This work was supported by a grant-in-aid from the Japan Society for the Promotion of Science (to Aya Miyagawa-Hayashino and Atushi Yoshizawa) and by the Funding Program for Next Generation World-Leading Researchers (to Satohiro Masuda), which was initiated by the Council for Science and Technology Policy of the Japan Society for the Promotion of Science.
An association between preformed donor-specific anti–human leukocyte antigen antibodies (DSAs) detected by conventional cytotoxic crossmatching1, 2 or a sensitive Luminex assay at the time of liver transplantation and early graft loss has been demonstrated.3-5 The detection of DSAs in the posttransplant period has been associated with lower graft survival in kidney transplantation.6, 7 In liver transplantation, a high incidence of preformed DSAs as well as human leukocyte antigen (HLA) antibodies developing after liver transplantation (de novo DSAs) with a high mean fluorescence intensity (MFI) has been found in patients experiencing acute or chronic rejection, but the role of de novo DSAs in long-term survivors after liver transplantation has been poorly investigated.5, 8-11
In the late period after transplantation, increasing evidence suggests that the humoral response to alloantigens plays an important role in the development of interstitial fibrosis and tubular atrophy in renal transplantation.6, 12 As for liver transplantation, graft fibrosis is frequently observed in late biopsy samples obtained from children with normal or nearly normal liver biochemistry, but specific diseases causing graft fibrosis should be sought.13-16 A recent report showed that central fibrosis was seen in stable pediatric recipients who had to reinitiate immunosuppression, but none was seen in patients with operational tolerance.17
Complement component 4d (C4d), a complement split product, has been used as a marker of antibody-mediated rejection (AMR) in liver allografts in the early posttransplant period.1, 18-20 Overall, 70% of patients with ductopenia had DSAs in a study,10 and 60% of the ductopenia cases had DSAs in association with diffuse C4d deposition. In the late posttransplant period, there are limited data on C4d staining in association with de novo DSAs in liver transplantation.5, 10
In this study, the association of donor-specific HLA antibodies at the time of protocol biopsy with histopathological findings as well as C4d immunostaining was examined in long-term pediatric liver transplant recipients with normal or nearly normal liver biochemistry. We hypothesized that subclinical AMR would manifest histologically as late graft fibrosis in long-term liver allografts. To emphasize centrilobular fibrosis, we scored graft fibrosis with a modified nonalcoholic fatty liver disease staging system.21
PATIENTS AND METHODS
This study was approved by the institutional review board of Kyoto University. Written informed consent was obtained from patients or their parents at the time of biopsy. A search of our pathology data system revealed 797 pediatric recipients who were less than 18 years old at the time of living donor liver transplantation (LDLT) between 1990 and December 2011 at Kyoto University Hospital; 537 survived more than 5 years after LDLT. Ninety patients underwent liver biopsy during the study period (July to December 2011). Stable patients were defined as those with liver enzyme levels (transaminase and total bilirubin) less than 1.5 times the upper limit of normal and with no clinical evidence of graft dysfunction or vascular/biliary complications within the year before the last biopsy. For 79 stable patients who underwent their last biopsy during the study period, there was information on both C4d immunostaining of liver tissue and DSAs (determined with Luminex at the time of the last biopsy). The remaining 11 patients were excluded from the study because of elevated liver enzymes due to chronic rejection (n = 3), persistent acute rejection (n = 3), hepatic vein stenosis (n = 2), a portal vein obstruction (n = 1), steatohepatitis (with transplantation for progressive familial intrahepatic cholestasis; n = 1), or pulmonary hypertension with a splenorenal shunt (n = 1).
The mean age at the time of LDLT was 2.3 years (median = 1 year, range = 0-12 years) for 79 patients. The patients included 46 females and 33 males, and all were Japanese. The indications for LDLT in the 79 patients were biliary atresia (n = 65), fulminant liver failure (n = 3), metabolic disease (n = 2), Alagille syndrome (n = 2), hepatoblastoma (n = 1), retransplantation for chronic rejection (n = 2), portal vein thrombosis (n = 2), and other indications (n = 2). The donors were the patients' mothers in 41 cases, their fathers in 37 cases, and an uncle in 1 case. The median donor age was 32 years (range = 23-53 years). The grafts were ABO-identical for 59 patients, ABO-compatible for 13 patients, and ABO-incompatible for 7 patients.
The liver biopsy specimens were fixed in 10% buffered formalin and were stained with hematoxylin and eosin, Masson's trichrome, and cytokeratin 7 (OV-TL 12/30, DakoCytomation, Glostrup, Denmark) at a 1:200 dilution. Slides were reviewed by pathologists (A.M.-H. and H.H.) blinded to the clinical history.
