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Background: Antibody-mediated rejection (AMR) remains associated with reduced kidney graft survival and no clear prognostic marker is available., Methods: We investigated whether donor-specific antibodies (DSA) ability to bind C1q in comparison with AMR C4d status, both indirect signs of complement activation, improve risk stratification at time of AMR. Hence, among 467 patients in whom 1 or more graft biopsies were performed between 2008 and 2015, we included 56 with AMR according to Banff '15 criteria. Using concurrent sera, we prospectively identified DSA by single-antigen beads (IgG and C1q) assays., Results: Antibody-mediated rejection C4d (+) (n = 28) was associated with preformed DSA (P = 0.007), whereas DSA C1q (+) (n = 25) cases had stronger IgG-DSA (P < 0.001). At AMR, graft function was similar between DSA C1q groups, but in the first year after, it improved in DSA C1q (-), whereas a steady decline was observed in DSA C1q (+) cases, remaining significantly lower from 1 year until 4 years after AMR. DSA C1q (+) was significantly associated with reduced graft survival (P = 0.021), whereas AMR C4d (+) was not (P = 0.550). Importantly, a similar negative impact of DSA C1q (+) on graft survival was observed within AMR C4d (+) (P = 0.040) and (-) (P = 0.036), cases. In multivariable analysis, DSA C1q (+) (hazard ratio, 3.939, P = 0.005) and de novo DSA (hazard ratio, 4.409, P = 0.033) were independent predictors of graft failure, but stronger IgG-DSA was not. Similar results were obtained considering C1q-DSA and IgG-DSA strength as continuous variables., Conclusions: C1q-DSA assessment at AMR can be a valuable tool in detecting patients with higher risk of graft failure.

Background: The progression from acute to chronic antibody-mediated rejection in kidney transplant recipients is usually not prevented by current therapeutic options. Here, we investigated whether the use of tofacitinib (TOFA), a Janus kinase 3 inhibitor, was capable of preventing the progression of allograft dysfunction in a Fisher-to-Lewis rat model of kidney transplantation., Methods: Rats were treated from the third week after transplantation to allow the development of rejection. Treatment was based on cyclosporin A, rapamycin or TOFA. Renal function was assessed at 1, 4, 8, and 12 weeks after transplantation, whereas rat survival, histological lesions, and infiltrating lymphocytes were analyzed at 12 weeks., Results: Tofacitinib prolonged graft survival, preserved tubular and glomerular structures and reduced humoral damage characterized by C4d deposition. Tofacitinib was able to reduce donor-specific antibodies. In addition, T and natural killer cell graft infiltration was reduced in TOFA-treated rats. Although rapamycin-treated rats also showed prolonged graft survival, glomerular structures were more affected. Moreover, only TOFA treatment reduced the presence of T, B and natural killer cells in splenic parenchyma., Conclusions: Tofacitinib is able to reduce the immune response generated in a rat model of kidney graft rejection, providing prolonged graft and recipient survival, better graft function, and less histological lesions.

Background: The differential pathogenicity of anti-HLA donor-specific antibodies (DSAs) is not fully understood. The presence of complement-binding DSAs helps in better defining the prognosis of acute antibody-mediated rejection (ABMR). The evolution of these antibodies after the treatment of ABMR is unknown., Methods: We included patients from the French multicenter RITUX ERAH study diagnosed with acute ABMR within the first year of renal transplantation, with circulating anti-HLA DSAs and treated randomly by rituximab or placebo (and intravenous immunoglobulins, plasma exchange). We centrally analyzed serum samples at the time of ABMR, 3 and 6 months after ABMR, with anti-HLA DSAs specificities and C1q-binding capacity assessment., Results: Twenty-five patients were included: 68% had C1q-binding DSAs at the time of ABMR. The presence of C1q-binding DSAs was associated with a poorer evolution of chronic glomerulopathy at 6 months (P = 0.036). The persistence of C1q-binding DSAs at 3 and/or 6 months after ABMR was associated with more severe chronic glomerulopathy (P = 0.006), greater C4d score deposition score at 6 months after ABMR (P = 0.008), and graft loss 5 years after ABMR (P = 0.029). C1q-binding capacity was associated with the DSA MFI but 5 C1q-binding DSAs in 4 patients had low MFI values without a prozone effect., Conclusion: The presence and persistence of anti-HLA C1q-binding DSAs after ABMR is a detrimental marker, leading to transplant glomerulopathy and graft loss. Assessment of the complement-binding capacities of DSAs could help decide treatment intensification.

