Errata: Correction Volume 53, Issue 4, 1415, Article first published online: 7 April 2011
Hepatitis C virus (HCV) causes chronic infection accompanied by a high risk of liver failure and hepatocellular carcinoma. CD8+ T cell responses are important in the control of viremia. However, the T cell response in chronic infection is weak both in absolute numbers and in the range of epitopes targeted. In order to explore the biology of this response further, we analyzed expression of a panel of natural killer cell markers in HCV compared with other virus-specific T cell populations as defined by major histocompatibility complex class I tetramers. We found that CD161 was significantly expressed on HCV-specific cells (median 16.8%) but not on CD8+ T cells specific for human immunodeficiency virus (3.3%), cytomegalovirus (3.4%), or influenza (3.4%). Expression was seen in acute, chronic, and resolved disease and was greatest on intrahepatic HCV-specific T cells (median 57.6%; P < 0.05). Expression of CD161 was also found on hepatitis B virus–specific CD8+ T cells. In general, CD161+CD8+ T cells were found to be CCR7− “effector memory” T cells that could produce proinflammatory cytokines (interferon-γ and tumor necrosis factor-α) but contained scanty amounts of cytolytic molecules (granzyme B and perforin) and proliferated poorly in vitro. Expression of CD161 on CD8+ T cells was tightly linked to that of CXCR6, a chemokine with a major role in liver homing. Conclusion: We propose that expression of CD161 indicates a unique pattern of T cell differentiation that might help elucidate the mechanisms of HCV immunity and pathogenesis. (HEPATOLOGY 2008;47:396–406.)
Hepatitis C virus (HCV) is a major human pathogen. Approximately 170 million people worldwide are chronically infected with HCV, and it is estimated to cause around 280,000 deaths per year. Although acute infection is usually asymptomatic, persistent infection results in approximately 80% of HCV-infected individuals; these individuals are at high risk of cirrhosis and hepatocellular carcinoma. Hepatitis C is now the major indication for liver transplantation in Europe and the United States.1
CD8+ T cell responses may play a role in determining the overall success of the immune response in acute infection.2 Not surprisingly, considerable effort has been applied to define the characteristics of successful CD8+ T cell immune responses. Successful responses (in acute infection) may be of higher magnitude, or broader, targeting a greater number of epitopes or target-specific epitopes.3 However, the outcome of infection also includes important contributions from innate CD4+ T cell and humoral responses.4
In chronic infection, the CD8+ T cell response has a narrow breadth and magnitude despite high levels of replication. Evidence suggests that there is progressive attrition of responses as chronic infection ensues.5 Thus, failure to maintain responses may be characteristic of chronic infection (as opposed to an inability to generate responses initially).
The function of T cells is reflected to some extent by the combination of molecular markers expressed on their surface—known as the T cell phenotype.6 HCV-specific CD8+ T cells during chronic infection have a phenotype that is described as “immature” or of a “central” memory phenotype.7, 8 This contrasts sharply with the phenotype of human immunodeficiency virus (HIV)- and cytomegalovirus (CMV)-specific T cells, which are consistently larger and more mature with effector potential.8 The low frequency of CD8+ T cells with their immature phenotype and narrow repertoire are the most salient features of HCV immunology and are currently unexplained.9
One possibility that might explain this is that the HCV-specific response develops along a distinctive paradigm of T cell differentiation and homeostasis compared with those in HIV and CMV. This may be because the site of infection is the liver, where the environment for antigen presentation and the surrounding cell types is unique. In an attempt to evaluate this hypothesis, we compared expression of a panel of natural killer (NK) receptors on different virus-specific CD8+ T cells as defined by major histocompatibility complex class I tetramer complexes specific for HCV, HIV, influenza, CMV, and hepatitis B virus (HBV).
