Loss of IL-7 receptor alpha-chain (CD127) expression in acute HCV infection associated with viral persistence

Authors

  • Lucy Golden-Mason,

    1. Division of Gastroenterology & Hepatology, Hepatitis C Center, and Integrated Program in Immunology, University of Colorado Health Sciences Center & National Jewish Hospital, Denver, CO
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  • James R. Burton Jr,

    1. Division of Gastroenterology & Hepatology, Hepatitis C Center, and Integrated Program in Immunology, University of Colorado Health Sciences Center & National Jewish Hospital, Denver, CO
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  • Nicole Castelblanco,

    1. Division of Gastroenterology & Hepatology, Hepatitis C Center, and Integrated Program in Immunology, University of Colorado Health Sciences Center & National Jewish Hospital, Denver, CO
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  • Jared Klarquist,

    1. Division of Gastroenterology & Hepatology, Hepatitis C Center, and Integrated Program in Immunology, University of Colorado Health Sciences Center & National Jewish Hospital, Denver, CO
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  • Salvador Benlloch,

    1. Hepatogastroenterology Department, Hospital Universitario La Fe, Valencia, Spain
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  • Chia Wang,

    1. Harborview Medical Center/University of Washington, Seattle, WA
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  • Hugo R. Rosen

    Corresponding author
    1. Division of Gastroenterology & Hepatology, Hepatitis C Center, and Integrated Program in Immunology, University of Colorado Health Sciences Center & National Jewish Hospital, Denver, CO
    • Division of Gastroenterology/Hepatology, University of Colorado Health Sciences Center GI Division, 4200 East Ninth Ave. #B-158, Denver, CO 80262
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    • fax: 303-315-5711.


  • Potential conflict of interest: Nothing to report.

Abstract

Interleukin-7 (IL-7) is required for the establishment and maintenance of memory CD4+ and CD8+ T lymphocytes, and cells lacking IL-7Rα (CD127) demonstrate impaired IL-2 secretion and have a short life-span. Chronic HCV is characterized by T cells that are functionally impaired and exhibit an immature phenotype. To investigate the potential role of IL-7/IL-7Rα in the outcome of HCV infection, we used multiparameter flow cytometry to characterize patients with acute infection (n = 24), long-term chronic infection (12) and normal subjects (13). HCV infection per se resulted in downregulation of CD127 on total CD4+ and CD8+ T lymphocytes as compared to normal controls. Total expression was lowest in those patients who subsequently developed persistence and intermediate in those patients with acute-resolving infection. This reduction affected both naïve and effector/memory T cells. CD127 correlated phenotypically with upregulation of chemokine receptors CCR7 and CXCR4, expression of the anti-apoptotic molecule B cell leukemia/lymphoma 2 (Bcl-2), and enhanced IL-2 production. In six HLA A2-positive patients, we longitudinally tracked tetramer responses to HCV and CMV epitopes; at baseline, reflecting the expression of CD127 on whole T cell populations, viral-specific CTLs in patients who became chronic demonstrated lower CD127. In conclusion, CD127 is a useful marker of functional CD4+ and CD8+ T cells and its expression correlates with virologic outcome of acute HCV. These data provide a mechanistic basis for the observation that CTLs generated in early infection rapidly decline as chronicity is established; CD127 expression should be considered in the design of novel immunotherapeutic approaches. (HEPATOLOGY 2006;44:1098–1109.)

The host immune response following pathogenic challenge is associated with a robust expansion of antigen-specific T cells that differentiate into effector populations, migrate from secondary lymphoid organs, and contribute to pathogen elimination by perforin-dependent cytolysis and secretion of cytokines.1, 2, 3 This phase is usually followed by rapid contraction of T cell responses due to apoptosis; the third phase involves the generation and maintenance of a stable memory population that mediates protection from subsequent challenge.1 Memory cell qualities include the ability to expand during recall responses, to produce IL-2 when re-stimulated, to express enhanced levels of the anti-apoptotic molecule B cell leukemia/lymphoma 2 (Bcl-2)4 and lymph node-homing molecules CCR7 and L-selectin (CD62L), and to survive because of cytokine-dependent homeostatic proliferation.1

Evidence derived from a number of models indicates that the expression of the IL-7R α-chain (CD127) is a marker of activated effector CD8 T cells that are more likely to survive and differentiate into protective memory T cells.5 Following infection with lymphocytic choriomeningitis virus (LCMV), cytotoxic T lymphocytes (CTLs) expressing high levels of IL-7Rα survive for a long period and differentiate into central memory cells, whereas those lacking CD127 exhibit effector function but have a short half-life.6 In LCMV, the emergence of CD127high T cells is mediated by both the presence of viral-specific CD4 cell help and containment of virus.7 In humans, recent data demonstrate that CD127 is a marker of early viral-specific CTLs destined to become memory CTLs8 and that its expression is lower on CD8+ T cells specific for persistent viruses [e.g., cytomegalovirus (CMV) and Epstein-Barr virus (EBV)] than for viruses cleared from the host (e.g., influenza and respiratory syncytial virus). Moreover, HIV infection is characterized by a robust expansion of CD127-negative CD8+ T cells that correlates with plasma viremia and CD4+ T cell depletion.9, 10

