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J Virol. Apr 2006; 80(7): 3532–3540.
PMCID: PMC1440406

Expression of the Interleukin-7 Receptor Alpha Chain (CD127) on Virus-Specific CD8+ T Cells Identifies Functionally and Phenotypically Defined Memory T Cells during Acute Resolving Hepatitis B Virus Infection

Abstract

Virus-specific CD8+ T cells play a central role in the outcome of several viral infections, including hepatitis B virus (HBV) infection. A key feature of virus-specific CD8+ T cells is the development of memory. The mechanisms resulting in the establishment of T-cell memory are still only poorly understood. It has been suggested that T-cell memory may depend on the survival of virus-specific CD8+ T cells in the contraction phase. Indeed, a population of effector cells that express high levels of the interleukin-7 receptor alpha chain (CD127) as the precursors of memory CD8+ T cells has recently been identified in mice. However, very little information is currently available about the kinetics of CD127 expression in an acute resolving viral infection in humans and its association with disease pathogenesis, viral load, and functional and phenotypical T-cell characteristics. To address these important issues, we analyzed the HBV-specific CD8+ T-cell response longitudinally in a cohort of six patients with acute HBV infection who spontaneously cleared the virus. We observed the emergence of CD127 expression on antigen-specific CD8+ memory T cells during the course of infection. Importantly, the up-regulation of CD127 correlated phenotypically with a loss of CD38 and PD-1 expression and acquisition of CCR7 expression: functionally with an enhanced proliferative capacity and clinically with the decline in serum alanine aminotransferase levels and viral clearance. These results suggest that the expression of CD127 is a marker for the development of functionally and phenotypically defined antigen-specific CD8+ memory T cells in cleared human viral infections.

It is widely accepted that virus-specific CD8+ T cells play an essential role in immune responses to viral infections. In a successful immune response, three distinct phases can be defined (2, 31). Initially, the effector phase is characterized by a clonal expansion of activated CD8+ effector T cells that eliminate virus-infected host cells. This phase is followed by a contraction phase, when CD8+ T cells massively die by apoptosis; in a third phase, a CD8+ memory T-cell population is established. Memory CD8+ T cells are characterized by their ability to survive homeostatically in the absence of antigen and by their ability to proliferate vigorously upon antigenic reencounter (2, 13, 31). Indeed, memory CD8+ T cells are easily activated upon antigen rechallenge, in which case they quickly produce antiviral cytokines or cytotoxic molecules. Several models have been proposed that characterize the CD8+ memory T-cell generation on the basis of the differential expression of a set of surface markers (e.g., the lymph node homing receptor CCR7 or the T-cell costimulatory receptor CD27) (1, 2, 13, 23, 31). However, it is still unclear precisely how the memory pool is established. For example, memory CD8+ T cells could simply represent the “survivors” of the contraction phase (being derived from the effector cell population) or a distinct population primed early during the immune response with a separate differentiation program.

Recently, Kaech et al. demonstrated that antigen-specific CD8+ T cells displayed differential interleukin-7 (IL-7) receptor α chain (CD127) expression during acute lymphochoriomeningitis virus (LCMV) infection in mice (12). Low numbers of CD127+ antigen-specific CD8+ T cells could be identified in acute infection, but antigen-specific CD8+ T cells analyzed in the memory phase expressed CD127, suggesting that CD127 is a marker that identifies early CD8+ T cells destined to become memory CD8+ T cells (9, 12). Indeed, adoptively transferred CD127+ CD8+ T cells but not CD127 CD8+ T cells survived in the absence of antigen by homeostatic proliferation and thus maintenance via CD127 (12). The biological role of CD127 is further supported by the finding that CD8αα is selectively expressed by memory precursors and required for CD8+ memory T-cell generation and survival and that CD8αα expression is linked to the up-regulation of CD127 (16). Thus, these results support the important role of IL-7 and its receptor in mediating homeostasis of memory CD8+ T cells (24). During viral infections, the emergence of CD127 expression on virus-specific CD8+ T cells occurs only when the antigen load is contained and sufficient CD4+ T-cell help is available (3, 5, 12). Persistent viral antigen suppresses CD127 expression on primed T cells and correlates with exhaustion of a previously stable primed T-cell population (14).