Background: Candidates for vascularized composite allotransplantation (VCA) are frequently sensitized, putting them at risk for antibody-mediated rejection. Current desensitization strategies are imperfect and require a living-donor setting. Here we investigated the impact of sensitization on and the efficacy of a desensitization protocol utilizing syngeneic hematopoietic stem cell transplantation (HSCT) to prevent antibody-mediated rejection in VCA., Methods: Skin transplants from Dark Agouti to Lewis rats were performed for sensitization. Orthotopic hind limb transplants from Dark Agouti donors were performed to sensitized and nonsensitized recipients, and the animals were treated with either daily tacrolimus or no immunosuppression. A desensitization protocol consisting of total body irradiation, fludarabine, and syngeneic HSCT was applied to sensitized animals. Graft rejection was monitored by clinical assessment and histological analysis. Serum levels of donor-specific antibodies (DSA IgG) were measured using flow cytometry., Results: Sensitized recipients exhibited accelerated rejection by 5.5 ± 1.2 days without immunosuppression and 10.2 ± 3.6 days with daily tacrolimus compared with 8.7 ± 1.2 days and longer than 30 days in nonsensitized recipients, respectively. Serum levels of DSA IgG were markedly elevated (37.3 ± 3.34-fold from baseline) in sensitized recipients after VCA and correlated with histologic evidence of rejection and C4d deposition. Desensitization significantly reduced DSA compared with sensitized controls (2.6 ± 0.5-fold vs 6.0 ± 1.2-fold, P < 0.01) and along with daily tacrolimus led to improved VCA survival longer than 30 days without evidence of C4d deposition (n = 6)., Conclusions: In summary, sensitization leads to accelerated rejection of VCA, and syngeneic HSCT combined with conventional immunosuppression effectively reduces DSA and improves allograft survival in sensitized rats.

Background: Recent data have shown an increased risk for rejection, fibrosis progression, and death in liver transplantation (LT) recipients with preformed or de novo HLA donor-specific alloantibodies (DSA). However, the role of non-HLA autoantibodies and the interaction between HLA DSA and non-HLA autoantibodies remains uncharacterized., Methods: We analyzed 1269 primary LT recipients from 1 of 2000 to 4 of 2009 with known HLA DSA status for angiotensin II type-1 receptor and endothelin-1 type A receptor autoantibodies pre-LT, and year 1 post-LT., Results: Preformed non-HLA autoantibodies alone did not impact outcomes. In multivariable modeling, the combination of preformed non-HLA autoantibodies and HLA-DSA were associated with an increased risk for death (hazard ratio [HR], 1.66; P = 0.02) especially if the HLA DSA was of the IgG3 subclass (HR, 2.28; P = 0.01). A single de novo non-HLA autoantibody was associated with an increased risk for T cell-mediated rejection or antibody-mediated rejection (68% vs 41%, P = 0.01) and fibrosis progression (HR, 1.84; P = 0.02). Biopsies with de novo non-HLA autoantibodies revealed a new sinusoidal C4d staining pattern when compared with HLA DSA (71% vs 3%; P < 0.001). Liver sinusoidal endothelial cell activation and stellate cell activation was increased in patients with non-HLA autoantibodies in the location of C4d positivity., Conclusions: A non-HLA autoantibody combined with a preformed HLA DSA is associated with an increased mortality risk. Isolated de novo anti-angiotensin II type-1 receptor and anti-endothelin-1 type A receptor autoantibodies are associated with an increased risk of rejection and fibrosis progression. The novel location of C4d staining in proximity to liver sinusoidal endothelial cell capillarization and stellate cell activation demonstrates allograft injury in proximity to non-HLA autoantibody binding.

Background: Antibody-mediated rejection (AMR) is a major cause of kidney allograft loss. Currently, AMR diagnosis relies on biopsy which is an invasive procedure. A noninvasive biomarker of acute AMR could lead to early diagnosis and treatment of this condition and improve allograft outcome. Microvesicles are membrane-bound vesicles released from the cell surface after injury. We hypothesized that because AMR is associated with allograft endothelial injury and C4d deposition, plasma microvesicles positive for endothelial (CD144) marker and C4d are increased in this condition., Methods: We studied microvesicle concentration in the plasma of 95 kidney transplant patients with allograft dysfunction and compared with 23 healthy volunteers. Biopsy diagnosis and scoring was performed using Banff classification., Results: In the 28 subjects with AMR, the density of C4d+/CD144+ microvesicles was on average 11-fold (P = 0.002) higher than transplant recipients with no AMR and 24-fold (P = 0.008) than healthy volunteers. Densities of C4d+ and C4d+/annexin V+ (C4d+/AVB+) microvesicles were also increased in AMR patients compared with no AMR and healthy subjects. C4d+/AVB+ microvesicles correlated with AMR biopsy severity. Nine patients with acute AMR that received treatment showed a mean 72% decrease (P = 0.01) in C4d+/CD144+ microvesicle concentration compared with pretreatment values., Conclusions: Quantification of plasma C4d+ microvesicles provides information about presence of AMR, its severity and response to treatment in transplant patients.

