SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Cluster of differentiation (CD)56+ lymphocytes are believed to play important roles in the innate immune response to viral infections by production of interferon (IFN)-γ and/or the recognition of virally infected cells, but their role in liver transplantation (LT) has not been characterized. Here, for the first time, we examine the phenotypic and functional features of these cells in patients undergoing LT for hepatitis C virus (HCV)-related liver failure. The study was comprised of four patient groups: patients with mild HCV recurrence (n = 9), severe HCV recurrence (n = 10), patients with non-HCV-related liver failure (n =10), and normal healthy subjects (n = 10). Pre-LT, the frequency of circulating CD56+ lymphocytes was significantly lower in patients who subsequently developed severe HCV recurrence, relative to those patients who developed mild histologic recurrence, as well as non-HCV controls. HCV was associated with impaired lymphokine-activated killing and natural cytotoxicity. We found that natural T (NT) cells that coexpressed CD4/CD8 or expressed CD8 alone were more frequent in patients who subsequently developed severe recurrence. In contrast, NT cells that expressed only CD4 appeared to be depleted in HCV infection relative to controls. A significantly higher percentage of NTs in both HCV groups expressed the inhibitory receptor NKG2A relative to HCV-negative controls with liver disease. In conclusion, these results demonstrate a previously unappreciated association between pretransplantation CD56+ lymphocytes and outcome of HCV recurrence and provide novel mechanistic insights into the immunopathogenesis of HCV recurrence, as well as potential targets for therapeutic manipulation. Liver Transpl 14:31–40, 2008. © 2007 AASLD.

Liver disease related to hepatitis C virus (HCV) is the single leading indication for orthotopic liver transplantation (OLT) throughout the world. HCV infection significantly impairs patient and allograft survival following liver transplantation (LT) (hazard ratio 1.30 for allograft failure after adjusting for potential confounders)1 and is unique among indications for LT in that recurrence is nearly universal. However, the spectrum of histologic injury related to HCV recurrence is highly variable, ranging from mild histologic abnormalities to allograft cirrhosis in 20 to 30% of recipients by the fifth postoperative year.2 Unfortunately, in contradistinction to other viral infections (e.g., cytomegalovirus), no tests accurately and consistently predict who will develop progressive HCV recurrence prior to LT.

Although the National Institute of Diabetes and Digestive and Kidney Diseases liver transplant database found that high viral loads correlate with diminished graft and patient survival following transplantation,3 therefore providing a rationale for preemptive antiviral treatment, much of the experience to date suggests that interferon (IFN)-based treatment in this setting is fraught with significant morbidity and lack of tolerance.4 Thus, identifying a group of variables before OLT that predicts more aggressive HCV recurrence and a therapeutic approach that targets only patients at risk is of paramount importance.

As reviewed recently, the human LT model provides a unique opportunity and theoretical framework to study HCV immunopathogenesis for a number of reasons.5 Although a number of groups have demonstrated that the presence of HCV-specific effector responses correlate with attenuated histologic severity,6, 7 considerable gaps exist in our understanding of how the immune response shapes HCV recurrence post-OLT. In particular, the role of innate immunity, as represented by natural killer (NK) and natural T (NT) cells,8 the same cell populations that are usually associated with the very early stages of an acute immune response, has not been examined. We hypothesized that the nature of the innate immune response prior to OLT would correlate with the severity of HCV recurrence following LT. In experiments aimed at elucidating the frequency, phenotype, and function of (CD)56+ lymphocytes, we examined patients with HCV who subsequently developed severe histologic recurrence or demonstrated mild histologic recurrence, as well as patients with end-stage liver disease unrelated to HCV and normal healthy controls. Our results indicate a previously unappreciated role for these innate lymphocytes and provide novel mechanistic insights into the immunopathogenesis of HCV recurrence.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Study Patients

The study group was comprised of healthy subjects and patients undergoing LT in Portland, OR. Informed consent was obtained in all cases and Internal Review Board approval was granted at the Portland Veterans' Administration Medical Center and the Oregon Health and Science University. The HCV-infected patients underwent OLT between November 1999 and May 2004 and typically underwent protocol liver biopsies at 6, 12, 18, and 24 months and annually thereafter. Modified hepatitis activity index and fibrosis scores were determined in all specimens9 and used to define the severity of HCV recurrence. Severe HCV recurrence was defined as the presence of grade 3 or 4 inflammation, stage ≥2 fibrosis, and/or evidence of the cholestatic variant. All patients were initially maintained on a double or triple immunosuppressive regimen including tacrolimus, steroids, and/or purine antagonists. Levels of HCV-ribonucleic acid levels in serum were determined by a branched-chain deoxyribonucleic acid signal amplification assay (Quantiplex HCV RNA 2.0 Assay; Bayer Diagnostics, Emeryville, CA). The lower limit of this assay is 0.2 and the upper limit is 120 mEq/mL (equal to 40 million copies/mL).

