Fine phenotypic and functional characterization of effector cluster of differentiation 8 positive T cells in human patients with primary biliary cirrhosis

Authors


  • Potential conflict of interest: Nothing to report.

  • Financial support for this work was provided by the National Institutes of Health (grant DK39588; Bethesda, MD).

Abstract

In primary biliary cirrhosis (PBC), patients develop a multilineage response to a highly restricted peptide of the E2 component of pyruvate dehydrogenase (PDC-E2) involving autoantibody and autoreactive cluster of differentiation (CD)4+ and CD8+ T-cell responses. Recent data from murine models have suggested that liver-infiltrating CD8+ cells play a critical role in biliary destruction in PBC. We hypothesized that chronic antigen stimulation of CD8+ T cells alters effector memory T cell (TEM) frequency and function similar to that seen with chronic viral infections, including failure to terminally differentiate and relative resistance to apoptosis. We have rigorously phenotyped CD8+ T-cell subpopulations from 132 subjects, including 76 patients with PBC and 56 controls, and report a higher frequency of TEM cells characterized as CD45ROhighCD57+CD8high, but expressing the gut homing integrin, α4β7, in peripheral blood mononuclear cells of PBC. These CD8high TEM cells have reduced expression of Annexin V after TCR stimulation. Consistent with a TEM phenotype, CD45ROhighCD57+CD8high T cells express higher levels of granzyme A, granzyme B, perforin, CCR5 and α4β7, and lower levels of CCR7 and CD28 than other CD8high T cells. Furthermore, interleukin (IL)-5 produced by CD8+CD57+ T lymphocytes upon in vitro T-cell receptor stimulation are increased in PBC. Histologically, CD8+CD57+ T cells accumulate around the portal area in PBC. Moreover, CD8+CD57+ T cells respond specifically to the major histocompatibility class I epitope of PDC-E2. Conclusion: In conclusion, our data demonstrate that CD45ROhighCD57+CD8high T cells are a subset of terminally differentiated cytotoxic TEM cells, which could play a critical role in the progressive destruction of biliary epithelial cells. (HEPATOLOGY 2011;54:1293–1302)

Primary biliary cirrhosis (PBC) is a female-predominant, organ-specific autoimmune disease characterized by destruction of intrahepatic small bile duct biliary epithelial cells (BECs).1 The serological hallmark of PBC is the presence of antimitochondrial autoantibodies (AMAs) directed against the pyruvate dehydrogenase E2 complex (PDC-E2) located in the inner membrane of mitochondria.2-4 High frequency of cluster of differentiation (CD)4+ and CD8+ T-cell infiltrates have been noted within the portal tracts of the PBC liver, which strongly suggests that these cells are involved in the pathogenesis of PBC.5 Indeed, PDC-E2-specific autoreactive CD4+ T and CD8+ T cells have been identified both in peripheral blood and, at much higher levels, in the liver of PBC patients.6-8 The dominant CD4+ and CD8+ T-cell epitopes on PDC-E2 have been mapped.6-8

Although both CD4+ and CD8+ T cells are present within portal tract infiltrates, there is a growing body of data that suggests a more direct role of cytotoxic CD8+ T cells in biliary destruction.9-11 The study of effector pathways is a particularly challenging problem in human autoimmunity. For one thing, the majority of effector pathways are likely to be mediated by nonspecific bystander cells recruited during inflammation. For another, it has been difficult to identify subpopulations of cells by phenotype and thence link such data to functionality. Our laboratory has focused attention on effector T-cell populations, using a variety of technologies, and has highlighted the important role of T cells in this and similar pathways.

