Antibody‐mediated rejection (AMR) after pancreas and pancreas–kidney transplantation

Antibody‐mediated rejection (AMR) requires specific diagnostic tools and treatment and is associated with lower graft survival. We prospectively screened C4d in pancreas (n = 35, in 27 patients) and kidney (n = 33, in 21 patients) for cause biopsies. Serum amylase and lipase, amylasuria, fasting blood glucose (FBG) and 2‐h capillary glucose (CG) were also analysed. We found that 27.3% of kidney biopsies and 43% of pancreatic biopsies showed C4d staining (66.7% and 53.3% diffuse in peritubular and interacinar capillaries respectively). Isolated exocrine dysfunction was the main indication for pancreas biopsy (54.3%) and was followed by both exocrine and endocrine dysfunctions (37.1%) and isolated endocrine dysfunction (8.6%). Laboratorial parameters were comparable between T‐cell mediated rejection and AMR: amylase 151.5 vs. 149 U/l (P = 0.075), lipase 1120 vs. 1288.5 U/l (P = 0.83), amylasuria variation 46.5 vs. 61% (P = 0.97), FBG 69 vs. 97 mg/dl (P = 0.20) and 2‐h CG maximum 149.5 vs. 197.5 mg/dl (P = 0.49) respectively. Amylasuria values after treatment correlated with pancreas allograft loss (P = 0.015). These data suggest that C4d staining should be routinely investigated when pancreas allograft dysfunction is present because of its high detection rate in cases of rejection.     

Antibody‐mediated rejection despite inhibition of terminal complement

Terminal complement blockade has been shown to decrease the incidence of early acute antibody‐mediated rejection (eAMR) in the first month after positive cross‐match kidney transplant recipients, yet some patients still develop eAMR. The current study investigated possible mechanisms of eAMR despite eculizumab treatment. Of the 26 patients treated with eculizumab, two developed clinical eAMR and another patient developed histologic signs of eAMR without graft dysfunction (‘subclinical eAMR’). Twenty‐three did not have histologic injury on early surveillance biopsies. All 26 patients had therapeutic levels of eculizumab and showed complete blockade of complement in hemolytic assays. High levels of donor‐specific alloantibody (DSA) including total IgG, IgG3, and C1q+ DSA were present in patients with and without eAMR, and none correlated well with eAMR. In contrast, IgM DSA was present in only four patients after transplantation: the two patients with clinical eAMR, one patient with subclinical AMR, and one patient without eAMR (P = 0.006 correlation with eAMR). Both clinical eAMR episodes were easily treated with plasma exchange which removed IgM more completely and rapidly than IgG, resulting in normalization of function and histology. These data suggest a possible role of antidonor IgM DSA in the pathogenesis of eAMR in patients treated with terminal complement blockade (ClinicalTrials.gov Identifier: NCT00670774).     

Near‐infrared lymphography as a minimally invasive modality for imaging lymphatic reconstitution in a rat orthotopic hind limb transplantation model

Wider application of vascularized composite allotransplantation (VCA) is limited by the need for chronic immunosuppression. Recent data suggest that the lymphatic system plays an important role in mediating rejection. This study used near‐infrared (NIR) lymphography to describe lymphatic reconstitution in a rat VCA model. Syngeneic (Lewis–Lewis) and allogeneic (Brown Norway–Lewis) rat orthotopic hind limb transplants were performed without immunosuppression. Animals were imaged pre‐ and postoperatively using indocyanine green (ICG) lymphography. Images were collected using an NIR imaging system. Co‐localization was achieved through use of an acrylic paint/hydrogen peroxide mixture. In all transplants, ICG first crossed graft suture lines on postoperative day (POD) 5. Clinical signs of rejection also appeared on POD 5 in allogeneic transplants, with most exhibiting Grade 3 rejection by POD 6. Injection of an acrylic paint/hydrogen peroxide mixture on POD 5 confirmed the existence of continuous lymphatic vessels crossing the suture line and draining into the inguinal lymph node. NIR lymphography is a minimally invasive imaging modality that can be used to study lymphatic vessels in a rat VCA model. In allogeneic transplants, lymphatic reconstitution correlated with clinical rejection. Lymphatic reconstitution may represent an early target for immunomodulation.     

