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Development of donor-specific antibodies (DSA) after renal transplantation is known to be associated with worse graft survival, yet determining which specificities in which recipients are the most deleterious remains under investigation. This study evaluated the relationship of the complement binding capacity of post-transplant de novo anti-human leukocyte antigen (HLA) antibodies with subsequent clinical outcome. Stored sera from 265 recipients previously identified as having de novo DSA were retested for DSA and their C3d binding capacity using Luminex-based solid-phase assays. Most recipients had anti-HLA class II-reactive DSA (class I = 12.5%, class II = 68.7%, class I and class II = 18.9%). The recipients that had C3d binding DSA (67.5%) had a significantly higher incidence of antibody-mediated rejection and any rejection. They also had significantly lower kidney survival, with the lowest survival in those that had both anti-HLA class I and class II C3d binding DSA. Concurrent biopsy comparison revealed a 96.2% positive predictive value and 47.4% negative predictive value for C4d peritubular capillary (Ptc) deposition. Anti-HLA class I and class II C3d binding DSA carried a twofold and 1.5-fold increased risk of kidney loss, respectively, in multivariate analysis., (© 2017 Steunstichting ESOT.)

Peritubular capillary C4d (ptc-C4d) usually marks active antibody-mediated rejection, while pseudolinear glomerular capillary C4d (GBM-C4d) is of undetermined diagnostic significance, especially when seen in isolation without concurrent ptc-C4d. We correlated GBM-C4d with structural GBM abnormalities and active antibody-mediated rejection in 319 renal transplant and 35 control native kidney biopsies. In kidney transplants, ptc-C4d was associated with GBM-C4d in 97% by immunofluorescence microscopy (IF) and 61% by immunohistochemistry (IHC; P < 0.001). Transplant glomerulopathy correlated with GBM-C4d (P < 0.001) and presented with isolated GBM-C4d lacking ptc-C4d in 69% by IF and 40% by IHC. Strong isolated GBM-C4d was found post year-1 in repeat biopsies with transplant glomerulopathy. GBM-C4d staining intensity correlated with Banff cg scores (rs = 0.45, P < 0.001). Stepwise exclusion and multivariate logistic regression corrected for active antibody-mediated rejection showed significant correlations between GBM duplication and GBM-C4d (P = 0.001). Native control biopsies with thrombotic microangiopathies demonstrated GBM-C4d in 92% (IF, P < 0.001) and 35% (IHC). In conclusion, pseudolinear GBM-C4d staining can reflect two phenomena: (i) structural GBM changes with duplication in native and transplant kidneys or (ii) active antibody-mediated rejection typically accompanied by ptc-C4d. While ptc-C4d is a dynamic 'etiologic' marker for active antibody-mediated rejection, isolated strong GBM-C4d can highlight architectural glomerular remodelling., (© 2017 The Authors. Transplant International published by John Wiley & Sons Ltd on behalf of Steunstichting ESOT.)

After kidney transplantation, C4d is an incomplete marker of acute antibody-mediated rejection (AMR) and C1q-binding donor-specific antibodies (DSA) have been associated with allograft survival. However, the impact on allograft survival of C1q+ DSA after clinical AMR has not been studied yet. We analysed retrospectively in clinical AMR C4d staining and C1q-binding impact on allograft survival. We compared clinical, histological and serological features of C4d- and C4d+ AMR, C1q+ and C1q- DSA AMR and analysed C4d and C1q-binding impact on allograft survival. Among 500 for-cause kidney allograft biopsies, 48 fulfilled AMR criteria. C4d+ AMR [N = 18 (37.5%)] have significantly higher number class I DSA (P = 0.02), higher microvascular score (P = 0.02) and more transplant glomerulopathy (P = 0.04). C1q+ AMR [N = 20 (44%)] presented with significantly more class I and class II DSA (P = 0.005 and 0.04) and C4d+ staining (P = 0.01). Graft losses were significantly higher in the C4d+ group (P = 0.04) but similar in C1q groups. C4d+ but not C1q+ binding was an independent risk factor for graft loss [HR = 2.65; (1.11-6.34); P = 0.028]. In our cohort of clinical AMR, C4d+ staining but not C1q+ binding is an independent risk factor for graft loss. Allograft loss and patient survival were similar in C1q+ and C1q- AMR., (© 2016 Steunstichting ESOT.)