Infection with hepatitis C virus (HCV) is associated with viral persistence in the majority of patients and spontaneous resolution in only a minority; although precise mechanisms governing outcome remain incompletely defined, abundant data demonstrate that the vigor and breadth of the cellular immune response in the earliest stages of infection is critical.11–16 Acute HCV infection is frequently associated with a strong virus-specific CTL response that declines exponentially over time, particularly in those who develop chronic infection.17 In accord with the model that repeated or persistent antigenic stimulation does not allow effector CTLs to mature to a population of long-lived memory cells, chronic HCV is characterized by CTLs that are functionally impaired (decreased antiviral cytokine production, cytotoxicity, and proliferative capacity)18, 19 or anergic, and may exhibit phenotypic features of early stages of differentiation.20, 21 Moreover, the effect of HCV on T cell phenotype extends beyond the T cells that target this virus; in this regard, HCV-infected individuals have CMV-specific CTLs cells that display a more immature phenotype (reduced expression of Fas and perforin)21 as compared to healthy controls.

In this prospective study of patients with acute HCV infection, we demonstrated that the loss of CD127 expression on total naïve and memory/effector populations and on viral-specific CTLs is a prelude to viral persistence. Patients with acute-resolving HCV infection express levels of CD127 on CD4+ and CD8+ T cells intermediate to normals and patients with acute-to-chronic infection. The differences in CD127 expression in the 2 groups of acutely infected patients were apparent even before virologic outcome had been determined, indicating that early downregulation of CD127 is a marker of viral persistence. CD127 expression was also associated with IL-2 production and increased levels of the anti-apoptotic molecule Bcl-2. These results provide insight into the mechanisms governing development of protective immunity versus persistence in HCV infection.

Abbreviations

IL, interleukin; Bcl-2, B cell leukemia/lymphoma 2; CTL, cytotoxic T lymphocyte; CMV, cytomegalovirus; EBV, Epstein-Barr virus; HCV, hepatitis C virus.

Patients and Methods

Study Population.

The study group comprised acutely HCV-infected patients recruited from multiple sites, and the study protocol was approved by all appropriate institutional review boards. Acute HCV was diagnosed based on HCV Ab seroconversion in a subject with previously negative HCV testing, seroconversion in a subject with new-onset risk factors and alanine aminotransferase level greater than 10-fold normal, or HCV RNA positivity with HCV Ab negativity. Twenty four treatment-naïve patients (12 male and 12 female; mean age 36 years) with acute HCV infection were recruited for this study. The majority of patients were Caucasian (91%). Spontaneous viral resolution (n = 11) and chronicity (n = 13) were defined as the absence or presence of HCV RNA at 6 months after enrollment with at least 2 viral determinations. Demographic and clinical details of the acute HCV study population are described in Table 1. Thirteen healthy, non-HCV-infected subjects (5 male and 8 female; mean age 33.5) and 12 patients with long-term chronic HCV (5 male and 7 female; mean age 43; all with HCV diagnosis more than 5 years) were used as controls.

Table 1. Demographic and Clinical Features of Patients With Acute HCV Infection
C/RPatient IDGenotypeVL BaselineVL 6 MonthRisk FactorAgeGenderALT Baseline
  • Abbreviations: C, chronic; R, resolved; VL, viral load (IU/mL); ALT, alanine aminotransferase; M, male; F, female.

  • *

    HCV Serotype;

  • 9 month;

  • 12 month.

CHS1021b619,756104551IDU45M682
CHS1111a52,8162,526,131IDU20MNA
CHS1162b1,483,424>7692316IDU24M603
CHS1181b<615705Unknown46F21
CHS1221a8932,498,935Unknown40M84
CHS1241b52,159137,089IDU19F248 (-1.5m)
CPD10611,009,1782,537,333IDU45M187
CPD1081a5,180166,863Sexual46F589
CPD1101a<615152,545IDU42M1565
CPD1111a<6151222IDU24M54
CTN1011a/1b25,1071,746,654Sexual38FNA
CTN1051a323,4666,147Unknown30M208
CTN1081a651,1051,445,738Unknown48M68
RHS1011a1,073<615IDU41M222
RHS1041b5,318,925<615Needle stick53F3,190
RHS1121a199,414<615IDU24M197
RHS1142<615<615IDU17F22
RLA2021*<615<615Unknown48M675 (-1.5m)
RPA1011*<615<615Surgery36F37
RPA1031*<615<615IDU25F15
RPD1021a<615<615Surgery52F19
RPD1031a<615<615IDU27F214
RPD1041a<615<615Needle stick27F18
RTN1061a5,185,599<615IDU48F14 (4m)

Sample Preparation and Storage.