Sample Preparation and Storage

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using cellular preparation tubes (Becton-Dickinson, Franklin Lakes, NJ; anticoagulant sodium citrate). PBMCs were viably frozen in 80% fetal bovine serum (FBS; BioWhittaker, Walkersville, MD), 10% dimethylsulfoxide, and 10% Roswell Park Memorial Institute (RPMI) 1640 Media (Life Technologies, Grand Island, NY) in liquid nitrogen. Following thaw, PBMCs were cultured in medium consisting of RPMI 1640 Media supplemented with 10% fetal bovine serum (FBS), 50 μg/ml gentamicin sulfate (BioWhittaker), 5 × 10−5 M 2 ME (Sigma, St. Louis, MO), and 2 mM glutamine (Life Technologies).

Plasma preparation tubes (PPT tubes; BD Biosciences, San Jose, CA) were used to isolate plasma from whole blood, which was frozen and later thawed for viral load and genotype testing.

Flow Cytometric Analysis of Cell Surface Antigens

Four-color multiparameter flow cytometry was performed using a BD FACSCalibur instrument (BD Biosciences) compensated with single fluorochromes and analyzed using CellQuest software (BD Biosciences). Fluorochrome-labeled (fluorescein isothiocyanate/PE/PerCP/APC) monoclonal antibodies specific for CD3, CD4, CD8, CD16, CD161, CD56, CD94, CD95, and CD95L were obtained from BD Biosciences. Anti-NKG2A-PE was obtained from Immunotech (Beckman Coulter, Fullerton, CA). PBMCs (2.5 × 105) were stained for cell surface antigen expression at 4°C in the dark for 30 minutes, then washed twice in 2 mL phosphate-buffered saline containing 1% bovine serum albumin and 0.01% sodium azide (FACS Wash) and subsequently fixed in 200 μL of 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO). Isotype-matched fluorescently-labeled control antibodies were used to determine background levels of staining. Lymphocytes were identified by characteristic forward scatter and side scatter parameters and populations of interest were gated on specific patterns of staining for CD3 and CD56 within the lymphocyte population (Fig. 1A). Results are expressed as % positive of gated populations.

thumbnail image

Figure 1. Levels of CD56+ lymphocytes in chronic HCV patients and HCV-negative liver disease patients prior to liver transplantation as well as normal healthy controls. Multi-parameter flow cytometric analysis was used to estimate the levels of CD56 lymphocytes, NK (CD56+CD3) and NT(CD56+CD3+) cells in chronic HCV infection prior to liver transplantation (A). Total CD56+ lymphocyte levels are significantly decreased in all chronic HCV patients compared to normal uninfected control subjects. This reflects a deficiency in both NK and NT cells. A decrease in total CD56+ cells was also observed for control non-HCV chronic liver disease patients due to a significant reduction in NT but not NK cells. Chronic HCV patients were stratified into two groups depending on severity of disease recurrence post liver transplantation. Of interest, all CD56+ lymphocyte populations were decreased in the patient group with subsequent severe outcome compared to those who had mild recurrence of HCV liver disease. Compared to non-HCV liver disease, this reduction was significant for total CD56+ and NK (but not NT) cells only for the severe group (B). *p < 0.05.

Download figure to PowerPoint

Cytokine Analysis

Antibodies for measurement of intracellular IFN-γ were supplied by BD Pharmingen. Thawed mononuclear cell suspensions were stimulated with phorbol myristate acetate (PMA; 10 ng/mL: Sigma-Aldrich) and ionomycin (1 μg/mL; Sigma-Aldrich) for 4 hours at 37°C in the presence of brefeldin A (Sigma-Aldrich). After stimulation cells were stained for surface antigens (as above), fixed for 30 minutes at 4°C in 100 μL Fix and Perm Medium A (Caltag, Burlingame, CA), permeabilized using 100 μL Fix and Perm Medium B (Caltag) and incubated with anti-cytokine monoclonal antibodies for 1 hour. Cell suspensions were then washed in phosphate-buffered saline–bovine serum albumin–sodium azide and fixed in 200 μL 1% PFA and acquired after 1 hour. Cells cultured under the same conditions in the absence of PMA and ionomycin served as controls.

Cytotoxicity Assays

Natural and interleukin-2-induced cytotoxicity by PBMCs against the target cell line K562 were assayed in 4-hour 51chromium-release assays. All PBMCs were cryopreserved immediately after preparation. Upon recovery they were cultured for 48 hours in complete RPMI medium (RPMI 1640 containing 25 mM 4-2-hydroxyethyl-1-piperazineethanesulfonic acid, 2 mM L-glutamine, 50 μg/mL streptomycin, 50 U/mL penicillin, and 10% fetal calf serum) at a density of 0.5 × 106 cells/mL in the absence or presence of 50 units/mL recombinant human interleukin-2 (Biological Resources Branch, National Cancer Institute at Frederick [NCI-Frederick], Frederick, MD), to measure natural and interleukin-2-induced cytotoxicity, respectively. The PBMCs were then incubated with 2,000 51Cr-sodium chromate-labeled target cells (MP Biochemicals, Mechelen, Belgium) at PBMC/target ratios of 1, 5, 25, and 50 in triplicate wells of 96-well plates. Specific lysis was calculated from the amounts of 51Cr released into supernatants using the formula: % specific lysis = (cpm of sample − cpm of spontaneous release) × 100/(cpm of maximum release − cpm of spontaneous release). Spontaneous release was determined by incubating the target cells in the absence of effector cells and maximum release was obtained by incubation of targets with 0.1% Triton X-100. In all experiments, cytotoxicity by an internal control sample consisting of cryopreserved PBMCs from a healthy donor, was assayed in parallel to monitor for day-to-day variation in target cell viability and specific activity of 51Cr.

Statistical Analyses

Results are expressed as median (range). Nonparametric Mann Whitney U test was used to compare differences between study groups. Spearman's test was used for correlation analyses. Significance was defined as a P value of less than 0.05. The JMP 6.0 (SAS Institute, Inc., Cary, NC) statistical package was used.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The study was comprised of four groups: patients with mild HCV recurrence (M1-M9), severe HCV recurrence (S1-10), patients with non-HCV related liver failure (n = 10), and normal healthy subjects (n = 10). The demographic and virologic features of the 19 patients who underwent LT from deceased donors for HCV-related liver failure and from whom PBMCs were collected prior to transplantation are shown in Table 1. All patients received tacrolimus-based basal immunosuppression.

Table 1. Demographic and Clinical Features of HCV-Infected Liver Transplant Recipients
PatientPatient IDRecurrenceGenotypeViral loadGenderAge
At transplant1 month post2 months post
  1. Abbreviations: na, not available; ABP, atypical band pattern; ND, not detectable.

M1303Mild2348013027196naMale49
M2416Mild1bna5792150>7,692,316Male58
M3497Mild1a308,93231,96861,129Male48
M4508Mild12,712,702642,1662,688,132Male52
M5770Mild3a380,26187,80057,216Male50
M61102Mild1a13,461>7,692,316naMale42
M71119Mild3a274,547160,680533,029Male53
M81145Mild1a63,891517,711556,948Female55
M91503Mild1b74,133na357,523Male55
S160Severe1b290,058>7,692,319>7,692,319Male44
S2307Severe1a371,7807,692,3166,474,199Female57
S3318Severe1b127,0201,910,1492,969,454Female57
S4387Severe1aND7,692,3167,507,318Female59
S5440Severe1a608,8147,181,4242,418,234Male49
S6501Severe1a11,9101,913,9243,801,471Female46
S7535Severe1a204,1801,298,9222,480,154Male50
S8636SevereABP1,9205,714,8313,981,605Male52
S9698Severe2915,2843,238,7746,506,995Male52
S101506Severe1a138,2282,262,1045,425,980Male40