Background: Antibody-mediated rejection is a leading cause for renal transplant loss. Rodent models are useful to dissect pathomechanisms and to develop treatment strategies. Although used for decades as a model, glomerular histopathological findings of Fischer-344 kidneys transplanted into Lewis rats have never been comprehensively described., Methods: Kidneys from Fischer-344 rats were transplanted into Lewis rats as life-sustaining allografts without immunosuppression. Lewis isografts and normal Fischer-344 kidneys served as controls. Grafts were harvested at 9 days, 6 and 26 weeks. Histopathological examination included light microscopy, immunohistochemistry, and morphometry. Findings were compared with 51 human biopsies with transplant glomerulopathy., Results: Most glomerular findings in rat allografts resembled human acute and chronic antibody-mediated rejection with glomerulitis, microthrombosis, microaneurysms, glomerular hypertrophy, podocyte loss, glomerular basement membrane splitting, and secondary focal and segmental glomerulosclerosis. In line with previous reports on nonendothelial antigens, glomerular immunoglobulin and C4d deposition was mostly nonendothelial. Only in 26-week allografts, we found mesangial and subendothelial immune complex-type electron-dense deposits. Similar deposits were found in 8 of 51 human biopsies with transplant glomerulopathy after rigorous exclusion of immune complexes of other cause, particularly recurrent glomerulonephritis and hepatitis C., Conclusions: Thus, our model closely reflects the glomerular changes of acute antibody-mediated rejection in humans and of a special subset of human transplant glomerulopathy. The significance of alloimmune immune complex-type deposits in human transplants deserves further investigation.

The lung transplant community continues to struggle with the diagnosis and management of antibody-mediated rejection. The four diagnostic tenets of donor-specific antibodies, C4d staining, histopathologic changes, and allograft dysfunction, which were largely derived from the early Banff meetings on renal transplantation, have somewhat arbitrarily been applied to lung transplantation. With the passage of time, it is increasingly apparent that merits of these diagnostic pillars are less robust in lung transplantation. In this article, we summarize some of the controversies and challenges surrounding the diagnosis of antibody-mediated rejection in lung transplantation.

Background: Recipients of incompatible allografts are at increased risk of graft loss. We hypothesized that analysis of sequential biopsies from these grafts could define progression of graft lesions and identify features predictive of progression., Methods: We studied the time course of histologic injury in 745 kidney graft biopsies from 129 patients transplanted with a positive crossmatch human leukocyte antigen-incompatible kidney between 2000 and 2010 (follow-up of 1-9 years)., Results: Graft survival was 98% at 1 year and 80% at 5 years after transplantation. Throughout follow-up, 70% of patients experienced rejection, with 52% showing subclinical rejection in the first year. Cell-mediated rejection was more frequent than antibody-mediated rejection throughout follow-up. Transplant glomerulopathy (TxGN; cg≥1) developed in 47% of patients over the period of the study, as early as 3 months in a few patients. TxGN was preceded by glomerulitis in more than 90% of cases, with a median time interval of 12 months. Glomerulitis and detectable posttransplantation donor-specific antibodies were risk factors for TxGN (P<0.0001 and P<0.05). C4d-negative antibody-mediated rejection manifesting as capillaritis (g≥1 and ptc≥1) with detectable donor-specific antibodies was observed in some recipients (<20%). There was progressively higher average tubulointerstitial scarring (ci+ct) from 3 to 6 to 12 months (P<0.001)., Conclusions: Despite good graft survival, a significant incidence of biopsy-proven rejection occurred in this subset of closely monitored human leukocyte antigen-incompatible recipients throughout follow-up. Microcirculation inflammation, particularly glomerulitis, irrespective of C4d, is associated with a high risk of development of TxGN at 1 year.