Peripheral blood and plasma were drawn at baseline, 2, 6, and 12 months later. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (Amersham Biosciences, Piscataway, NJ) density gradient centrifugation and cryopreserved for subsequent analyses. For patients HS116, PD106, PD110, TN101, and TN108, the first PBMCs available for study were from the 2-month time point so the difference between early and later time points was 4 months (6 months for all other patients). Plasma preparation tubes (PPT tubes, BD Biosciences, San Jose, CA) were used to isolate plasma from whole blood, which was frozen and later thawed for IL-7 measurement, viral load, and genotype testing. HCV genotyping (LiPA) and viral level determination (HCV RNA 3.0 bDNA, lower limit 615 copies/mL) were performed by Bayer Reference Testing Laboratory (Berkeley, CA). Plasma IL-7 level was measured using the Quantikine HS high sensitivity ELISA (R&D Systems, Minneapolis, MN). Samples were assayed in duplicate following the manufacturer's instructions.

Flow Cytometric Analysis of Cell Surface Antigens.

Four-color multiparameter flow cytometry was performed using a BD FACSCalibur instrument (BD Biosciences) compensated with single fluorochromes and analyzed using CellQuest Pro 4.0.2 software (BD Biosciences) Fluorochrome-labeled (FITC/PE/PerCP) monoclonal antibodies (MAbs) specific for CD3, CD4, CD8, CD45RA, CD45RO, CD62L, HLA-DR, CCR5, and CXCR4 were obtained from BD Biosciences. Anti-CD127-APC (IL-7 receptor α-chain) and anti-CCR7-FITC MAbs were supplied by R&D Systems (Minneapolis, MN). Cryopreserved PBMCs (2.5 × 105) were stained for cell surface antigen expression at 4°C in the dark for 30 minutes. Then, PBMCs were washed twice in 2 mL phosphate-buffered saline (PBS) containing 1% bovine serum albumin and 0.01% sodium azide (Facs Wash) and subsequently fixed in 200 μL of 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO). Isotype-matched control antibodies were used to determine background levels of staining.

Analysis of Antigen-Specific CD8+ T Cell Responses.

Six of the patients in the study cohort who were A2 positive were assessed over time for antigen-specific responses to HCV by tetramer staining. PE-labeled tetramers for HCV NS3 1073-1081 (CINGVWCTV), NS3 1406-1415 (KLVALGINAV), NS4B 1992-2000 (VLSDFKTWL), and NS5 2594-2602 (ALYDVVTKL) were supplied by Beckman Coulter (Fullerton, CA). Cells were stained in conjunction with CD3-FITC, CD8-PerCP and CD127-APC following the manufacturer's instructions. The CMVpp65 (NLVPMVATV)-PE tetramer (Beckman Coulter) was used as a non-HCV control. For flow cytometric analysis of CD127 expression on tetramer-positive cells, 1 × 105 CD8+ events were acquired for each tetramer stain.

Intracellular Staining of Bcl-2.

Intracellular staining for Bcl-2 was performed following membrane permeabilization of surface-antigen-stained fixed cells with 0.2% saponin (Sigma-Aldrich). The anti-human Bcl-2 antibody from BD Biosciences was used for analysis of Bcl-2 levels according to the manufacturer's instructions. The level of expression of Bcl-2 in CD127+ T cell subsets was taken as 100%. Shifts (% change) in the median fluorescence intensity, which correlates directly with the number of molecules expressed on a per-cell basis, was used to compare expression of Bcl-2 between T cell subpopulations. Isotype-matched control antibodies confirmed that all T cells were positive at a low level.

Intracellular Cytokine Analysis.

Antibodies for measurement of intracellular IFN-γ, TNF-α, and IL-2 were supplied by BD Pharmingen (see Supplemental Materials, available at the HEPATOLOGY website: http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

Statistical Analyses.

Results are expressed as mean (range). Student's t test was used to compare differences between patient groups and Spearman rank for correlation analysis; a P value of less than 0.05 was taken as significant. The JMP 6.0 (SAS Institute, Inc, Cary NC) statistical package was used.

