Adoptive transfer of immunity to hepatitis B virus (HBV) through organ transplantation was first documented in patients who received bone marrow transplant from donors with HBV immunity.1 Donor-derived immunity may help to clear hepatitis B surface antigen (HBsAg) in bone marrow recipients with chronic HBV infection1, 2 and to maintain a long-term immune memory against HBV in these immunosuppressed individuals.3 Based on the potential benefit in prevention of HBV infection, the related donors of chronic myeloid leukemia patients were advised to receive vaccinations against HBV before bone marrow donation.4 This phenomenon of adoptive HBV immunity transfer has recently been observed in liver transplantation. An initial study has shown the spontaneous development of antibody against hepatitis B surface antigen (anti-HBs) in recipients with chronic HBV infection on lamivudine monoprophylaxis after liver transplantation that may be attributed to adoptive transfer of donors' immunity against HBV through the liver grafts.5 Dahmen et al.6 have also demonstrated the adoptive transfer of anti-HBs immunity through liver transplantation in a rat model. In a woodchuck model, the adoptive transfer of donor-derived immunity after active vaccination of donors with woodchuck hepatitis virus surface antigen was effective in reducing and delaying hepatitis virus reinfection of grafts after liver transplantation.7 We hypothesized that this phenomenon results from an adoptive transfer of HBV-specific lymphocytes through a liver graft.5 Intrahepatic T cells specific for HBV antigens have been well characterized in HBV-infected patients,8, 9 and the HBV-specific immunocompetent cells can last for decades following resolution of HBV infection10 or active immunization.11 In addition, it has been shown that the human liver graft contains a considerable number of resident lymphocytes that were often described as passenger lymphocytes in liver transplantation setting,12, 13 but a direct demonstration of lymphocytes with specific functional relevance has never been attempted. To elucidate the mechanism for the immunity transfer, we characterized the resident lymphocytes in human liver grafts, and quantified the intrahepatic HBV-specific lymphocytes in relation to the donor and subsequent recipient immunity.
Both animal and human studies have demonstrated the adoptive transfer of immunity against hepatitis B virus (HBV) through liver transplantation that may be attributed to the presence of HBV-specific immunocompetent cells of donor origin in liver grafts. In this study, we characterized the resident lymphocytes in 41 human liver grafts by immunohistochemical staining and flow cytometry and directly identified the intragraft HBV-specific lymphocytes in relation to the donor's and subsequent recipient's immunity using enzyme-linked immunospot assay. A significant number of HBV-specific T and B cells were detectable in 59.4% (19/32) and 28.1% (9/32), respectively, of liver grafts from HBV-immune donors. The presence of various HBV-specific lymphocytes was closely associated with each other and with a higher serum titer of antibody against hepatitis B surface antigen (anti-HBs) in donors (P < 0.05). After liver transplantation, 17 of 35 (48.6%) patients with chronic HBV infection showed a spontaneous anti-HBs production, which was significantly associated with a higher number of donor-derived T lymphocytes specific for hepatitis B surface antigen (P = 0.043). In conclusion, the presence of considerable numbers of donor-derived HBV-specific immunocompetent cells in grafts may account for the adoptive transfer of HBV immunity through liver transplantation. Liver Transpl, 2006. © 2006 AASLD.
PATIENTS AND METHODS
Liver Donors and Recipients
From April 2003 to August 2004, wedge biopsies were obtained from 41 liver grafts from live donors (n = 33) or deceased donors (n = 8) after perfusion with preservation solution on the back-table. Forty donors were ethnic Chinese, and 1 was Pakistani. There were 18 men and 23 women, with a median age of 37 years (range, 21–67 years). The cause of death in all deceased donors was cerebrovascular accident. The 33 live donor grafts were right lobe grafts with the middle hepatic vein. Liver biochemistry was normal in all donors, and there was no evidence of any viral, autoimmune, metabolic, or drug-associated liver disease. The liver grafts were divided into 4 groups according to the HBV-serologic status of the donors: 22 donors (group 1) were positive for both anti-HBs and antibody against hepatitis B core antigen (anti-HBc), 10 (group 2) were positive for anti-HBs only, 2 (group 3) were positive for anti-HBc only, and 7 (group 4) were negative for both anti-HBs and anti-HBc.