Background: Mixed rejection in kidney transplantation consists of histologic and/or serological evidence of both cellular and humoral components. As it is not confined to a distinct category in the Banff classification, how to best manage these patients is not clear. The aim of this study was to determine the incidence and outcome of morphological T-cell-mediated rejection (TCMR) with a humoral component, defined as the presence of either DSA or C4d, compared with the outcome of pure TCMR., Methods: We retrospectively studied 922 consecutive renal transplant recipients and analyzed patients with TCMR according to the evidence of a humoral component., Results: A total of 147 cases of morphological TCMR were analyzed. Of these, 92 (62.6%) had "pure" TCMR and 55 (37.4%) had "mixed" TCMR on the index biopsy. On univariant analysis, diffuse C4d (odds ratio [OR]=10.9, 95% confidence interval [CI]=1.8-66.9, P=0.01) and DSA positivity at the time of index biopsy (OR=2.8, 95% CI=1.2-6.6, P=0.02) were associated with allograft loss, whereas arteritis (OR=0.5, 95% CI=0.2-1.2, P=0.11) and glomerulitis (OR=0.9, 95% CI=0.4-2.1, P=0.8) were not. Arteritis was associated with subsequent antibody-mediated rejection (OR=4.9, 95% CI=1.1-20.8, P=0.03), and glomerulitis was associated with the development of transplant glomerulopathy (OR=10.7, 95% CI=3.1-37.1, P<0.01). On the multivariate analysis, only patients with C4d and DSA were at risk of graft failure (OR=4.9, 95% CI=2.0-12.0, P<0.01) in the medium term., Conclusion: TCMR with a humoral component has a worse prognosis when compared with pure TCMR. As such, it is important to test for alloantibody in cases of morphological TCMR to optimize patient management. Such cases might benefit from more aggressive immunotherapy.

Background: Efforts to increase the specificity and sensitivity of human leukocyte antigen (HLA) antibody detection assays recently led to the establishment of two novel Luminex bead-based assays to detect complement-activating antibodies by the assessment of complement products C1q or C4d. Here, we present a systematic comparison of the four methods, complement-dependent lymphocytotoxicity (CDC) and C1q-, C4d-, and IgG-Luminex, to assess or predict the complement-binding capability of HLA IgG antibodies., Methods: Forty-five sera of highly immunized patients have been assessed by in-house modified C1q- and C4d-Luminex assays and compared with standard CDC and IgG-Luminex., Results: Antibody specificities assigned by the C1q- and C4d-Luminex assay revealed an excellent concordance of 94% and 97% for HLA class I and II, respectively. Complement-fixing HLA class II antibodies were found less frequently among IgG antibodies compared with class I. Both C1q- and C4d-Luminex detected, on average, three times more specificities than CDC. Although we found a high correlation of mean fluorescence intensity values between C1q- and C4d-Luminex assays, IgG mean fluorescence intensity was not a suitable surrogate marker for the prediction of complement binding., Conclusions: C1q- and C4d-Luminex assays are characterized by an increased sensitivity and specificity compared with CDC, the current standard in detecting complement-fixing HLA antibodies. Pretransplantation risk assessment for transplantation but also posttransplantation monitoring are important applications for both assays to improve overall allograft survival.

Background: 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., Methods: 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., Results: 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., Conclusions: 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.

Background: The causes of endothelial dysfunction after cardiac transplantation are unknown. Here, we have investigated whether the indirect alloimmune response mediates endothelial dysfunction in a major histocompatibility complex class I mismatch model., Methods: PVG.RT1 rat hearts were transplanted into thymectomized CD8 T-cell-depleted allogeneic (PVG.R8) or syngeneic (PVG.RT1) recipients. Alloantibody was assessed at 2, 4, and 8 weeks. Cardiac allograft vasculopathy, the nature of the inflammatory infiltrate, and origin of endothelial cells were examined at 1, 2, 4, and 8 weeks. Endothelial function was assessed by Langendorff preparations at 1, 2, and 4 weeks., Results: Recipients produced alloantibody and showed luminal occlusion at 1 (17.7%±8.0%), 2 (23.2%±4.9%), 4 (34.3%±5.0%), and 8 weeks (58.1%±1.8%) posttransplantation. The major inflammatory features of the allografts consisted of CD11b monocytes, CD4 T cells, and C4d deposition. At 1 week, the basal coronary flow and the vasodilator response to 5-hydroxytrytamine of syngeneic and allografted hearts were inhibited compared with normal hearts. At 4 weeks, the basal coronary flow of allografts was 54% lower than syngrafts (P<0.01), and 5- hydroxytrytamine and sodium nitroprusside did not evoke an increase in coronary flow in the allograft heart compared with syngeneic controls (P<0.01). Culture of aortic rings with antibody to major histocompatibility complex class I inhibited endothelium-dependent vasodilation to acetylcholine., Conclusion: Transient microvascular endothelial dysfunction occurred in syngeneic and allogeneic cardiac grafts after transplantation. Syngeneic but not allogeneic grafts recovered, suggesting the indirect immune response, consisting of CD4 T cells, monocytes, and antibody, mediates endothelial dysfunction. A possible role for alloantibody in endothelial dysfunction is discussed.