Protocol liver biopsies were performed at 6, 12, 18, and 24 months and annually thereafter; histology was used to define HCV severity. Like many transplant programs, the strategy we use with regard to initiation of antiviral therapy is to treat when clinically significant evidence of recurrence exists (grade 3 or 4 inflammation, stage ≥2 fibrosis and/or there is evidence of the cholestatic variant).10 Patient S4 had undetectable HCV ribonucleic acid by branched-chain deoxyribonucleic acid testing pre-OLT, but by the first month had >7,692,316 IU/mL. All 10 patients with severe HCV were treated with antiviral therapy, but none in the mild group. Patients S2, S8, and S10 had cholestatic HCV recurrence. A total of 6 of the severe HCV patients are deceased (60%), on the average dying 844 days after their transplantation. All patients in the mild HCV group are alive. Table 2 shows the demographic characteristics of the non-HCV controls, including those with advanced liver disease (pre-OLT) and healthy subjects.

Table 2. Demographic Features of HCV-Negative Controls (± Liver Disease)
Patient IDEtiologyGenderAge (yr)
  1. Abbreviations: EtOH, ethyl alcohol (ethanol); HCC, hepatocellular carcinoma; NASH, nonalcoholic steato- hepatitis.

146EtOH, HCCMale56
1494EtOHFemale54
1533EtOHMale53
1636NASHFemale40
1657NASHMale65
1762NASHMale56
1765EtOHMale67
1768EtOHMale59
1776EtOHMale57
1548HealthyMale24
1560HealthyMale45
1685HealthyFemale60
1751HealthyFemale28
1773HealthyFemale35
1775HealthyMale24
1778HealthyFemale56
1779HealthyFemale22
1787HealthyMale21
1790HealthyMale38

Pretransplantation CD56+ Levels Correlate With Severity of HCV Recurrence

Multiparameter flow cytometric analysis was used to determine baseline conventional T (CD56CD3+), NK (CD56+CD3), and CD56+ NT cells. CD56+ lymphocytes, NK, and NT cells comprise the innate lymphocyte populations that can recognize conserved structures and signal viral invasion, thus providing an important first line of defense against viral infection.11, 12 We hypothesized that the level and function of these innate lymphocytes pretransplantation might predict the outcome of HCV reinfection and histologic recurrence.

As shown in Figure 1, the frequency in total CD56+ lymphocytes in peripheral blood prior to OLT was significantly lower in patients who subsequently developed severe HCV recurrence relative to those patients who developed mild histologic recurrence, as well as non-HCV liver disease controls. The same pattern was demonstrable when the frequency of NK cells (CD56+CD3) cells was analyzed (Fig. 1, mid-panel). Moreover, the frequency of NT (CD56+CD3+) cells was significantly lower in HCV-positive patients as compared to normal controls, and the median frequency of NT cells was higher in patients who later demonstrated only mild histologic HCV recurrence (vs. severe HCV).

Next, we examined whether there were differences in phenotypic and functional features among the 3 transplant groups. The phenotypic markers included CD16, CD161 (both activating receptors), and NKG2A (inhibitory receptor). Compared to the normal healthy controls, expression of CD16 on NK cells was significantly downregulated in the HCV patients but not different between mild and severe (Fig. 2A); these data suggest that HCV infection per se impairs antibody-dependent cell cytotoxicity of NK cells. It has been previously shown that the subset of human NK cells that express CD8 are more cytotoxic and more likely to survive after target cell lysis, making them “serial killers.”13 We found that these CD8+CD56+ lymphocytes were depleted in HCV infection although there were no differences between the HCV groups (Fig. 2B). No differences were observed with respect to NK cell expression of CD161, NKG2A, or total CD94 (data not shown).

thumbnail image

Figure 2. NK cell phenotype pre-transplantation. Flow cytometry was used to characterize the phenotype of NK cells. CD16 on NK cells is significantly down-regulated in the HCV patients but not different between mild and severe patient groups (A). CD8+ NK cells are less frequent in the HCV groups as compared to normal controls (B). *P < 0.05.