Of 41 corresponding recipients, 35 underwent liver transplantation for HBV-associated liver disease and were positive for HBsAg before transplantation; 4 for hepatitis C virus-associated liver disease (1 positive for both anti-HBs and anti-HBc; 1 positive for anti-HBc only; 2 negative for both anti-HBs and anti-HBc); 1 for Wilson's disease, positive for both anti-HBs and anti-HBc; and 1 for primary biliary cirrhosis, was positive for anti-HBs only. The median levels of pretransplantation viral load determined by the quantitative polymerase chain reaction assay (Cobas Amplicor, Roche Diagnostics, Basel, Switzerland) was 35,550 copies/mL (range, 304 to 30,500,000 copies/mL) in 33 HBV-infected patients, and HBV DNA titer was less than the cutoff level (300 copies/mL) in the remaining 2 patients. Lamivudine (100 mg per day) was started before transplantation and continued indefinitely afterward, and adefovir dipivoxil (10 mg per day) was added if viral mutations at the YMDD (tyrosine, methionine, aspartate, and aspartate) motif were identified. None of the patients received hepatitis B immunoglobulin. All liver donors and recipients gave written informed consent to the study. The study was conducted according to the ethical guidelines of the Helsinki Declaration and was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster.
Five-micrometer sections were prepared from liver biopsy samples that had been formalin-fixed and paraffin-embedded. Sections were stained with antibodies to a pan T cell marker CD3 (dilution 1:100, DAKO, Glostrup, Denmark) and a pan B cell marker CD20 (dilution 1:200, DAKO) using the Avidin-Biotin Complex immunoperoxidase method. The isotype-matched immunoglobulin G control antibody was used instead of the primary antibody to control nonspecific staining (negative control).
Isolation of Resident Lymphocytes in Liver Grafts
Intrahepatic lymphocytes were isolated according to the method described by Hata et al.14 Briefly, wedge biopsies of liver grafts were taken after extensive perfusion with University of Wisconsin solution for those from deceased donors or histidine-tryptophan-ketoglutarate solution for those from live donors and were gently dissected into 1-mm3 pieces in complete Roswell Park Memorial Institute (RPMI) 1640 medium containing 0.5 mg/mL type IV collagenase (312 U/mg, Sigma-Aldrich, St. Louis, MO), 0.02 mg/mL DNase I (Sigma-Aldrich), 10% fetal calf serum, penicillin (100 U/mL), streptomycin (100 μg/mL) and 2 mmol/L L-glutamine. The resulting mixture was incubated with gentle agitation at 37°C for 30 minutes and passed through a nylon mesh filter (40-μm diameter) to remove undissociated tissue clumps, centrifuged at 300g for 10 minutes, resuspended in Hank's Balanced Salt Solution, and washed twice. The hepatocyte-rich matrix was removed by centrifugation for 1 minute at 30g. The final pellet was resuspended in complete RPMI 1640 medium. The mononuclear cell suspension was recovered by centrifugation over Ficoll-Hypaque density gradient at 300g for 10 minutes. Cell viability and numbers were assessed by a hemocytometer-based trypan blue dye exclusion test. Peripheral blood was obtained from all donors before liver harvest to isolate the peripheral blood mononuclear cells using standard Ficoll-Hypaque density centrifugation separation techniques. The peripheral blood mononuclear cells were inactivated by 25 μg/mL of mitomycin C (Sigma-Aldrich) and served as autologous antigen-presenting cells in enzyme-linked immunospot (ELISPOT) assay for the detection of HBV-specific T cells.
Flow Cytometric Analysis
Lymphocytes were gated on the basis of both forward and side scatter on a FACScan cytofluorometer (Becton Dickinson FACS System, San Jose, CA), and 10,000 gated cells were analyzed. The percentage of positive cells was calculated using CELLquest software (Becton Dickinson). Double immunofluorescence staining was performed with the following combinations of conjugated antibodies: CD3-FITC, CD4-Allophycocyanin, CD8-Peridinin chlorophyll protein, CD56-PE, CD20-PE (all monoclonal antibodies, BD Biosciences Pharmingen, San Jose, CA). Irrelevant monoclonal antibodies of appropriate isotype served as negative controls.