First evidence suggests that CD127 might also be a marker for T-cell differentiation in viral infection in humans, because all CD8+ T cells specific for viruses that are being cleared, such as influenza virus and respiratory syncytial virus, express this marker (29). By contrast, cytomegalovirus- and Epstein-Barr virus-specific CD8+ T cells lack significant CD127 expression during acute and latent infection (29). Human immunodeficiency virus infection is consistently associated with the expansion of CD127 CD8+ antigen-specific CD8+ T cells that correlates precisely with markers of disease progression and the overall level of immune system activation (20). However, because of the persistent nature of these viruses, no information is currently available about the kinetics of CD127 expression in an acute resolving human viral infection. Specifically, the kinetics of CD127 expression on antigen-specific CD8+ T cells and its association with disease pathogenesis, viral load, and functional and phenotypical T-cell characteristics has not been studied previously. To address these important issues, we analyzed the hepatitis B virus (HBV)-specific CD8+ T-cell response longitudinally in a cohort of six acutely infected patients who subsequently cleared the virus. Acutely HBV-infected patients are usually identified between weeks 10 and 15 after infection, when they become symptomatic (22, 30). At that time, multispecific activated CD8+ T cells are readily detectable and associated with liver disease and subsequent viral clearance (17, 28). The central role of HBV-specific CD8+ T cells for viral clearance and disease pathogenesis has further been directly demonstrated by performing depletion studies of CD8+ T cells in acutely infected chimpanzees (26).

In this study, we demonstrate the emergence of CD127 expression on functionally and phenotypically defined antigen-specific CD8+ memory T cells in acute resolving HBV infection in humans. CD127 expression could be identified in 2% to 6% of HBV-specific CD8+ T cells during acute infection. In contrast, HBV-specific CD8+ T cells of all patients showed that CD127 expression after viral clearance increased markedly, with increases ranging from 42% to 86%. Importantly, the up-regulation of CD127 correlated phenotypically with the loss of CD38 and PD-1 expression and acquisition of CCR7 expression, functionally with enhanced proliferative capacity, and clinically with the decline of serum alanine aminotransferase (ALT) levels and viral clearance. These results suggest that CD127 expression is a marker for the development of antigen-specific CD8+ memory T cells in viral infection in humans.

MATERIALS AND METHODS

Subjects.

Six HLA-A2-positive, acutely HBV-infected patients were included in this study after informed consent. The study was approved by the local medical ethics committee. The diagnosis of acute hepatitis B was based on at least 10-fold-elevated serum ALT levels and detection of hepatitis B surface antigen and anti-hepatitis B core antigen antibodies (immunoglobulin M). All patients were negative for antibodies to hepatitis A virus, hepatitis C virus (HCV), hepatitis D virus, and human immunodeficiency virus type 1 and type 2. The clinical and virological data of the patients at the first time point of clinical appearance and at a later time point after viral clearance are summarized in Table Table11.

TABLE 1.
Study population

PBMC.

Peripheral blood mononuclear cells (PBMC) were isolated from EDTA anticoagulated blood samples on a Ficoll-Histopaque density gradient (PAA, Vienna, Austria). After isolation, cells were washed twice in phosphate-buffered saline (Gibco, Auckland, NZ) and were studied immediately or were cryopreserved in medium containing 80% fetal calf serum (FCS) (Gibco, Auckland, NZ), 10% dimethyl sulfoxide (Sigma-Aldrich, Germany), and 10% RPMI 1640 (Gibco, Auckland, NZ). The results of functional assays performed with fresh and cryopreserved PBMC from the same bleed date were comparable.

Synthetic peptides and HLA-A2 multimer complex.

HBV-derived peptides previously shown to be HLA-A2-restricted HBV epitopes were synthesized with free N and C termini (Biosynthan, Berlin, Germany). The amino acid sequences of the HLA-A2-restricted HBV and influenza virus epitopes are shown in Table Table2.2. These peptides were initially dissolved in 100% dimethyl sulfoxide (Sigma-Aldrich, Germany) at 20 mg/ml and were further diluted to 1 mg/ml with RPMI 1640 (Gibco, Auckland, NZ) before use. HLA-A2 tetramers or pentamers corresponding to the HBV peptides were obtained either from ProImmune (Oxford, UK) or from the National Tetramer Core Facility at Emory University (Atlanta, GA).

TABLE 2.
Study peptides

Antibodies.