Results

To determine whether the outcome of acute HCV infection is associated with differential expression of CD127 (IL-7 receptor α-chain), multiparameter flow cytometric analysis was used to characterize lymphocytes derived from a cohort of acutely infected HCV patients (n = 24) compared to healthy uninfected controls (n = 13) and patients with long-term chronic HCV infection (n = 12). The clinical and demographic features of the study patients with acute HCV are shown in Table 1. Thirteen of the patients developed chronic infection, defined by serum HCV RNA positivity on at least 2 occasions beyond 120 days of enrollment, and 11 spontaneously resolved their infection. Of note, a number of patients (TN106, HS104, HS112) who spontaneously cleared HCV at a later time had detectable viremia when the flow cytometric analyses were performed; conversely, some patients had undetectable viremia (e.g., PD111) at the time analyses were performed but later became persistently infected.

Overall Levels of CD127+ Lymphocytes at Baseline.

In acutely HCV-infected patients, there is a lower level of CD127-expressing lymphocytes (mean 44.06%, % of total lymphocytes, range 23.12%-62.68%, n = 24) compared to normal controls (51.74%, range 42.7%-67.09%, n = 13, P = .046). Of interest, in long-term chronic HCV infection, levels of CD127-expressing lymphocytes are further reduced (36.46%, 19.6%-60.35%, n = 12), this reduction being significant compared to both normal controls (P = .0018) and the acute HCV-infected group who subsequently cleared the virus (P = .027), but not compared to the chronically evolving group. In the acutely infected cohort, the decrease in CD127 expression is most pronounced in patients who subsequently develop chronic infection (40.12%, 23.12%-55.79%, n = 13, P = .0032 compared to controls); patients who spontaneously resolve their infection have intermediate levels (48.72%, 25.45%-62.68%) of CD127+ lymphocytes, not significantly different from either controls or patients with chronically evolving infection. This suggests that responsiveness to IL-7 may influence the outcome of acute HCV infection.

CD127 Expression on CD4+ and CD8+ Subsets of T Cells.

As both CD4+ helper T cells and CD8+ CTLs have been shown to be involved in HCV clearance, we determined if decreased CD127 expression was characteristic for defined T cell subsets. At the earliest time point for which PBMCs were available, in the acute patient group who subsequently develop chronic infection, expression of CD127 was decreased in both the CD4+ T cell subset (77.43%, % of CD4+ T cells, 62.56%-88.33%, P = .0010) compared to the control HCV-uninfected group (88.88%, 77.9%-94.63%) but not to the spontaneously resolved patient group (82.0%, 55.93%-91.37%). Total expression was lower on CD8+ T cells in all groups and a similar pattern of downregulated expression in the chronic group (32.38%, 16.03%-65.01%) was observed as compared to controls (45.38%, 26.16%-65.13%, P = .018), whereas the resolved group was not significantly different from controls (35.99%, 12.97%-70.92%). This reduction is maintained over time as evidenced by the significantly lower levels of both CD127+CD4+ T cells (mean 69.44%) and CD127+CD8+ T cells (mean 15.39%) in patients with long-term HCV infection (Fig. 1A-B).

Figure 1.

Decreased CD127 expression on T cell subsets in acute HCV infection. (A) Baseline CD127 expression is relatively reduced on both CD4+ and CD8+ subsets of T cells in acutely infected patients who subsequently fail to clear the virus, as well as in patients with long-term chronic HCV infection (mean + SEM) (see also supplementary Figure 1). (B) Representative flow cytometry plots are from 1 control, and 2 acute HCV-infected patients who subsequently became chronic or who spontaneously resolved infection and 1 patient with long-term chronic infection. Percentages represent the percent of CD4+ or CD8+ cells expressing CD127. (C) There was a strong positive correlation (P < .0001, Spearman) between the levels of CD127 expression on CD8+ and CD4+ T cells, suggesting that CD127 expression is not preferentially lost in the CTL or helper T cell subset. (D) Naïve T cells were defined by the co-expression of CD45RA and CD62L (upper right quadrant, inset), the remaining 3 quadrants represent antigen-experienced effector/memory T cells. CD127+CD4+ and CD127+CD8+ T cells are decreased in both the naïve and effector/memory compartments in chronically evolving infection compared to HCV-uninfected controls. In patients who subsequently resolved HCV infection, significant differences in CD127+ T cells were observed only in the naïve compartment. * P < .05; ** P < .005.