We found that the C-type lectin CD161 is expressed on a significant subset of HCV-specific T cells, but not on CMV-, HIV-, or influenza-specific responses. It was, however, found on HBV-specific CD8+ T cells. Exploration of the properties of CD161+CD8+ T cells in uninfected patients showed that these are effector cells that produce proinflammatory cytokines, such as interferon-γ (IFN-γ) and tumor necrosis factor-α, but express low levels of granzyme B and perforin, perhaps indicating reduced ability to lyse virus-infected cells. CD161 was linked strongly to CXCR6, a chemokine receptor implicated in liver homing.10–13 These findings suggest another dimension to T cell biology beyond the central and effector memory dichotomy6 with implications for mechanisms of virus persistence.
CMV, cytomegalovirus; FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; IFN-γ, interferon-γ; IL, interleukin; NK, natural killer; PBMC, peripheral blood mononuclear cell; PE, phycoerythrin; SE, standard error; TGF-β, transforming growth factor-β.
Materials and Methods
We used fresh buffy coats of healthy laboratory volunteers, from the National Blood Transfusion Service and from routine clinical hematology samples as described previously.14 HIV+ responses were obtained from 16 HIV+ patients with human leukocyte antigen A2–restricted responses to SLYNTVATL undergoing study in Oxford, UK. HCV-specific responses were obtained by screening patients at the John Radcliffe Hospital in Oxford, UK, and Massachusetts General Hospital in Boston, MA. HBV+ patients and studies of intrahepatic HCV-specific T cell responses were enrolled as described.15
Tetramers were either obtained commercially (Beckman Coulter, Fullerton, CA; Pro-immune, Oxford, UK) or synthesized as described.2 Staining was performed on whole blood14 or on fresh or previously frozen peripheral blood mononuclear cells (PBMCs)7 as described. After preparation, cells were incubated with tetramer at 37°C for 20 minutes before staining for surface antibodies.
Antibodies and Phenotyping.
A panel of phycoerythrin (PE)-conjugated anti-KIR receptor antibodies was obtained from Dr. Neil Young (KIR2DL2/L3/S2, KIR3DL1, KIR3DL2) and Beckman Coulter (EB6). These antibodies were mixed to detect any staining in the same stain. CD85j(ILT-2/LIR-1) was obtained from Beckman Coulter (PE-conjugated) or BD Biosciences (Franklin Lakes, NJ) [fluorescein isothiocyanate (FITC)-conjugated]. CD161 antibody was obtained from Beckman-Coulter (clone 191B8-PE) or BD Biosciences (DX12 clone conjugated to FITC or PE). IFN-γ-FITC, CCR7-FITC, anti-CXCR6/Bonzo-PE, anti-mouse immunoglobulin G-Fab-allophycocyanin, and immunoglobulin G-Fab-FITC was obtained from R&D Systems (Minneapolis, MN). CD103-FITC, β7-integrin-PE, Ki67-FITC, perforin-FITC, IFN-γ-FITC, CD3-FITC, CD3- and CD8-peridinin-chlorophyll protein complex, CD8-allophycocyanin, and CD45RA-allophycocyanin antibodies were obtained from BD Biosciences. Unconjugated anti-CD3 was obtained from Beckman Coulter. Anti-granzyme B-allophycocyanin antibody was obtained from Caltag/Invitrogen.
Cells were stained with antibody, incubated for 20 minutes at 4°C, washed, and fixed in 1% paraformaldehyde solution (Sigma-Aldrich). Phenotyping of bulk CD8+ T cells was performed using whole blood from HIV/HCV-uninfected patients as described.14
Microbead cell sorting (CD8+ T cell Isolation Kit II, anti-PE microbeads, and LS columns; Miltenyi Biotec, Surrey, England) was performed using freshly isolated PBMCs from buffy coats or laboratory volunteers according to the manufacturer's instructions.