Download figure to PowerPoint

We also examined the expression of Fas ligand (FasL), previously demonstrated to be a primary mechanism of tumor necrosis factor–dependent hepatotoxicity after activation of innate lymphocytes.14 The NK cells from both HCV-positive groups overexpressed FasL relative to the HCV-negative liver disease control group. Moreover, FasL expression on NK cells pretransplantation was highest in those subjects who subsequently developed severe HCV recurrence (Fig. 3A). FasL was also upregulated on CD56+CD3+ NT and T cells in the HCV group prior to LT compared to the non-HCV LT patient group (Fig. 3B and C). Of interest, increased FasL expression has been previously shown to induce T cell-dependent hepatic inflammation.15 Fas (CD95) was similarly expressed on NK, NT, and NT cells in all study groups (data not shown).

thumbnail image

Figure 3. FasL expression on peripheral NK, NT and T cells is significantly upregulated in HCV infection. The expression of FasL was examined on NK (A), NT (B) and T (C) cells by flow cytometry. Upregulation of FasL was evident on all lymphocyte populations examined in the HCV-infected patients compared to normal controls. In contrast, non-HCV liver disease was characterized by downregulation of FasL on the same lymphocyte populations relative to HCV liver disease and normal controls. *P < 0.05.

Download figure to PowerPoint

NK Cells and Viral Level

Because hepatitis C viral level previously has been associated with increased risk of developing severe HCV recurrence, we determined whether lower NK levels might be explained by higher viral level. As shown in Figure 4, there were no statistically significant relationships between NK levels and viral level at baseline or 1 month posttransplantation, 2 months posttransplantation, or a serial increase in viral level (data not shown). Thus, the association between NK level and severity of HCV recurrence appears to be independent of viral level.

thumbnail image

Figure 4. Lack of correlation between NK levels and viral load. The levels of NK (CD56+CD3−) cells as a percent of total lymphocytes (% lymphs) detected by flow cytometry were correlated with circulating viral levels. There was no association between pre-transplant NK cell levels either before transplantation (simultaneous comparison) or at one month post transplantation.

Download figure to PowerPoint

Functional Analysis of NK cells

Previous studies in immunocompetent individuals have suggested that chronic HCV persistence may be associated with defective NK or NT function.16–19 By secreting cytokines and killing infected targets, both NK and NT cell populations can provide an immediate response, making them poised for early defense. Therefore, diminished function prior to OLT might be hypothesized to be a risk for development of severe HCV recurrence.

Percent specific lysis was assessed for natural cytotoxicity (K562) and lymphokine-activated killing, and as shown in Figure 5, both were diminished in HCV-infected patients as compared to non-HCV controls (including patients with and without liver disease). There were no significant cytotoxic differences among the HCV severity groups, but the numbers of patients were relatively small; HCV per se was associated with significant diminution in natural cytotoxicity and lymphokine-activated killing. Next, we determined whether there was an association between NK frequency and cytolytic activity. Figure 6 demonstrates that in HCV-uninfected liver disease controls, there is a trend toward statistically significant correlation between NK cell frequency and natural cytotoxicity (at an E:T ratio of 50:1). In contrast, HCV infection was characterized by dissociation between NK cell frequency and cytolysis (Fig. 6B). Moreover, NK cell frequency was not associated with lymphokine-activated killing (data not shown).

thumbnail image

Figure 5. Cytotoxicity is diminished in chronic HCV compared to uninfected controls. Natural cytotoxicity and lymphokine (Il-2) activated killing (LAK) were asssayed in 4-hour 51chromium-release assays as described in the Methods section. HCV infection per se is associated with diminished natural cytotoxicity (A) and LAK activity (B) compared to uninfected controls. *p < 0.05.

Download figure to PowerPoint

thumbnail image

Figure 6. Correlation of natural cytotoxicity and circulating NK levels prior to transplant. In non-HCV liver disease controls, there is a correlation R2 = 0.59, p = 0.07 (A); in HCV positive patients, the correlation coefficient relating NK cell percentage to lysis was very low indicating only a weak relationship between these two parameters (B).

Download figure to PowerPoint

Experiments designed to measure intracellular cytokine production following short-course PMA stimulation are shown in Figure 7. Comparison of the patients with HCV patients to normal controls showed a reduced frequency of IFN-γ-producing NT cells, however this reduction was also observed in non-HCV-related liver disease.

thumbnail image

Figure 7. Intracellular IFNγ production by NK, NT and T Cells in 4 study group. PBMCs stimulated with phorbol myristate acetate (PMA) and ionomycin for 4 hours at 37°C in the presence of brefeldin A were assessed for intracellular interferon-γ (IFN-γ) production by flow cytometry. NK cells staining positive for IFN-γ were similar in all study groups (A). Comparison of the patients with HCV to normal controls showed a reduced frequency of IFN-γ producing NT cells; however, this reduction was also observed in non-HCV related liver disease (B). IFn-γ production by CD56 negative T cells was similar in all groups (C). *p < 0.05.