In this study, we took advantage of newer reagents, including cell-surface markers, that are associated with CD8high effector memory T cells (TEM), organ- and tissue-specific homing, and alterations in susceptibility to apoptosis. Indeed, we report that patients with PBC not only have an increased frequency of CD45ROhighCD57+CD8high T cells, compared to controls, but also that such cells have increased α4β7 expression with concurrently increased expression of CCR5 and decreased expression of CCR7 and CD28, compared to other CD8high T cells. Furthermore, this T-cell subset has increased the production of granzyme A, granzyme B, and perforin, compared with other CD8high T cells, and, interestingly, have decreased stimulation-induced apoptosis. Furthermore, interferon gamma (IFN-γ) and interleukin (IL)-5 produced by CD8+CD57+ T lymphocytes upon in vitro T-cell-receptor (TCR) stimulation are increased in PBC patients. Histologically, CD8+CD57+ T cells accumulate around the portal area in the liver of PBC patients. Moreover, purified CD8+CD57+ T cells from PBC patients specifically respond to the major histocompatibility class I restricted epitope of PDC-E2. These data have implications for understanding CD8 effector pathways in this autoimmune disease. We submit that CD45ROhighCD57+CD8high T cells are a subset of cytotoxic memory cells, which play a critical role in the chronic, progressive destruction of BECs in PBC.

Abbreviations

AMA, antimitochondrial antibodies; APC, allophycocyanin; BEC, bile duct biliary epithelial cell; BSA, bovine serum albumin; CD, cluster of differentiation; CVH, chronic viral hepatitis; EDTA, ethylenediaminetetraacetic acid; ELC, EBI1-ligand chemokine; ELISA, enzyme-linked immunosorbent assay; FcR, Fc receptor; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; HLA, human leukocyte antigen; HNK-1, human natural killer-1; HPLC, high-performance liquid chromatography; IFN-γ, interferon gamma; IP-10, IFN-γ-inducible protein 10; IL, interleukin; IgG, immunoglobulin G; LGL, large granular lymphocyte leukemia; mAb, monoclonal antibody; MAdCAM-1, mucosal addressin cell adhesion molecule-1; MFI, mean fluorescent intensity; NASH, nonalcoholic steatohepatitis; NK, natural killer; PBC, primary biliary cirrhosis; PBMCs, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; PD-1, programmed death 1; PDC-E2, E2 component of pyruvate dehydrogenase; PE, phycoerythrin; RPMI, Roswell Park Memorial Institute; SEM, standard error of the mean; SLC, secondary lymphoid tissue chemokine; TCR, T-cell receptor; TECK, thymus-expressed chemokine; TEM, effector memory T cell; thymus-expressed chemokine CCL25, CC chemokine ligand 25; TIM-3, T-cell immunoglobulin mucin-3.

Materials and Methods

Subjects.

Heparinized (Vacutainer; BD Biosciences, Franklin Lakes, NJ) peripheral blood samples were obtained from 76 PBC patients (59.0 ± 1.0 years; mean ± standard error of the mean [SEM]) and 56 age-matched healthy controls (54.8 ± 1.5 years). The diagnosis of PBC was based on internationally accepted criteria.12 Stage of disease was established according to Ludwig et al..13 In the present study, 50 of 76 (65.8%) patients with PBC were stage I or II and 22 of 76 (28.9%) were III or IV, whereas 5 of 76 (6.6%) patients were AMA negative (Table 1). We did not observe any difference between AMA-positive and -negative patients; hence, the data are combined herein. The study was approved by the Institutional Review Board of the University of California at Davis (Davis, CA), and all subjects provided written, informed consent prior to enrollment.

Table 1. Clinical Characteristics of PBC Patients*
PBC (n = 76)AMA PositiveAMA NegativeAge: 59.0 ± 1.0
  • *

    There were 56 controls (age, 54.8 ± 1.5 years).

  • Abbreviations: PBC, primary biliary cirrhosis; AMA, antimitochondrial antibodies.

Stage In = 32n = 1Early stage (65.8%)
Stage IIn = 15n = 2
Stage IIIn = 9n = 1Late stage (28.9%)
Stage IVn = 11n = 1
Unknownn = 4 

Peripheral Blood Mononuclear Cell Isolation.

Peripheral blood mononuclear cells (PBMCs) from all subjects were isolated by density gradient using Histopaque-1077 (Sigma Chemical Co., St. Louis, MO) under endotoxin-free conditions. PBMCs were resuspended in phosphate-buffered saline (PBS) (Mediatech Inc., Herndon, VA), containing 0.5% bovine serum albumin (BSA) (Fraction V, OmniPur; EMD Chemicals Inc., Gibbstown, NJ) and 0.05% ethylenediaminetetraacetic acid (EDTA). The viability of cells was >98%, which was confirmed using trypan blue dye exclusion.

Evaluation of Cell Phenotypes.

The polychromatic phenotypic analysis of PBMCs was carried out on a FACScan flow cytometer (BD Immunocytometry Systems, San Jose, CA) upgraded for the detection of five colors by Cytek Development (Fremont, CA). Cells were stained with different combinations of fluorochrome-conjugated monoclonal antibodies (mAbs), including CCR5, CD8b, CCR7, and CD45RO (BD Pharmingen, San Diego, CA), CCR9 (R&D Systems, Minneapolis, MN), CD56, CXCR3, CD57, CD8a, CD45RO, CD28, and CD16 (BioLegend, San Diego, CA), and CCR7 (eBioscience, San Diego, CA). The allophycocyanin (APC)-conjugated anti-α4β7 was produced in our laboratory. Immunoglobulin G (IgG) isotype controls with matching conjugates for each antibody were used as negative controls. PBMCs were resuspended in staining buffer (0.2% BSA, 0.04% EDTA, and 0.05% sodium azide in PBS), divided into 25-μL aliquots, and incubated with antihuman Fc receptor (FcR) blocking reagent (eBioscience) for 15 minutes at 4°C. Cells were then washed and stained with the antibody cocktails for 30 minutes at 4°C. Cells were washed once with PBS containing 0.2% BSA. For intracellular staining, cells were first stained with phycoerythrin (PE)-anti-CD57 (BioLegend), PerCP-anti-CD8a (BioLegend), APCe780-anti-CCR7 (eBioscience), and APC-anti-CD45RO (BioLegend), then fixed and permeabilized with BD Cytofix/Cytoperm solution (BD Pharmingen) for 15 minutes at 4°C. Subsequently, intracellular staining was performed with AF488-labeled antigranzyme A (BioLegend), AF488-labeled anti-Perforin (BioLegend), or fluorescein isothiocyanate (FITC)-labeled antigranzyme B (BD Pharmingen) or IgG isotype controls. After staining, cells were washed and fixed with 1% paraformaldehyde in PBS. Acquired data were analyzed with Cellquest PRO software (BD Immunocytometry Systems).

Isolation and Culturing of CD57+CD8+ T Cells.

CD8+ T cells were isolated with RosetteSep™ human CD8+ T cell enrichment cocktails (StemCell Technologies, Vancouver, British Columbia, Canada), following the manufacturer's instructions, then resuspended in PBS containing 2% fetal bovine serum (FBS). The CD57+ CD8+ T-cell subset was isolated from the enriched CD8+ T cells using human CD57 MicroBeads (Miltenyi Biotec, Auburn, CA). An aliquot of the isolated CD57+ population was analyzed for purity with flow cytometry, which was always >92%. Aliquots of CD57+CD8+ T cells (2 × 105) were cultured in 96-well, round-bottomed plates in 200 μL of Roswell Park Memorial Institute (RPMI) medium with 10% heat-inactivated FBS (Gibco-Invitrogen Corp., Grand Island, NY), 100 μg/mL of streptomycin, 100 U/mL of penicillin, and 0.5 μg/mL each of anti-CD3 (BioLegend) and anti-CD28 (BioLegend). Cells were incubated for 5 days at 37°C in a humidified 5% CO2 incubator, then centrifuged. The supernatant was collected for cytokine analysis.

Cytokine Detection.

Supernatant from cultured CD57+ CD8+ T cells was analyzed with enzyme-linked immunosorbent assay (ELISA) kits for IFN-γ (R&D Systems), granzyme A (Bender MedSystems, Vienna, Austria), and IL-5 (BioLegend).

Apoptosis.

To assess the relative susceptibility of in vitro stimulated CD45ROhighCD57+CD8high T cells to apoptosis, 1 × 106 PBMCs were cultured in 48-well, flat-bottomed plates in 1 mL of RPMI 1640 (Gibco-Invitrogen Corp.), supplemented with 10% heat-inactivated FBS, 100 μg/mL of streptomycin, 100 U/mL of penicillin, 5 μg/mL of anti-CD3 (BioLegend), and 5 μg/mL of anti-CD28 (BioLegend). Cultures were incubated at 37°C in 5% CO2. After 48 hours of culturing, cells were washed twice with 0.2% BSA in PBS, and the frequency of cells undergoing apoptosis was determined with flow cytometry, using FITC-conjugated anti-Annexin-V (BD Pharmingen), following the manufacturer's instructions. Cells were also stained with Fas (CD95), programmed death 1 (PD-1) (Biolegend), and T-cell immunoglobulin mucin-3 (TIM-3) (eBioscience) after culture.

Synthetic Peptide Assay.

PBMC from a nested study, including 3 patients with PBC (PBC 1-3) who were human leukocyte antigen (HLA) A2.1 and 6 other patients with PBC (PBC 4-9) who had other class I alleles, were isolated. As controls, PBMCs from 4 healthy HLA A2.1 controls and 5 HLA A2.1 negative were collected. The peptide, 159-167 of PDC-E2 (KLSEGDLLA), was synthesized by F-moc chemistry (Model Synergy; Applied Biosystems Inc., Foster City, CA). This peptide was purified by reverse-phase high-performance liquid chromatography (HPLC), and the purity was more than 80% as determined by HPLC analysis. Aliquots of CD57+CD8+ T cells or CD57CD8+ T cells (2 × 105) were cultured in the presence of autologous irradiated (3,000 rad) APC (2 × 105), with or without the 159-167 synthetic peptide, for 5 days. The supernatant was collected for cytokine analysis. Controls were used throughout all assays. Supernatant from the cultured cells was analyzed by ELISA for IFN-γ (R&D Systems).

Immunohistochemistry.

Liver sections were immunostained using our standard microwave protocol, as previously described.14, 15 All tissues were fixed in 10% neutral buffered formalin and embedded in paraffin, then 4-μm-thick sections were cut from each paraffin block from 4 patients with PBC, 3 with chronic viral hepatitis (CVH), and 1 with nonalcoholic steatohepatitis (NASH). The following antibodies were used for the detection of CD8 and CD57 in human liver specimens: rabbit polyclonal antibody against CD57 (Novus Biologicals, Littleton, CO), antihuman CD8 antibody (mAb; DAKO, Glostrup, Denmark) and Envision-peroxidase (DAKO). In all samples, predetermined optimal dilutions were used, and positive and negative samples were included with each assay, and the data were interpreted by a “blinded” pathologist (K.T.).

Statistical Analysis.

The percentages of CD8high T-cell subsets that express individual cell markers in PBC patients and healthy controls were expressed as mean ± SEM and compared with the two-tailed Mann-Whitney U test. A P value <0.05 was considered statistically significant. Percentages of the CD45ROhighCD57+CD8high T-cell subset and other CD8high T-cell subsets were analyzed using a two-tailed Wilcoxon matched-pairs test.

Results

Increased Frequency of CD45ROhighCD57+CD8high T Cells in Peripheral Blood of PBC Patients.

There were no significant differences observed in the mean frequencies of CD8high T cells in the PBMC and CD57+ cells in CD8high T cells of PBC patients, compared with control subjects (Fig. 1). However, the frequency of CD45ROhighCD57+ cells were significantly higher in CD8high T cells of PBC patients (7.15% ± 0.77%), in particular patients at earlier disease stages (8.25% ± 1.16%), compared with healthy controls (4.10% ± 0.37%; P < 0.0005) (Fig. 2). The CD45ROhighCD57+CD8high population did not include natural killer (NK) cells, as the vast majority of these cells were CD3+ (99.9% ± 0.14%) and CD16 (98.69% ± 0.69%) (data not shown).

Figure 1.

Analysis of the frequencies of CD8highCD57+ cells in lymphocyte population from PBC patients and healthy subjects. Left panels: representative dot plot of gated lymphocyte population. Right panels: percentage of the gated CD8high in PBMC (upper panel) or CD57+ in CD8high lymphocytes (lower panel) populations in healthy controls (cont), all PBC patients (PBC), and PBC patients at early stages (I/II) and late stages (III/IV). Bars denote mean percentages.

Figure 2.

Analysis of the frequency of the CD57+ and CD45ROhigh cells in CD8high lymphocytes from PBC patients and healthy subjects. Top panels: representative dot plots of CD8high gated lymphocyte population from a healthy control and a patient with PBC. Bottom panel: comparison of the mean percentages of the gated CD45ROhighCD57+ population in CD8high lymphocytes, among healthy controls (cont), all PBC patients (PBC), and PBC patients at early stages (I/II) and late stages (III/IV). Bars denote mean percentages. ***P < 0.001.

Increased Expression of α4β7high and Decreased CD28 on CD45ROhighCD57+CD8highT Cells in PBC Patient.

To further characterize the CD45ROhighCD57+CD8high T-cell subset, these cells were analyzed for their expression of a panel of phenotypic markers, including multiple chemokine receptors, α4β7 integrin, and the costimulatory molecule, CD28. Results of these studies (Fig. 3) demonstrate that whereas the frequency of the CD45ROhighCD57+CD8high subset expressing the gut homing α4β7high integrin was significantly higher in PBC patients (18.51% ± 1.94%) than that in controls (11.69% ± 1.41%; P < 0.03) and decreased the expression of CD28 in early-stage PBC patients (25.01% ± 5.81%) than controls (58.76% ± 9.36%; P < 0.03), there were no significant differences in the frequencies of the cells expressing the chemokine receptors, CXCR3 (a receptor for IFN-γ-inducible protein 10 [IP-10] and the monokine, Mig), CCR5 (a receptor for RANTES and MIP-1α,β), and CCR7 (a receptor for EBI1-ligand chemokine [ELC] and secondary lymphoid tissue chemokine [SLC]), compared with controls. Of interest, there was no difference in the frequencies of this CD8high subset that expressed the gut-homing chemokine receptor, CCR9, a receptor for thymus-expressed chemokine (TECK)/CC chemokine ligand 25 (thymus-expressed chemokine CCL25).

Figure 3.

Analysis of the expression of CXCR3, CCR5, CCR7, CCR9, CD28, and α4β7 on CD45ROhighCD57+CD8high gated T lymphocytes from healthy controls (cont), all PBC patients (PBC), and PBC patients at early stages (I/II) and late stages (III/IV). Bars denote the mean percentages. *P < 0.05.

Altered Granzyme and Perforin Expression in CD45ROhighCD57+CD8high T Cells.

In PBC patients, we have compared phenotypes of CD45ROhighCD57+CD8high T cells to other CD8high T cells. In addition to the analysis of homing and chemokine receptors, we also studied the cytotoxic potential of the CD45ROhighCD57+CD8high subset of T cells. Interestingly, though there was a significant increased expression of CCR5 and α4β7high, and significantly decreased expression of CCR7 and CD28 in CD45ROhighCD57+CD8high T cells, compared to other CD8high T cells in PBC patients, there was no difference observed in α4β7high and CD28 expressions in healthy controls (Fig. 4A,C). CCR5, CCR7, granzyme A, granzyme B, and perforin demonstrated a similar phenotypic pattern in PBC and healthy controls (data not shown). In addition, relative to other CD8high T cells, CD45ROhighCD57+CD8high T cells had increased production of granzyme A (P < 0.001), granzyme B (P < 0.001), and perforin (P < 0.001), suggesting their strong cytotoxic effector functions (Fig. 4B).

Figure 4.

Comparison of phenotypes between CD45ROhighCD57+CD8high T cells and other CD8high T cells. The other CD8high T cells include CD45RO− to lowCD57+CD8high and CD57CD8high cells. Bars denote mean percentages. *P < 0.05; **P < 0.01; ***P < 0.001. (A) Cell-surface markers in PBC. (B) Intracellular markers in PBC. (C) Cell-surface markers in healthy control.

The CD45ROhighCD57+CD8high T Cells Are Not Susceptible to Apoptosis Upon Anti-CD3 Stimulation.

CD57+ T cells have previously been demonstrated to be susceptible to apoptosis during chronic antigenic stimulation.16 Therefore, we determined the relative susceptibility of CD45ROhighCD57+CD8high T cells from PBC patients to undergo apoptosis. PBMCs from PBC patients and controls were stimulated by anti-CD3/28 for 48 hours, then examined for Annexin V expression by the CD45ROhighCD57+CD8high T cells. Interestingly, Annexin V expression was decreased in CD45ROhighCD57+CD8high T cells from PBC patients (35.23% ± 3.07%), specifically those with early disease stages (32.68% ± 4.02%), compared with healthy controls (46.18% ± 1.51%; P < 0.03) (Fig. 5A). We further investigated the expression of Fas, TIM-3, and PD-1, and found that PD-1 expression was significantly decreased in PBC patients (51.8% ± 6.54%), compared to that in healthy controls (75.03% ± 7.12%; P < 0.02) (Fig. 5A). This reduced susceptibility to stimulation-induced apoptosis was not observed in other CD8high T cells, suggesting a unique apoptosis resistance in the CD45ROhighCD57+CD8high T cells from PBC patients.

Figure 5.

Comparison of the expression of Annexin V, Fas, TIM-3, and PD-1 on CD45ROhighCD57+CD8high T cells and other CD8high T lymphocytes between PBC patients and healthy subjects. PBMCs were stimulated with anti-CD3/28, then stained for Annexin V, Fas, TIM-3, and PD-1. (A) Percentage of Annexin V, TIM-3, and PD-1-positive cells or mean fluorescent intensity (MFI) of Fas in CD45ROhighCD57+CD8high T cells from healthy controls (cont), all PBC patients (PBC), and PBC patients at early stages (I/II) and late stages (III/IV). (B) Percentage of Annexin V, TIM-3, and PD-1 positive cells or MFI of Fas in other CD8high T cells. See Fig. 4 legend for the definition of other CD8high T-cell populations. Bars denote mean percentages. *P < 0.05.

The CD57+CD8+ T-Cell Subset From PBC Patients Produce Increased Levels of IL-5, IFN-γ, and Granzyme A.

To investigate the cytokine profile of CD57+CD8+ T cells, CD57+CD8+ T cells isolated from PBC patients and healthy controls were stimulated in vitro with anti-CD3/28 for 5 days. Supernatants were collected and analyzed for levels of secreted IFN-γ, IL-5, and granzyme A. As shown in Fig. 6, CD57+CD8+ T cells from PBC patients secreted increased levels of IL-5 (157.4 ± 47.7 pg/mL), compared with controls (30.0 ± 7.7 pg/mL; P < 0.001). No significant difference was observed in the levels of IFN-γ and granzyme A.

Figure 6.

Production of IFN-γ, IL-5, and granzyme A by CD57+CD8+ T cells from PBC patients and healthy controls. Aliquots of isolated CD57+CD8+ T cells from PBC patients (n = 7) and healthy controls (n = 8) were cultured in duplicates in the presence of anti-CD3/28 (0.5 μg/mL) for 5 days. ***P < 0.001.

CD8+CD57+Cells Were Infiltrated in Hepatic Portal Track and Respond to the HLA-A2-Restricted CTL Epitope, PDC-E2.

CD57+cells coexisted in the area of CD8+ infiltration (i.e., were CD8+CD57+ double positive). In contrast, it was uncommon to have CD57+ coexpression detected in CD8+-infiltrating control livers (Fig. 7A). To further assess the prevalence of autoreactive T cells, we investigated the difference in response with the HLA-A2-restricted cytotoxic T lymphocyte (CTL) epitope. CD8+CD57+cells from HLA-A2.1-positive PBC patients (124.3 ± 16.5 pg/ml) had increased production of IFN-γ upon PDC-E2 stimulation, compared to CD8+CD57+cells from HLA-A2.1-positive healthy controls (39.7 ± 10.1 pg/ml); as expected, there was no significant difference between PBC non-HLA-A2.1 patients versus controls (Fig. 7B).

Figure 7.

(A) Liver immunohistochemistry from representative PBC and CVH patients. Top and middle rows demonstrate 100% near complete (top row) to 70% (middle row) CD57-positive staining within the CD8-positive area in PBC; only minor CD57-positive staining was detected within the CD8-positive area in CVH, as shown in the bottom row. (B) IFN-γ production upon PDC-E2 peptide stimulation. Data from HLA-A2.1-positive PBC patients (n = 3) and healthy controls (n = 4) are shown. Left bar graphs are without peptide stimulation, and right bar graphs are with peptide stimulation.

Discussion

In this study, we have carried out a comprehensive phenotypic and functional characterization of CD8+ T cells, which is believed to be directly responsible for the destruction of BECs in PBC. Our results demonstrate the following: (1) PBC patients had an increased frequency of CD45ROhighCD57+ cells in CD8high T cells, compared with age-matched healthy controls; (2) CD45ROhighCD57+CD8high T cells from PBC patients more frequently expressed α4β7 and demonstrated reduced CD28 expression, compared with controls; (3) in PBC patients, the CD45ROhighCD57+CD8high subset had increased frequency of CCR5+ and α4β7high cells, decreased frequency of CCR7+ and CD28+ cells, and expressed increased levels of granzyme A, B, and perforin, in comparison to other CD8high T cells, consistent with an effector memory phenotype; (4) upon CD3 stimulation, CD57+CD8+ T cells from PBC patients were less prone to apoptosis while having secreted increased levels of IL-5 than healthy controls; and (5) CD57+CD8+ T cells infiltrate the PBC liver portal area; this cell population demonstrates autoreactivity against the HLA-A2.1, the restricted epitope.

It is of interest to note that the CD57+CD8+ T-cell subset has been previously described as possessing both cytotoxic and regulatory functions.17-21 In our present study, the results suggest that a subpopulation of the CD57+CD8+ T cells, namely CD45ROhighCD57+CD8high T cells, is a subset of cytotoxic effector memory cells that could be critical in cell-mediated immune response in PBC. The CD57 antigen is a glycoepitope that was first described on human NK 1 (HNK-1) cells.22 An increase in the frequency of CD57+ T cells has been reported in patients after bone marrow and solid organ transplants,23, 24 in rheumatoid arthritis,25, 26 and acquired immune deficiency patients.27 These studies have suggested a role for such CD57+ T cells in the immunological abnormalities manifested in such diseases. Although the frequency of the CD8+CD57+ T cells in normal hosts ranges from 5% to 20%,28 the frequency of this subset increases with aging.29, 30 Although several studies have suggested an augmented cytotoxic ability of the CD8+CD57+ T cell,19-21 there is a paucity of data on this CD8+CD57+ T-cell population in PBC.

The present results provide further insights into the potential mechanisms by which CD8+ cytotoxic T cells serve as effector cells in the pathogenesis of PBC.7, 9, 11 We demonstrate herein that the CD45ROhigh subset of CD57+CD8high cells were more resistant to stimulation-induced apoptosis, as compared to their counterparts, in the control subjects, which is similar to the finding that the CD57+CD8+ T-cell population in PBMCs from patients with large granular lymphocyte leukemia (LGL) were resistant to Fas-stimulated apoptosis (31).31 This resistance to apoptosis is demonstrated by lower expression of Annexin V and PD-1. PD-1/PD-L interaction plays a critical role in CD8+ T-cell tolerance32; previous work has demonstrated that a decrease in PD-1 signaling can generate murine autoimmune hepatitis.33

We reasoned that examination of chemokine receptors and the integrin, α4β7, which provide homing signals for circulating leukocytes to migrate to disease-specific tissues, would provide evidence that these circulating cells reflect the immune response in the target organ.34 T-cell recruitment to the liver is orchestrated by a series of adhesion molecules and homing chemokines.35 We demonstrate herein that the CD45ROhigh subset of CD57+CD8high cells more frequently expressed the homing integrin, α4β7. Although the integrin, α4β7, and chemokine receptor, CCR9, are typically associated with gut-homing phenotypes, they have also been shown to mediate the adhesion of liver-infiltrating lymphocytes through the expression of their cognate ligands. Specifically, hepatic expression of the α4β7 ligand, mucosal addressin cell adhesion molecule-1 (MAdCAM-1), has been demonstrated in a variety of liver diseases, including PBC.35 The CCR9 ligand and the presumed gut-specific chemokine, CCL25, is expressed primarily by epithelial cells that line the small intestine and has also been shown to be expressed on hepatic endothelial cells of patients with primary sclerosing cholangitis, but, in contrast to MAdCAM-1, not in PBC.36 Thus, it is not surprising that, in our study, there was an increased frequency of CD45ROhighCD57+CD8high T cells expressing α4β7, but not CCR9.

In a murine model of graft-versus-host disease, which develops both portal hepatitis and nonsuppurative destructive cholangitis similar to PBC, CCR5-expressing CD8+ T cells migrate into the portal areas of the liver and play a significant role in causing liver injury.37 The increased expression of CCR5 by CD45ROhighCD57+CD8high T cells, but not other CD8high T cells, suggests that CD45ROhighCD57+CD8high T cells play an active role as effector cells during bile duct destruction in PBC. The expression of CD28 decreases when CD8+ T cells differentiate from memory to effector CD8+ T cells.38 The decrease in CD28 expression observed in PBC, especially in early-stage PBC patients, implicates strong effector function with autoreactive properties of CD45ROhighCD57+CD8high T cells.

Also, our observation of decreased CCR7 expression on CD45ROhighCD57+CD8high T cells, compared with other CD8high T cells, is consistent with the theory that these cells are effector memory cells, as opposed to CCR7+ central memory cells, which express lymph-node homing receptors and lack immediate effector function.39 It is reasoned that the lymph-node homing CD8high T cells may become mobilized to the periphery and acquire a different spectrum of cell-surface molecules while decreasing the levels of CCR7 expression during this process.

Our data demonstrate an increased secretion of IL-5 by CD57+CD8+ T cells, compared with a similar population from controls. Increased IL-5 has also been found in other studies of CD8+CD57+ T lymphocytes.40 The transcripts for both Th1- and Th2-type cytokines, such as INF-γ, IL-2, and IL-5, are up-regulated in the blood and liver of PBC,41, 42 and IL-5 promotes the differentiation of activated B cells into Ig-producing cells and augments both IgM and IgA production.43, 44 Moreover, IL-5 has potent, specific effects on eosinophil activation and degranulation.45, 46 Eosinophilia has been demonstrated in PBC patients, and eosinophil cytotoxic products, such as major basic protein, have been localized to the periportal regions of the patient liver.42, 47 Our data demonstrate that CD57+CD8+ T cells are a potential source of IL-5 during the chronic stages of PBC, exacerbating the destruction of BECs. CD57+ CD8+, in particular CD45ROhighCD57+CD8high, T cells may also contribute to continuous AMA production in PBC.

Collectively, our data demonstrate that CD57+ CD8high T cells are a subset of cytotoxic memory T cells that include specific autoreactive CD8+ T cells. Our results demonstrate, for the first time, the increased frequency of CD45ROhighCD57+CD8high T cells with the unique increased expression of α4β7 integrin and CCR5 as well as resistance to apoptosis in PBC PBMCs; this reflects a role of CD45ROhighCD57+CD8high T cells as a CD8+ subpopulation contributing to the progressive destruction of small bile ducts. We do not imply that the data herein will be unique only to patients with PBC and, indeed, may well be a property of multiple other autoimmune diseases, obviously with different antigenic specificity and tissue-specific homing receptors. We do suggest, however, that further studies focused on these effector mechanisms will enable the dissection of the role of CD8+ subpopulations in PBC.

Acknowledgements

The authors thank Dr. Kazuhito Kawata, Dr. Katsunori Yoshida, and Dr. Yuki Moritoki for technical support in this experiment. We also thank Ms. Nikki Phipps for support in preparing this article.

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