Phorbol 12-myristate 13-acetate (1 μg/mL)/ionomycin (1μg/mL) was used to stimulate cytokine secretion. We used a cytometric bead array (BD Biosciences) assay on supernatants of purified CD161+ or CD161−CD3+CD8+ T cells; the median purity (percentage of all events acquired) was 76.9% [standard error (SE) 9.9] and 81% (SE 8.4), respectively. Cells were plated in a 96-well round-bottomed plate at 0.1 − 0.5 × 106 cells in R10 [RPMI 1640 (Sigma-Aldrich, Dorset, UK) supplemented with 10% heat-inactivated fetal bovine serum, penicillin, and streptomycin]. Cells were stimulated overnight at 37°C.
For intracellular cytokine staining, unsorted PBMCs were sorted on CD161 as above [purity (gated on CD3+CD8+ cells) consistently >95%] and placed in a 96-well plate as above. Cells were incubated for 5 hours. Golgiplug (BD Biosciences) was added after 1 hour.
Cells sorted on CD161 were placed with CD8-depleted PBMCs [<1% CD8+ T cells − CD8-DYNA beads (Invitrogen, Paisley, UK)] at a ratio of 1:2 (PBMCs sorted on CD161/CD8-depleted PBMCs). Addition of CD8-depleted cells provided accessory cells. Interleukin (IL)-2 (Chiron, Emeryville, CA) was added at 200 U/mL. Cells were then incubated at 37°C for 3 days before staining for intracellular Ki67 (a marker of cell proliferation).
Effect of Cytokines on Expression of CD161.
An ex vivo restimulation assay for an influenza response was performed in 1 patient known to have a response to the influenza peptide GILGFVFTL. After 2 × 106 cells/mL were placed in a 48-well plate, 10 μg/mL of influenza-peptide was added along with 100 IU/mL of IL-2. Either recombinant IL-12 (2 or 20 ng/mL) or transforming growth factor-β (TGF-β) (2 or 10 ng/mL) (R&D Systems) was added as indicated. Cells were left for 10 days with fresh R10 and IL-2 every 3 to 4 days. Collected cells were stained as described above.
Intracellular Cytokine and Ki67 Staining.
Cells were stained with surface antibodies as above and resuspended in 4% paraformaldehyde (Sigma-Aldrich) in phosphate-buffered saline and left in the dark at room temperature for 30 minutes. Cells were then washed and resuspended in permeabilization buffer (eBiosciences, San Diego, CA), and intracellular antibody to CD3 and either perforin or IFN-γ or Ki67 was added. Cells were then incubated with antibody and permeabilization buffer at room temperature for 15 minutes, washed, and fixed with 1% paraformaldehyde. (Intracellular staining for granzyme B and perforin was performed on sorted cells, because this was found to allow more precise determination of staining of CD161+ cells, due to the occasional low background frequency of this population.)
Data Acquisition and Statistical Analysis.
Samples were acquired using a FACScalibur fluorescence-activated cell sorting (FACS) machine (BD Biosciences) using Cell Quest software or an LSR II cytometer (BD Biosciences) with FACS Diva software (BD Biosciences). Files were analyzed using Flowjo software (Tree Star, Ashland, OR). Data acquisition and statistical analysis were performed using GraphPad Prism Version 4 (GraphPad Software, San Diego, CA). Wilcoxon's matched pairs test was used for analyzing paired data. The Mann-Whitney U test was used to compare unpaired data.
Expression of NK Receptors on HCV-, Influenza-, HIV-, and CMV-Specific T Cells.
CD161 and LIR-1 (CD85j/ILT-2) showed significant expression on HCV-specific T cells (median 16.85%, SE 4.9, and median 10.53%, SE 2.9, respectively) (Fig. 1A). KIRs were not expressed on HCV-specific cells (median 0.2%, SE 0.64). Unlike LIR-1 (CD85j/ILT-2), which is known to be expressed on CMV-specific T cells, only HCV-specific cells expressed significant levels of CD161 (range 0%–74.9%) (Fig. 1B-C), indicating that HCV-specific responses frequently contain a significant CD161+ subset.