IVIG and rituximab for treatment of chronic antibody‐mediated rejection: a prospective study in paediatric renal transplantation with a 2‐year follow‐up

Chronic antibody‐mediated rejection (AMR) is the major cause of late renal allograft loss. There is, however, no established treatment for this condition. We report the results of a prospective pilot study on an antihumoral therapy (AHT) consisting of high‐dose intravenous immunoglobulin G (IVIG) and rituximab in 20 paediatric renal transplant recipients. Donor‐specific HLA antibodies (HLA DSA) were quantified by Luminex‐based bead array technology. Loss of eGFR decreased significantly from 7.6 ml/min/1.73 m² during 6 months prior to AHT to 2.1 ml/min/1.73 m² (P = 0.0013) during 6 months after AHT. Fourteen patients (70%) responded: nine of nine patients (100%) without and five of 11 (45%) with transplant glomerulopathy (P = 0.014). C4d positivity in PTC decreased from 40 ± 18.5% in the index biopsy to 11.6 ± 12.2% (P = 0.002) in the follow‐up biopsy. In four of nine biopsies (44%) C4d staining turned negative. During 2 years of follow‐up, the median loss of eGFR in each of the four 6‐month periods remained significantly lower compared with prior to AHT. Class I DSA declined in response to AHT by 61% (p = 0.044), class II DSA by 63% (p = 0.033) 12 months after intervention. AHT with IVIG and rituximab significantly reduces or stabilizes the progressive loss of transplant function in paediatric patients with chronic AMR over an observation period of 2 years, apparently by lowering circulating DSA and reducing intrarenal complement activation.     

Long‐Term Follow‐Up in Composite Tissue Allotransplantation: In‐Depth Study of Five (Hand and Face) Recipients

Composite tissue allotransplantations (CTAs) have clinically shown little, if any, evidence of chronic rejection. Consequently, the effect of chronic rejection on bones, joints, nerves, muscles, tendons and vessels may still have undescribed implications. We thoroughly assessed all allograft structures by histology, magnetic resonance imaging, ultrasonography and high resolution peripheral quantitative computed tomography scan in four bilateral hand‐grafted patients (10, 7, 3 and 2 years of follow‐up, respectively) and in one facial allotransplantation (5 years of follow‐up). All the recipients presented normal skin structure without dermal fibrosis. Vessels were patent, without thrombosis, stenosis or intimal hyperplasia. Tendons and nerves were also normal; muscles showed some changes, such as a variable degree of muscular hypotrophy, particularly of intrinsic muscles, accompanied by fatty degeneration that might be related to denervation. In the majority of hand‐grafted patients graft radius and recipient tibia showed a decrease in trabecular density, although in the graft radius the alterations also involved the cortices. No deterioration of graft function was noted. In these cases of CTA no signs of chronic graft rejection have been detected. However, the possibility that chronic rejection may develop in CTA exists, highlighting the necessity of close continuous follow‐up of the patients.     

Intravenous immunoglobulins and rituximab therapy for severe transplant glomerulopathy in chronic antibody‐mediated rejection: a pilot study

Outcome of patients with transplant glomerulopathy (TG) is poor. Using B‐cell targeting molecules represent a rational strategy to treat TG during chronic antibody‐mediated rejection. In this pilot study, 21 patients with this diagnosis received four doses of intravenous immunoglobulins and two doses of rituximab (IVIG/RTX group). They were retrospectively compared with a untreated control group of 10 patients. At 24 months post‐biopsy, graft survival was similar and poor between the treated and the untreated group, 47% vs. 40%, respectively, p = 0.69. This absence of response of IVIG/RTX treatment was observed, regardless the phenotype of TG. Baseline estimated glomerular filtration rate (eGFR) and decline in eGFR during the first six months after the treatment were risk factors associated with 24‐month graft survival. The IVIG/RTX therapy had a modest effect on the kinetics of donor‐specific alloantibodies at M24, compared to the untreated group, not associated with an improvement in graft survival. The mean number of adverse events per patient was higher in the IVIG/RTX group than in the control group (p = 0.03). Taken together, IVIG/RTX treatment for severe TG during chronic antibody‐mediated rejection does not seem to change the natural history of TG and is associated with a high incidence of adverse events.     

De novo donor‐specific anti‐HLA antibodies mediated rejection in liver‐transplant patients

The incidence and consequences of de novo donor‐specific anti‐HLA antibodies (DSAs) after liver transplantation (LT) are not well known. We investigated the incidence, risk factors, and complications associated with de novo DSAs in this setting. A total of 152 de novo liver‐transplant patients, without preformed anti‐HLA DSAs, were tested for anti‐HLA antibodies, with single‐antigen bead technology, before, at transplantation, at 1, 3, 6 and 12 months after transplantation, and thereafter annually and at each time they presented with increased liver‐enzyme levels until the last follow‐up, that is, 34 (1.5–77) months. Twenty‐one patients (14%) developed de novo DSAs. Of these, five patients had C1q‐binding DSAs (24%). Younger age, low exposure to calcineurin inhibitors, and noncompliance were predictive factors for de novo DSA formation. Nine of the 21 patients (43%) with de novo DSAs experienced an acute antibody‐mediated rejection (AMR). Positive C4d staining was more frequently observed in liver biopsies of patients with AMR (9/9 vs. 1/12, P < 0.0001). Eight patients received a B‐cell targeting therapy, and one patient received polyclonal antibodies. Only one patient required retransplantation. Patient‐ and graft‐survival rates did not differ between patients with and without DSAs. In conclusion, liver‐transplant patients with liver abnormalities should be screened for DSAs and AMR.     

