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The outcome of liver transplantation for hepatitis B virus (HBV)–related liver disease depends on the prevention of graft reinfection.1 In the absence of prophylactic therapy, the vast majority of patients develop recurrent HBV infection and severe hepatitis leading to graft failure and death.2–5 Since the 1990s, prophylactic strategies using hepatitis B immunoglobulin (HBIG) and antiviral agents such as lamivudine have been developed to dramatically reduce the incidence of HBV recurrence and improve patient survival.3, 6–9 However, neither HBIG nor antiviral agents can completely eradicate the virus,10, 11 and the optimal protocol and duration of prophylaxis remain controversial.1, 12
The host cellular immune response is responsible not only for viral clearance and pathogenesis of liver disease during HBV infection13 but also for control of viral reactivation.14, 15 The HBV-specific immune response in patients with chronic HBV infection may be modified after liver transplantation because of the removal of the major reservoir of virus in the explanted liver and ongoing antiviral and immunosuppressive treatment. The possibility of adoptive transfer of donor immunity has also been reported.16 In this study, we examined the changes in HBV-specific CD4 T cell immune response after liver transplantation for chronic hepatitis B.
ALT, alanine aminotransferase; anti-HBc, antibody against hepatitis B core antigen; anti-HBs, antibody against hepatitis B surface antigen; HBcAg, hepatitis B core antigen; HBeAg, hepatitis B e antigen; HBIG, hepatitis B immunoglobulin; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HLA, human leukocyte antigen; IFN, interferon; OLT, orthotopic liver transplantation; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin.
PATIENTS AND METHODS
Fifty-two patients (48 men and 4 women) who had undergone liver transplantation for HBV-related liver disease under antiviral prophylaxis were enrolled from the liver transplant clinic at Queen Mary Hospital (Hong Kong). They included 40 patients who had no evidence of HBV recurrence [seronegative for hepatitis B surface antigen (HBsAg) and HBV DNA] for at least 1 year (median, 575 days; range, 365–3324 days) and 12 with HBV recurrence (seropositive for HBsAg with or without HBV DNA; median, 624 days; range, 356–3042 days) after transplantation. The demographic, clinical, and virologic profiles of these patients are shown in Table 1. At the time of enrollment, all 40 patients without HBV recurrence had been persistently seronegative for HBsAg and HBV DNA as determined by the Amplicor quantitative polymerase chain reaction assay (lowest detection limit, 300 copies/mL) for at least 6 months. Two patients were positive for serum antibody against hepatitis B surface antigen (anti-HBs; 24 and 13.6 mIU/mL, respectively). Thirty-nine of 40 patients had a normal serum alanine aminotransferase (ALT) level, and 1 had a slightly elevated level of 57 IU/L. For the 12 patients who had developed HBV recurrence, the median time of HBsAg reappearance in serum was 405 days (range, 96–2649 days) after transplantation, and the median time from HBsAg reappearance to enrollment was 300 days (range, 182–1344 days). Eight patients had the YIDD (tyrosine, isoleucine, aspartate, and aspartate) mutant detected by direct sequencing of HBV polymerase gene after the reappearance of serum HBsAg. At the time of enrollment, 2 (17%) were seropositive for hepatitis B e antigen (HBeAg), and 7 (58%) were seropositive for HBV DNA in serum with a median titer of 140,000 copies/mL (range, 1040–5,070,000 copies/mL). The median level of ALT was 26 IU/L (range, 13–173 IU/L).
Table 1. Demographic, Clinical, and Virologic Profiles of 52 Patients Who Underwent Liver Transplantation for Hepatitis B–Related Liver Disease
Without HBV Recurrence (n = 40)
With HBV Recurrence (n = 12)
Abbreviations: anti-HBc, antibody against hepatitis B core antigen; anti-HBs, antibody against hepatitis B surface antigen; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus.