No significant difference in CD161 expression was seen in patients with chronic or resolved HCV (Fig. 1D) or acute hepatitis C (Figs. 1E, 2). Expression was sustained at similar levels until at least 4 months postinfection (Fig. 2).
Analysis of CD161 Expression on HBV-Specific T Cells.
Because CD161 expression appeared to be selectively elevated on HCV-specific T cells, we next addressed whether CD161 expression was a feature of T cells targeting another hepatitis virus: hepatitis B (Fig. 3A). We looked for CD161 expression in 5 patients with acute HBV infection (in patients with persistent infection, tetramer staining is typically below the level of detection ex vivo from blood).16 Expression varied from 3.3% to 27.3% of antigen-specific T cells, indicating significant CD161 expression in some cases on HBV-specific cells. These expression levels were significantly higher than those on CMV- and HIV-specific T cells (P < 0.05), and not significantly different from those found on HCV-specific CD8+ T cells in acute disease (Fig. 3B). Thus, CD161 expression may reflect a more general feature of T cells specific for liver-associated infections.
Study of Expression of CD161, CCR9, and CD103 on Intrahepatic T Cells.
We next determined expression of CD161 on HCV-specific T cells in the livers of 4 patients (Fig. 4A–B). The median expression was 57.6% (SE 14.39%; total of 6 responses) compared with 16.85 (SE 5%) in peripheral blood (P = 0.03). Two patients also had intrahepatic influenza-specific responses that showed low levels of CD161 expression (5.2% and 2.7%) (Fig. 4B). Thus, a “step up” in CD161 expression between peripheral blood and the liver was observed.
CD161 has been linked with the gut and with expression of gut homing markers.17–19 Therefore, HCV-specific T cells might express other markers related to T cell homing to the liver and intestine. Therefore, we first looked at expression of CCR9, which may be linked to gut20 and possibly liver homing.21 We found extremely low levels of CCR9 expression on HCV-specific cells in the liver and in peripheral blood. CD103 has also been linked to CD8+ T cell homing to the liver in HCV.22 Although CD103 showed limited expression on HCV-specific T cells in peripheral blood [median 4%, SE 3.1% (n = 5)], intrahepatic HCV-specific CD8+ T cells showed relatively high expression (16.8%–43%), which is consistent with previous reports.22
TGF-β up-regulates CD10323 and may be produced by regulatory T cells in the liver. We found that CD161+CD8+ T cells (bulk populations from peripheral blood of normal donors) expressed more CD103+ cells than CD161− cells (median 12.2 [SE 2.7] and 3.1 [SE 0.63], respectively; P < 0.005). This suggests that peripheral blood CD161+CD8+ T cells are more likely to have recently been exposed to TGF-β and/or trafficked through gastrointestinal tissues. CD161+ cells were also relatively high in expression of the β7 chain of the integrin heterodimer α4/β7 (P = 0.065) (data not shown).
Linkage Between CD161 Expression and CXCR6 Expression on CD8+ T Cells.
The strongest link with CD161 and liver homing was found by analysis of CXCR6 expression. CXCR6 and its ligand CXCL16 have been strongly implicated in liver homing.10–13 There was a striking association between the presence of CD161 and the presence of CXCR6 in a large series of HCV− donors (n = 30) (Fig. 5A,B). Similar findings were seen in the peripheral blood of 7 HCV+ donors and a single donor with acute hepatitis B (P = 0.02) (Fig. 5A).
Phenotype of CD161+CD8+ T Cells.
