-negative and those who had C4d-positive AMR. These findings suggest that C4d deposition may not be a necessary criterion for the diagnosis of AMR.

Presentation and Outcomes of C4d-Negative Antibody-Mediated Rejection After Kidney Transplantation

The updated Banff classification allows for the diagnosis of antibody-mediated rejection (AMR) in the absence of peritubular capillary C4d staining. Our objective was to quantify allograft loss risk in patients with consistently C4d-negative AMR (n = 51) compared with C4d-positive AMR patients (n = 156) and matched control subjects without AMR. All first-year posttransplant biopsy results from January 2004 through June 2014 were reviewed and correlated with the presence of donor-specific antibody (DSA). C4d-negative AMR patients were not different from C4d-positive AMR patients on any baseline characteristics, including immunologic risk factors (panel reactive antibody, prior transplant, HLA mismatch, donor type, DSA class, and anti-HLA/ABO-incompatibility). C4d-positive AMR patients were significantly more likely to have a clinical presentation (85.3% vs. 54.9%, p < 0.001), and those patients presented substantially earlier posttransplantation (median 14 [interquartile range 8-32] days vs. 46 [interquartile range 20-191], p < 0.001) and were three times more common (7.8% vs 2.5%). One- and 2-year post-AMR-defining biopsy graft survival in C4d-negative AMR patients was 93.4% and 90.2% versus 86.8% and 82.6% in C4d-positive AMR patients, respectively (p = 0.4). C4d-negative AMR was associated with a 2.56-fold (95% confidence interval, 1.08-6.05, p = 0.033) increased risk of graft loss compared with AMR-free matched controls. No clinical characteristics were identified that reliably distinguished C4d-negative from C4d-positive AMR. However, both phenotypes are associated with increased graft loss and thus warrant consideration for intervention.

C4d Negative Antibody Mediated Rejection(AMR) Has a Similar Prognosis as C4d Positive AMR in Desensitized Live Donor Kidney Transplant Recipients.

INTRO:AMR is characterized by graft dysfunction, donor specific antibodies(DSA), microcirculatory inflammation, and evidence of complement activation in peritubular capillaries(C4d). Many of our positive crossmatch patients have histologic features of AMR but are C4d neg. We reviewed our live donor +XM cohort to identify whether AMR outcomes differed by C4d pos and C4d neg status. METHODS/RESULTS: We performed 58 +XM kidney transplants from 9/2004 to 12/2013. All received a minimum of two pre- and post- txp plasmapheresis with IVIG replacement (100mg/kg) and maintenance immunosuppression with tacrolimus, mycophenolate, and steroids. Induction therapy varied, and included either an IL-2 receptor blocker or Thymoglobulin® with or without intra-op rituximab, and IV methylpred with taper to pred 20 mg by post-op day 4. AMR was defined as graft dysfunction, PTCitis and +DSA testing by solid phase assay or XM. 30 +XM patients met these criteria for AMR as their first rejection episode -- 15 were C4d neg and 15 were C4d pos.(see table) Both groups were similar in terms of age, gender, sensitization history, and CDC vs flow +XM. Time to rejection was shorter in the C4d neg cohort (6 vs. 10 days, p=0.01), however, response to treatment, cellular rejections, recurrent rejections, and renal function were similar between groups. More C4d neg AMR grafts failed, but this difference was not statistically significant.Table: No Caption available.CONCLUSION: Our data suggest that desensitized patients with AMR have similar outcomes regardless of C4d staining. Patients with features of AMR and negative C4d stain should be considered for DSA testing. Diagnostic criteria of AMR may need modified to include patients with negative C4d testing.