Analyses of protocol liver biopsy samples taken during the long-term follow-up of pediatric liver transplant recipients have revealed a high frequency of centrilobular fibrosis.16 The systems commonly used for grading and staging chronic viral hepatitis (the Ishak fibrosis score and the Metavir fibrosis score) show fibrous expansion of the portal tracts as the disease progresses. They are very insensitive for describing changes in centrilobular fibrosis.22 The fibrosis score used in this study was a modification of the nonalcoholic fatty liver disease staging system devised by the pathology committee of the Nonalcoholic Steatohepatitis Clinical Research Network.21 For semiquantitative grading, the fibrosis was graded as follows: stage 0.5, mild zone 3 perisinusoidal fibrosis (originally stage 1a); stage 1, moderate zone 3 perisinusoidal fibrosis (originally stage 1b); stage 2, zone 3 perisinusoidal fibrosis and periportal fibrosis; stage 3, bridging fibrosis; and stage 4, cirrhosis (Fig. 1A-E).
Early acute cellular rejection (ACR) was defined as graft dysfunction accompanied by histological findings consistent with ACR within 1 year of transplantation. Late-onset acute rejection (LAR) was defined as ACR appearing more than 1 year after transplantation. Mild LAR could be present as perivenular inflammation involving a majority of central veins with patchy perivenular hepatocyte loss. Moderate LAR was defined as a finding of mild LAR with at least focal confluent hepatocyte dropout. When perivenular inflammation involved a minority of central veins, the diagnosis of indeterminate LAR was made.23 When a biopsy sample showed mild, nonspecific inflammation without rejection-related inflammatory changes, the findings were described as minimal inflammation.
C4d staining was performed on paraffin-embedded liver tissue with a polyclonal rabbit anti-human C4d antibody (BI-RC4D, Biomedia, Vienna, Austria) at a 1:50 ratio. Because a recent study5 showed an excellent correlation between the total numerical C4d score and semiquantitative C4d scoring (minimal/negative, focal, and diffuse), C4d staining was evaluated semiquantitatively for practical reasons. Diffuse staining was defined as C4d deposition in more than 50% of the portal tracts of the hepatic artery, portal vein, or capillary endothelium (Fig. 2A). Portal C4d immunolabeling of less than 50% of the portal tracts was considered focal (Fig. 2B). Sinusoidal or central vein C4d deposition was analyzed separately. C4d deposition on elastic fibers within arteries and portal stroma without endothelial staining (Fig. 2C) was regarded as a nonspecific finding without clinical significance.10
The last biopsy sample for each patient was included for assessing histological findings and C4d immunostaining. When the last biopsy sample was negative or equivocal for C4d immunostaining, the previous biopsy sample was subsequently stained. The presence of at least 2 C4d-negative stains for a patient was considered a negative finding for C4d in that case. When the last biopsy sample was negative for C4d but the previous biopsy sample was positive, the case was considered positive for C4d (overall C4d staining).
Biochemical Liver Tests and Detection of Anti-HLA Antibodies
Blood samples for performing liver biochemistry tests and for determining the presence of HLA antibodies were taken concurrently with the liver biopsy samples. Laboratory data at the time of the last biopsy included the following: serum aspartate aminotransferase (AST; normal range = 13-29 IU/L), alanine aminotransferase (ALT; normal range = 8-28 IU/L), total bilirubin (normal range = 0.2-1.0 mg/dL), gamma-glutamyl transpeptidase (GGT; normal range = 7-29 IU/L), immunoglobulin G (IgG; normal range = 826-1840 mg/dL), and anti-nuclear antibodies (ANAs). Screening anti-HLA tests were performed with multiplex technology (LABScreen mixed test, One Lambda, Canoga Park, CA), and when the results were positive, specificity was determined with the LABScreen single-antigen test (One Lambda).6 HLA antibody specificities were considered positive with a normalized MFI value greater than 1000.11 The pretransplant HLA antibody status was assessed with a complement-dependent lymphocytotoxic assay or flow T and B cell crossmatches.1 Donor typing for DP was not performed.
DSA-Positive Group Versus DSA-Negative Group
The patients were divided into 2 groups: DSA-positive and DSA-negative. DSA information was available for 67 patients. Twelve patients were positive for anti–class I or II antibodies, but there was no information about whether they were DSAs or non-DSAs; these patients were excluded from further analysis. Demographic data, laboratory data, histological findings, and C4d staining results were compared for the 2 groups.
The initial immunosuppressive treatment was based on tacrolimus. The type of immunosuppression used at the time of the last biopsy (monotherapy, double therapy, or triple therapy) and the tacrolimus trough level were investigated.