Background: Production of antibodies against donor-specific antigens is one of the central mechanisms of allograft rejection. This antibody-mediated rejection (AMR) is evidenced by the presence of circulating donor-specific antibodies and deposition of complement component C4d on renal endothelium. Although anti-human leukocyte antigen (HLA) antibodies account for a high proportion of AMR, in many cases anti-HLA antibodies cannot be demonstrated. In liver transplant, antibodies against glutathione-S-transferase T1 (GSTT1) expressed on the graft may induce an antibody response leading to a severe graft dysfunction. In addition, presence of antibodies against major-histocompatibility-complex class I chain-related gene A (MICA) has been associated with a poor graft survival in kidney transplantation., Methods: Pre- and posttransplantation sera from 19 patients fulfilling the criteria for AMR including C4d deposition in renal biopsies were included. Donor-specific antibodies against HLA-I and -II and MICA were studied using Luminex. Anti-GSTT1 antibodies were analyzed by indirect immunofluorescence and by an ELISA method. A control group of 39 patients with graft dysfunction negative for C4d was also included., Results: At the time of the biopsy, 4 (21%) patients had only anti-HLA class I antibodies; 3 (15.8%) had anti-GSTT1, 2 (10.5%) had anti-HLA-class II, and 2 (10.5%) had anti-MICA; four patients had combination of antibodies: HLA-I + MICA (n=1), HLA-I + GSTT1 (n=2), and GSTT1+MICA (n=1). No antibodies were found in 4 (21%) patients. In total, 6 (31.6%) C4d+ patients had anti-GSTT1 antibodies, whereas, among the 39 C4d-negative patients, only 3 (7.7%) had anti-GSTT1 antibodies (P=0.027)., Conclusion: Besides anti-HLA antibodies, donor-specific antibodies against MICA and GSTT1 antigens could be responsible for the occurrence of antibody-mediated kidney graft rejection.

Background: Chronic rejection (CR) is an important cause of kidney graft loss. Some studies have suggested the role of antibodies mediating chronic graft dysfunction. In this context, C4d identification is an important tool to evaluate antibody-mediated rejection., Method: This is a retrospective study that analyzed 80 patients with histological diagnosis of chronic allograft nephropathy (CAN) according Banff 97 and no evidence of transplant glomerulopathy. These patients had renal biopsies available for C4d immunoperoxidase staining at the time of diagnosis. Cases were reclassified by the presence of C4d in peritubular capillaries., Results: C4d was negative in 30 cases (37.5%) and positive in 50 (62.5%). C4d+ group had more female and highly sensitized patients (PRA) at transplant. All variables were similar between C4d- and C4d+ cases at diagnosis time, but more C4d+ patients presented proteinuria (>0.3 g/L). Patients were submitted to various immunosuppression regimens after the CAN diagnosis. Four years after the diagnosis, death-censored graft survival was 87% for C4d- and 50% for C4d+ (P=0.002). In the multivariate Cox regression analysis, C4d+, PRA>10%, and vascular intimal proliferation were the variables that present higher relative risk for graft loss., Conclusion: These data indicate that C4d positive chronic rejection is very common, associated with proteinuria, and has a poor outcome. A larger study is warranted to identify which immunosuppressive regimen may modify the poor course of this entity.

It is suggested that non-HLA endothelial antigens may also cause C4d-positive acute rejection, but this is very rare. We report on three renal allograft recipients who developed C4d-positive acute rejection with detectable circulating antiendothelial cell antibodies (AECAs). All patients had severe dialysis-dependent graft dysfunction. Histologic manifestations include neutrophils infiltration on peritubular capillaries and glomeruli. Endoarteritis can be seen in all the patients. Two patients lost grafts after the rescue therapy including immunoadsorption, mycophenolate mofetil with or without tacrolimus. The titer variation of AECAs might be associated with the graft outcome. In patients those who lost grafts, AECAs titer increased from 1:10 to 1:80 in one patient, and was kept positive during the treatment in the other patient. In the recovered patient, however, the titer became negative from 1:40. From our report, it appears that persisting circulating AECAs during the rescue treatment of C4d-positive acute rejection may be associated with a poor outcome.