There was a strong positive correlation (R = .635, P < .0001, Spearman's coefficient) between the levels of CD127 expression on CD4+ and CD8+ T cells (Fig. 1C), indicating that CD127 expression is not preferentially lost in CTL or helper T cell subsets. Because it was suggested that loss of CD127 accompanies transition to an effector/memory phenotype,4–6 we next asked if loss of CD127 expression was confined to the antigen-experienced T cell compartment. By convention, T cells (CD3+) were further defined as naïve if they coexpressed CD45RA and CD62L; T cells expressing CD62L in the absence of CD45RA, negative for both CD45RA and CD62L, or expressing CD45RA alone were defined as antigen-experienced effector/memory cells (Fig. 1D, inset). CD127+ T cells were decreased in both the CD4+ (39.79%, % of CD4+ naïve T cells, 13.87%-77.09%, P = .0002) and CD8+ (11.93%, percentage of CD8+ naïve T cells, 1.06%-44.41%, P = .0015) naïve, as well as in the CD4+ (36.14%, 11.32%-69.02%, P < .0001) and CD8+ (15.6%, 1.95%-49.24%, P = .0062) effector/memory T cell compartments in chronically evolving infection compared to HCV-uninfected controls (66.69%, 44.86%-87.78%, percentage of CD4+ naïve; 33.55%, 12.25%-67.22%, % of CD8+ naïve; 65.65%, 54.17%-82.56%, percentage of CD4+ effector/memory; 34.28%, 9.92%-66.26%, percentage of CD8+ effector/memory). Again, the resolved group had intermediate expression. In the effector/memory compartment, although levels were reduced in both the CD4+ (47.87%, 18.49%-65.75%) and CD8+ (27.81%, 5.07%-55.12%) T cell subsets, they were not statistically different from the chronic or control uninfected groups. Patients who subsequently resolved HCV infection demonstrated significantly fewer CD127+CD4+ naïve cells (47.36%, 16.67%-70.13%, P = .0008) than uninfected controls and significantly more CD127+CD8+ naïve cells (29.44%, 2.47%-49.75%, P = .01) than the acute HCV patient group who subsequently failed to clear the virus (Fig. 1D).

Plasma IL-7 Levels and IL-7Rα Expression.

IL-7 has been shown to downregulate CD127 expression on CD4 and CD8 T cells in vivo and in vitro.22 In order to determine whether the noted differences in CD127 expression levels were related to serum levels of IL-7, we used an ELISA assay to measure circulating IL-7 in 35 subjects. Levels of IL-7 were comparable in normals, acutes with evolving chronicity, acutes-resolving, and those with long-term chronic infection (Fig. 2A). Furthermore, we found that plasma IL-7 did not correlate with the frequency of CD4+ or CD8+ T cells that were CD127+/− (Fig. 2B-C), nor with the naïve and memory subsets that were CD127+/− (data not shown). Thus, in accord with recent HIV data, CD127 expression was altered in virally infected subjects but there was no correlation with plasma IL-7 levels.9

Figure 2.

(A) Plasma IL-7 levels do not differ significantly between study groups (10 normal controls, 10 acute chronic patients, 10 acute resolved patients, and 5 long-term chronic patients); open circles represent individual patients and horizontal bars represent the mean value. (B and C) The level of IL-7 detected in plasma (n = 35) does not correlate with levels of CD127CD8+/CD4+ T cells for all subjects tested for both (B) and (C).

Comparison of CD127+ and CD127 T Cell Subsets: Immunophenotypic and Functional Features

Phenotype:

The previously described properties of CD8+ T cells expressing CD127 in other infectious models7, 9 have implications for successful immunity to acute HCV infection. We therefore examined the phenotype and function of both CD8+ and CD4+ T cell subsets defined by the presence or absence of CD127 and evaluated if these populations were changed in acute HCV infection. As expected, expression of the activation antigen HLA-DR and chemokine receptors CCR7 and CXCR4 differed significantly between CD8+CD127+ and CD8+CD127 T cell populations (Fig. 3A, Supplemental Table 1).

Figure 3.

Comparison of CD127-positive and CD127-negative subsets of CD8+/CD4+ T cells. (A) At baseline, CD8+CD127+ T cells express significantly less HLADR and significantly higher levels of chemokine receptors CCR7 and CXCR4. A similar phenotype was seen for CD4+ cells; however, overall CD4+ T cells express more CCR7 than their CD8+ counterparts regardless of the presence (P = .0001) or absence (P = .0004) of CD127. The CD127-negative subset of CD8+ T cells expressed more HLADR in chronically evolving (P = .0241) and resolving (P = .002) acute HCV infection compared to normal controls. There are no significant differences in phenotype across the 3 groups with respect to expression of CCR7 and CXCR4. (B) Functional analysis of cytokine production confirmed that CD8+CD127+ cells express significantly higher levels of IL-2 but similar levels of IFN-γ and TNF-α than their CD127 counterparts. This was not perturbed by acute HCV infection, although CD127-negative CD8+ T cells from patients who subsequently developed a chronic course of infection produced more TNF-α than both the resolving (P = .0363) and normal control (P = .0123) groups. Analysis of CD4+CD127+ T cells showed that this subset also produces more IL-2. However, in contrast to CD8+CD127+ T cells CD4+CD127+ T cells produce more TNF-α than the CD127 subset in the acute HCV infection setting. (C) Flow cytometric analysis of BCL-2 expression showed that the downregulation observed in CD8+CD127 T cells, as assessed by percent decrease in median fluorescent intensty (MFI), was also evident for CD4+ T cells in all study groups tested. *CD127+ population significantly different from CD127 population; †significantly different from normal control group.