Immunosuppressive medications consisted of tacrolimus alone in 51 patients and mycophenolate mofetil alone in 1 patient at the time of study. None of the patients had received anti-CD3 therapy for rejection. All patients were receiving lamivudine monoprophylaxis (100 mg per day) with the addition of adevofir dipivoxil rescue in those with viral breakthrough due to the YIDD mutant. None of these patients had received HBIG. In addition to these 52 posttransplant patients, 11 HBV-naïve healthy adults without any serologic marker of HBV infection or vaccination, 23 HBV-immune healthy subjects with previous self-limited HBV infection [seropositive for both anti-HBs and antibody against hepatitis B core antigen (anti-HBc)], and 40 patients with chronic HBV infection (seropositive for HBsAg) awaiting liver transplantation served as control groups. There was no evidence of hepatitis C virus or human immunodeficiency virus infection or metabolic and autoimmune liver disease in the study subjects. The study was approved by the local institutional review board, and informed consent was obtained from each subject.
T Cell Proliferation Response
Peripheral blood mononuclear cells (PBMCs) were isolated from fresh heparinized venous blood by a standard Ficoll-Hypaque density gradient centrifugation assay. The T cell proliferation response was determined by the 3H-thymidine uptake assay as described elsewhere.16, 17 In brief, PBMCs (2 × 105/well) were cultured in 96-well round-bottomed plates (Nunclon, Gibco BRL, Glasgow, Scotland) for 3 days in the presence of 1 μg/mL phytohemagglutinin (PHA; Sigma-Aldrich, St. Louis, MO) or for 6 days in the presence of the different antigens. The dose titration experiments were performed, and the final concentrations of the antigens were 2 μg/mL for recombinant HBsAg (Biodesign International, Saco, ME) derived from Saccharomyces cerevisiae yeast (>98% pure), 1 μg/mL for recombinant hepatitis B core antigen (HBcAg; Biodesign International) derived from Escherichia coli (>95% pure), and 1 μg/mL for tetanus toxoid (Sigma-Aldrich). Each well was pulsed with 0.5 μCi of 3H-thymidine (Amersham Biosciences, Buckinghamshire, England) 12 hours before harvesting. The amount of radiolabel incorporated into DNA was measured by a beta-counter (MicroBeta Trilux Counter, Wallac, Turku, Finland). All proliferation assays were performed in triplicate. The results were expressed as the stimulation index, which was calculated by the division of the mean counts per minute incorporated in the presence of the mitogen or antigens by those obtained in the control cultures (PBMCs with a culture medium only). A stimulation index of greater than 3 (higher than the mean plus 2 standard deviations for HBV-naïve subjects) was considered positive.
HBV-Specific Interferon-γ (IFNγ)–Secreting CD4 T Cell Response
The CD4 T cell response against HBV was assessed by the determination of the frequency of IFNγ-secreting T cells after in vitro challenge with HBsAg or HBcAg with the enzyme-linked immunosorbent spot assay as described18, 19 with some modifications. Briefly, polyvinylidene-difluoride membrane–bottomed 96-well plates (MAIP S4510, Millipore, Billerica, MA) were coated at 4°C overnight with anti-human IFNγ capture antibody at 5.0 μg/mL (BD Biosciences Pharmingen, San Diego, CA). After washing, plates were preincubated with complete Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal calf serum for 1 hour at 37°C. In parallel, PBMCs at 2 × 105/well were cultured in triplicate with 4 μg/mL recombinant HBsAg or 2 μg/mL recombinant HBcAg for 48 hours at 37°C and 5% CO2 in a humidified atmosphere. After washing, biotinylated anti-human IFNγ detection antibody (BD Biosciences Pharmingen) at 2 μg/mL was added to the plates, which were then incubated for 2 hours at room temperature; this was followed by washing and the addition of avidin–horseradish peroxidase (BD Biosciences Pharmingen) for 1 hour. An enzyme reaction with a freshly prepared 3-amino-9-ethyl-carbazole–containing substrate solution (BD Biosciences Pharmingen) was carried out for 20 minutes, and the wells were rinsed with water to stop the reaction. After air drying, dark red spots reflecting IFNγ-secreting cells were automatically enumerated with the ImmunoSpot Image Analyzer (Cellular Technology, Ltd., Cleveland, OH). The mean number of HBV-specific spot-forming cells per 106 PBMCs was calculated by subtraction of the number of positive spots in wells without antigen (control) from that in wells with HBsAg or HBcAg. The result was considered positive if the number of HBV-specific spot-forming cells was greater than 50 (higher than the mean plus 2 standard deviations for HBV-naïve subjects).