Identification and quantification of anti-HBs-secreting B cells and HBV-specific interferon (IFN)-γ-secreting T cells in intrahepatic lymphocytes were performed by ELISPOT assay.11, 15 For the detection of anti-HBs-secreting B cells, polyvinylidene-difluoride membrane-bottomed 96-well plates (MAIP S4510 Millipore, Billerica, MA) were coated at 4°C overnight with 100 μL/well of a solution containing 5 μg/mL recombinant HBsAg ad subtype (Biodesign International, Saco, ME) derived from yeast Saccharomyces cerevisiae (>98% pure). Complete RPMI 1640 medium supplemented with 10% fetal calf serum was used as negative control. After washing with phosphate-buffered saline, plates were preincubated with complete RPMI 1640 medium for 1 hour at 37°C before intrahepatic lymphocytes were added in triplicates in concentration of 4.0 × 105 per well. After incubation for 24 hours at 37°C and 5% carbon dioxide, plates were washed with phosphate-buffered saline and incubated with horseradish peroxidase–linked rabbit immunoglobulin G antibodies (1:1000; DAKO) for 1 hour at room temperature followed by washing and staining with the 3-amino-9-ethyl-carbazole-containing substrate (Sigma-Aldrich) solution for 20 minutes. After washing and drying, dark red spots reflecting anti-HBs-secreting B cells were automatically enumerated using the ImmunoSpot Image Analyzer (Cellular Technology Ltd., Cleveland, OH). No positive spot was detected in the negative control group. Results were expressed as means of triplicates of number of spot-forming cells per 106 intrahepatic lymphocytes.
HBV-specific IFN-γ-secreting T cells were studied following a similar method by coating with anti-human IFN-γ capture antibody at 5.0 μg/mL (BD Biosciences Pharmingen) at 4°C overnight. Two × 105 intrahepatic lymphocytes and 1 × 105 autologous antigen-presenting cells treated with 25 μg/mL of mitomycin C were added in triplicates and incubated for 48 hours in presence of 4 μg/mL of recombinant HBsAg, 2 μg/mL recombinant hepatitis B core antigen (HBcAg) (Biodesign International) derived from E. coli (>95% pure) or medium only (control). After washing, the plates were added with biotinylated anti-human IFN-μ detection antibody (BD Biosciences Pharmingen) at 2.0 μg/mL and incubated for 2 hours followed by washing and adding avidin–horseradish peroxidase (BD Biosciences Pharmingen) for 1 hour. The staining and spot counting procedure was performed as described above. The number of HBV-specific IFN-μ-secreting cells was calculated by subtracting the number of spots in wells without HBV antigen (control) from that of spots in wells with HBsAg or HBcAg. A result was considered positive if the number of HBV-specific spots was >3 (greater than the mean number + 1 SD of the control).
Serum samples were collected from the donors and recipients before transplantation, and from recipients at day 7, day 14, day 21, day 28, month 3, month 6, and year 1 after transplantation. The samples were tested for the anti-HBs and anti-HBc by microparticle enzyme immunoassay using commercially available kits (IMx System, Abbott Laboratories, Chicago, IL). The anti-HBs titer was determined by a standard curve obtained using calibrators containing anti-HBs with concentrations standardized against the World Health Organization reference standards and was expressed as mIU/mL. Donors were considered immune if the serum anti-HBs titer was >10 mIU/mL. The spontaneous development of anti-HBs by the recipient was determined according to the criteria as described previously.5 These were, in brief, (1) anti-HBs titer >10 mIU/mL in 2 or more consecutive samples; (2) a >100% increase in anti-HBs titer as compared with that of day 7 after liver transplantation; and (3) absence of transfusion of any blood products associated with the increase in anti-HBs titer.
Continuous variables were expressed as mean ± SD. The statistical tests used included the Mann-Whitney U test, chi-square test, Fisher exact test, and linear regression test. Statistical analysis was conducted with standardized biomedical statistical program (SPSS/PC+, SPSS, Inc., Chicago, IL).