Anti-CD8-phycoerithrin (anti-CD8-PE), anti-CD8 Cy7-PE, anti-CD38-PE, anti-gamma interferon (anti-IFN-γ), and anti-PD-1 antibodies and isotype fluorescein isothiocyanate, PE, Cy7, and allophycocyanin controls were obtained from BD Bioscience-Pharmingen (San Jose, CA). Anti-CD127 antibody was purchased from Immunotech (Marseille, France), anti-CCR7-PE antibodies were purchased from R&D Systems (Minneapolis, MN), and anti-CD27-PE antibody was purchased from Hoelzel Diagnostika (Cologne, Germany). All antibodies were used for immunostaining and fluorescence-activated cell sorter (FACS) analysis according to the instructions of the manufacturers.

Multimer and antibody staining.

A total of 1 × 106 cells per well on a 96-well plate were incubated with allophycocyanin-conjugated HLA-A2 tetramers or pentamers (ProImmune, Oxford, UK) corresponding to the peptides shown in Table Table2.2. Incubation was performed at 37°C and 5% CO2 for 15 min. Cells were washed three times with phosphate-buffered saline containing 1% FCS, blocked with pure immunoglobulin G1 for 10 min, and stained with an anti-CD8 antibody for 15 min. Cells were washed three times followed by surface staining with anti-CD127, anti-PD-1, anti-CD38, anti-CCR7, or anti-CD27 antibodies for 15 min at 4°C, washed again, and fixed in 100 ml CellFIX (BD Bioscience Pharmingen) per well. As a positive control for PD-1 staining, 4 × 106 PBMC were resuspended in 1 ml RPMI medium (Gibco) containing 10% FCS, 1% streptomycin-penicillin, and 1.5% HEPES buffer (1 M), stimulated with 100 U recombinant IL-2 (rIL-2) and 0.4 μg/ml anti-human CD3 monoclonal antibody (Immunotech, Marseilles, France), and cultured for 72 h in the presence of 2 × 106 irradiated autologous PBMC. FACS analysis was performed using a BD FACSCalibur flow cytometer and either Cell Quest software (BD Biosciences) or FlowJo software (Tree Star, Inc., Ashland, OR).

Intracellular IFN-γ staining.

Procedures were performed essentially as described previously (25). Briefly, cells (0.2 × 106 per well, 96-well plate) were stimulated with peptides (10 μl/ml) in the presence of 50 U/ml human rIL-2 (Hoffmann La Roche, Basel, Switzerland) and 1 μl/ml brefeldin A (BD Pharmingen). CD8+ T cells stimulated with phorbol-12-myristate 13-acetate (Sigma-Aldrich (Seelze, Germany) served as positive controls. After 5 h of incubation (37°C, 5% CO2), cells from each well were blocked and stained with anti-CD8 antibodies. Prior to staining with intracellular antibodies against IFN-γ, cells were fixed and permeabilized by adding Cytofix-Cytoperm (BD Pharmingen). Cells were washed three times and fixed in 100 μl CellFIX (BD Pharmingen) per well. FACS analysis was performed on a BD FACSCalibur flow cytometer using either Cell Quest software (BD Biosciences) or FlowJo software (Tree star, Inc., Ashland, Or, USA).

Antigen-specific cell proliferation.

A total of 4 × 106 PBMC were resuspended in 1 ml RPMI medium (Gibco) containing 10% FCS, 1% streptomycin-penicillin, and 1.5% HEPES buffer (1 M), stimulated with 10 μg/ml of synthetic HBV peptide and 0.5 μg/ml anti-human CD28 (BD Pharmingen), and cultured in a 48-well plate. On day 4, 20 U/ml human rIL-2 (Hoffmann La Roche, Inc., Basel, Switzerland) was added. After 7 days, cells were restimulated with 1 ×106 irradiated PBMC. On day 10, 20 U/ml human rIL-2 was again added. Cells were stained for antigen-specific responses on day 14. FACS analysis was performed using a BD FACSCalibur flow cytometer and either Cell Quest software (BD Biosciences) or FlowJo software (Tree Star, Ashland, OR).

Multimer calculation.

Multimer responses are shown as the percentage of multimer-positive T cells of all CD8+ T cells. The severalfold expansion was calculated as follows: percentage of multimer-positive CD8+ T cells after 14 days in culture divided by percentage of multimer-positive CD8+ T cells ex vivo.

RESULTS

HBV-specific CD8+ T-cell responses during acute resolving infection.