Tests for serum HBsAg, HBeAg, anti-HBs, antibody against HBeAg, and anti-HBc were performed by microparticle enzyme immunoassay with commercially available kits (IMx System, Abbott Laboratories, Chicago, IL). Subjects were considered immune to HBV if the serum anti-HBs titer was >10 mIU/mL. The serum HBV DNA titer was measured with quantitative polymerase chain reaction assay (Cobas Amplicor, Roche Diagnostics, Basel, Switzerland) with a lower detection limit of 300 copies/mL.
The Mann-Whitney U test was used to compare continuous variables, and proportions were compared by the chi-square test. Correlation analysis was performed with the Spearman test. Statistical analysis was conducted with a standardized biomedical statistical program (SPSS/PC+, SPSS, Inc., Chicago, IL), and a P value of less than 0.05 was considered significant.
T Cell Proliferation Response to the Mitogen (PHA) and Recall Antigen (Tetanus Toxoid)
When compared to HBV-naïve or HBV-immune healthy subjects, pretransplant patients with chronic HBV infection had significantly lower levels of T cell proliferation response to PHA (P = 0.029 and 0.019, respectively), and this suggested an impaired overall T cell reactivity in these patients with chronic hepatitis B. There were no statistically significant differences in the proliferation response to PHA between pretransplant patients with chronic HBV infection and posttransplant patients with or without HBV recurrence (Fig. 1). T cell proliferation responses to tetanus toxoid tended to be lower in pretransplant patients with chronic HBV infection and in posttransplant patients with or without HBV recurrence, but the difference was not statistically significant.
T Cell Proliferation Response to HBV Antigens
Figure 2 illustrates the T cell proliferation response to in vitro stimulation with recombinant HBsAg or HBcAg. The T cell proliferation response to HBsAg was significantly lower in posttransplant patients without HBV recurrence than in HBV-immune healthy controls (P <0.001) or pretransplant patients with chronic hepatitis B (P <0.001). It was positive in only 2 of 40 (5%) posttransplant patients without HBV recurrence versus 9 of 23 (39%) HBV-immune healthy subjects (P = 0.001) and 11 of 40 (28%) pretransplant patients with chronic hepatitis B (P = 0.006). The differences in the T cell proliferation response to HBcAg were more remarkable, the response again being significantly lower in the posttransplant patients without HBV recurrence than in HBV-immune healthy controls (P <0.001) or pretransplant patients with chronic hepatitis B (P <0.001). Only 5 of 40 (13%) posttransplant patients without HBV recurrence showed a positive T cell proliferation response to HBcAg versus 15 of 23 (65%) HBV-immune healthy subjects and 21 of 40 (53%) pretransplant patients with chronic hepatitis B (P < 0.001). The HBV-immune subjects with previous self-limited infection mounted a particularly intense response to HBcAg.
In contrast, in the posttransplant patients with HBV recurrence, the T cell proliferation response to HBsAg or HBcAg was comparable to that of HBV-immune healthy subjects (P = 0.754 and 0.651, respectively) or pretransplant patients with chronic hepatitis B (P = 0.385 and 0.905, respectively) and was significantly higher than that of posttransplant patients without recurrence (P < 0.001 in both comparisons). It was positive in 4 of 12 (33%) patients to HBsAg and in 7 of 12 (58%) patients to HBcAg (P = 0.021 and 0.003, respectively, in comparison with posttransplant patients without recurrence).
The proliferation response to HBV antigens was significantly inhibited in a dose-dependent manner when the antibody against human leukocyte antigen DR (HLA-DR) was added at the initiation of the cell culture but not when addition was delayed for 5 days (data not shown). This confirmed that the HBV-specific proliferation response was HLA class II–restricted.