The phenotype of these CD8+ subsets was similar in all groups; however, higher HLA DR expression was observed in the CD8CD127 cells from the acutely infected cohort compared to normal controls irrespective of subsequent outcome. With respect to phenotype, a similar pattern was observed for CD4+ cells (supplementary Table 1); however, CD4+ T cells express more CCR7 than their CD8+ counterparts regardless of the presence (P = .0001) or absence (P = .0004) of CD127.

Function:

Cytokine production by PMA/ionomycin-stimulated cells was used to examine the function of CD127+/− CTLs and helper T cells. As reported for normal and HIV-infected subjects,9 CD8+ CD127+ T cells produce more IL-2 than do their CD127 counterparts (Fig. 3B, first panel). We found this to be true also for the CD4+ T cells in acute HCV infection irrespective of outcome. IFN-γ production was not significantly different in CD127+/− subsets of CTLs or helper T cells (Fig. 3B, second panel). In chronically evolving acute HCV infection, CD8+CD127 T cells produce more TNF-α compared to the resolved group and normal controls. In contrast to CD8+ T cells, CD4+CD127+ T cells produce more TNF-α in both chronic and resolved acute HCV infection (Fig. 3B, third panel).

Chronicity has been associated with a decline in number and proliferative capacity of T cells which may in part be due to the levels of CD127. IL-7 is a well described T cell survival factor that activates STAT5 and upregulates the anti-apoptotic molecule Bcl-2.23 Previous studies4, 9 show that CD8+CD127+ cells express a higher level of Bcl-2. In the current study, intracellular FACS staining of Bcl-2 confirmed this was the case in our normal control group and demonstrated that this was also true for the acutely infected HCV patient cohort, as well as for CD4+ T cells. For CD8+ T cells, there was a 18.87% reduction (as measured by percent decrease in median fluorescent intensity) in the control group, 10.68% in the acute-to-chronic group, and 19.85% in the resolved group. For CD4+ T cells, similar reduction was found (controls 24.19%, acute-to-chronic 11.99%, resolved 13.81%) (Fig. 3C). Taken together, these results suggest that while higher levels of CD127 correlate with spontaneously resolved acute HCV infection, the CD8+CD127+/− subsets themselves are not dysregulated. We also show that CD4+ CD127+/− T cell subsets are in many ways phenotypically and functionally similar to CD8+ T cells (with the exception of TNF-α production).

Longitudinal Kinetics Analyses of CD127 Expression on HCV-Specific CTLs in Acute HCV.

Next, we prospectively studied the proportional expression of CD127 in six HLA A2-positive patients (3 who became chronically infected and 3 who resolved spontaneously without antiviral therapy). We used 4 HCV-specific tetramers loaded with peptides previously noted to stain CTLs in a large proportion of HCV-exposed individuals,18 as well as an immunodominant CMV peptide (Table 2). Our results indicate that the expression of CD127 on viral-specific CTLs reflects the expression on bulk T cell subsets described above, i.e., relatively greater CD127 expression at the earliest time point in those who subsequently resolved. At baseline, the mean CD127 expression for the 4 HCV tetramers was 40.26% in those with acute resolving infection versus 23.31% in those with acute, chronically evolving infection (Table 3). The difference was most remarkable for the NS3 1073 tetramer, where 51.49% and 14.41% in the resolved and chronic groups, respectively, demonstrated CD127 expression at baseline (Fig. 4). However, over time, the differences between the acute resolved and acute chronic groups became less apparent. Indeed, as shown in Table 3, the relative percentage of NS3 1073-reactive CTLs that expressed CD127 increased in 2 patients with chronic infection; for example, in patient HS118 the expression of CD127 of CTLs peaked at 94% at month 4 after enrollment. Moreover, the 3 patients with acute resolving infection (HS101, TN106, HS112) did not consistently demonstrate an increase in CD127 expression as serum HCV RNA was becoming negative. These data indicate that although CD127 is a useful marker of functional CD4+ and CD8+ T cells that predicts virologic outcome after acute infection, the kinetics of its expression on HCV-specific CTLs fluctuate significantly over time, and there is not a direct contemporaneous correlation with viral level.