To determine the frequency and hierarchy of HBV-specific CD8+ T cells during acute HBV infection, we analyzed PBMC from six patients (Table (Table1)1) with four multimers specific for dominant HLA-A2-restricted core-, envelope-, and polymerase-derived epitopes (Table (Table2).2). As previously described, we observed multispecific CD8+ T-cell responses upon clinical presentation for all patients (Fig. (Fig.1A)1A) (17, 28, 30). Of note, no clear hierarchy of the different epitope-specific CD8+ T cells could be identified. The weakest initial responses were observed in patients 4 and 5, who nevertheless cleared the virus. The magnitude of the HBV-specific CD8+ T-cell response declined during the course of infection, with markedly reduced frequencies of multimer-positive cells after resolution of infection (Fig. (Fig.1B).1B). This reduction is particularly striking for patients 1, 3, and 5, in whom frequencies dropped for all specificities; e.g., patient 1 lost 92% of her pol-455-specific CD8+ T cells. Representative density plots are displayed in Fig. Fig.1C.1C. Taken together, these results support the notion that multispecific, HBV-specific CD8+ T-cell responses correlate with liver disease and that they are associated with viral clearance, after which they decline (22).

FIG. 1.
Multispecific CD8+ T-cell responses during acute resolving HBV infection. (A and B) PBMC of six patients with acute HBV infection were analyzed for CD8+ T-cell responses to four immunodominant HBV epitopes in the early phase (A) and after ...

Emergence of CD127+ HBV-specific CD8+ T cells during acute resolving infection.

In the mouse model of LCMV infection, it has been shown that only a fraction (9% to 15%) of antigen-specific CD8+ T cells are CD127+ during acute infection, but after viral clearance CD127+ T cells preferentially survive the contraction phase and dominate the memory pool (12). To investigate whether a similar pattern can be observed during acute viral infection in humans, we determined the expression of CD127 on HBV-specific CD8+ T cells at the onset of infection and after viral clearance. Importantly, at initial presentation, CD127 could be identified only in 2% to 6% of HBV-specific CD8+ T cells whereas all patients showed markedly increased CD127 expression after viral clearance (Fig. (Fig.2A).2A). Overall, the mean CD127+ expression increased from 2% to 69% (Fig. (Fig.2A),2A), ranging from 42% in patient 6 to 86% in patient 1. The increase of CD127 expression over time is also shown for patients 1, 2, and 3 (Fig. 3J, K, and L). For example, in patient 3, CD127+ expression of HBV-specific CD8+ T cells started at 2%, rose to 35% at week 2 and to 42% at week 4, and peaked at 73.5% at week 10 (Fig. (Fig.3L).3L). These results clearly demonstrate the emergence of CD127-expressing HBV-specific CD8+ T cells during acute resolving infection.

FIG. 2.
Phenotypic characterization of HBV-specific CD8+ T cells. For each patient, surface staining of HBV-specific CD8+ T cells was performed using an HBV multimer with a strong ex vivo response (Table (Table1)1) and antibodies to CD127 ...
FIG. 3.
Courses of acute resolving HBV infection. Clinical and virological data are shown for three patients at several time points during and after acute HBV infection. (A, B, and C) Viral load (VL), ALT levels, and dominant multimer response. GE, genome equivalents; ...

Expression of CD127 correlates with distinct patterns of surface expression.