Download figure to PowerPoint

Pretransplantation T Cell Frequencies and Phenotype Also Correlate With Severity of HCV Recurrence

As an important comparator group to the CD56+ populations, we examined the frequency and phenotype of CD3+ T cells. Unlike NK cells, T cells were not depleted in HCV-infected patients; indeed, the circulating frequency of T cells was greater in the HCV-infected patients awaiting OLT as compared to HCV-uninfected liver transplantation patients (Fig. 8A). Among the patients who subsequently developed severe HCV recurrence, a significantly lower percentage expressed CD4+ (median % for mild vs. severe) and a significantly higher percentage were CD8+ (Fig. 8B) or “double positive,” i.e., expressed both CD4 and CD8 (Fig. 8B). Although relatively rare, the double-positive population has recently been shown to constitute a distinct T-cell population exhibiting typical CD4 and CD8 T-cell functions with a predominant T helper 1/T cytotoxic 1 (Th1/Tc1) profile in chronic HCV.20

thumbnail image

Figure 8. T cell frequency and co-receptor expression in 4 study groups. In contrast to NK cells, the circulating frequency of T cells detected by flow cytometry was greater in the HCV-infected patients awaiting OLT as compared to HCV-uninfected liver transplant patients (A). Among the patients who subsequently developed severe HCV recurrence, a significantly lower percentage expressed CD4+ and a significantly higher percentage was CD8+ (B) or “double positive”, i.e., expressed both CD4 and CD8 (C). DP = double positive; DN = double negative. *p < 0.05.

Download figure to PowerPoint

NT Cells

NT cells coexpress CD56 and CD3 (Fig. 1A) and can be activated through either receptor. We found that NT cells that coexpressed CD4/CD8 or expressed CD8 alone were more frequent in patients who subsequently developed severe recurrence. In contrast, NT cells that expressed only CD4 appeared to be depleted in HCV infection (relative to non-HCV liver controls) (Fig. 9A). A significantly higher percentage of NT cells in both HCV groups expressed the inhibitory receptor NKG2A relative to HCV-negative controls with or without liver disease (Fig. 9B). No differences were observed with respect to expression of CD16, CD161, or CD94 on NT cells between the study groups (data not shown).

thumbnail image

Figure 9. NT (CD56+CD3+) cell phenotype in 4 study groups. Before liver transplantation, NT cells that co-expressed CD5/CD8 (DP) or expressed CD8 alone were more frequent in patients who subsequently developed severe recurrence. HCV infection per se was associated with depletion of CD4+ NT cells (A). A significantly higher percentage of NT cells in both HCV groups expressed the inhibitory receptor NKG2A relative to HCV-negative controls with or without liver disease (B). DP = double positive; DN = double negative. *p < 0.05.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

This is the first study to characterize the potential role of CD56+ lymphocytes in patients undergoing LT for HCV-related liver failure. The rationale for studying these lymphocytes is that they are believed to play important roles in the innate immune response to viral infections by production of IFN-γ and/or the recognition of virally-infected cells.8 These functions are an important aspect of the immediate response of the host to the virus and are believed to control the initial infection. Accordingly, injection drug users who remain human immunodeficiency virus-1 uninfected despite many years of high-risk exposure demonstrate significantly augmented NK cell lytic activities and cytokine secretion when compared to human immunodeficiency virus-1 infected injection drug users.21

Our results can be summarized as follows: 1) total CD56+, NK, and NT cells are depleted in the peripheral blood of patients with HCV awaiting LT as compared to patients with other causes of liver failure and healthy controls; 2) NK cells from HCV-positive patients overexpress FasL relative to the HCV-negative liver disease control group and are highest in those subjects who subsequently developed severe HCV recurrence; 3) cytotoxicity is decreased in HCV-positive patients as compared to HCV-negative liver disease and healthy controls; 4) in contrast, the circulating frequency of T cells is greater in the HCV-infected patients, including a double-positive (CD4/CD8) subset more frequent in patients who developed severe recurrence; 5) pre-OLT, patients with chronic HCV demonstrate lower production of IFN-γ by NT cells (but not NK or T cells); and 6) NT cells differentially expressed phenotypic markers associated with HCV infection and severity post-OLT.