The paired Student t test or the Mann-Whitney U test was used to compare continuous variables. The chi-square test or Fisher's exact test was used to compare the frequencies of categorical variables. A regression analysis was used to test relationships between quantitative variables. The statistical analysis was performed with Stata SE 9.0 (StataCorp., College Station, TX). A P value less than 0.05 was considered significant.
The median time from LDLT to the time of last liver biopsy and DSA analysis was 11 years (range = 5-20 years). In the last biopsy samples of the 79 patients, a high percentage of graft fibrosis was detected. The time since transplantation correlated with the degree of fibrosis according to a regression analysis (R2 = 0.07, P = 0.02; Fig. 3).
No correlation was found between the severity of fibrosis and donor age (P = 0.35). As for outcomes, no patients developed graft failure and required retransplantation.
Anti-HLA Antibodies in the Study Population
No patients had a positive crossmatch before transplantation. Overall, HLA antibodies were found in 49 of the 79 patients (62%). Three had anti–class I antibodies alone, 31 had anti–class II antibodies alone, and 15 had anti–class I and anti–class II antibodies. The remaining 30 patients (38%) had no evidence of HLA antibodies.
Thirty-two of the 79 patients (41%) had DSAs: 2 had anti–class I antibodies, and 30 had anti–class II antibodies. Many patients had more than 1 DSA. The MFI value was 1383 for class I DSAs and 17,681 for class II DSAs. Twelve patients had class II DSAs specific to DR and DQ, 14 had class II DSAs specific to DR, and 4 had class II DSAs specific to DQ. One patient had class I DSAs specific to A, and another had class I DSAs specific to B.
One patient had non–donor-specific class I antibodies, 2 patients had non–donor-specific class II antibodies, and 2 had non–donor-specific class I and class II antibodies. Three had non–donor-specific class I antibodies, but class II DSAs were detected (Table 1).
Table 1. Anti-HLA Antibodies in the Study Population (n = 79)
DSA Locus (n)
Including 3 cases with class II DSAs and class I non-DSAs.
There were 32 patients in the DSA-positive group and 35 patients in the DSA-negative group. Twelve patients were not examined for DSAs because their donor HLA typing was not available (8 with anti–class II antibodies only and 4 with anti–class I and anti–class II antibodies). There was no association between the time since LDLT and the presence of DSAs (P = 0.58). There was no apparent relationship between the severity of fibrosis and the DSA MFI value (P = 0.56).
Comparison of the DSA-Positive and DSA-Negative Groups
The baseline characteristics recorded for the DSA-positive group (n = 32) and the DSA-negative group (n = 35) are listed in Table 2. In the DSA-positive group, 2 patients had class I DSAs, and 30 had class II DSAs. The patients in the 2 groups did not differ with respect to the age at LDLT, the indication for LDLT, the time since LDLT, the donor age or sex, or the ABO blood type compatibility status. There were no differences between the 2 groups with respect to serum transaminase levels, GGT levels, IgG levels, the presence of ANAs, previous episodes of ACR and LAR, or previous episodes of moderate LAR. The ANA titer was 1:160 in 1 patient in the DSA-negative group and 1:40 in the remaining 6 ANA-positive patients. The DSA-positive group had elevated total bilirubin levels (P = 0.01). Male recipients were significantly associated with DSA positivity (P = 0.02).
Table 2. Clinical Characteristics of DSA-Positive and DSA-Negative Patients
DSA-Positive (n = 32)
DSA-Negative (n = 35)
The data are presented as means and standard deviations.
C4d immunostaining was performed for 162 liver biopsy samples (which included the last biopsy samples for 67 patients), with a median of 2 stains per patient (range = 1-6). When the last biopsy samples for C4d staining were taken, the median time after transplantation was 11 years (range = 5-20 years). When previous biopsy samples were included, biopsy samples for C4d immunostaining were taken 1.4 to 20 years after transplantation (overall C4d staining). The median time after transplantation for biopsy samples showing positive C4d staining was 10.2 years (range = 4.2-18.6 years).
Focal or diffuse C4d deposition was seen in the hepatic artery, portal vein, or capillary endothelium, and there was no sinusoidal or central vein deposition in any of the cases.