Next, we correlated the pattern of CD127 expression with other markers of T-cell activation and differentiation by monitoring HBV-specific CD8+ T cells for the expression of the activation marker CD38 and the differentiation and memory markers CD27 and CCR7 (Fig. 2B, C, D). Not surprisingly, and as described previously, we observed a highly activated phenotype at the time of clinical presentation, with CD38 expression on HBV-specific CD8+ T cells of over 90% in all 6 patients (17, 28). The activation status of the HBV-specific CD8+ T cells correlated with ongoing significant liver disease and high viral loads. By contrast, HBV-specific CD8+ T cells were primarily CD38 negative after resolution of infection, with CD38+ frequencies of 10% or lower (Fig. (Fig.2B).2B). The down-regulation of CD38 occurred rapidly during the course of infection, as shown in detail for patients 1, 2, and 3 (Fig. 3D, E, and F). The decrease in CD38 expression correlated with the decrease in serum ALT activity and viral load in all patients, suggesting that CD38 expression is antigen driven. The expression of CD27 on HBV-specific CD8+ T cells was high at all time points tested, ranging from 75% to 100% (Fig. (Fig.2D).2D). Of note, the expression of the lymphoid homing receptor CCR7 increased in all patients during the acute phase of infection. We observed a mean fivefold increase of CCR7 expression between the initial presentation (3.6% to 41.5% CCR7-positive HBV-specific CD8+ T cells) and resolution of infection (17.3% to 87% CCR7-positive HBV-specific CD8+ T cells). The kinetics of CCR7 expression is displayed for patients 1, 2, and 3 (Fig. 3G, H, and I). In patients 1 and 3, CCR7 expression among HBV-specific CD8+ T cells rose from 21% and 9% at presentation, respectively, to 86% and 65% after resolution of infection (Fig. 3G and I). Interestingly, patient 2, the oldest person in our study cohort, showed an only slight increase in CCR7 expression during the course of infection (Fig. (Fig.3H).3H). Overall, our results indicate a clear switch in the phenotype of HBV-specific CD8+ T cells during acute resolving infection, with a predominantly activated CD127 CCR7 CD38+ CD27+ CD8+ effector memory T-cell population during acute infection that is associated with high viral loads and liver disease and a CD127+ CCR7+ CD38 CD27+ CD8+ HBV-specific central memory T-cell population after resolution of HBV infection. Of note, this switch was observed in all epitope-specific CD8+ T-cell responses of a given patient (data not shown).

CD127+ HBV-specific CD8+ T cells show increased proliferative capacity.

Next, we assessed the functional properties of the different HBV-specific CD8+ T-cell populations by determination of intracellular cytokine production and in vitro proliferative capacity after peptide-specific stimulation in five of the six patients. Of note, we observed an impaired ability of HBV-specific CD8+ T cells to produce IFN-γ throughout the infection. Indeed, the majority of HBV-specific CD8+ T cells were unable to produce significant amounts of IFN-γ upon stimulation either at the peak or after resolution of infection (Fig. 4A and B). At the onset of infection only 10/19 (52.6%) multimer-positive CD8+ T-cell responses were also detectable by intracellular IFN-γ staining and only one epitope-specific response showed IFN-γ production above 50% (patient 4; polymerase-specific response) (Fig. (Fig.1A1A and and4A).4A). After resolution of the infection, 12/17 (70.6%) multimer-positive CD8+ T-cell responses were able to produce IFN-γ after peptide-specific stimulation and the response was >50% in 3 of those (Fig. (Fig.1B1B and and4B).4B). Thus, a slight tendency towards stronger IFN-γ production after resolution was observed. Overall, however, the ability of HBV-specific CD8+ T cells to produce IFN-γ was weak and often absent. Interestingly, this impaired IFN-γ production is specific for HBV-specific CD8+ T cells because Flu-specific CD8+ T cells readily secreted IFN-γ after peptide-specific stimulation, as shown for patient 2 (Fig. (Fig.4C),4C), for example, and all CD8+ T cells produced this cytokine after stimulation with phorbol-12-myristate 13-acetate (data not shown). It is also important to note that the HBV-specific CD8+ T cells did not produce other cytokines such as IL-2 and tumor necrosis factor alpha (data not shown).

FIG. 4.
Functional capacities of HBV-specific CD8+ T cells. (A and B) PBMC of five patients with acute HBV infection were analyzed for HBV-specific CD8+ T-cell IFN-γ production after stimulation with four immunodominant HBV epitopes in ...

Next, we measured the proliferative capacity of the HBV-specific CD8+ T cells by peptide-specific stimulation, focusing on dominant initial epitope-specific CD8+ T-cell responses (Table (Table1).1). There was a clear difference in the expansion capacities of virus-specific CD8+ T cells at the peak of infection, when the HBV-specific CD8+ T-cell population was largely CD127, as compared to after resolution of infection, when most HBV-specific CD8+ T cells expressed CD127. Indeed, the proliferative capacity of HBV-specific CD8+ T cells stimulated in vitro was at least 10 times stronger after resolution than during acute hepatitis B (Fig. (Fig.4D).4D). The proliferative capacity increased in parallel with the expression of CD127 and CCR7 (Fig. 2A and C and and4D),4D), as shown longitudinally for patients 1, 2, and 3 (Fig. (Fig.3).3). Depletion experiments revealed that the proliferative capacity was indeed increased in the CD127+ population (data not shown). The expansion kinetics did not depend on initial T-cell frequency, as patients with reduced initial multimer-positive frequencies (e.g., patient 5) showed antigen-specific proliferation comparable to that of patients with high initial frequencies (e.g., patient 2). Of note, we did not observe a consistent correlation between IFN-γ production and peptide-specific expansion, suggesting that these CD8+ T-cell effector functions are regulated differentially. Since PD-1 has previously been shown to be associated with an impaired ability of virus-specific CD8+ T cells to proliferate (11), we tested HBV-specific CD8+ T cells for their PD-1 expression. Interestingly, the increase in T-cell proliferation for HBV-specific CD8+ T cells correlated with the down-regulation of PD-1. We observed a marked decrease in PD-1 mean fluorescence intensity on HBV-specific CD8+ T cells between early and late time points for all patients (Fig. 5A and B). Of note, this effect was HBV specific, since the entire CD8+ T-cell population and the Flu-specific CD8+ T cells did not show a similar induction of PD-1 (Fig. 5C and D).