The finding that NK and NT levels, phenotype, and function before OLT are associated with divergent outcomes of HCV recurrence is noteworthy because this topic has not been studied previously. NK and NT cells recognize virally-infected cells before major histocompatibility complex class I expression is upregulated, a particularly important property for the control of infections of certain cell types, e.g., hepatocytes, that normally express little or no major histocompatibility complex class I. Moreover, class I antigen recognition required for T cell stimulation is limited by the fact donor and recipients are frequently human leukocyte antigen mismatched in LT.22

Our data indicate that HCV-infected subjects have fewer circulating NK cells as well as total CD56+ cells compared to HCV-uninfected controls, consistent with results published recently by Meier et al.23 The fact that higher levels of CD56+ lymphocytes is protective, i.e., associated with milder HCV recurrence, corroborates the original hypothesis of our study. NK cells mediate the lysis of virus-infected cells via natural cytotoxicity and antibody-dependent cellular cytotoxicity and are controlled by positive and negative cytolytic signals. We found that HCV infection per se results in impairment of cytolytic ability; these results contrast with a recent study that used NK-enriched populations,24 but the study cohorts were different (ours was comprised entirely of patients with advanced liver disease). We examined both natural and lymphokine-activated killing, and found dissociation between NK frequency and K562 cytolytic function in HCV that was not readily demonstrable in the HCV-uninfected patients. This suggests that the functional defect in HCV infection may be even more disproportionately profound than appreciated simply by enumeration of NK frequency. Taken together, our data demonstrating decreased CD16 expression, decreased circulating frequency of CD8+CD56+ lymphocytes, and increased FasL on CD56+ lymphocytes depict a model of global dysfunction in HCV infection with impaired antibody-dependent cellular cytotoxicity and natural cytotoxicity as well as enhanced apoptosis.

