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2Graft reinfection in patients receiving an orthotopic liver transplant (LT) due to hepatitis C virus (HCV)-induced chronic liver failure is ubiquitous, and severe HCV-related graft hepatitis occurs in about 50% of these patients and up to 30% develop cirrhosis.1–6 Recently acquired knowledge of liver-grafted HCV positive patients indicated that HCV and graft recurrences as well as development of fibrosis are multifactorial and may depend upon several factors like pre–or post–hepatitis C viremia levels, renal function, HCV genotype, rejection treatment, and donor as well as recipient age.7–14
Following LT, the virus load increased up to >10-fold of the pretransplant levels,15 which is thought to reflect suppression of those host effector immune responses usually controlling HCV replication. These observations underline the hypothesis that long-term immunosuppression after liver transplantation may play a major role in the rapid progression of recurrent HCV infection after LT. In this context, human leukocyte antigen (HLA) mismatches may be a possible factor involved in the development and course of recurrent HCV.16
The influence of HLA matching on outcome in liver transplant recipients is being discussed controversially. A number of studies show an adverse dualistic effect of HLA matching, namely that on the one hand HLA matching reduces the frequency of rejection and on the other hand it may enhance recurrent underlying diseases like hepatitis B/C.
Manez et al. reported in 1995 that the incidence of recurrent hepatitis B virus/HCV infection after LT was significantly higher for HLA-B-compatible liver transplant recipients.17, 18 On the contrary, Belli and coworkers recently observed a beneficial effect of HLA-B14 and HLA-DRB1*04 matching on outcome in patients transplanted for hepatitis C.19 Furthermore, recent reports have shown that major histocompatibility complex (MHC)-I restricted T cells may be involved in the control of postoperative HCV spread.20, 21 HLA-DR may also play a role in the occurrence of cytomegalovirus infection,18 which has recently been shown to increase the severity of HCV recurrence after liver transplantation.6, 22, 23
This issue has been addressed only marginally in the context of posttransplant HCV recurrence. HLA matching may influence the outcome of LT in HCV-positive patients due to 2 separate mechanisms. The reduced incidence of rejection in better matched grafts may lead to a reduction of antirejection treatment, which has shown to increase HCV related graft losses. In contrast, the lack of MHC I/II restricted T-cell response in patients with a complete HLA mismatch may lead to a less severe recurrent hepatitis.
The aim of this retrospective study was to examine whether HLA matching affects the incidence of HCV recurrence, the incidence of rejection, and the outcome of patients after LT for chronic HCV-related liver failure. We therefore examined in our patient population the incidence of rejection, the severity of graft hepatitis using histological scores, graft failure rate, and patient survival depending on the degree of HLA mismatches.
LT,liver transplantation; HCV, hepatitis C virus; HLA, human leukocyte antigen; HLA-B, human leukocyte antigen B; MHC, major histocompatibility complex; CsA, cyclosporine A; Tac, tacrolimus; HLA-DR, human leukocyte antigen DR; HLA-A, human leukocyte antigen A; anti-HBc, anti-hepatitis B core antigen.
PATIENTS AND METHODS
Between January 1999 and April 2002 we identified 165 liver transplants in 153 HCV-positive patients undergoing liver transplantation. All patients were followed-up for a minimum period of two years after OLT and had suitable donor/recipient HLA typing. One hundred twenty-nine functioning transplants underwent liver biopsies after 1 year and routinely thereafter, and were followed for a median of 74.5 months (range, 12 to 166.8 months). In 29 patients a protocol biopsy was not possible, due to graft loss within the first year after LT, and 7 patients had no liver biopsy due to other reasons. Follow-up evaluations of all 153 recipients were performed regularly. The median age of the 55 women and 110 men was 51.3 years (range, 15–72 years). Graft losses were divided in hepatitis C–related and non-hepatitis C–related cases. Additionally, we investigated the occurrence of recurrent cholestatic hepatitis with consecutive graft loss.24 A total of 30 out of the 165 recipients with recurrent hepatitis C underwent treatment with interferon-α 2a (Roferon-A, Roche, Basel, Switzerland) for a minimum of 6 months. None of the recipients were, however, treated within the first year posttransplant.