FACS analysis with costaining for CCR7 and CD45RA24 in 11 uninfected patients enabled us to determine what “memory” subtype CD161+CD8+ T cells are (i.e., bulk populations in peripheral blood of normal patients). We found that CD161 expression was very low on naïve T cells (median 0.89%) and was only weakly expressed on TCM cells (8.41%). However, significant expression was seen on effector memory subtypes TEMRAand TEM (25.4% and 23.7%, respectively) (Fig. 6A). Thus, CD161+CD8+ T cells are primarily a subpopulation of effector memory cells. This finding is consistent with the linkage between CD161 and CXCR6 expression, because CXCR6 is known to be expressed on effector memory (CCR7−) populations.10
CD161 Expression Is Not Up-regulated by IL-12 or TGF-β.
In order to explore further what the determinants of CD161 expression might be, we explored if exposure to specific cytokines might induce CD161 expression. PBMCs from a donor already defined as positive for an influenza-specific tetramer-positive (CD161−) population were restimulated with peptide in vitro as described in Materials and Methods (Fig. 6B–D). IL-12 has been reported to up-regulate CD161 on NK cells,25 and TGF-β may be secreted by regulatory T cells that might modulate HCV-specific T cell function.26 However, none of these cytokines were able to modulate CD161 expression. Thus, we found no evidence that exposure to these specific cytokines could be responsible for CD161 expression—at least in the short term—in vitro.
CD161 Expression Is Not Modulated by T Cell Activation.
In order to address whether CD161 is modulated by T cell activation, we looked at CD161 expression on sorted CD161+ or CD161− T cells (mixed with CD8− PBMCs as above) for 3 days with CD2/CD3/CD28-coated beads (Fig. 6E). No alteration in CD161 expression was seen for CD161− cells (99%) or CD161+ cells (97.6%). In addition, the lack of CD161 expression after restimulation of influenza-specific cells strongly suggests that CD161 is not simply an activation marker.
Function of CD161+CD8+ T Cells.
We next explored the cytokine-secreting potential of CD161+ T cells (Fig. 7). Supernatants of CD161+ and CD161−CD3+CD8+ T cells from 5 patients (sorted bulk populations from the peripheral blood of uninfected patients) showed that they could elaborate IFN-γ, tumor necrosis factor-α, and IL-2 but not IL-10 or IL-4 (Fig. 7A). Intracellular cytokine staining yielded similar findings (Fig. 7B). Like CXCR6,10 therefore, CD161 expression is linked to a Tc1 phenotype. In contrast to the clear capacity of CD161+ T cells to produce proinflammatory cytokines, CD161+ T cells were low in expression of lytic granule components perforin (P < 0.05) and granzyme B (P < 0.05) compared with CD161− cells, suggesting that they may have diminished capacity to kill virally infected cells through this pathway, or require further activation to do so (Fig. 7C–D). Sorted CD161+CD3+CD8+ T cells also showed reduced capacity to proliferate compared with sorted CD161− cells (as indicated by staining for Ki67) in response to nonspecific stimuli even when accessory cells and additional IL-2 were provided (Fig. 7E).
CD161 (KLRB1/NKRP1A) is a C-type lectin that shows high expression on NK and NK T cells.25, 27 The cell surface ligand for CD161 has been recently identified as lectin-like transcipt 1.28 Unlike other NK receptors, therefore, CD161 does not interact with class 1 molecules. Interactions between CD161 and lectin-like transcipt 1 may have marked effects on multiple cell processes through effects on acid sphingomyelinase activity,29 including NK and T cell function.27, 30
This study examined CD161 expression on virus-specific T cells. The findings have been illuminating, because CD161-expressing cells account for a significant subset of HCV-specific cells, whereas virtually no expression is seen in other prototype antiviral T cell responses. Trogocytosis (acquisition of CD161 from other cells in the liver31) seems an unlikely explanation, because other NK markers (notably KLRGI) show low expression in HCV.32 In addition, an HCV-specific cell line also showed expression of CD161 (data not shown), whereas trogocytosis would predict dilution and loss of expression after proliferation in culture.