Table 2. Levels of Tetramer-Positive Cells
Patient IDTimeCMV pp65HCV 1073HCV 1406HCV 1992HCV 2594
  • Abbreviation: neg, negative.

  • *

    Percent of CD8+ T cells (levels greater than .02 are considered positive).

HS101M00.36*1.360.720.100.31
Acute resolvedM20.410.740.250.220.40
 M40.240.490.150.090.32
 M60.340.460.120.060.19
TN106M00.080.06neg0.170.08
Acute resolvedM20.180.06neg0.120.16
 M40.030.04neg0.110.07
 M60.040.16neg0.090.04
HS112M00.030.05neg0.100.15
Acute resolvedM40.060.06neg0.050.05
 M60.070.03neg0.100.07
 M120.040.03neg0.120.03
HS118M00.030.03negnegneg
Acute chronicM20.090.06negnegneg
 M40.040.07negnegneg
 M60.040.04negnegneg
 M120.040.06negnegneg
HS122M0neg0.06neg0.05neg
Acute chronicM6neg0.01neg0.05neg
 M12neg0.03neg0.04neg
TN105M00.253.15neg0.210.10
Acute chronicM20.212.10neg0.080.04
 M60.233.29neg0.140.08
 M120.222.42neg0.130.10
Table 3. CD127 Expression on Antigen-Specific Cells
PatientTimeCMVHCVHCVHCVHCVMeanVirus
IDpp651073140619922594Tet+CD127+IU/mL
  • *

    Percentage of specific tetramer-positive cells expressing CD127.

  • Mean of all HCV-specific tetramer-positive cells expressing CD127.

HS101M07.28*5.266.548.7513.618.531,073
ResolvedM210.69.057.6940.7817.0818.65<615
 M47.399.959.8644.2622.5421.65689
 M67.568.243.3922.9232.516.76<615
TN106M06877.78neg42.8645.1655.275,185,599
ResolvedM278.1272.23neg31.5880.9561.5960,774
 M42550neg47.3436.3644.57<615
 M65061.66neg47.062544.57<615
HS112M066.6771.43neg23.0846.4346.98199,414
ResolvedM482.3572.73neg35.2963.6457.22<615
 M680.8580neg21.4361.2954.24<615
 M1256.5243.75neg48.2841.6744.57<615
HS118M021.7425negnegneg25.00<615
ChronicM277.7855.56negnegneg55.56<615
 M42594.44negnegneg94.44<615
 M661.1164.29negnegneg64.29705
 M1246.8844.23negnegneg44.2354,852
HS122M0neg16.67neg19.44neg18.06893
ChronicM6neg55.56neg25neg40.282,498,935
 M12neg46.67neg45.45neg46.06901,266
TN105M011.631.56neg42.4236.6726.88323,466
ChronicM26.031.58neg44.4428.5724.864,226
 M65.710.67neg37.546.6728.286,147
 M125.190.65neg29.0334.7821.4934,025
Figure 4.

CD127 Expression on antigen-specific CD8+ T cells. Six of the acutely HCV-infected patient cohort who were A2+ are shown. For this group, CD127 expression on tetramer-positive cells was followed over time. All patients demonstrated reactivity to at least 1 of the 4 HCV-specific tetramers tested (range 1-4) and all patients tested had detectable levels of HCV-1073 tetramer positive cells. Five of the 6 patients also tested positive for CMV-specific cells. (A) Mean expression of CD127 on HCV-1073 tetramer-positive cells is shown for a given group of patients at baseline (M0) and at 2 and 6 months later. Mean viral load at each of the time points is shown on the secondary y axis. In the resolved group, the expression of CD127 does not change over time despite a dramatic decrease in viral load. Conversely, an increase is seen in the chronic group at 2 months accompanied by a dramatic increase in viral load. (B) The expression of CD127 on CMV-specific cells is shown in 5 of the same patients in whom responses were detected. (C) Flow cytometry plots are shown for CMV-pp65 and HCV-1073 tetramer-positive cells for 1 resolved and 1 chronic patient over time. (D) CD127 expression is shown on all HCV-tetramer-positive cells for the same patients at baseline.

Discussion

This study of HCV infection examined the expression of IL-7Rα (CD127), considered to serve as a “signature” for cellular fitness because it demarcates effector cells that survive in the long term and retain functional responsiveness.1 Given the fact that IL-7 supports the emergence and survival of memory CD4+ and CD8+ T lymphocytes and that HCV is characterized by impaired proliferation, IL-2 secretion, and cytolytic function of viral-specific T cells,18, 19, 21 we hypothesized that differential CD127 expression in the early stages of infection would be associated with persistence versus recovery. Accordingly, we found that HCV infection per se downregulated expression on both CD4+ and CD8+ T cells irrespective of naïve/memory phenotype and that downregulation of CD127 expression preceded the loss of viral control in patients with acute infection. CD127 expression inversely correlated with the activation molecule HLA DR. Thus, it is possible that upon antigenic stimulation, IL-7Rα-negative cells differentiated from the pool of IL-7Rα+ cells, as supported by the finding that HLA DR expression on CD127-negative CTLs was greater in patients with acute HCV as compared to normals (Fig. 3A). Reduction in CD127 expression was most pronounced among those who ultimately developed chronic infection, underscoring a potential role for IL-7 responsiveness in determining the outcome of acute HCV infection.