Donor Selection and Surgery
Only grafts from Caucasian brain death donors were analyzed in the study. All grafts were negative for hepatitis C and anti-hepatitis B core antigen. Overall, 12 patients underwent liver retransplantation. Mean donor age was 36.1±14.2 years. Grafts were preserved exclusively using the University of Wisconsin solution. LT was performed in standard technique using a venous-venous bypass and completion of all vascular anastomosis prior to reperfusion, as previously described. The bile duct anastomosis was performed as side-to-side choledochocholedochostomy.
Protocol liver biopsies were performed after 1, 3, 5, 7, and 10 years. In the case of elevated alanine aminotransferase levels or other signs of graft dysfunction, additional liver biopsies were performed according to standard methods. A semiquantitative histological score was applied based on the histological classification and quantitation of the Scheuer scoring system using a scale of 0–3 for the inflammatory activity (0, absent; 0.5, minimal; 1, mild; 2, moderate; 3, severe), and 0–4 for the evaluation of fibrosis (0, absent; 1, mild; 2, moderate without septa; 3, moderate with septa; 4, cirrhosis).25 Patients developing stage 3 and 4 fibrosis within 1 year after liver transplantation were defined as having fast and severe fibrosis progression.
Immunosuppression and Rejection Treatment
Standard immunosuppression consisted either of cyclosporine A (CsA)- or tacrolimus (Tac)-based regimens. CsA-based protocols included triple (CsA, azathioprine/mycophenolate mofetil, steroids) or quadruple drug induction regimens with CsA, azathioprine/mycophenolate mofetil, steroids and (Fresenius Bad Homburg, Germany), or a monoclonal anti-interleukin 2-receptor antibody. Tacrolimus-based immunosuppressive regimens consisted of dual (Tac, steroids), triple (Tac, azathioprine/mycophenolate mofetil, steroids) or quadruple drug induction including Tac, azathioprine/mycophenolate mofetil, and steroids.
Signs of acute rejection episodes of liver grafts were rising liver serum enzymes, fever, or clinical deterioration, and they were confirmed by graft biopsies. The criteria used to distinguish between acute rejection and recurrent HCV were originally developed for hepatitis B virus–positive patients.26 Acute rejection was diagnosed in the case of portal inflammation with inflammatory bile duct damage or mononuclear perivenular inflammation involving terminal hepatic venules, associated with hepatic necrosis. However, a histopathological diagnosis of acute rejection and recurrent hepatitis C remains difficult.27 Initial therapy of acute cellular rejection episodes consisted of elevation of the base immunosuppressant or 500 mg methylprednisolone for 3 days. In the case of steroid-resistant rejection episodes, recipients received monoclonal CD3 antibodies (OKT 3, 5 mg/day, Orthoclone, Janssen-Cilag GmbH, Neuss, Germany) for 5–7 days. Since 1992, tacrolimus was used as primary treatment for steroid-resistant rejection episodes in CsA-treated recipients, whereas OKT3 treatment was avoided whenever possibly. If the patient did not respond to additional immunosuppressive treatment, a liver biopsy was once again performed to identify patients with acute rejection or recurrent hepatitis C.
Detection of Viral Markers and HCV Genotyping
The diagnosis of hepatitis C was based on the detection of anti-HCV (enzyme linked immunosorbent assay II, Chiron Corp., Emeryville, CA) and HCV RNA in the serum (HCV Amplicor kit; Roche Diagnostic Systems, Inc., Branchburg, NJ) of patients prior to OLT. For the genotyping of HCV, HCV-Amplicor products were employed in the Inno-LiPa assay (Innogenetics N.V., Zwynderecht, Antwerpen, Belgium). Protocol determinations of HCV RNA were done after the liver transplant.