Our study indicated reduced levels of granzyme B and perforin in CD161+ T cells. HCV-specific T cells also show low expression (although maturation can occur upon in vitro culture).7, 8, 33 It is possible that CD161+ T cells, though they are effector memory cells, may have relatively defective killing independent of HCV status. The mechanisms involved in the killing of HCV-infected cells in the liver have not been fully elucidated, but this may be relevant to the intrahepatic environment. A number of studies have indicated the importance of IFN-γ secretion over perforin as a mediator of control over intrahepatic pathogens.33, 34 We have recently shown using in vitro models that hepatocytes are relatively resistant to non-Fas–mediated killing after exposure to inflammatory stimuli.35 Further study is needed to define the cytolytic function of these cells in vivo, especially after further activation and/or ligation of the CD161 receptor.
Using in vitro assays, we observed that CD161+ T cells do not proliferate as well as their CD161− counterparts. This finding is consistent with previous work.36 We did carefully control for numbers of CD8− accessory cells and supplemented IL-2—though, as a caveat, CD161− cells are enriched for central memory cells, which have high proliferative capacity compared with effector memory cells.37 The issue of proliferative capacity is important, because amplification of priming and recall T cell responses to HCV may be defective. Some studies have suggested limited proliferative capacity of HCV-specific CD8+ T cells,38 and this—together with our findings—could help explain the low frequency of HCV-specific T cells in chronic infection.
Consistent with the phenotypic data showing an effector memory phenotype (CCR7lo), CD161+ T cells did retain other effector functions. They were able to produce proinflammatory cytokines (notably IFN-γ and tumor necrosis factor-α). They did not produce any type II cytokines (e.g., IL-10 and IL-4), however, suggesting that they are not involved in anti-inflammatory processes. IL-10 secretion from HCV-specific T cells has been observed in the liver,39 so further analysis of CD161+ cell function—especially among antigen-specific cells—in the liver will be of interest to establish if this phenotype/function link is maintained. The expression of IFN-γ, as described above, could play a potential role in clearing hepatotropic viruses.40
We found CD161 expression in acute disease, in chronic infection, and on memory responses once HCV infection had resolved. We were unable to show that CD161 expression could be modulated by T cell activation or by TGF-β and IL-12. This may indicate that CD161 is hardwired into the basic properties of T cells, and expression (or a lack thereof) is relatively consistent over time for different T cell populations. This stability is in contrast to many other markers that may be either modulated by short-term (CD38) or long-term (CD127, CD27, CD28, CD85j) antigenic exposure. Clearly, specific signaling pathways must contribute to the up-regulation of CD161 early in T cell maturation. Further studies to establish the signaling pathways active in CD161+ cells and their link to CXCR6 expression are underway.
Expression of CD161 may relate to the site of HCV infection (the liver), as enrichment of CD161+ cells is seen here and elsewhere.17, 18 In addition, there is evidence of some CD161 expression on HBV-specific cells, indicating that its expression is not restricted to HCV-specific cells. Although the number of acute HBV+ patients studied remains limited, the CD161 level was nevertheless significantly higher than in the larger CMV and HIV groups studied (P < 0.05). Finally, the strong link of CD161 to CXCR6 observed here—and to a lesser extent with CD103 and β7 integrin—suggests a link with liver-associated T cell pools, because these markers, particularly CXCR6, have been linked to liver and gut homing. It will be important to assess whether signaling through CXCR6 affects CD161 expression and vice versa or whether expression of both is linked to a common signaling history.
In conclusion, we propose that CD161 expression is the tip of the iceberg with regard to biological properties that discriminate HCV-specific responses from those to other commonly studied pathogens. Some features, such as the relationship to hepatic T cell pools and linkage to CXCR6, the potential for T cell modulation through CD161 (by interaction with lectin-like transcipt 1), and the low expression of cytolytic markers (e.g., perforin and granzyme B) clearly have implications for the pathogenesis and persistence of HCV infection and its treatment. The findings provide a tangible handle with which to explore the enigmatic nature of T cell responses to chronic HCV infection.