There is consensus that CD56+ cells are regulated by a fine balance between positive and negative signals, as reflected by the higher expression of the inhibitory receptor NKG2A on NT cells of patients with HCV (Fig. 9B) in the current study. Future studies will focus on understanding how these components are functionally orchestrated to mediate or protect against HCV-related allograft injury, including the role of recipient/donor immune interactions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank John Ham and Sharlene Winters for their excellent care of the patients and the kind provision of follow-up data. We are also grateful to the patients for their willingness to participate in this study.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  • 1
    Forman LM, Lewis JD, Berlin JA, Feldman HI, Lucey MR. The association between hepatitis C infection and survival after orthotopic liver transplantation. Gastroenterology 2002; 122: 889896.
  • 2
    Gane EJ, Portmann BC, Naoumouv NV, Smith HM, Underhill JA, Donaldson PT, et al. Long-term outcome of hepatitis C infection after liver transplantation. N Engl J Med 1996; 334: 815820.
  • 3
    Charlton M, Seaberg E, Wiesner R, Everhart J, Zetterman R, Lake J, et al. Predictors of patient and graft survival following liver transplantation for hepatitis C. Hepatology 1998; 28: 823830.
  • 4
    Crippin JS, McCashland T, Terrault N, Sheiner P, Charlton MR. A pilot study of the tolerability and efficacy of antiviral therapy in hepatitis C virus-infected patients awaiting liver transplantation. Liver Transpl 2002; 8: 350355.
  • 5
    Rosen HR. Hepatitis C virus in the human liver transplantation model. Clin Liver Dis 2003; 7: 107125.
  • 6
    Weston SJ, Leistikow RL, Reddy KR, Torres M, Wertheimer AM, Lewinsohn DM, et al. Reconstitution of hepatitis C virus-specific T cell-mediated immunity following liver transplantation. Hepatology 2005; 41: 7281.
  • 7
    Gruener NH, Jung MC, Ulsenheimer A, Gerlach JT, Zachoval R, Diepolder HM, et al. Analysis of a successful HCV-specific CD8+ T cell response in patients with recurrent HCV-infection after orthotopic liver transplantation. Liver Transpl 2004; 10: 14871496.
  • 8
    Doherty DG, O'Farrelly C. Innate and adaptive lymphoid cells in the human liver. Immunological Rev 2000; 174: 520.
  • 9
    Batts KP, Ludwig J. Chronic hepatitis: an update on terminology and reporting. Surg Pathol 1995; 19: 14091417.
  • 10
    Gopal DV, Rosen HR. Duration of antiviral therapy for cholestatic HCV recurrence may need to be indefinite. Liver Transpl 2003; 9: 348353.
  • 11
    Biron CA, Nguyen KB, Pien GC. Innate immune responses to LCMV infections: natural killer cells and cytokines. Curr Top Microbiol Immunol 2002; 263: 727.
  • 12
    Kelly-Rogers J, Madrigal-Estebas L, O'Connor T, Doherty DG. Activation-induced expression of CD56 by T cells is associated with a reprogramming of cytolytic activity and cytokine secretion profile in vitro. Hum Immunol 2006; 67: 863873.
  • 13
    Addison EG, North J, Bakhsh I, Marden C, Haq S, Al-Sarraj S, et al. Ligation of CD8alpha on human natural killer cells prevents activation-induced apoptosis and enhances cytolytic activity. Immunology 2005; 116: 354361.
  • 14
    Nakashima H, Inui T, Habu Y, Kinoshita M, Nagao S, Kawaguchi A, et al. Activation of mouse natural killer T cells accelerates liver regeneration after partial hepatectomy. Gastroenterology. 2006; 131: 15731583.
  • 15
    Cruise MW, Melief HM, Lukens J, Soguero C, Hahn YS. Increased Fas ligand expression of CD4+ T cells by HCV core induces T cell-dependent hepatic inflammation. J Leukoc Biol 2005; 78: 412425.
  • 16
    Bonavita MS, Franco A, Paroli M, Santilio I, Benvenuto R, De Petrillo G, et al. Normalization of depressed natural killer activity after interferon-alpha therapy is associated with a low frequency of relapse in patients with chronic hepatitis C. Int J Tissue React 1993; 15: 1116.
  • 17
    Par G, Rukavina D, Podack ER, Horanyi M, Szekeres-Bartho J, Hegedus G, et al. Decrease in CD3-negative-CD8dim+ and Vδ2/Vγ9 TcR+ peripheral blood lymphocyte counts, low perforin expression and the impairment of natural killer cell activity is associated with chronic hepatitis C virus infection. J Hepatol 2002; 37: 514522.
  • 18
    Ahmad A, Alvarez F. Role of NK and NKT cells in the immunopathogenesis of HCV-induced hepatitis. J Leukoc Biol 2004; 76: 743759.
  • 19
    Kawarabayashi N, Seki S, Hatsuse K, Ohkawa T, Koike Y, Aihara T, et al. Decrease of CD56(+)T cells and natural killer cells in cirrhotic livers with hepatitis C may be involved in their susceptibility to hepatocellular carcinoma. Hepatology 2000; 32: 962969.
  • 20
    Nascimbeni M, Shin EC, Chiriboga L, Kleiner DE, Rehermann B. Peripheral CD4(+)CD8(+) T cells are differentiated effector memory cells with antiviral functions. Blood 2004; 104: 478486.
  • 21
    Scott-Algara D, Truong LX, Versmisse P, David A, Luong TT, Nguyen VN, et al. Cutting edge: increased NK cell activity in HIV-1 exposed but uninfected Vietnamese intravascular drug users. J Immunology 2003; 171: 56635667.
  • 22
    Rosen HR, Hinrichs DJ, Leistikow RL, Callender G, Wertheimer AM, Nishimura MI, Lewinsohn DM. Cutting edge: selection of HCV-specific CD8+ T cells by donor HLA alleles following liver transplantation. J Immunology 2004; 173: 53555359.
  • 23
    Meier U-C, Owen RE, Taylor E, Worth A, Naoumov N, Willberg C, et al. Shared alterations in NK cell frequency, phenotype, and function in chronic human immunodeficiency virus and hepatitis C virus infections. J Virol 2005; 79: 1236512374.
  • 24
    Morishima C, Paschal DM, Wang CC, Yoshihara CS, Wood BL, Yeo AE, et al. Decreased NK cell frequency in chronic hepatitis C does not affect ex vivo cytolytic killing. Hepatology 2006; 43: 573580.