Lymphoid neogenesis in skin of human hand,nonhuman primate,and rat vascularized composite allografts

The mechanisms of skin rejection in vascularized composite allotransplantation (VCA) remain incompletely understood. The formation of tertiary lymphoid organs (TLO) in hand transplantation has been recently described. We assess this phenomenon in experimental and clinical VCA rejection. Skin biopsies of human (n = 187), nonhuman primate (n = 11), and rat (n = 15) VCAs were analyzed for presence of TLO. A comprehensive immunohistochemical assessment (characterization of the cell infiltrate, expression of adhesion molecules) including staining for peripheral node addressin (PNAd) was performed and correlated with rejection and time post‐transplantation. TLO were identified in human, nonhuman primate, and rat skin samples. Expression of PNAd was increased in the endothelium of vessels upon rejection in human skin (P = 0.003) and correlated with B‐ and T‐lymphocyte numbers and LFA‐1 expression. PNAd expression was observed at all time‐points after transplantation and increased significantly after year 5. In nonhuman primate skin, PNAd expression was found during inflammatory conditions early and late after transplantation. In rat skin, PNAd expression was strongly associated with acute rejection and time post‐transplantation. Lymphoid neogenesis and TLO formation can be uniformly found in experimental and human VCA. PNAd expression in vascular endothelium correlates with skin rejection and T‐ and B‐cell infiltration.     

Diagnosing skin rejection in vascularized composite allotransplantation: advances and challenges

Refinements in microsurgical techniques coupled with advances in immunosuppressive and immunomodulatory protocols have enabled broader clinical application of vascularized composite allotransplantation (VCA) with encouraging immunological, functional, and esthetic results. However, skin rejection remains a significant obstacle and a serious complication for VCA recipients. Clinical and histopathological features of rejection in VCA have been described in a number of studies, which led to the development of an international consensus on the classification guidelines of rejection in the context of VCA. Nevertheless, currently available diagnostic modalities still have several limitations and shortcomings that can pose a significant diagnostic challenge, particularly when signs of rejection are found to be equivocal. In this review, we provide a critical analysis of these advances and challenges in diagnosing skin rejection. Specifically, we highlight the gaps in understanding of rejection mechanisms, the shortfalls in correlating cellular, molecular, and clinicopathologic markers with rejection grades, deficiencies in defining chronic rejection, and antibody‐mediated rejection after VCA, as well as providing an outlook on novel concepts, such as the utilization of advanced computational analyses and cross‐disciplinary diagnostic approaches.     

B cells: a rational target in alloantibody-mediated solid organ transplantation rejection

Loss of allografts over time remains a barrier to achieving successful outcomes in solid organ transplantation. Although the role of donor-specific alloantibody-mediated mechanisms in hyperacute rejection is well known, research and management of early and late post-transplant rejection have traditionally focused on T cell-mediated mechanisms. However, available agents that affect T-cell pathways have minimal impact on long-term graft survival, suggesting that other effector mechanisms are involved. A growing body of evidence now supports a role for alloantibody-mediated mechanisms in early and late graft rejection, which can significantly impact on long-term graft survival. The important role of B cells in generating and perpetuating alloantibody production provides a rationale for B-cell depletion therapy as an approach to prevent or reduce alloantibody formation before transplantation and to treat or prevent early and late alloantibody-mediated rejection. The use of monoclonal antibodies that directly target B cells, in combination with standard alloantibody-depleting therapies and/or immunosuppression, has been investigated in several small non-controlled studies. Promising results suggest that this strategy warrants further investigation in larger controlled studies.     