High Frequency of Graft Fibrosis and Inflammation in the DSA-Positive Group
Patients in the DSA-positive group had a higher degree of fibrosis than patients in the DSA-negative group (Fig. 4). The mean fibrosis score was 3.1 in the DSA-positive group and 1.1 in the DSA-negative group. There was a high incidence of severe fibrosis (score ≥ 3) among patients in the DSA-positive group (28/32 or 88%) versus the DSA-negative group (6/35 or 17%, P < 0.001). Two of the 6 patients with stage 3 or higher fibrosis in the DSA-negative group had a previous episode of idiopathic posttransplant hepatitis that was treated with triple immunosuppression, 2 patients had a previous episode of portal or hepatic vein stenosis that was resolved by an intervention at the time of the last biopsy, and the last 2 patients had no known cause for their graft fibrosis. Four patients had a previous episode of portal or hepatic vein stenosis in the DSA-positive group, but the stenosis resolved early after transplantation; 3 of these 4 patients had stage 3 fibrosis, and 1 had stage 4 fibrosis at the time of the last biopsy. There were no other causes of fibrosis, such as a viral etiology or idiopathic posttransplant hepatitis, in the DSA-positive group.
In the DSA-positive group, 6 patients were diagnosed with mild LAR, and 9 were diagnosed with indeterminate LAR at the time of the last biopsy. In the DSA-negative group, 4 showed mild LAR, and 1 showed indeterminate LAR. Thus, DSA-positive patients had a higher frequency of mild or indeterminate LAR than DSA-negative patients (47% in the DSA-positive group versus 14% in the DSA-negative group, P = 0.004). Twelve of the DSA-positive patients and 16 of the DSA-negative patients had no inflammation at the time of the last biopsy (P = 0.50).
Significant C4d Positivity in the DSA-Positive Group According to Overall C4d Staining
According to overall C4d staining, 25 of the 32 patients (78%) in the DSA-positive group showed C4d positivity (focal or diffuse endothelial staining), with 5 showing diffuse endothelial staining, whereas 8 of the 35 patients (23%) in the DSA-negative group showed C4d positivity, with all showing focal endothelial staining (P < 0.001; Fig. 5A). As for the last biopsy samples, there was no significant difference in C4d positivity between the DSA-positive group (11/32 or 34%) and the DSA-negative group (7/35 or 20%, P = 0.10; Fig. 5B). There were 14 patients whose previous biopsy samples showed focal C4d staining at a median of 2.5 years (range = 100 days to 7 years) before the last C4d-negative biopsy sample.
Diffuse C4d Staining Pattern
When C4d positivity was restricted to diffuse endothelial staining, DSA-positive patients had a significantly higher frequency of diffuse endothelial C4d staining, which was seen in 5 DSA-positive patients and in no DSA-negative patients according to overall C4d staining (P = 0.02). These 5 patients underwent ABO-identical/compatible LDLT. According to the last C4d staining results, 4 DSA-positive patients had diffuse endothelial C4d staining, whereas no DSA-negative patients did (P = 0.03). One patient was DSA-positive but had C4d-negative staining in the last biopsy sample 8.1 years after LDLT. The previous biopsy sample, which was taken 3.1 years before the last biopsy sample, was stained with C4d, and this turned out to be diffuse C4d staining.
Association of C4d Positivity With Graft Fibrosis and DSAs
Overall, C4d-positive biopsy samples (focal and diffuse C4d staining) showed higher fibrosis scores, higher rates of DSA positivity, lower donor ages, and higher AST levels than C4d-negative cases (Table 3). In the DSA-positive group, the DSA MFI for patients with diffuse C4d staining (MFI = 23,238) did not significantly differ from the MFI for patients with focal C4d staining (MFI = 16.800, P = 0.09). There was no difference in the MFIs for overall C4d-positive cases (diffuse or focal C4d staining; MFI = 20,017) and C4d-negative cases (MFI = 18,352, P = 0.60).
Table 3. Characteristics of Patients According to Overall C4d Staining Results for Biopsy Samples: Diffuse C4d Staining Versus Focal C4d or Negative Staining and Diffuse or Focal C4d Staining Versus C4d-Negative Staining
Diffuse C4d Staining (n = 5)
Focal C4d or Negative Staining (n = 62)
Diffuse or Focal C4d Staining (n = 33)
C4d-Negative Staining (n = 34)
The data are presented as means and standard deviations.
The use of immunosuppression was similar in the DSA-positive and DSA-negative groups (Table 2) and in the C4d-positive and C4d-negative groups at the time of the last biopsy (Table 3). There were no differences in the use of triple or double immunosuppression with tacrolimus, prednisone, and mycophenolate mofetil or in the use of tacrolimus monotherapy between the 2 groups. Four DSA-negative patients were off immunosuppression, but no patients in the DSA-positive group were (P = 0.048). These 4 DSA-negative patients were all C4d-negative. The mean tacrolimus trough levels did not differ between the 2 groups.