FIG. 5.
PD-1 expression on HBV- and Flu-specific CD8+ T cells. Virus-specific CD8+ T cells were detected via multimer staining and analyzed for PD-1 expression. (A) PD-1 expression on HBV-specific CD8+ T cells was analyzed at clinical ...

DISCUSSION

In our longitudinal study, we show the emergence of CD127-expressing HBV-specific CD8+ T cells during acute resolving HBV infection. In acute infection, the majority of HBV-specific CD8+ T cells were CD127, while after viral clearance, most HBV-specific CD8+ T cells displayed a CD127+ phenotype. Of note, a similar kinetics of CD127 expression on virus-specific CD8+ T cells has previously been described by Kaech et al. using a mouse model of LCMV infection (12): low numbers of CD127+ antigen-specific CD8+ T cells could be identified in acute infection, but antigen-specific CD8+ T cells analyzed in the memory phase expressed CD127. Thus, these combined results suggest that CD127 is a marker that identifies early CD8+ T cells destined to become memory CD8+ T cells (9, 12), not only in the mouse model but also in humans with acute resolving viral infection.

Importantly, the emergence of CD127 expression was closely associated with other phenotypic characteristics. Indeed, at the onset of clinical symptoms, CD127 HBV-specific CD8+ T cells expressed the activation marker CD38 and were primarily negative with respect to the lymph node homing receptor CCR7. The presence of these activated virus-specific CD8+ T cells correlated closely with the extent of liver disease and the viral load. The up-regulation of CD127, however, was closely associated with the down-regulation of CD38 and an increase in CCR7 expression. Both occurred in concert with resolution of liver disease and containment of viral antigen, supporting the notion that the emergence of CD127 is governed by withdrawal of antigenic stimulation (5, 12). This hypothesis is also supported by recent studies that showed high CD127 expression on human virus-specific CD8+ T cells specific for cleared viruses (e.g., influenza virus, respiratory syncytial virus) and low CD127 expression on human virus-specific CD8+ T cells specific for persisting viruses (e.g., cytomegalovirus, human immunodeficiency virus, Epstein-Barr virus) (20, 29).

Interestingly, we found a correlation between CD127 and CCR7 expression in all patients but one. Patient 2, the oldest patient in this study, maintained a low level of expression of CCR7 on HBV-specific CD8+ T cells at all time points monitored. Interestingly, aging has been shown to be associated with a decrease in CCR7 expression on CD8+ T lymphocytes (8). This finding can be explained either by “memory inflation,” e.g., the accumulation of mature, differentiated CCR7-negative CD8+ memory T cells over the years (19), or by altered kinetics of CCR7 expression on T cells, as has been demonstrated for murine CD4+ T cells (18). However, due to the small sample size in our cohort it is difficult to confirm whether age may play a factor in the expression profile of CCR7.

The overall close association between CD127 and CCR7 expression is of significance, since CCR7 has previously been used to divide antigen-experienced cells into central (CCR7+) and effector (CCR7) memory cell categories (23). Although some studies have questioned this simple distinction (21, 27), our results support the notion that up-regulation of CD127 in combination with CCR7 expression may be a good marker for the development of long-living memory CD8+ T cells in patients who cleared the viral infection (9, 12). They also suggest that CD127 CD8+ T cells have phenotypic and functional features of effector memory cells and that CD127+ CD8+ T cells have phenotypic and functional features of central memory cells. Indeed, the emergence of CD127 expression was accompanied by functional developments in our study because CD127+ HBV-specific CD8+ T cells proliferated vigorously after peptide-specific proliferation. By comparison, primarily CD127 virus-specific CD8+ T cells expanded poorly (Fig. (Fig.4C).4C). These results are in agreement with recent studies indicating a high potential of antigen-driven proliferation of CD127+ memory CD8+ T cells in humans and mice (5, 12).