HLA typing was performed by standard lymphocytotoxicity test with confirmation of polymerase chain reaction. HLA-DR typing was performed by cytotoxicity during the early part of the study and then performed by polymerase chain reaction for most of the typings.28, 29 Analyses were performed for HLA-A, -B, -DR and for cumulative HLA matches as well as for the group with 6 mismatches, but not for patients with less than 6 mismatches.
Graft losses were divided in 3 groups depending on the reason of graft loss. The first group consisted of patients with graft loss not related to recurrent HCV (n = 35), the second group were patients with graft loss due to recurrent cirrhosis (n = 15), and the third group were patients with graft losses due to fibrosing cholestatic hepatitis(n = 6). Cholestatic hepatitis was defined as previously described. In all cases liver biopsy was performed and all patients suffered from high viremia level, progressive cholestasis, and hepatocyte ballooning in the liver biopsy.30, 31 Patients were treated with reduction of immunosuppression and antiviral agents if possible. Chronic rejection was diagnosed using the Banff criteria.26, 32 Radiological investigations were performed to exclude other reasons for cholestasis such as ischemic-type biliary lesions.
Data were prospectively collected in a database (Microsoft Access 2.0, Microsoft Corporation, Redmond, WA). Statistical analyses were performed using SPSS for Windows 6.1 (SPSS for Windows, statistical software 6.1. SPSS, Inc., Chicago, IL). Data were given as median and range. Yates-corrected chi square test and the Fisher exact test were performed for dichotomous variables and likelihood ratio chi square test for higher dimensional (2 × 3) tables. The unpaired nonparametric Mann-Whitney U test was used to compare differences in the medians of continuous variables. Multivariate analysis of risk factors for the development of fibrosis stage 3/4 within 1 year after liver transplantation was done with logistic regression analysis. Cumulative risk for recurrent HCV and cumulative survival were assessed by applying Kaplan-Meier technique. Differences were defined as significant when P < 0.05.
Rejection episodes occurred in 77/165 transplants (46 %). Of 77 patients, 65 (84%) were steroid sensitive and 12 (16%) patients required OKT3 treatment. A total of 15/165 (9.1%) patients developed more than one rejection episode. Overall rejection probability declined with decreasing numbers of HLA mismatches (P < 0.05; Table 1; Fig. 1). However, there was no influence of the cumulative HLA-A, -B, and -DR matching on the number of recurrent or steroid-resistant rejection episodes (Table 1). The incidence of acute rejection was significantly increased in patients with less HLA-B and -DR matches. Signs of chronic rejection occurred in 6/165 (3.6%). There was no significant correlation between HLA-A, -B, -DR and cumulative HLA matching and chronic rejection. Rejection treatment correlated with the severity of rejection in the 10-year biopsy (P < 0.05). No correlations between HLA matching and fibrosis progression could be observed 5 and 7 years after liver transplantation.
Table 1. Analysis of the Influence of HLA-Matches on Rejection Probability in Patients After Liver Transplantation for HCV Cirrhosis
Acute rejection (%)
Repeated rejection (%)
Steroid resistant rejection (%)
Indicates significant differences between two groups (p<0.05).
Severity of Graft Hepatitis Using Histological Scores
Mean stage of fibrosis after 1, 3, 5, 7, and 10-years was 1.27, 1.84, 2.02, 2.1, and 2.2, respectively. Influence of HLA-A, -B, and -DR matching on fibrosis progression was analyzed separately for 1-year fibrosis progression and severe recurrent fibrosis progression of stage 3/4 (n = 28) within 1 year after LT (Table 2). HLA-A and -DR had no influence on fibrosis progression after liver transplantation. In contrast to this, HLA-B matching correlated significantly with higher fibrosis stage after 1 year and with the development of severe fibrosis within the first year (P < 0.05). Additionally, the progression towards fibrosis stage 3 and 4 was significantly enhanced in the group of patients with a minimum of one HLA-B match compared to patients with 2 mismatches (Table 1; P < 0.05). This data were confirmed by multivariate logistic regression analysis including donor/recipient age, virus genotype, and type of primary immunosuppressant (Table 3). The overall survival of these patients developing stage 3/4 fibrosis after 1 year was significantly decreased (P < 0.05).
