CD127 - Wiley Online Library

Loss of IL-7 Receptor Alpha-Chain (CD127) