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The Journal of Immunology, 2006, 177: 729-738.
Copyright © 2006 by The American Association of Immunologists

Detailed Analysis of Intrahepatic CD8 T Cells in the Normal and Hepatitis C-Infected Liver Reveals Differences in Specific Populations of Memory Cells with Distinct Homing Phenotypes1

Mathis Heydtmann2,*, Debbie Hardie{dagger}, Philip L. Shields*, Jeff Faint{dagger}, Christopher D. Buckley{dagger}, James J. Campbell{ddagger}, Michael Salmon{dagger} and David H. Adams2,*

* Liver Research Laboratories and {dagger} Department of Rheumatology, Medical Research Council, Centre for Immune Regulation, University of Birmingham, United Kingdom; and {ddagger} Children’s Hospital, Boston, MA 02115


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In hepatitis C virus (HCV) infection the immune response is ineffective, leading to chronic hepatitis and liver damage. Primed CD8 T cells are critical for antiviral immunity and subsets of circulating CD8 T cells have been defined in blood but these do not necessarily reflect the clonality or differentiation of cells within tissue. Current models divide primed CD8 T cells into effector and memory cells, further subdivided into central memory (CCR7+, L-selectin+), recirculating through lymphoid tissues and effector memory (CCR7–, L-selectin–) mediating immune response in peripheral organs. We characterized CD8 T cells derived from organ donors and patients with end-stage HCV infection to show that: 1) all liver-infiltrating CD8 T cells express high levels of CD11a, indicating the effective absence of naive CD8 T cells in the liver. 2) The liver contains distinct subsets of primed CD8+ T cells including a population of CCR7+ L-selectin– cells, which does not reflect current paradigms. The expression of CCR7 by these cells may be induced by the hepatic microenvironment to facilitate recirculation. 3) The CCR7 ligands CCL19 and CCL21 are present on lymphatic, vascular, and sinusoidal endothelium in normal liver and in patients with HCV infection. We suggest that the recirculation of CCR7+/L-selectin– intrahepatic CD8 T cells to regional lymphoid tissue will be facilitated by CCL19 and CCL21 on hepatic sinusoids and lymphatics. This centripetal pathway of migration would allow restimulation in lymph nodes, thereby promoting immune surveillance in normal liver and renewal of effector responses in chronic viral infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
More than 170 million people worldwide are infected with the hepatitis C virus (HCV),3 which results in chronic infection in >80% of cases (1). Chronic infection is characterized by persistent infection of hepatocytes, associated with progressive liver injury as a consequence of chronic inflammation driven by a frustrated immune response. Virological factors play a part in disease progression, but the host immune response is the primary determinant of viral clearance and the crucial factor in progression to chronic hepatitis, fibrosis, and cirrhosis in chronic disease (2). Both CD4 and CD8 T cell-mediated responses against HCV can be detected in infected patients by studying PBL but, despite their importance, local intrahepatic immune responses are relatively poorly understood (3, 4). The majority of T cells in the liver are not specific for HCV Ags; these bystander cells secrete cytokines such as IFN-{gamma} and modulate the local immune responses to allow chronic inflammation to become established (5, 6).

Lymphoid neogenesis, in which lymphoid follicles develop in portal areas, is a characteristic feature of chronic HCV infection and has led to the suggestion that naive T cells are recruited to the liver. Although most T cells in the inflamed liver express a differentiated phenotype characteristic of memory T cells (CD11ahighCD45RBdimCD45RObright), a significant proportion express CD45RA, a marker associated with naive T cells but which is also found on subsets of highly differentiated effector and some long-term memory CD8 T cells (7, 8, 9, 10). The liver is a site of clearance for terminally differentiated effector T cells but it also contains a large population of lymphocytes comprising NK cells, NK T cells, and conventional T cells which include viral-specific CD8 T cells (11, 12, 13). These T cells presumably recirculate through the liver to provide continuing immune surveillance (14) but a mechanism for their return from the parenchyma to local portal associated lymphoid tissues and draining lymph nodes has not been found.

Different models have been proposed to explain the differentiation of Ag-experienced CD8 lymphocytes. The linear differentiation model suggests that differentiation from naive into effector cells in response to Ag directly expands the T cell pool. Subsequent activation-induced death kills most of the effector cells, but some survive to form the long-term central memory pool and these cells show enhanced proliferative potential (15). In contrast, the parallel differentiation model suggests that effector and memory T cells develop independently after encounter with Ag (16). A recent analysis (17) of human blood T cells indicated that naive cells differentiate initially into memory then effector populations. Loss of CD45RA and expression of CD45RO is the classical marker of primed memory CD8 T cells (8), which can be further divided into CCR7+CD62L+ central memory and CCR7–CD62L– effector memory cells (18, 19) based on the assumption that expression of CD62L and CCR7 will facilitate entry of central memory cells to secondary lymphoid tissue from blood, whereas effector memory cells home to peripheral tissues.

Effector cells have been characterized within the CD45RA+ subset based on their cytolytic activity or cytokine secretion in response to Ag (20, 21, 22, 23, 24) and similar CD45RO+ CD8 T cells; they express high levels of LFA-1 and lack the costimulatory molecules CD27 and CD28 (8, 21, 25). CD45RA+ effector cells are the most potent producers of IFN-{gamma}, and are thus likely to play a significant role in viral infections such as chronic HCV (10). Reversion to the CD45RA+ state and loss of CD7, CD28, and CD27 occur late in the process of differentiation (25, 26, 27) and have been used to define distinct subsets of virus-specific CD8 T cells in blood (27). Furthermore, there are virus-specific enrichments of particular subsets in the chronic phase of persistent HIV, EBV, and HCV infections (27, 28). Thus, whereas CD45RA– memory cells display strong Ag-induced proliferation, express high levels of the costimulatory markers CD27 and CD28, and produce a wide array of cytokines including IL-2 and IL-4 CD45RA+, effector cells produce perforin and granzymes, express CD95 ligand, and mediate spontaneous cytolysis (reviewed in Ref. 9).

Most of the work on antiviral responses in humans has been performed on peripheral blood-derived cells, but it is clearly critical to understand the nature of the response within the liver in chronic HCV (29). We, therefore, performed a detailed analysis of effector and memory CD8 T cells infiltrating the human liver in end-stage HCV infection and, where possible, compared intrahepatic T cells with those in blood and secondary lymphoid tissue. We used high LFA-1 expression to define all Ag-experienced CD8 T cells regardless of their Ag specificity (10, 30) and to discriminate them from naive cells. To simplify the nomenclature in this study, we refer to CD11alowCD45RA+CD8+ cells as naive cells and divide the Ag-experienced CD8 T cells (all of which are CD11ahigh) into CD45RA– (conventional memory) and CD45RA+ (CD45RA+ memory).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Tissue studied and patient characteristics

Samples of liver were obtained within 1 h of explantation from 20 patients with end-stage hepatitis C cirrhosis, 5 patients with primary biliary cirrhosis, and 5 patients with primary sclerosing cholangitis undergoing orthotopic liver transplantation. Samples (1 cm3) were snap frozen in liquid nitrogen for later immunohistochemical studies (samples from all patients) and processed for lymphocyte isolation (HCV patients only). Matched peripheral blood was obtained at the time of transplantation (before any blood transfusions) from the same patients (n = 16), all of whom had evidence of active HCV infection (HCV RIBA+ve, HCV RNA+ve by PCR using the Roche Amplicor assay; Roche) and biopsy-confirmed cirrhosis. Normal donor liver was obtained from surplus liver tissue removed from surgically reduced grafts used for pediatric liver transplantation (n = 2) or from resected undiseased liver removed at hemihepatectomy for metastatic liver disease (n = 6). Hepatic lymph nodes were obtained from the porta hepatis at the time of organ retrieval in four organ donors without liver disease and from three HCV-infected livers removed at transplantation. The lymph nodes were stored in RPMI 1640 (Sigma-Aldrich) at 4°C for a maximum of 12 h before lymphocyte isolation. Liver donors had no evidence of either hepatitis B or C infection (negative by HCV ELISA and HCV RIBA; hepatitis B surface Ag, and hepatitis B core Ab negative). Donor organs had been perfused with and were stored in University of Wisconsin preservation fluid 12–18 h before cell isolation. Material was collected in accordance with guidelines for human experimentation and approved by our local research ethics committee.

Isolation of liver-infiltrating (LIL) lymphocytes

Hepatic tissue was collected in RPMI 1640. Tissue was diced using sterile blades into 1-mm3 pieces in RPMI 1640 and 1 mg/ml collagenase (type 1a; Sigma-Aldrich). Liver was incubated at 37°C for 90 min and passed through a 100-µm nylon mesh filter to remove cell clumps and undissociated tissue. Because we found that some markers are susceptible to cleavage by collagenase, we also used a nonenzymatic technique to isolate cells from some of the livers to confirm the stability of the markers. Liver was diced into 5-mm3 pieces and enzymatic digestion was replaced with further mechanical dissociation using a Seward stomacher 400 (230 rpm for 5 min). Cells were washed three times in SM2 (PBS with 10% heat-inactivated FCS; Invitrogen Life Technologies), 1 mM CaCl2, 0.5 mM MgCl2, and 0.1% sodium azide (all three from Sigma-Aldrich). The cell suspension was then layered on a Percoll (Amersham Biosciences) density gradient and centrifuged for 30 min at 868 x g. The lymphocyte band was removed from the interface between 30 and 70% Percoll (Amersham Biosciences) and further washed three times in SM2. Cell viability was assessed by trypan blue exclusion to ensure >95% viability and cells were frozen in FCS with 10% DMSO (Sigma-Aldrich) in a temperature-controlled freezing container (Cryo 1°C freezing container; Fisher Scientific), according to the manufacturer’s instructions.

Isolation of autologous PBL

Venous blood was collected into bottles containing EDTA at the time of operation or organ retrieval. Blood was mixed 1:1 with SM2, layered onto Lymphoprep (Nycomed Pharma), and centrifuged at 403 x g for 30 min. The PBL layer was removed, washed twice with SM2, pelleting the cells at 258 x g for 10 min, and resuspended in SM2 for immediate flow cytometry or in FCS with 10% DMSO (Sigma-Aldrich) for freezing.

Isolation of lymph node T cells

Lymph node tissue was diced using sterile blades, placed in cold PBS at 4°C for 1 h, and then filtered through a 100-µm nylon mesh to remove debris. The cells were pelleted from the suspension at 258 x g for 10 min and resuspended in FCS with 10% DMSO for freezing.

Abs for flow cytometry and immunohistochemistry

The following primary Abs were used for immunohistochemistry and flow cytometry: CD3 FITC, CD4 FITC, CD3 PE, CD4 PE, CD8 PE, CD56 PE, CD8 biotin, CD27 FITC, CD27 PE, L-selectin (CD62L); IgG1 unconjugated and PE conjugated; CD11a biotinylated and CD11a IgG2b unconjugated (all from BD Biosciences); CD3 ECD, CD4 ECD, CD8 ECD, CD45RA ECD, CD8beta PE, CD11a FITC, CD11a PE, CD45RA PE-TR, and CD56 allophycocyanin (all Beckman Coulter); and CD11a PE-Cy7, CD3 PE-Cy7, and CD8 allophycocyanin-Cy7 (all Caltag Laboratories). None of the CD11a Abs used detect conformation-dependent epitopes. CD45RO FITC (a gift from Prof. P. C. L. Beverley, University College Hospital, London, U.K.), CD45RA FITC (a gift from Dr. A. Akbar, Royal Free Hospital, London, U.K.), CD18 unconjugated, IgG1 (a gift from Prof. N. Hogg, Imperial Cancer Research Fund, London, U.K.). Chemokine receptors (all unconjugated): CCR4 IgG1 (R&D Systems), CCR5 IgG1 (Leukosite), CCR7 IgG2a (clone 7H12), CCR7 IgM (clone 3D9 from Dr. L. Wu; both from Millenium Pharmaceuticals), CCR7 IgG2a (clone 150503), and CXCR4 IgG2a (both from R&D Systems). Secondary Abs used were streptavidin PE-Cy7, goat anti-mouse IgG (H + L) Cy5, and goat anti-mouse IgM Cy5 (all from Cedarlane Laboratories).

For immunohistochemistry, including fluorescence, the following Abs were used: CCL19, 5 µg/ml; CCL21 5, µg/ml (R&D Systems), CD8beta-PE, 1/5 (Beckman Coulter), CD45RA-FITC, 1/400, CD34 1:50 (both from BD Biosciences), CK19 (1/25; PROGEN Biotechnik, or 200 µg/ml from DakoCytomation), von Willebrand factor (2 µg/ml; DakoCytomation), LYVE-1 (5.85 µg/ml; a gift from Dr. D. G. Jackson, Medical Research Council Human Immunology Unit, Oxford, U.K.), and CD31 (4.32 µg/ml; Pierce, and 1/50; BD Biosciences). Secondary Abs used for immunofluorescence were goat anti-mouse IgG2a FITC (20 µg/ml), goat anti-mouse IgG2b FITC (20 µg/ml; both from Southern Biotechnology Associates), and goat anti-mouse IgG1 Alexa 633 (8 µg/ml; Invitrogen Life Technologies).

Flow cytometry

Up to six-color flow cytometry was performed on autologous PBL and liver-infiltrating cells from organ donors and also from patients with hepatitis C cirrhosis. Anti-CD3 along with anti-CD8 or anti-CD8beta was used to detect CD8 T cells, and anti-CD11a or anti-CD18 was used to differentiate between naive and Ag-experienced cells. The expression of a range of differentiation-associated markers, CD45RA, CD45RO, CD27, and CD56, was studied. mAbs to the chemokine receptors CCR4, CCR5, CCR7, and CXCR4 were used to assess the migratory potential of T cells in HCV patients and normal liver donors.

Flow cytometry was performed using standard techniques; all incubation steps and washes were performed on ice in cold SM2. Briefly, where an unconjugated or a biotinylated primary Ab (raised in mouse) was used, lymphocytes were incubated for 45 min with the primary Ab (0.25 µg/106 cells in 100 µl) in Ig (1 mg/ml) from the species from which the secondary Ab was raised (mouse). Cells were washed and then incubated for 45 min with secondary Ab. After a further wash, the suspension was blocked with mouse Ig (3 mg/ml) for 5 min to saturate free binding sites on the FITC-conjugated F(ab')2 and the directly conjugated Abs were added in the last incubation step. FITC-, PE-, ECD-conjugated, and PE-Cy5-conjugated Abs were used to mark T cell populations in four-color assays. In six-color assays, biotin conjugated CD27 or CD11a were used as primary Abs, and streptavidin-PE-Cy7 was used as secondary Ab, before washing and incubation with the directly conjugated Abs. After final incubation, cells were washed with SM2, fixed with 1% paraformaldehyde, and resuspended in SM2 after a final wash. Whereas only conjugated Abs were used in four-color analysis, the cells were stained using a one-step method in 100 µl of SM2 at predetermined Ab concentrations for 45 min and then washed in cold SM2 before acquisition.

On flow cytometry, the lymphocyte population was gated using forward and side scatter parameters to exclude debris and dead cells. CD8 T cells were then gated using CD3 and CD8 or CD8beta markers and then the three populations of CD11alowCD45RA+ (naive); CD11ahighCD45RA– (memory), and CD11ahighCD45RA+ (CD45RA+ memory) cells were analyzed separately (Table I). An isotype-matched control was analyzed in one of the samples per donor of blood, liver, and lymph node because in pilot experiments we did not find a difference in control fluorescence of lymphocytes from these sites. Isotype-matched controls were gated in the same way as test Abs, but for clarity only one of the controls is displayed in the figures. Analysis was performed using a Beckman Coulter flow cytometer for four-color assays and a MoFlo cytometer (DakoCytomation) for six-color assays, and the data were analyzed using Summit software (DakoCytomation).


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  		Table I. Classification of CD8 T cells and their markers used for detection

 
The effect of collagenase isolation on chemokine receptor and adhesion molecule expression by T cells

PBMC were cultured in RPMI 1640 with or without PHA (10 µg/ml) for 24 h and then with collagenase 1A (1 mg/ml; Sigma-Aldrich) for 1 h. Cells were washed once in PBS/50% FCS and then twice in PBS. These cells were then stained using three-color FACS analysis with a panel of Abs. Median channel fluorescence (MCF) and percentage of positive cells were calculated. Expression of T cell markers and chemokine receptors was also examined on freshly isolated T cells incubated with collagenase for 0, 45, or 90 min, and there was no difference in expression of any of the chemokine receptors with the different techniques used.

Immunohistochemistry

Immunohistochemistry for CCL19 and CCL21 was done on 6-µm cryostat sections using serial sections stained with isotype-matched control for comparison as previously described (31, 32). The same technique was used to stain for CCL19 using 5 µg/ml primary Ab. Briefly, sections were fixed in acetone for 10 min and then incubated with primary mouse anti-human Ab followed by secondary rabbit anti-mouse (IgG, H + L) Abs (1/100; DakoCytomation). Endogenous peroxidase was blocked using sodium azide as described (32). Up to four-color stains for fluorescence microscopy were performed using unconjugated and directly conjugated primary Abs detected by confocal microscopy (single-image sections). All incubations were conducted at room temperature for 45 min and sections were washed for 30 min in TBS buffer (pH 7.4) between incubations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Virtually all liver-infiltrating CD8 T lymphocytes are Ag experienced

Ag-experienced CD8 T cells express high levels of the integrin LFA-1 which reliably distinguishes them from naive CD8 T cells (10, 30). We used four-color flow cytometry to determine the expression of CD11a and CD18 (the two components of LFA-1) on CD45RA– and CD45RA+ CD8 T cells. Preliminary experiments showed that CD11a and CD18 invariably demonstrate the same staining pattern; therefore, CD11a expression alone was used for subsequent analyses. In normal peripheral blood, ~25% of CD8 T cells are naive, being CD11alowCD45RA+ (23.29% SEM: 9.68, Fig. 1 and Tables I and II). This population was either completely absent or present at very low levels (<2%) in LIL (Figs. 1A and 2A). The level of CD11a expression in liver-derived lymphocytes (MCF, 117.27; SEM, 5.16) was similar to that detected on the CD11ahigh cells in matched blood samples (MCF, 107.54; SEM, 7.95) and 10-fold higher than that of naive blood CD8 T cells. High expression of CD11a was found in both normal and chronic HCV-infected livers, both of which were negative for the lymph node homing receptor L-selectin which is expressed at high levels on naive cells (Fig. 1B). The absence of L-selectin on LIL was also confirmed by immunohistochemistry (data not shown). Thus, virtually all CD45RA+ CD8 T cells in the liver are CD11ahigh/ L-selectin–, allowing us to conclude that very few, if any, naive CD8 T cells exist in the liver. Because CD11ahighCD45RA+ and CD45RA– CD8 T cells in tissue have not been well characterized, we analyzed these subsets of T cells in peripheral blood, lymph nodes and the liver using up to six-color flow cytometry to detect markers of T cell differentiation, function, and homing potential.


Figure 1
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  		FIGURE 1. A, CD45RA+LFA-1high CD8 T cells are enriched in the liver compared with peripheral blood and lymph node. Shown are the lymphocyte populations gated on CD3 and CD8 analyzed by CD11a and CD45RA. A naive population of CD11alowCD45RA+ cells can be detected in peripheral blood but is absent from the liver. CD11ahighCD45RA– memory cells can be detected in both compartments. A third population of CD45RA+CD11a high CD8 T cells is enriched in the liver. Numbers are percentages of the naive, CD45RA+CD11ahigh, and CD45RA–CD11a high CD8 T cells in the gated quadrants. B, L-selectin is undetectable on LIL from both normal and HCV livers. Shown is the percentage (of total) of CD8 T cells expressing L-selectin (mean and SD) on peripheral blood (PBL) and LIL, in normal liver donors (N), and patients with end-stage hepatitis C cirrhosis (HCV).

 

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  		Table II. Naive, conventional memory, and CD45RA+ memory CD8 populations in normal donors and HCV patients

 

Figure 2
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  		FIGURE 2. Expression of CD56, CD27, and CCR7 in peripheral blood as well as in normal and HCV-infected liver. A, Lymphocytes from peripheral blood or liver were gated on CD8beta and subsequently on CD11a and CD45RA to define the naive (green), CD45RA+ memory (blue), and conventional memory (black) CD8 T cells. Isotype-matched control is red. CCR7 is expressed at high levels on naive CD8 T cells in blood but this subset is absent from the liver. A subset of Ag-experienced CD45RA+ and CD45RA– memory CD8 T cells in the liver express CCR7. B, PBL were gated on CD8, CD3 as well as CD45RA, and CD11a to compare the CD45RA+ memory population (CD11ahigh, blue line) with the naive (CD11alow, green line) and the conventional memory (CD11ahigh, black line) population. MCF (CD56 and CD27) or percentages (CCR7) of samples and histograms of a representative patient are given. Isotype-matched controls are in red. There is comparable CD56 expression in all three cell types (left panels). The heterogeneous expression of CD27 and CCR7 in the CD45RA+ memory cells resembles that of the CD45RA– memory CD8 T cells and both receptors are expressed at lower levels than on naive cells. C, Analysis of lymphocytes isolated from undiseased liver (top row) and chronically HCV-infected cirrhotic liver (bottom row). Lymphocytes were gated on CD8beta and CD11ahigh to allow a comparison of the CD45RA+ with the CD45RA– memory CD8 populations. Plots from representative patients and the table report mean and SD of MCF for CD56 (left panel) and CD27 (middle panel) and percentage for CCR7 (right panel) for five samples (NL, normal liver). A lower percentage of CD45RA– CD8 T cells are CCR7+ in the HCV-infected livers compared with undiseased control livers.

 
Liver tissue is a repository of CD8+CD45RA+CD11ahigh T cells in healthy individuals

To investigate the principal site of residence of CD45RA+CD11ahigh CD8 T cells in vivo, we determined the proportion of this subset among all CD8 T cells and among Ag-experienced T cells in blood, lymph node, and liver tissue in normal donors (Table II). CD45RA-expressing B cells and NK cells were excluded by gating on either CD3 or CD8beta+, the T cell-specific component of CD8. The greatest proportion of CD8+CD45RA+CD11ahigh cells was seen within the liver (18.9% of CD8 T cells) with decreasing amounts within peripheral blood (11.0%) and lymph node (3.2%; see Figs. 1 and 2 and Table II). We then analyzed the CD8+CD11ahighCD45RA+ T cell population in detail to determine their likely origin and function in the normal and HCV-infected liver.

Characterization of CD45RA+CD11ahigh CD8 T cells in peripheral blood, lymph node, and liver

CD45RA+CD11ahigh CD8 T cells in peripheral blood have a similar phenotype in normal liver donors and HCV-infected patients (Table III). We used expression of CD56, CD27, and CCR7 to further characterize CD8 T cells. On circulating cells levels of CD27 and CCR7 were significantly lower on both CD45RA– and CD45RA+ Ag-experienced (CD11ahigh) CD8 cells compared with naive (CD11alow) CD45RA+ CD8 cells. Naive cells displayed consistently high levels of CCR7, whereas Ag-experienced cells expressed little or intermediate levels of CCR7 (Fig. 2B). All naive cells expressed high levels of CD27, whereas Ag-experienced (CD11ahigh) cells were heterogeneous for CD27 expression (Fig. 2B). MCF of CD56 was not significantly different between CD45RA+CD11ahigh cells and naive CD8 T cells in blood. These patterns of CCR7 and CD27 expression were similar in normal donors and patients infected with HCV. Furthermore, for each of the three populations naive (CD45RA+CD11alow), conventional memory (CD11ahighCD45RA–), and CD45RA+ memory (CD11ahighCD45RA+) CD8 T cells and each of the three markers (CD56, CD27, and CCR7), the nine comparisons revealed no statistical difference between normal donors and HCV patients in peripheral blood (data not shown). We then analyzed the same subsets within LIL.


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  		Table III. CD56 expression in CD8 subsets in normal liver donorsa

 
Intrahepatic CD45RA+ CD8 T cells can be divided into subpopulations according to CD27 expression

Because levels of CD27 have been shown to define different subsets of blood CD8 T cells in viral infections, we analyzed CD27 expression of matched peripheral blood and LIL. In normal subjects, Ag-experienced CD8+ T cells showed similar levels of CD27 in blood (MCF, 60.3; SEM, 10.18) and liver (MCF, 58.17; SEM, 5.56). We detected CD27high and CD27low subsets within the conventional memory and the CD45RA+ memory CD8 T cell populations (Fig. 2). Expression of CD27 was generally lower on CD45RA+ Ag-experienced cells compared with CD45RA– cells in both peripheral blood (MCF, 49.07; SEM, 6.54 vs 62.65 SEM, 10.54; p = 0.06) and liver (MCF, 43.74 SEM, 3.87 vs 59.78 SEM, 5.98; p = 0.04), consistent with these cells being terminally differentiated (Fig. 2, B and C). However, six-color flow cytometry of CD8+CD45RA+ T cells in the liver and matched peripheral blood of three donors revealed a small but significant population of lymphocytes (1.7% of the CD3+CD8+CD45RA+ T cells) within the liver, which expressed levels of CD27 comparable to those seen on naive CD8 T cells in peripheral blood (data not shown). These CD27high cells were not detected in the conventional memory (CD45RA–) cells in LIL and they differed from naive CD45RA+ T cells in matched PBL because they were L-selectinlowCD11ahigh, had lower levels of CCR7 (MCF, 93.9 ± 2.33 vs 127.1 ± 3.15; p < 0.025), and higher levels of CD56 (MCF, 94.41 ± SEM 1.92 vs 76.88 ± 5.51; p < 0.01).

In end-stage HCV infection the intrahepatic CD11ahigh CD8 T cell population is skewed toward CD45RA– cells

In chronic HCV infection, the proportion of CD45RA+CD11ahigh CD8 T cells in peripheral blood was significantly greater than that seen in the normal controls (22.00% vs 11.03%; p = 0.04; Table II). Despite this, in all nine matched liver and blood samples from end-stage HCV, a lower proportion of CD11ahigh CD8 cells in the liver was CD45RA+, compared with peripheral blood (LIL, 8.87% vs PBL, 30.24%; p = 0.003; see Table II). This was in contrast to the findings with normal donors, in which a higher percentage of these cells was found in the liver (18.9%) than in PBL (14.4%).

Distinct patterns of homing receptors on CD8 T cells in the liver

Naive and primed T cells undergo distinct pathways of recirculation determined in part by differences in their expression of chemokine receptors. We compared expression of several chemokine receptors on the different CD8 subsets from matched blood, lymph node, and liver. True naive cells (CD11alowCD45RA+) in blood and lymph node expressed high levels of CCR7 and CXCR4 and low levels of CCR5, as expected from previous studies (33). A high proportion of CD8+CD11ahigh LIL were CCR5+ in both HCV-infected livers and normal livers in both the CD45RA– and CD45RA+ populations (Fig. 3). We used the skin-homing molecule CCR4 as a receptor that we would not expect to be involved in recruitment to the liver. In undiseased liver, both CD45RA+ and CD45RO+ memory/effector populations expressed similar levels of CCR4 compared with peripheral blood, whereas in HCV very few CCR4+ cells were found in the liver or in lymph nodes. In normal and diseased liver, we detected a distinct subset of CCR7+ cells in both the CD45RA– and the CD45RA+ effector/memory populations. The lack of L-selectin (Fig. 1 and data not shown) on the liver-infiltrating CD45RA+ and CD45RA– CD8 T cells precludes these intrahepatic CCR7+ T cells from being classic central memory cells. We further characterized the CCR7+ liver-infiltrating CD8 T cells and compared them with matched peripheral blood in five normal donors. In peripheral blood, all naive CD8beta+CD45RA+CD11alow T cells expressed high levels of CCR7, whereas the Ag-experienced CD8beta+CD11ahigh cells could be divided into CCR7– and CCR7low populations in both CD45RA– and CD45RA+ cells (Figs. 2 and 3 and Table IV). We divided the Ag-experienced cells further into the four groups CD45RA+CD27–, CD45RA+CD27+, CD45RA–CD27–, and CD45RA–CD27+ using all events in the CD3+CD8+CD11ahigh gate of a six-color flow cytometry analysis. Fewer LIL were CCR7+ compared with matched PBL for all four subsets of Ag-experienced CD8 T cells (paired comparison of the means of each subgroup: p = 0.01 for difference in MCF and p = 0.03 for difference in percent positive cells (data not shown).


Figure 3
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  		FIGURE 3. Chemokine receptor expression in normal and HCV-infected peripheral blood, lymph node, and LIL. CCR4 (left panels), CCR5 (middle panels), and CCR7 (right panels) expression on Ag-experienced CD8 T cells in the liver, in peripheral blood, and in lymph node in HCV transplant patients and normal donors. All histograms are gated on CD8beta and CD11ahigh expression. Histograms for CD45RA+ (blue) and CD45RA– (black) memory CD8 T cells are compared with isotype-matched controls (red). Samples are representative of three or more samples and percentages of chemokine-positive cells are given for the CD45RA+ and CD45RA– populations. The peripheral blood samples of normal liver donors and HCV-infected patients show comparable chemokine receptor expression. Normal and HCV-infected livers contain high numbers of CCR5+ cells in both CD45RA+ and CD45RA– populations compared with peripheral blood and lymph node. In HCV-infected liver samples, the CD45RA– memory cells contain a low proportion of CCR4- and CCR7-expressing cells.

 

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  		Table IV. CXCR4 and CCR7 expression in CD8 subtypes in normal donors and HCV patientsa

 
Analysis of primed CD8 T cells according to CCR7 and CD27 expression

Next, we analyzed the CCR7– and CCR7+ CD8+ T cells separately, divided into the same four groups of CD27–CD45RA+, CD27+CD45RA+, CD27–CD45RA–, and CD27+CD45RA– and determined the MCF of CD56 on each subset as a marker of effector activity (Table III). We found that CD56 expression was higher in the liver regardless of CD45RA, CCR7, or CD27 expression. We also found that in each of the four subgroups of CD8+CD11ahigh T cells, CD56 expression was greater in CCR7+ cells compared with CCR7– cells in peripheral blood, as well as in LIL (Table III; p < 0.01). In this setup, we took great care that the effect seen was not due to lack of compensation of the flow cytometer by using fluorochromes excited by different lasers (PE at 488 nm for CD56 and allophycocyanin-Cy5 at 633 nm for CCR7).

The CCR7 ligands CCL19 and CCL21 are present in normal and HCV-infected liver

The expression of CCR7 on intrahepatic CD8 T cells is likely to determine their routes of migration. We, therefore, stained normal and diseased liver tissue for CCL19 and CCL21 (Table V and Fig. 4). In normal liver, CCL21 was confined to sinusoidal endothelium whereas CCL19 was also detected on small vessels and biliary epithelium in portal tracts and on hepatocytes (Fig. 4). In inflammatory liver disease, including HCV, CCL19 staining was increased in portal tracts and sinusoids in all of the inflammatory liver diseases studied. Detailed analysis of HCV liver tissue using confocal microscopy localized CCL19 expression in the portal tracts to CK19+ bile ducts and ductules and to two distinct populations of vessels which we defined as vascular neovessels (CD31+ and CD34+) or lymphatic vessels (LYVE-1+). CCL19 staining was detected on most of the LYVE-1+ lymphatic vessels and a subset of the CD34+ neovessels (Fig. 4C and data not shown). Many of the LYVE-1+ lymphatics were observed to contain lymphocytes.


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  		Table V. CCL19 and CCL21 expression in normal and diseased livera

 

Figure 4
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  		FIGURE 4. CCL19 and CCL21 are expressed in normal and HCV-infected liver. A, Immunohistochemical expression of CCL19 (a–e), CCL21 (g and h) in the liver of normal liver donors (a, b and g), and patients with chronic hepatitis C (c–e and h); isotype-matched control shown in HCV liver only (f). Positive cells stain brown with the peroxidase technique. In normal liver, CCL19 is expressed in portal fields (a) on small vessels (arrowheads) and bile ducts (arrow). In the parenchyma (b), in addition to a generalized membranous staining of hepatocytes, there is patchy staining of sinusoidal endothelium (arrow) and occasional lymphocytes (arrowheads). In hepatitis C liver, CCL19 stains similar structures (Table V). In a number of HCV livers, strong staining was detected in the basement membrane of small bile ducts (c). A number of small portal vessels stained strongly for CCL19. Some of these vessels had morphological characteristics of high endothelial venules (d) and subsequent analysis revealed them to include CD34+ neovessels and LYVE-1+ lymphatic vessels (Bc). Staining of sinusoidal endothelium was seen in HCV (e, arrow) and normal livers and occasional lymphocytes stained positive for CCL19 (b, arrowhead). CCL21 stains show sinusoidal staining in both normal (g) and HCV-infected (h) liver (arrows). B, Localization of CCL19 to cholangiocytes, lymphatics, and neovessels in HCV by confocal microscopy. Liver samples from patients with chronic HCV (a and c) and uninfected liver donors (b) were stained with four-color immunofluorescence confocal microscopy. a and b, CCL19 (green) was visualized along with CK19 to mark cholangiocytes (red) and CD34 to mark neovessels (blue). CCL19 was expressed on CD34+ vessels (costaining in magenta) and small bile ducts and ductules (costaining in yellow) mainly on the abluminal side (original magnification, x10). CCL19 (green) with von Willebrand’s factor to mark vascular endothelium (blue) and LYVE-1 to mark lymphatic vessels (red). CCL19 staining was also found on the abluminal side of LYVE-1+ small lymphatic vessels (costaining in yellow; original magnification, x40).

 
In chronic hepatitis C liver-infiltrating memory (CD45RA–) CD8 T cells show a relative lack of CCR7 and CXCR4

Using four-color flow cytometry, we quantified the number of CCR7+ cells in the different CD8 subsets in peripheral blood and liver of patients with chronic HCV infection (Table IV). In the CD45RA+ memory CD8 T cell population in both nondiseased and HCV liver, similar numbers of cells were CCR7+ (21%; Table IV and Fig. 3). However, in chronic HCV-infected livers, significantly fewer of the CD45RA–CD11ahigh CD8 T cells expressed CCR7 (8.6%) compared with normal liver (39%) or matched peripheral blood (40%).

In normal liver tissue and lymph nodes, CD11ahigh CD8 T cells expressed high levels of CXCR4 comparable to those seen on naive cells in blood, suggesting that CXCR4 may be increased as a consequence of liver infiltration. CXCR4 expression in peripheral blood was generally lower in patients with HCV compared with normal donors and in contrast to the situation in the normal liver, <50% of CD8 T cells infiltrating the liver in HCV were CXCR4+ (Table IV).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
CD45RA+CD11ahigh CD8 T cells in blood have defining characteristics of Ag-experienced T cells (10, 30) and include suppressor cells (34), terminally differentiated effector cells (8, 18, 21), and long-lived memory cells (10). Studies using MHC class I tetramers have shown that they also include CD8 T cells with specificity against several persistent viral infections including HCV, CMV, EBV, and HIV (11, 27, 28). Whereas MHC class I tetramers only bind to the subset of primed cells that recognize a specific epitope, CD11a is increased on all primed CD8 T cells regardless of their specificity, making it a particularly useful marker in the context of hepatitis C infection, in which the immune response includes numerous different epitopes, none of which is immunodominant (35). Furthermore, previous studies (5) using tetramers showed that despite a 30-fold higher percentage of HCV-specific T cells in the liver compared with blood, the vast majority of intrahepatic CD8 T cells are not virus-specific, implying that bystander-primed but non-HCV-specific CD8 T cells contribute to liver damage. The ability of such primed bystander T cells to contribute to tissue damage has been described (36) in Listeria monocytogenes infection, during which Ag-experienced CD8 T cells secrete IFN-{gamma} in response to IL-12 and IL-18 in the absence of cognate Ag.

We found a higher percentage of CD8+CD45RA+C11ahigh T cells in liver compared with those in peripheral blood and lymph node. These cells could be either activated effector cells, that retain expression of CD45RA, or long-lived (posteffector) memory CD8 T cells. It has been reported that effector T cells are rapidly removed from the circulation by the liver where they die by Fas-mediated apoptosis (37). However, this may not apply to the Ag-experienced CD45RA+ CD8 T cell population, because these cells express low levels of Fas and high levels of Bcl-2, making them relatively resistant to apoptosis (8, 25). These cells are highly stable within individuals for up to 10 years after primary infection (10, 22), suggesting that at least a subset of them are long-term memory cells that preferentially localize to nonlymphoid tissues, such as the liver, even in the absence of inflammation (11, 38, 39). Ag-experienced CD45RA+CD8 cells show a biased repertoire reflecting specificity for persistent viruses and recruitment to relevant infected tissue, rather than secondary lymphoid tissue, is likely to be crucial for their function. Furthermore, CD45RA+CD8 cells include those with high cytotoxic potential (8, 21, 27) likely to be important for the intrahepatic response against HCV (8, 21, 40). We found the proportion of CD45RA+CD8+ T cells in the liver to be lower in HCV compared with undiseased controls, suggesting that CD45RA– T cells are either preferentially recruited or proliferate within the liver in HCV. The relative lack of CD45RA+ effectors could contribute to viral persistence in HCV infection.

Our data add to the complexity of differentiated CD8 T cell populations defined by expression of CD27 and CD45 isoforms. In particular, we describe a novel subset of tissue-infiltrating CD45RA+ CD8 memory T cells, which expresses CD27. The presence of the costimulatory marker CD27 on these cells argues against them being terminally differentiated effector cells and suggests they are able to respond to costimulatory signals and thus may provide long-term memory (10, 41). It is unclear what differentiation pathway would lead to the development of these cells. Instead of developing from classical CD45RA– proliferating memory, CD8 T cells might develop in a linear model from the CD45RA+CD27– effector cells by regaining CD27. These cells are compatible with posteffector, long-term memory cells, which are responsive to costimulation. On encountering Ag they might lose CD45RA expression and develop into CD45R0+ proliferating memory CD8 T cells with subsequent costimulation resulting in CD27 loss and redifferentiation into effector cells. This would be compatible with a circular model of differentiation in which effector function and/or efficiency increases with each round of Ag exposure and costimulation. Recent studies (42) have proposed a role for CD27 in the local activation of effector T cells in the gut and it is possible that a similar mechanism operates in the liver.

We report CCR7 expression on a subset of LIL. The expression of CCR7 and L-selectin on central memory cells enables them to traffic to lymph nodes via high endothelial venules (43, 44), however, the liver-infiltrating CCR7+ CD8 T cells we describe were all low in L-selectin, which distinguishes them from classical central memory cells and precludes them entering lymph node via high endothelial venules. They could represent a population of resident liver cells but this would not explain why they express CCR7. An alternative explanation is that CCR7 is involved in trafficking to and from the liver. This possibility is supported by our finding the CCR7 ligands CCL19 and CCL21 on CD31+CD34+ neovessels and portal lymphatic vessels. CCL19 staining of the lymphatic vessels was predominantly abluminal, consistent with a role in promoting reverse transmigration from tissue into the lymphatics (Fig. 4Bc). We have previously reported (31) CCL21 expression on lymphatics in portal-associated lymphoid tissue and, in this study, we added the presence of CCL21 on sinusoidal endothelium (Table V and Fig. 4, g and h). CCR7 ligands on sinusoids and lymphatic endothelium could provide a route for CCR7+ lymphocytes to exit the liver to regional lymph nodes. A similar CCR7-dependent pathway has been proposed for dendritic cells in which activated dendritic cells emigrate from the liver to lymph nodes via a parasinusoidal route to the portal tracts and then via lymphatics to draining nodes (45, 46, 47, 48, 49). Recently, CCR7 has been shown to be involved in T cell egress from the skin (40) and lung (50) via afferent lymphatics to draining lymph nodes, observations that are consistent with our findings in this study. The existence of such a pathway would explain the high proportion of CCR7+/L-selectin–-primed cells we saw in the lymph nodes.

Reconciling the data on CCR7 expression with the concept that CD27– cells are of the effector type (8, 21) and CD27+ cells represent long-term memory cells (43, 51), the CD27 and CCR7 expression would give four different CD45RA+CD11ahigh CD8 T cells with potentially different functions. First, CD27–CCR7– cells could be effector T cells in the liver parenchyma destined to die rapidly by apoptosis (12, 52). Second, CD27–CCR7+ cells are likely to migrate to regional lymph nodes or be retained in portal-associated lymphoid tissue. The third population of CD27+CCR7– T cells is compatible with local long-term memory cells that transform into proliferating memory and effector cells after Ag challenge. Finally, CD27+CCR7+ cells may represent memory cells that can respond to restimulation and provide immune surveillance through a migratory pathway from blood to liver and then back via lymphatics to secondary lymphoid tissues.

The proportion of CCR7+ cells in hepatitis C-infected liver was less than that in normal liver tissue despite expression of both CCR7 ligands CCL19 and CCL21 in HCV liver. This suggests that either CCR7– CD8 T cells are preferentially recruited to the inflamed liver, which could result in increased bystander inflammation driven by effector T cells in the CCR7– CD8 T cell population, or, alternatively, CCR7 might be down-regulated after ligand engagement of CCR7 or Ag encounter in the liver. CXCR4 expression was also reduced in T cells from HCV-infected liver, which could reflect preferential recruitment of CXCR4low cells to the inflamed liver or a loss of CXCR4 within the liver (27, 53, 54). Normal human liver expresses high levels of CXCL12 constitutively but low levels of CCR5 and CXCR3 ligands (55, 56, 57). Thus, CXCR4 may be critical for trafficking through the noninflamed liver, whereas in HCV increased expression of CCR5 and CXCR3 ligands would promote the recruitment of CD8 T cells expressing these receptors at the expense of CXCR4high cells (32).

In summary, we report that CD11ahigh CD8 cells in the liver are not a homogeneous population. Consistent with their tissue distribution, they lack L-selectin and show high levels of chemokine receptors associated with tissue infiltration, including CCR5. They include a subset of CD45RA+ CD8 cells with an activated effector phenotype that lack CD27 and CCR7 (8, 18, 21) and, surprisingly, a significant number of CCR7+ cells. We propose that the CD11ahighCD45RA+CCR7+CD27+ cells may act as long-term memory cells able to undergo centripetal migration from liver to lymph node, in response to CCL19 and CCL21 on sinusoidal and lymphatic endothelium. The potential for these cells to re-enter lymph nodes and undergo further restimulation and differentiation (58) suggests a role in immune surveillance and in the renewal of responses to chronic viral infection.


   Acknowledgments
 
We thank the patients and families of donors for the donated blood and tissue. We also thank our clinical colleagues, in particular, the nurses and medical staff of the Birmingham Liver Unit for their help in obtaining patient samples. We also thank Drs. L. Wu and M. Briskin (Millenium Pharmaceuticals) and D. G. Jackson (University of Oxford, Oxford, U.K.) for providing Abs.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was sponsored by a grant from the Medical Research Council and the Wellcome Trust. Back

2 Address correspondence and reprint requests to Dr. Mathis Heydtmann and Dr. David H. Adams, Liver Research Laboratories, Institute for Biomedical Research, Birmingham University, Birmingham, B15 2TH, U.K. E-mail addresses: m.heydtmann{at}bham.ac.uk and d.h.adams{at}bham.ac.uk Back

3 Abbreviations used in this paper: HCV, hepatitis C virus; LIL, liver-infiltrating lymphocytes; MCF, median channel fluorescence. Back

Received for publication July 6, 2005. Accepted for publication April 12, 2006.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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