HBV-Specific IFNγ-Secreting T Cell Response
The HBV-specific IFNγ-secreting T cell response showed a similar pattern to the T cell proliferation response to HBV antigens. Posttransplant patients without HBV recurrence had a significantly lower frequency of HBsAg-specific or HBcAg-specific circulating IFNγ-secreting T cells in comparison with HBV-immune healthy controls and pretransplant patients with chronic hepatitis B (P < 0.001 in all comparisons; Fig. 3). The IFNγ-secreting T cell response to HBsAg was positive in only 2 of 40 (5%) posttransplant patients without HBV recurrence versus 11 of 23 (48%) HBV-immune healthy subjects and 17 of (43%) 40 pretransplant patients with chronic hepatitis B (P < 0.001 in both comparisons). The difference in response to HBcAg was again more obvious, the response being positive in only 8 (20%) of the 40 posttransplant patients without HBV recurrence versus 18 of 23 (78%) HBV-immune healthy subjects and 27 of 40 (68%) pretransplant patients with chronic hepatitis B (P < 0.001 in both comparisons).
Similar to the pattern of the T cell proliferation response, the frequency of HBsAg-specific or HBcAg-specific circulating IFNγ-secreting T cells in the posttransplant patients with HBV recurrence was significantly higher than that in those without recurrence (P <0.001 and 0.015, respectively) and was comparable to that in HBV-immune healthy subjects (P = 0.848 and 0.281, respectively) or pretransplant patients with chronic hepatitis B (P = 0.474 and 0.745, respectively; Fig. 3). Seven (58%) and 8 (67%) of the 12 posttransplant patients with HBV recurrence showed a positive IFNγ-secreting T cell response to HBsAg and HBcAg, respectively, versus 2 (5%) and 8 (20%) of the 40 patients without recurrence (P < 0.001 and P = 0.004, respectively).
HBV-Specific T Cell Response and Viral Load
Among the 12 patients with HBV recurrence, 5 had significant viremia with an HBV DNA titer ≥ 104 copies/mL, and all 5 had a positive T cell proliferation and IFNγ-secreting T cell response to HBcAg. Their HBcAg-specific T cell proliferation and IFNγ-secreting T cell responses were significantly higher than those of the remaining 7 patients with a serum HBV DNA titer < 104 copies/mL (P = 0.005 in both comparisons by the Mann-Whitney U test; Fig. 4). There was a significant correlation between the HBV DNA titer and the level of HBcAg-specific T cell responses in the 12 posttransplant patients with HBV recurrence (HBcAg-specific T cell proliferation response, R2 = 0.718, P = 0.009; HBcAg-specific IFNγ-secreting T cell response, R2 = 0.653, P = 0.021, by the Spearman correlation test). In turn, the patients with a significant HBV-specific T cell response had higher ALT levels than those without it, although the difference did not reach statistical significance (P = 0.074).
Resolution of HBV infection is dependant on a cellular immune response that provides long-term viral suppression rather than eradication.20 CD4 T cells are an important subset because these cells are needed to support the priming of virus-specific cytotoxic T cells,21, 22 to help the production of neutralizing antibodies by HBV-specific B cells,23, 24 and to maintain an anamnestic response against viral reactivation.14 Patients with chronic HBV infection have a less vigorous CD4 T cell response than those with acute self-limited infection, and this lack of cellular immunity is considered a major reason for the failure of viral clearance.17, 25 After liver transplantation, the cellular immunity may be suppressed further by the immunosuppressive treatment. Effective prophylaxis against reinfection requires the long-term use of passive immunoprophylaxis and/or antiviral agents, but the ultimate cure may depend on the recovery of the host immune response.