Pathology of C4d-negative antibody-mediated rejection in renal allografts

To summarize current evidence supporting the existence of C4d-negative antibody-mediated rejection (AMR) in renal allografts, its potential to cause chronic graft injury, and whether histopathologic features of C4d-negative AMR differ from those of C4d-positive AMR. Recently published molecular, clinicopathologic, and ultrastructural studies provide strong evidence that microvascular injury in the presence of donor-specific alloantibodies (DSA) has the potential to cause interstitial fibrosis/tubular atrophy, transplant glomerulopathy, and graft loss, whether or not peritubular capillary (PTC) C4d is present. Although C4d-positive AMR may represent a more severe form of AMR, recent studies have found that in patients with DSA, microvascular injury (glomerulitis, peritubular capillaritis) is more strongly associated with graft loss than C4d deposition. Our data suggest that C4d-positive and C4d-negative AMR show similar degrees of glomerulitis and peritubular capillaritis, similar frequencies of concurrent cell-mediated rejection, and that both may occur early or late posttransplantation. In renal allografts, microvascular injury in the presence of DSA but with negative C4d staining in PTC nonetheless is indicative of humorally mediated graft injury that has the potential to cause tubular atrophy/interstitial fibrosis, transplant glomerulopathy, and graft loss. Prompt treatment for AMR may prevent or at least delay subsequent development of transplant glomerulopathy.

Solid phase detection of C4d-fixing HLA antibodies to predict rejection in high immunological risk kidney transplant recipients

Protocols for recipient desensitization may allow for successful kidney transplantation across major immunological barriers. Desensitized recipients, however, still face a considerable risk of antibody-mediated rejection (AMR), which underscores the need for risk stratification tools to individually tailor treatment. Here, we investigated whether solid phase detection of complement-fixing donor-specific antibodies (DSA) has the potential to improve AMR prediction in high-risk transplants. The study included 68 sensitized recipients of deceased donor kidney allografts who underwent peritransplant immunoadsorption for alloantibody depletion (median cytotoxic panel reactivity: 73%; crossmatch conversion: n=21). Pre and post-transplant sera were subjected to detection of DSA-triggered C4d deposition ([C4d]DSA) applying single-antigen bead (SAB) technology. While standard crossmatch and [IgG]SAB testing failed to predict outcomes in our desensitized patients, detection of preformed [C4d]DSA (n=44) was tightly associated with C4d-positive AMR [36% vs. 8%, P=0.01; binary logistic regression: odds ratio: 10.1 (95% confidence interval: 1.6-64.2), P=0.01]. Moreover, long-term death-censored graft survival tended to be worse among [C4d]DSA-positive recipients (P=0.07). There were no associations with C4d-negative AMR or cellular rejection. [C4d]DSA detected 6months post-transplantation were not related to clinical outcomes. Our data suggest that pretransplant SAB-based detection of complement-fixing DSA may be a valuable tool for risk stratification.

Concurrent Acute Cellular Rejection Is an Independent Risk Factor for Renal Allograft Failure in Patients With C4d-Positive Antibody-Mediated Rejection

Identification of risk factors for renal allograft failure after an episode of acute antibody-mediated rejection (AMR) may help the outcome of this difficult-to-treat complication. During December 2003 to February 2011, 833 kidney graft recipients underwent 1120 clinically indicated biopsies at our center. We reviewed the biopsy results and identified 87 biopsy specimens from 87 patients positive for the degradation product of complement component 4 (C4d) and acute AMR. We generated Kaplan-Meier survival curves and performed a multivariable analysis using the Cox proportional hazards regression model to identify risk factors for allograft failure after C4d+ acute AMR. Among the 87 patients, 26 had a diagnosis of acute AMR according to the Banff '09 classification schema, 29 had acute AMR and chronic active AMR, 18 had acute AMR and acute T-cell mediated rejection (TCMR), and 14 had acute AMR, chronic active AMR, and acute TCMR. Kaplan-Meier survival estimates showed that concurrent acute TCMR (P=0.001, Mantel-Cox log-rank test), concurrent chronic active AMR (P=0.03), and time to biopsy (P=0.04) are associated with graft survival. The Cox proportional hazards regression analysis identified that concurrent acute TCMR (hazard ratio, 2.59 [95% confidence interval, 1.21-5.55]; P=0.01) and estimated glomerular filtration rate (hazard ratio, 0.65 [95% confidence interval, 0.48-0.88]; P=0.01) are independent risk factors for allograft loss. Concurrent chronic active AMR or time to biopsy was not associated with graft failure by the multivariable Cox analysis. Our single-center study has elucidated that concurrent acute TCMR in kidney transplant recipients with C4d+ acute AMR is an independent risk factor for graft failure. Level of allograft function at the time of diagnosis was also an independent predictor of graft loss.