Determining donor‐specific antibody C1q‐binding ability improves the prediction of antibody‐mediated rejection in human leucocyte antigen‐incompatible kidney transplantation

Detrimental impact of preformed donor‐specific antibodies (DSAs) against human leucocyte antigens on outcomes after kidney transplantation are well documented, however, the value of their capacity to bind complement for predicting antibody‐mediated rejection (AMR) and graft survival still needs to be confirmed. We aimed to study DSA characteristics (strength and C1q binding) that might distinguish harmful DSA from clinically irrelevant ones. We retrospectively studied 60 kidney‐transplanted patients with preformed DSA detected by single antigen bead (SAB) assays (IgG and C1q kits), from a cohort of 517 kidney graft recipients (124 with detectable anti‐HLA antibodies). Patients were divided into DSA strength (MFI < vs. ≥ 15 000) and C1q‐binding ability. AMR frequency was high (30%) and it increased with DSA strength (P = 0.002) and C1q+ DSA (P < 0.001). The performance of DSA C1q‐binding ability as a predictor of AMR was better than DSA strength (diagnostic odds ratio 16.3 vs. 6.4, respectively). Furthermore, a multivariable logistic regression showed that C1q+ DSA was a risk factor for AMR (OR = 16.80, P = 0.001), while high MFI DSAs were not. Graft survival was lower in high MFI C1q+ DSA in comparison with patients with C1q? high or low MFI DSA (at 6 years, 38%, 83% and 80%, respectively; P = 0.001). Both DSA strength and C1q‐binding ability assessment seem valuable for improving pretransplant risk assessment. Since DSA C1q‐binding ability was a better predictor of AMR and correlated with graft survival, C1q‐SAB may be a particularly useful tool.     

Long-term outcome after hand and forearm transplantation – a retrospective study

Between 2000 and 2014, five patients received bilateral hand (n = 3), bilateral forearm (n = 1), and unilateral hand (n = 1) transplants at the Innsbruck Medical University Hospital. We provide a comprehensive report of the long-term results at 20 years. During the 6–20 years follow-up, 43 rejection episodes were recorded in total. Of these, 27.9% were antibody-related with serum donor-specific alloantibodies (DSA) and skin-infiltrating B-cells. The cell phenotype in rejecting skin biopsies changed and C4d-staining increased with time post-transplantation. In the long-term, a change in hand appearance was observed. The functional outcome was highly depending on the level of amputation. The number and severity of rejections did not correlate with hand function, but negatively impacted on the patients´ well-being and quality of life. Patient satisfaction significantly correlated with upper limb function. One hand allograft eventually developed severe allograft vasculopathy and was amputated at 7 years. The patient later died due to progressive gastric cancer. The other four patients are currently rejection-free with moderate levels of immunosuppression. Hand transplantation remains a therapeutic option for carefully selected patients. A stable immunologic situation with optimized and individually adopted immunosuppression favors good compliance and patient satisfaction and may prevent development of DSA.     

Graft vasculopathy in the skin of a human hand allograft: implications for diagnosis of rejection of vascularized composite allografts

Whereas vascularized composite allografts often undergo acute rejections early in the postgraft period, rejection manifesting with severe vascular changes (graft vasculopathy) has only been observed on three occasions in humans. We report a hand‐allografted patient who developed severe rejection following discontinuation of the immunosuppressive treatment. It manifested clinically with erythematous maculopapules on the skin and pathologically with graft vasculopathy that affected both large vessels and smaller cutaneous ones. The observation that graft vasculopathy can affect skin vessels shows that it is amenable to diagnosis with usual skin biopsy as recommended for the follow‐up of these allografts. Graft vasculopathy developing in the setting of vascularized composite allografts likely represents chronic rejection due to under‐immunosuppression and, if confirmed, should be included in a future update of the Banff classification of vascularized composite allograft rejection.     

Rituximab is associated with improved survival in cardiac allograft patients with antibody‐mediated rejection: a single center review

Antibody‐mediated rejection (AMR) after cardiac transplantation is associated with significant mortality, and the optimal treatment of this condition is poorly defined. Rituximab has been used successfully for the treatment for antibody‐mediated diseases; however, its role in AMR is unclear. We review our experience with rituximab in patients with cardiac allograft AMR. We conducted a retrospective analysis of cardiac transplant patients with a diagnosis of AMR from 2001 to 2011. Inclusion criteria were clinical suspicion of rejection with the presence of C4d complement staining on endomyocardial biopsy and the absence of cellular rejection of grade 2R or greater. Patients were divided into Rituximab and NoRituximab groups. The primary endpoint was all‐cause mortality. Secondary endpoints were infection, change in ejection fraction (EF), and rehospitalization. Thirty‐three patients met inclusion criteria, of whom 13 received rituximab and 20 did not. Baseline characteristics were similar between groups. Kaplan–Meier curves for a three‐yr follow‐up period demonstrate improved survival in the Rituximab group (p = 0.0089). There were no differences in secondary endpoints. We found that rituximab therapy was associated with improved survival in cardiac allograft AMR. Further prospective, randomized studies in larger patient populations are needed to confirm this finding and to define ideal timing for rituximab administration.