Antibody-mediated rejection (AMR) occurs in 10-20% of patients after heart transplantation. C4d immunostaining is one parameter used in its diagnosis. This study aimed to determine whether C4d staining has prognostic significance for mortality, coronary allograft vasculopathy (CAV), cell-mediated rejection (CMR), and graft dysfunction in patients post-transplantation. Consecutive patients receiving an endomyocardial biopsy between 2007 and 2008 were selected. Left ventricular function, angiography, episodes of AMR/CMR, and death were noted. C4d was graded from 0 to 3 (immunostaining). Cox proportional models (recurrent events analysis) were used to evaluate C4d staining with mortality, graft dysfunction, CAV (≥grade 2), and episodes of ≥2R-CMR. We analyzed 2525 biopsy specimens (n = 217). During a follow-up of 4.5 ± 2 years, 35 died, 49 had graft dysfunction, seven had ≥grade 2 CAV, and 95 episodes of CMR occurred. A one-grade increase in C4d staining was associated with an increase in mortality (HR 1.57; 95% CI 1.0-2.5), a higher risk of CAV (HR 2.4, 95% CI 1.04-5.4), and a trend toward graft dysfunction (HR 1.42; 95% CI 1.0-2.09). C4d was not associated with CMR. C4d immunostaining was a significant predictor of CAV and death but not subsequent episodes of CMR. There was also a trend toward increased graft failure., (© 2015 Steunstichting ESOT.)

Histological evaluations of renal allograft biopsies are essential for diagnosis, but still show a low predictive value for long-term allograft function. One limitation relies on the fact that the analysis is usually based on a single biopsy sample, and therefore, no dynamic changes are considered. Using two distinct approaches, we evaluated the evolution of fibrosis and related markers in 36 stable kidney transplant patients under calcineurin inhibitor therapy with two indication biopsies each, prior and at least 6 months after substitution by mTORi (N = 18), or maintenance on CNI (N = 18). In the method comparison, both Banff chronicity score and the digitally assessed fibrosis were correlated with allograft function at biopsy (r = -0.36 and r = -0.72, P = 0.002 and P < 0.0001, respectively). However, only the progression of fibrosis digitally assessed was correlated with allograft function loss, not only within the time between biopsies (r = -0.47, P = 0.004) but also in the 60-month follow-up (r = -0.47, P = 0.006). In the group analysis, despite of a higher incidence of C4d positivity (P = 0.05), progression of fibrosis, TGF-β1 expression, and allograft function decline were significantly lower after conversion to mTORi compared with maintenance on CNI (P = 0.05, P = 0.02 and P = 0.01, respectively). PDGF, VEGF, b-FGF, and HIF1A expressions remained stable over time regardless of therapy., (© 2015 Steunstichting ESOT.)

While linear C4d staining in peritubular capillaries (PTC) is established as a marker of antibody-mediated rejection, the significance of a distinct granular C4d deposition pattern has not yet been clarified. In this study, 329 renal allograft recipients who underwent indication biopsies were analysed for immunohistochemical C4d staining characteristics. Fifty-six (17%) recipients showed granular C4d in PTC, without any relationship to conventional risk factors and morphological features of rejection. We found a strong association with long-term overall graft survival (7-year survival: 41% vs. 66% in granular C4d-negative subjects, P=0.001), which was mainly driven by a greater risk of mortality [hazard ratio: 3.12 (95% confidence interval: 1.23-7.94); P=0.02]. Granular C4d was associated with delayed graft function [39% vs. 22% (C4d-negative subjects), P=0.007], higher 1-year serum creatinine [median 2.1 (interquartile range: 1.7-2.6) mg/dl vs. 1.6 (1.3-2.0) mg/dl, P=0.001] and a trend towards worse death-censored graft survival (P=0.07). In support of a role of capillary immune complex formation, granular C4d was associated with electron-dense deposits in PTC basement membranes, which were occasionally accompanied by focally distributed capillary IgG deposits. In conclusion, our study suggests clinical relevance of detecting capillary granular C4d deposition. Our results point to a pathogenetic role of alloimmune-independent immune complex deposition., (© 2013 Steunstichting ESOT.)

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 6 months 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., (© 2012 The Authors Transplant International © 2012 European Society for Organ Transplantation. Published by Blackwell Publishing Ltd.)

Antibody-mediated hyperacute rejection was the first rejection phenotype observed in human organ transplants. This devastating phenotype was eliminated by reliable crossmatch technologies. Since then, the focus was on T-cell-mediated rejection and de novo donor-specific antibodies were considered an epiphenomenon of cognate T-cell activation. The immune theory was that controlling the T-cell response would entail elimination of antibody-mediated rejection (ABMR). With modern immunosuppressive drugs, T-cell-mediated rejection is essentially treatable. However, this did not prevent ABMR from emerging as a significant phenotype in all types of organ transplants. It became obvious that both rejection types require distinct treatment and thus reliable diagnosis. This is the current challenge. ABMR, depending on stage, grade, time course, organ type or prior treatment, can present with a wide spectrum of phenotypes. This review summarizes the current diagnostic consensus for ABMR, describes unmet needs and challenges in diagnostics, and proposes new approaches for consideration., (© 2012 The Authors. Transplant International © 2012 European Society for Organ Transplantation.)