Resident Lymphocytes in Liver Grafts
Immunohistochemical staining with anti-CD3 revealed the presence of a large number of resident T lymphocytes scattered throughout the human liver graft. These cells were predominantly located around the portal tracts with fewer cells scattered throughout the sinusoids of the liver parenchyma. CD20+ B cells were detected in a much smaller number than T cells (Fig. 1).
Liver tissue samples of 0.78 ± 0.14 g were obtained. The mean yield per gram of liver tissue was 3.7 ± 1.9 × 106 mononuclear cells, and the viability was 86.4 ± 5.8%. Flow cytometry showed that natural killer cells bearing the classical phenotype (CD3−, CD56+) accounted for 27.6 ± 9.1% of total intrahepatic lymphocytes. The majority of CD3+ T cells expressed CD8 (67.3 ± 14.1%), whereas a smaller proportion expressed CD4 (23.3 ± 9.7%). Natural killer-T cells bearing both the T cell marker CD3 and the natural killer cell marker CD56 constituted 29.7 ± 18.1% of all CD3+ lymphocytes. B cells (CD20+) accounted for 6.3 ± 3.8% of the total intrahepatic lymphocytes.
HBV-Specific Lymphocytes in Liver Grafts
ELISPOT assay revealed the presence of IFN-γ-secreting T cells specific for HBsAg or HBcAg in 19 of 32 (59%) liver grafts from HBV immune donors seropositive for anti-HBs with or without anti-HBc (groups 1 and 2) (Fig. 2). HBsAg-specific and HBcAg-specific T cells were detected in 41% (9/22) and 55% (12/22), respectively, of liver grafts from donors positive for both anti-HBs and anti-HBc (group 1). In liver grafts from donors positive for anti-HBs only (group 2), 70% (7/10) had detectable HBsAg-specific T cells, but none had HBcAg-specific T cells. HBsAg-specific or HBcAg-specific T cells were not detectable in any of the liver grafts of group 3 or group 4. Anti-HBs-secreting B cells were detected in 28% (9/32) of liver grafts from HBV-immune donors, 32% (7/22) in group 1, and 20% (2/10) in group 2 (Fig. 3). No anti-HBs-secreting B cells were detected in liver grafts of group 3 and group 4. The mean numbers of detectable HBsAg-specific, HBcAg-specific T cells, and anti-HBs-secreting B cells in liver grafts were 6.2 ± 2.4, 5.8 ± 1.7, and 2.8 ± 1.2 per 106 intrahepatic lymphocytes, respectively.
To identify the factors that could correlate with the presence of HBV-specific lymphocytes in liver tissues from HBV immune donors, 4 variables related to donors' characteristics were compared among donors with and without detectable HBV-specific intrahepatic lymphocytes (Table 1). Statistical analysis showed that the presence of HBsAg-specific T cells and anti-HBs-secreting B cells in liver grafts was significantly associated with the serum anti-HBs level of the donors (P < 0.001 and P = 0.002, respectively). The presence of T cells specific for HBcAg was significantly related to the serum anti-HBs level and anti-HBc status of the donors (P = 0.009 and P < 0.001, respectively). For 22 liver donors seropositive for anti-HBs and anti-HBc (group 1), there was a significant correlation between the number of HBsAg-specific T cells and that of the HBcAg-specific T cells (R = 0.564; F = 9.307; P = 0.006). The frequencies of IFN-γ-secreting T cells specific for HBsAg or HBcAg were significantly higher when anti-HBs-secreting B cells were detectable (P < 0.001 and P = 0.017, respectively).