Endothelial transcripts uncover a previously unknown phenotype: C4d-negative antibody-mediated rejection

In the last decade, there has been a growing recognition of alloantibody responses in organ transplantation, but phenotypes related to antibody-mediated rejection (ABMR) remain incompletely defined. This article reviews recent molecular studies in kidney allograft tissues that decipher molecular burden and mechanisms of ABMR, leading to discovery of a new phenotype: 'C4d-negative ABMR'. High endothelial gene expression in kidney transplant biopsies with anti-human leukocyte antigen alloantibody indicates active antibody-mediated damage and poor graft survival, defining a previously unknown group of C4d-negative ABMR. C4d-negative ABMR is characterized by high intragraft endothelial gene expression, alloantibodies, histology of chronic ABMR (less frequently acute ABMR), and poor outcomes. Thus, endothelial molecular phenotype in biopsies with circulating antibody detects degree of active graft injury, and many of these transcripts reflect endothelial activation. C4d-negative ABMR is twice as common as C4d-positive ABMR. Recognition of this new phenotype reveals ABMR (C4d positive or negative) as the most common cause of late kidney transplant loss. C4d staining, although very useful, is insensitive for detecting ABMR. Measuring endothelial gene expression in biopsies from kidneys with alloantibody is a sensitive and specific method to diagnose ABMR and predict graft outcomes.

In Vitro Detection of C4d-Fixing HLA Alloantibodies: Associations With Capillary C4d Deposition in Kidney Allografts

Capillary C4d deposition is a valuable marker of antibody-mediated rejection (AMR). In this analysis, flow cytometric detection of alloantibody-triggered C4d deposition to HLA antigen-coated microparticles ([C4d]FlowPRA) was evaluated for its value as a marker for C4d deposition in renal allografts. For comparative analysis, 105 first renal biopsies performed for graft dysfunction and an equal number of concurrent sera were subjected to immunohistochemistry and [C4d] plus standard [IgG]FlowPRA, respectively. C4d deposition/fixation was detected in 17 biopsies and, applying [C4d]FlowPRA HLA class I and II screening, also in a small number of corresponding sera (N = 20). IgG reactivity detected by standard [IgG]FlowPRA was more frequent (49% of sera). Comparing [C4d]FlowPRA screening with capillary C4d staining, we found a high level of specificity (0.92 [95% confidence interval: 0.86-0.98]), which far exceeded that calculated for [IgG]FlowPRA (0.60 [0.50-0.70]). [IgG]FlowPRA screening, however, turned out to be superior in terms of sensitivity (0.94 [0.83-1.05] vs. 0.76 [0.56-0.97] calculated for C4d-fixing panel reactivity). Remarkably, posttransplant single antigen testing for identification of complement-fixing donor-specific alloreactivities failed to improve the predictive value of FlowPRA-based serology. In conclusion, our results suggest that detection of complement-fixing HLA panel reactivity could provide a specific tool for monitoring of C4d-positive AMR.

Immunoadsorption in Severe C4d-Positive Acute Kidney Allograft Rejection: A Randomized Controlled Trial

Antibody-mediated rejection (AMR) frequently causes refractory graft dysfunction. This randomized controlled trial was designed to evaluate whether immunoadsorption (IA) is effective in the treatment of severe C4d-positive AMR. Ten out of 756 kidney allograft recipients were included. Patients were randomly assigned to IA with protein A (N = 5) or no such treatment (N = 5) with the option of IA rescue after 3 weeks. Enrolled recipients were subjected to tacrolimus conversion and, if indicated, 'anti-cellular' treatment. All IA-treated patients responded to treatment. One death unrelated to IA occurred after successful reversal of rejection. Four control subjects remained dialysis-dependent. With the exception of one patient who developed graft necrosis, non-responders were subjected to rescue IA, however, without success. Because of a high graft loss rate in the control group the study was terminated after a first interim analysis. Even though limited by small patient numbers, this trial suggests efficiency of IA in reversing severe AMR.