This study demonstrated that a significant proportion of long-term survivors of pediatric LDLT displayed DSAs and graft fibrosis, which probably originated from centrilobular-based fibrosis. Limiting the analysis of lesions associated with DSAs to late biopsy samples raises the possibility that DSAs and graft fibrosis may be time-associated variables, and their association should not be directly linked.7 In this study, the time since transplantation correlated with advanced fibrosis, and this was consistent with a previous report13; however, the presence of DSAs was not correlated with the time since transplantation. The cause of graft fibrosis may be multifactorial and include immunological and nonimmunological factors, and it may not be the result of chronic AMR.23-25 Because most cases of ischemic damage seen as centrilobular fibrosis within the first 6 months after transplantation are resolved,26 the causes of late fibrosis may occur subsequently. Vascular and biliary complications were resolved by the time of the last biopsy for our patients.
Diffuse C4d staining and the documentation of circulating DSAs in the presence of late graft dysfunction are now accepted as evidence of active AMR in renal transplantation.27, 28 No consensus has developed about C4d staining patterns and the significance of the detection of AMR in late liver allografts.10, 29 Diffuse portal endothelial C4d staining and sinusoidal endothelial C4d staining have been associated with circulating DSAs, and DSA-positive/diffuse C4d-positive subjects have high frequencies of acute and chronic rejection.10 This is in line with a previous study showing that extensive portal C4d staining in crossmatch-positive patients was associated with AMR.1, 30 Our previous study1 revealed that focal endothelial C4d deposition may lack diagnostic specificity, and it has been seen in patients with various conditions after transplantation without clinical significance. In this study, when significant C4d positivity was restricted to diffuse portal capillary/venular C4d deposition, only 5 patients in the DSA-positive group showed diffuse C4d endothelial staining, and this was found to occur in association with positive DSAs and severe graft fibrosis, which may be evidence of chronic AMR. Because C4d binds to nearby proteins or carbohydrates covalently, it is expected to have variable tissue persistence,10 and it has been reported that C4d deposition precedes the development of chronic allograft glomerulopathy.31 We tried to perform C4d staining on previous serial biopsy samples. As a result, many DSA-positive cases were negative for C4d staining at the time of the last biopsy, but previous biopsy samples were positive (focal endothelial staining) in additional cases; this resulted in a significant difference in C4d positivity between the DSA-positive and DSA-negative groups. Because focal C4d staining with DSAs in addition to diffuse staining could distinguish between stable patients with late graft abnormalities and stable patients without late graft abnormalities, we thought that focal C4d staining might be a specific finding late after transplantation. The liver has a large absorption capacity for antibodies5; liver allografts may be able to accommodate DSAs at a low MFI, and class II DSAs may be clinically significant only at a very high MFI.11
Girnita et al.32 showed the development of anti-HLA antibodies in patients for whom drug withdrawal failed. DSAs were absent in tolerant recipients in their study. Our data showed that DSA-positive patients with stable graft function had higher frequencies of graft fibrosis and inflammation and significant C4d positivity in comparison with DSA-negative patients. DSAs were absent in all of our patients who were off immunosuppression. These findings suggest that serial monitoring of DSAs and C4d staining during drug withdrawal may help in identifying patients who require the reinstitution of immunosuppression. There were DSA-positive cases with almost normal histological findings in the present series, and the clinical importance remains to be clarified with future follow-up. Some DSA-negative patients had unexplained stage 3 fibrosis. This assumed that some DSA-negative patients could have had DSAs specific to Cw and DP, neither of which was assessed in this study.7
The outcomes of T cell–mediated rejection have improved with current immunosuppressive regimens,7 but whether HLA antibodies develop late after transplantation in association with late graft failure remains unknown in liver transplantation. DSA-positive patients had a higher frequency of indeterminate or mild LAR than DSA-negative patients at the time of the last biopsy. The presence of mild inflammation suggests that graft fibrosis may occur as a result of ongoing allograft injury.13 However, in this study, the effects of immunosuppressive strategies before and after biopsying on outcomes cannot be assessed.33 In addition, the DSA-positive and DSA-negative groups showed no differences in early or late cellular rejection rates. It is unknown whether late graft fibrosis can be resolved with current immunosuppression regimens. Strategies using plasmapheresis, intravenous immunoglobulin, and mycophenolate or tacrolimus may reverse late AMR.6 Recently, bortezomib has been used as a tool for DSA removal via the depletion of plasma cells from kidney allograft patients.34, 35 Further study is needed to determine whether de novo DSAs are associated with late graft fibrosis and graft dysfunction. Unexplained graft fibrosis may be reversible and amenable to treatment.