|Donors' Characteristics||HBsAg-Specific T Cells||HBcAg-Specific T Cells||Anti-HBs-Secreting B Cells|
|Age, years (range)*||37 (21–56)||36 (24–67)||0.758||36 (21–56)||42.1 (28–67)||0.115||36.5 (21–67)||38 (25–49)||0.518|
|Graft type (live/cadaveric)||19/6||14/2||0.448||24/5||9/3||0.672||24/8||9/0||0.164|
|Anti-HBs titer (mIU/mL)†||35.2 (0–142)||1400.1 (281–3067)||<0.001||92.7 (0–393)||1,108.5 (217–2,065)||0.009||79.1 (2.3–641.3)||1,495 (554–4,140)||0.002|
Serology of Liver Recipients
After liver transplantation, 34 of 35 (97%) HBsAg-positive recipients cleared HBsAg in circulation (median time, 7 days; range, 1–183 days), and 17 (49%) developed anti-HBs production after transplantation according to the criteria as described previously.5 Among these 17 patients, 13 had received liver grafts from donors seropositive for both anti-HBs and anti-HBc (group 1) and 4 from donors seropositive for anti-HBs only (group 2), but there was no statistical difference in the development of anti-HBs response between the 2 groups (P = 0.45). The serial changes of anti-HBs titer in the 17 liver recipients with anti-HBs production are shown in Figure 4. The increase of serum anti-HBs titer in the recipients was observed with a peak during 2 to 12 weeks posttransplantation in the absence of blood products transfusion. As compared with the anti-HBs level on day 7 posttransplantation, the increase was more than 100-fold in 2 recipients, 10- to 100-fold in 9 recipients, and 2- to 10-fold in 6 recipients. Statistical analysis by Mann-Whitney U test showed that the anti-HBs production was not associated with any of the recipients' characteristics or with the number of the HBcAg-specific T cells (P = 0.082) and anti-HBs-secreting B cells (P = 0.088) in the liver graft, but it was significantly associated with the number of HBsAg-specific T cells in the liver graft (P < 0.001) and the serum anti-HBs level of the liver donor (P < 0.001) (Fig. 5, 6). Multivariate analysis using a logistic regression model showed that only the number of intragraft HBsAg-specific T cells was the independent predictor for the production of anti-HBs in the recipients (odds ratio, 1.524; 95% confidence interval, 1.014 to 2.291; P = 0.043). None of the remaining 13 liver recipients, who were negative for HBsAg or received liver grafts from donors without HBV immunity, fulfilled the criteria for spontaneous anti-HBs production.
The liver has not been conventionally regarded as a lymphoid organ that can harbor large numbers of tissue-resident lymphocytes. The presence of lymphocytes in the liver tissue has largely been considered as inflammatory infiltrates that enter the liver from the blood in response to pathological stimuli.14 More recent studies, however, have shown that the normal liver contains a significant number of lymphocytes that appear to be resident.13, 16 The resident lymphocytes were estimated to constitute about 25% of the nonparenchymal cell pool in normal human liver tissues13, 17 and to play an important role in immunosurveillance and maintenance of immunological homeostasis.18 The total number of the resident lymphocytes, however, was a matter of considerable debate, and the estimated numbers vary, ranging from 109 to 1011 cells for a normal adult human liver graft.17, 18 The low-flow environment of liver sinusoid and high expression of adhesion molecules, such as intercellular adhesion molecule-1, vascular adhesion protein-1, on endothelial cells and Kupffer cells, may be responsible for the retention of lymphocytes that flow through the liver.19–21 Therefore, selective homing and retention of peripheral lymphocytes into the liver and subsequent expansion in response to the hepatic cytokine milieu may in part account for the presence of resident lymphocytes in human liver grafts.22, 23 In addition, a few studies have demonstrated that apart from cells derived from periphery some populations of intrahepatic lymphocytes might originate locally.24–26
These resident lymphocytes in the healthy human liver are believed to play an important role not only in regional immunity,18 but also in immunomodulation in the posttransplantation setting.27 Another consequence that might be expected after transfer of these allogeneic lymphocytes into a liver recipient is graft-vs.-host disease28 and, occasionally, other immune-mediated disease conditions, such as idiopathic thrombocytopenic purpura,29 peanut allergy,30 and immune-hemolytic anemia.31 In contrast to these adverse immunologic events, the adoptive transfer of immunity against HBV through liver transplantation can potentially promote the clearance of residual virus and protect the liver graft from HBV reinfection.5, 6 The phenomenon represents a transfer of HBV-specific immunocompetent cells in the liver grafts from HBV-immune donors. The presence of HBV-specific resident lymphocytes in liver grafts and its functional relevance, however, have not been determined yet.