The mechanisms underlying the impaired proliferative capacity of CD127 HBV-specific CD8+ T cells in early HBV infection are currently unknown. Note, however, that the suppression of T-cell proliferation was temporarily associated with the expression of PD-1. Signaling through the PD-1/PD-L1 pathway has been shown to inhibit CD8+ T-cell proliferation in vitro (11). PBMC from healthy donors typically have no significant PD-1 expression; however, PD-1 can be induced upon activation (4). Thus, it is tempting to speculate that the impaired effector function of activated HBV-specific CD8+ T cells may be due to the suppressive signaling via the upregulated PD-1 receptor. In the HBV transgenic mouse model, PD-1 is also up-regulated on virus-specific CD8+ T cells after activation in the liver within 1 to 2 days after infection and is associated with an impaired effector function (10). However, in this model PD-1 expression was associated with an impaired ability to secrete antiviral cytokines such as IFN-γ. In our study, however, we did not observe a clear correlation between PD-1 expression and IFN-γ production. Clearly, additional studies are needed to address the possible role of PD-1 in mediating T cell dysfunction in humans and mice.

Another interesting finding of our study was the weak ex vivo IFN-γ production of HBV-specific CD8+ T cells during the course of acute-resolving HBV infection. Indeed, in contrast to the regained ability of CD127+ memory CD8+ T cells to proliferate vigorously, the capacity to produce IFN-γ was weak, both at the onset of infection and after viral clearance, although we observed a slight tendency towards an increase in IFN-γ production after resolution of infection. Reduced IFN-γ production capacity has been shown for different antigen-specific CD8+ T cells that had recent antigen encounter (6, 32, 35). In addition, dysfunctional virus-specific CD8+ T cells, termed “stunned,” during acute HCV infection have also been described previously (15). However, in contrast to HBV-specific CD8+ T cells, most HCV-specific CD8+ T cells regain their ability to produce this cytokine during the course of resolving infection, and this regained ability is temporarily closely associated with a rapid decline in viral load (15, 25). The persistent weak IFN-γ production of HBV-specific CD8+ T cells during acute resolving HBV infection in humans is somewhat surprising, since IFN-γ gene expression in the liver correlates with viral clearance in acutely HBV-infected chimpanzees and since IFN-γ has been shown to inhibit HBV replication in the transgenic mouse model and in vitro (7, 33, 34). However, since our study, like most human studies, is limited by the fact that only peripheral blood and not liver-derived lymphocytes can be analyzed and that most patients are first diagnosed at the onset of clinical symptoms, several weeks after infection, we cannot investigate the likely possibility that IFN-γ is produced by intrahepatic HBV-specific CD8+ T cells and/or that IFN-γ production of virus-specific CD8+ T cells is a transient and early event in acute HBV infection, as has been suggested previously (7, 10, 34). In this context, an early loss of the ability of intrahepatic HBV-specific CD8+ T cells to produce IFN-γ in the transgenic mouse model has recently been described (10). Our results also indicate that the kinetics of CD8+ T-cell proliferation and that of IFN-γ production differ during the course of acute HBV infection, which may suggest that these effector functions are regulated differentially. They also suggest that the proliferation of virus-specific T cells is a better marker of HBV recovery than IFN-γ production.

Taken together, our results show that the emergence of CD127 on HBV-specific CD8+ T cells is a good marker for the identification of functionally competent memory CD8+ T cells during acute resolving human viral infection. Furthermore, our findings may have important implications for vaccine design because, for most vaccines, the induction of memory T cells with a high proliferative capacity is an important goal and the evaluation of CD127 expression of virus-specific CD8+ T cells may be particularly useful for identifying functionally competent memory T cells after prophylactic or therapeutic vaccination.

Acknowledgments

We thank Hanspeter Pircher for critically reading the manuscript and Nadine Kersting for excellent technical assistance.

This work was supported by grant DFG TH 719/2-3 (Emmy Noether-Programm) from the Deutsche Forschungsgemeinschaft to Robert Thimme.

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