Table 2. Univariate Analysis of the Influence of HLA-Matches on Fibrosis Progression in Patients After Liver Transplantation For HCV Cirrhosis. Data For Fibrosis Progression Within the First Year After LT Are Given As Mean and SEM
Mean Fibrosis stage ± SEM after 1-year
Stage 3/4 fibrosis after 1-year (n=28)
Indicates significant differences between two groups (p<0.05).
Table 3. Multivariate Analysis For Risk Factors Contributing Towards the Development of Stage 3 and 4 Liver Fibrosis Within 1 Year After Liver Transplantation in Hepatitis C Positive Patients
Fibrosis stage 1–4 RR (CB)
Cyclosporine vs. Tacrolimus
HCV-RNA before OLT (> 106mEq/mL)
HLA-B mismatches (0–1 vs.2)
Virus genotype (1 and 4 vs. 2 and 3)
Graft Failure Rate
Overall, 56 graft losses occurred after a median of 344 days (3–2,797 days). Of 56 graft losses, 35 (62.6%) were not related to recurrent hepatitis C. Of the remaining 21 patients, 15/56 (26.8%) developed recurrent cirrhosis, and 6/56 (10.7%) showed the typical clinical and histological features of severe fibrosing cholestatic hepatitis. Graft loss due to fibrosing cholestatic hepatitis (median, 277 days; range, 54–910 days) occurred significantly earlier compared to patients with recurrent HCV-related cirrhosis (median, 726 days; range, 275–2385 days; P < 0.05) (Fig. 2). Of 6 patients developing fibrosing cholestatic hepatitis, 5 had 6 HLA mismatches, whereas 14/15 patients with graft loss due to recurrent cirrhosis had a minimum of 1 HLA-match (P < 0.05).
Patient Survival Depending on the Degree of HLA Mismatch
Overall 1-, 5-, and 10-year patient and graft survival was 88.8%, 76.2%, 69.7% and 81.8%, 69.1%, 62.8%, retrospectively. We found no statistical correlation between graft or patient survival and number of HLA mismatches (Fig. 3). Additionally, there were no significant differences in cumulative HLA-A, -B, and -DR mismatches alone, and the donor or recipient HLA allele also did not significantly influence the outcome after OLT.
Risk factors for impaired outcome, such as a history of previous liver transplantation, United Network for Organ Sharing status 4, HCV as underlying disease, and prolonged ischemic time, are well established.3, 33 Immunological risk factors such as a positive T- or B-cell crossmatch have been verified in kidney and heart transplantations, but for liver transplantations the matter is still being discussed controversially.34–39
In contrast to kidney and heart transplants, the nature of the underlying disease may be related to specific HLA alleles in clinical liver transplantation, as described for primary sclerosing cholangitis, autoimmune hepatitis, and hepatitis B/C, and it may affect the severity of recurrence after LT.19, 38, 40 This suggests that in clinical liver transplantation, HLA matching may not only influence frequency of rejection but also may accelerate immunological processes of the underlying disease.
However, the complex interplay between virus pathogenicity and immunological host responses in HCV-positive liver transplant recipients are not well understood yet. A number of risk factors, such as genotype, donor age, and rejection treatment, have been shown to interact with the severity of HCV recurrence after LT, but the mechanism have still not been identified.6, 41–45
In this study we have shown for the first time that HLA matching has a dualistic effect on outcome in liver transplant recipients for hepatitis C–related liver failure. On the one hand, rejection episodes were significantly reduced. However, on the other hand, the severity of fibrosis progression within the first year after LT was enhanced. Interestingly, this contradiction did not result in impaired survival figures in better-matched grafts.