In the present study, we adopted the ELISPOT assay to directly detect the HBV-specific lymphocytes in liver grafts. The ELISPOT assay allows a direct detection of cytokines or antibody in an antigen-specific manner at a single cell level and is therefore useful in the identification of virus-specific T or B lymphocytes at very low frequencies.11, 15 Using this technique, we detected the presence of anti-HBs-secreting B cells in 28% of liver grafts from HBV-immune donors. Theoretically, there should always be some, albeit small, number of anti-HBs-secreting lymphocytes in the body of liver donors with positive anti-HBs. The liver, however, is not the major lymphoid organ, such as bone marrow,32, 33 to sustain the antibody response. Furthermore, the low proportion of B cells (6.3%) in the intrahepatic lymphocyte population, the limited size of the biopsy, and the detection limit of ELISPOT assay might be responsible for such a low detection rate. HBV-specific T cells, however, were detected in a greater proportion (59%) of liver grafts from HBV-immune donors, and this was compatible with the larger number of T cells shown by immunohistochemical staining and flow cytometry.
Our results indicated that there was a significant association between the donor's serum anti-HBs level and the number of various HBV-specific intrahepatic lymphocytes. The numbers of these HBV-specific lymphocytes were closely related to each other, and the number of HBs-Ag-specific T cells, in turn, significantly correlated with the anti-HBs production in the liver recipients. Further characterization of the HBV-specific lymphocytes derived from donors in the blood of liver recipients is difficult because of the small number of these cells. Moreover, these donor-derived lymphocytes are released from the liver grafts after transplantation and migrate preferentially at first to the host lymphoid organs.34, 35 The duration of survival of these immunocompetent cells in the liver recipient is not very clear. Schlitt et al.27 have shown that the donor-derived lymphocytes could not be detected in the liver recipient's circulation with flow cytometry 1 month after transplantation, but the phenomenon of the long-lasting microchimerism has been well described.35 This immunity transfer occurs not only from donors with naturally acquired immunity against HBV (seropositive for both anti-HBs and anti-HBc), but also from those with vaccine-induced immunity (anti-HBs seropositive only).
Although our results showed that there was a significant association between the number of HBV-specific lymphocytes in liver grafts and the development of anti-HBs in recipients after transplantation, the relatively low number of donor-derived lymphocytes seems not sufficient to maintain lasting anti-HBs immunity in recipients. We also recognized that the duration and level of the anti-HBs was not comparable with that following bone marrow transplantation.36 In a prior clinical study recruiting another group of posttransplant patients, we observed that active immunization with 2 courses of double-dose recombinant HBV vaccine was not able to induce a secondary anamnestic response in vast majority of recipients after anti-HBs disappearance.37 Therefore, we have postulated that the transient anti-HBs seroconversion and limited efficacy of booster vaccination in liver recipients appear to be the result of the clearance of most immunocompetent cells of donor origin.5, 37 This is consistent with the short-term presence of donor-derived isohemagglutinins in recipient after ABO-unmatched liver transplantation.31 Nevertheless, it may be worthwhile to investigate the possibility of booster immunization of living donors to augment the immunity transfer, since its efficacy was related to the number of HBV-specific lymphocytes in liver grafts and, in turn, to the anti-HBs titer of the donors. A study in a rat model by Li et al.38 showed that the liver recipients with co-transplantation of bone marrow from HBsAg-immunized donors are able to generate a secondary response to booster vaccination. Apart from the number of immunocompetent cells derived from the donor, other factors, such as the amount of immunosuppression and the extent of HLA compatibility between the donor and recipient, may also affect the efficacy of adoptive transfer of immunity against HBV. A better understanding of the mechanisms for the adoptive transfer of HBV immunity through liver transplantation might help to develop new immunologic strategies in prevention of HBV recurrence.
In conclusion, our study demonstrates for the first time the presence of HBV-specific immunocompetent cells in healthy liver grafts from HBV-immune donors by directly visualizing the cytokine and antibody produced by the virus-specific lymphocytes. The relationship between donors' anti-HBs titer, intrahepatic HBV-specific lymphocytes, and, eventually, recipients' anti-HBs production provides further evidence and a possible mechanism for the adoptive transfer of HBV immunity through liver transplantation.
The authors thank Dr. Wan Ching Yu, Ms. Wai Man Lee, and Ms. Ka Yiu Lam for their assistance in sample collection for this study.