CD127 expression has previously been shown to decrease as cells move from the naïve to the terminal differentiated stage, and this correlates with loss of CCR7 (Fig. 2A). Although prior studies suggest this is a major feature of CD8 lymphocytes and that CD4 lymphocytes express CD127 independently of their naïve or memory phenotype,24 we found that in patients who subsequently develop chronic infection, the proportion of CD127-expressing cells is lower in the different lymphocytes subsets, i.e., naïve and effector/memory CD4+ and CD8+ T cells. These findings suggest that HCV infection has a global effect on the expression of this cytokine receptor. The fact that patients with different outcomes of HCV have plasma IL-7 levels comparable to normals but altered receptor expression suggests that decreases in CD127 expression may precede increases of circulating IL-7,25 re-expression of CD127 might be impaired unless IL-7 is cleared,25 or alternatively, that the expansion of CD127-negative cells is the consequence of IL-7-independent mechanisms.9 Although the precise mechanism remains undefined, it is conceivable that specific HCV proteins modulate the expression of IL-7Rα on cells, as shown recently for HIV.26 In this regard, it might be hypothesized that the high HCV load during the earliest phase of infection might induce the expansion of IL-7Rα-negative cells. We recognize that one of the inherent limitations of our study is that the time interval between HCV acquisition and recruitment into our study cannot be controlled, and hence the earliest viral level is typically not known.

We found that CD127 expression in CD4+ and CD8+ T lymphocytes following a brief stimulation with PMA/ionomycin correlated with the ability to produce IL-2 (Fig. 3B), in accord with previous reports.9 Therefore, the downregulation of CD127 expression may contribute to the relative IL-2 deficiency in the acute phase of HCV infection27 that has been implicated in the primary failure of CD4+ T cell responses28 and weak CTL responses. We found no difference in IFN-γ production according to CD127 expression. These data are congruent with the model that T cells lose the ability to exert effector function in a hierarchal manner, with IL-2 lost first, followed by loss of IFN-γ secretion.29

IL-7 signaling plays a critical function in T cell survival by increasing the expression of anti-apoptotic molecules and through activation of multiple signal transduction pathways.1 We found that CD127-negative CD4+ and CD8+ T cells expressed significantly less Bcl-2 than their CD127-positive counterparts (Fig. 3C). These results provide a mechanistic basis for the observation that acute HCV patients who progress to chronicity demonstrate significantly greater contraction and loss of antigen-specific immunity over time compared to those with spontaneous resolution.17 Because IL-7 appears to be particularly important for the survival of antigen-experienced CTLs in other models,4, 7 we characterized the expression of CD127 on tetramer-positive cells at multiple time points. The majority of viral-specific T cells were negative for CD127 at baseline, but levels of CD127 expression at baseline were lower in those patients who subsequently developed chronicity. However, somewhat surprisingly, the loss of circulating viremia was not associated with the temporal emergence of CD127-expressing HCV-specific CTLs. For example, in TN106 the significant drop in viral level was not associated with a simultaneous increase in the expression of CD127 in any of the viral-specific CTLs. These results are in contradistinction to a recent analysis of 6 patients with acute HBV infection.30 In our study, the lack of correlation between CD127 expression and HCV level suggests that not all aspects of immune function are restored in parallel following spontaneous resolution of HCV infection. Alternatively, although the initially high viral burden was brought under control (as reflected by circulating serum levels), there could be recurrent stimulation from an undefined reservoir of virus as suggested by the recent demonstration that low-level HCV replication persists in PBMC of patients with therapy-induced or spontaneous resolution of HCV infection.31 This continued, subclinical antigenic stimulation might maintain the pool of IL-7R negative cells. Moreover, in a separate analysis of chronically infected patients who received antiviral therapy, CD127 was not restored in those with virologic clearance (data not shown), suggesting that HCV might irreversibly impair IL-7R signaling.

In summary, we report a previously unrecognized decline in CD127 expression early after acute HCV infection that predates viral persistence. CD127 expression might be an important clinical marker of memory cell precursors and development of protective immunity, and thus, should be considered in the design and assessment of efficacy of novel immunotherapeutic approaches.

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