The analysis of HCV-related graft losses showed that 2 different clinical and histomorphological patterns of severe recurrent hepatitis occurred. Severe cholestatic fibrosing hepatitis occurred earlier than recurrent cirrhosis during the course of the disease. The significant higher number of cholestatic fibrosing hepatitis in patients with a complete HLA mismatch may indicate direct cytopathic mechanisms of the virus due to a lack of specific MHC I/II related T-cell responses. In accordance with the literature, this might explain the high virus titers in these patients.24, 31 In contrast, all patients, with the exception of 1 with recurrent cirrhosis and consecutive graft loss, had a minimum of 1 HLA compatibility. Additionally, an increased number of HLA-B matches led toward a significant increase of stage 3 and 4 fibrosis after 1 year, which is associated with impaired survival as previously described. This group included a high number of patients with rapid progression toward fibrosis, despite less rejection episodes and decreased steroid bolus treatment.
In patients with hepatitis B virus and HCV, Manez et al. showed that MHC II matching was associated with severe recurrence of the underlying disease,17 which was however not found in a further study by Belli et al.19 Our study confirmed the detrimental effect of MHC I (HLA-B) matching on the severity of recurrent hepatitis C. However, in the study of Belli et al., no effect of Class I and Class II HLA matching on outcome was shown, which confirmed to another study.46 Only matching of single HLA alleles contributed to an improved outcome after liver transplantation in HCV-positive patients. Since liver transplant recipients are not matched for HLA antigens, the number of patients with a specific HLA allele match is very low. We could not identify any published HLA allele by Belli et al. with more than 10 patients with a match, which makes any statistical evaluation doubtful. Cellular immunity to HCV includes classic CD4+ T cells and CD8+ T lymphocytes.47 This involvement of Class I and II antigens in the recognition of HCV peptides may explain the host recognition of HCV peptides. However, it has been well shown that the graft is repopulated with recipient-derived antigen-presenting cells within 2 weeks after liver transplantation.48 This can explain the minor influence of Class II matching on rejection incidence and severity of recurrent hepatitis C. In contrast, Rosen et al. recently identified HCV-specific CD8+ T cells restricted by donor HLA alleles following liver transplantation.21 These cells were absent before liver transplantation, suggesting that the allograft is capable of expanding naive CD8+ T cells. CD8+ T cells are the primary effector lymphocytes for provision of protective immunity against intracellular pathogen infection of parenchymal cells. The in vitro specificity to donor allele-restricted CD8+ T cells antigens may explain the impact of HLA Class I matching after LT.
One major problem of older investigations regarding outcome of HLA matching after LT are the different typing methods including serological and polymerase chain reaction–based HLA determination. Since it is well known that serological HLA matching of the MHC II locus is inaccurate, new studies on this issue are required. Our study utilizing new typing techniques showed that HLA mismatches had no effect on patient and graft outcome but accelerated the progress of HCV recurrence after LT. Indeed, with the increased utilization of living donors and consecutively better-matched grafts, this issue will become more important in the future. Especially in living related transplantation, HLA matching could be used to develop individualized immunosuppressive protocols for HCV-positive patients.
In conclusion, the challenges in maintaining and improving the outcome of HCV-positive patients is rising, since in recent years progress of HCV recurrence after LT has increased.1, 49 In addition to the immunosuppressive treatment, viral factors and donor age, immunological factors like MHC I–restricted T-cell responses may be involved in the course of recurrent HCV. The lower risk for rejection in patients receiving a better-matched graft may led to individualized immunosuppressive protocols and therefore improved long-term outcome of HCV-positive patients after LT.