The true incidence of positive C4d staining in the peritubular capillaries of biopsies with chronic allograft nephropathy (CAN) and transplant glomerulopathy (TGP) remains controversial. We retrospectively reviewed all transplant biopsies performed at Saint Louis University Hospital between June 2002 and May 2004. We examined the incidence of positive C4d staining in the peritubular capillaries of biopsy specimens with pure CAN with or without features of TGP. We identified 54 biopsies in 43 patients showing CAN. The average age was 46 +/- 13 years. The average creatinine at the time of biopsy was 308 +/- 211 micromol/l (3.5 +/- 2.4 mg/dl). Twenty (37%) biopsies exhibited features consistent with TGP. Only two biopsies had positive C4d staining in the peritubular capillaries. The C4d positive biopsies were from two different patients; one patient had donor specific antibodies (DSA) against HLA class 1 at the time of biopsy and the other patient had no detectable DSA. None of the TGP biopsies showed peritubular C4d staining. C4d staining of the peritubular capillaries appears to be rare in patients with pure CAN with and without TGP features.

CXCR3, a chemokine receptor mainly expressed by T cells, is involved in animal transplant models and in human allograft rejection. CXCR3 expression was localized in formalin-fixed, paraffin-embedded renal allograft biopsies without signs of rejection (C4d-negative, Banff 0, n = 16), with C4d deposits as a sign of humoral rejection (C4d-positive, Banff 0, n = 8), with cellular rejection (C4d-negative, Banff I, n = 7) and with signs of both cellular and humoral rejection (C4d-positive, Banff 1, n = 5). Small, round infiltrating cells were CXCR3-positive. A high number of these cells was present in biopsies with cellular rejection (independent of C4d deposition). CXCR3-positive cells diffusely infiltrated the interstitium, including the tubular epithelium (tubulitis). CXCR3 scores and the area of CXCR3 staining were significantly higher in cellular rejection, when compared to biopsies without rejection, and with deposition of C4d alone. CXCR3-positive cells infiltrate renal allografts during cellular rejection, whereas C4d deposition is not associated with the recruitment of these cells.

Post-transplant circulating anti-human leukocyte antigens (HLA)-antibodies and C4d in allograft biopsies may be important in chronic rejection in renal transplant recipients (RTR). We determined the prevalence and significance of anti-HLA-antibodies and donor-specific antibodies (DSA). Sera were collected from 251 RTR >6 months post-transplant. Sera were tested using enzyme-linked immunosorbent assay (ELISA) screening for anti-HLA antibodies. Positive sera were retested with ELISA-specific panel for antibody specificity. A 11.2% of patients had anti-HLA antibodies and 4.4% had DSA. Anti-HLA antibodies were significantly associated with pretransplant sensitization, acute rejection and in multivariate analysis, higher serum creatinine (2.15 +/- 0.98 vs. 1.57 +/- 0.69 mg/dl in negative anti-HLA antibodies group). Allograft biopsies performed in a subset of patients with anti-HLA antibodies revealed that 66% had C4d in peritubular capillaries (0% in patients without antibodies). Anti-HLA antibodies were associated with a worse allograft function and in situ evidence of anti-donor humoral alloreactivity. Long-term RTR with an increase in creatinine could be screened for anti-HLA antibodies and C4d in biopsy.

Cardiac allograft vasculopathy (CAV) is a long-term threat in heart transplant recipients and its exact pathogenesis remains to be established. As complement activation contributes to early and late allograft dysfunction, we hypothesized that deposition of the complement fragment, C4d, in capillaries of cardiac allograft biopsies may be associated with CAV. A polyclonal anti-C4d antibody was used for immunohistochemistry on endomyocardial biopsies obtained from heart transplant recipients during the first year post-transplantation. CAV was assessed by intracoronary ultrasound performed at 1-year post-transplantation. We were able to show that CAV is highly associated with C4d deposition in capillaries of cardiac allografts and that serial C4d studies may predict development of CAV at 1-year post-transplantation.

Acute humoral rejection (AHR) is currently perceived as an immunological reaction against donor antigens mediated by complement-binding antibodies. C4d, a split product of complement activation and bound to endothelial cells of the peritubular capillaries, is used as a diagnostic marker for AHR. We report on three patients with biopsy-proven acute humoral rejection who were treated initially with plasmapheresis (PS). As two of the patients did not recover renal function, and biopsy showed persistent C4d staining after PS, immunoadsorption (IAS) was additionally performed on them. In all patients, renal function recovered, and follow-up biopsies in two patients showed complete disappearance of C4d, 29 days and 58 days after transplantation and only minimal residual C4d deposits in one patient 48 days after transplantation. We conclude that successful treatment of AHR is followed by complete resolution of serological and histological markers of AHR, displayed by the disappearance of C4d.