The present study shows that the frequency and magnitude of CD4 T cell responses against both HBV envelope and nucleocapsid antigens were indeed significantly lower in the posttransplant patients after HBV clearance with the use of antiviral prophylaxis alone without HBIG in comparison with those of pretransplant patients with chronic hepatitis B. Immunosuppression alone, however, could not account for this low HBV-specific response because T cell reactivity to the mitogen (PHA) and recall antigen (tetanus toxoid) was maintained. This is further supported by the fact that when HBV recurrence developed, posttransplant patients were able to mount a significant T cell response to HBV antigens despite the ongoing immunosuppression, and the response significantly correlated with the viral load in circulation. Hence, the disappearance of cellular immunity against HBV was the result of viral clearance after liver transplantation, and the CD4 T cell response is dependent on the presence of continuing antigenic stimulation. By virtue of the removal of the major reservoir of HBV in the explanted liver and the continuous antiviral treatment, liver transplantation represents the most drastic measure to reduce the viral load in chronically HBV-infected patients. Our results provide strong evidence to support the concept that the CD4 T cell–mediated immune response is an antigen-driven process, and the activation and expansion of virus-specific CD4 T cells depend on the recognition of viral peptides presented by HLA molecules.14, 26 It has been shown that long-term effective antiviral treatment may reduce the HBV-specific T cell immune responses in patients with chronic HBV infection, at least in some of them, to undetectable levels.27 In the present study, all patients received antiviral prophylaxis, and none received HBIG intraoperatively or postoperatively. Our findings contrast with those of a recent study reporting that recipients with successful HBIG prophylaxis showed a sustained and focused HBcAg-specific T cell response pattern similar to that of patients with nonviremic chronic HBV infection,29 and this may be attributed to the difference in the prophylactic strategy or methodology for the T cell response assay. However, the presence of active proliferative CD4 T cell responses in recipients with HBV recurrence and liver damage was in line with the finding of another study.30
The low response with viral clearance and the high response after recurrence in the current study indicate that the CD4 T cell immunity is unlikely to be the major cause of the HBV clearance after liver transplantation under antiviral prophylaxis. Instead, the HBV clearance is attributable mainly to the removal of the native liver and the ongoing antiviral therapy. The general lack of effective CD4 T cell immunity after HBV clearance suggests that the risk of HBV reinfection continues and that long-term antiviral prophylaxis is necessary. Nonetheless, our results show some liver transplant recipients with cellular immunity, preferentially nucleocapsid-specific, comparable to individuals with self-limited hepatitis B. Further studies may identify those recipients whose cellular immunity can afford long-term HBV suppression with or without the use of HBV vaccination, with the eventual goal that all antiviral prophylaxis could be terminated.
The reconstitution of the HLA class II–restricted T cell immune response has been documented in patients with recurrent HBV or hepatitis C virus infection after liver transplantation and is independent of the HLA matching between the donor and recipient.29-33 The reconstitution of the HBV-specific immune response in this setting may be due to the redundancy and pleiotropic nature of the immune system. Our results suggest that the CD4 T cell immunity of these posttransplant patients resembles that of pretransplant patients with chronic hepatitis B. The magnitude of the cellular immune response is dependant on the viral load and is associated with the pathogenesis of chronic liver injury.29
We recognize several shortcomings in the current study. The source of immunocompetent cells used in the present ex vivo study was taken from the peripheral blood compartment, which might only partially reflect the immune events mounted in vivo upon viral antigen presentation to the immune system. Moreover, we assessed the HBV-specific T cell response only at 1 time point more than 1 year after liver transplantation, and the interval from transplantation to the study ranged from 1 to 9 years. The T cell immune response against viral infection is a dynamic event and varies greatly from person to person and even within a given person from time to time.13, 34 For patients with HBV recurrence, for example, data on HBV cellular immunity before recurrence were not available for comparison. The possibility of adoptive transfer of a donor's cellular immunity could not be assessed because a previous study indicated that the adoptive transfer of humoral immunity through liver transplantation lasted for an average duration of 7 months only.16 Hence, in order to understand the kinetics and role of the cellular immune response against HBV after liver transplantation, a prospective study on serial changes in HBV-specific cellular immunity at standardized time points before and after liver transplantation would be necessary.
In summary, this study shows that HBV-specific CD4 T cell immunity is unlikely to be responsible for the viral clearance after liver transplantation under antiviral prophylaxis. The cellular immunity is antigen-driven and evanesces with viral clearance, hence providing a favorable milieu for viral reactivation once prophylaxis is withdrawn. The cellular immunity to HBV after liver transplantation is not significantly different from that of individuals with chronic hepatitis B. When recurrence develops, the enhanced cellular immunity, which is dependant on the viral load, is not effective in providing HBV resolution but may contribute to graft damage.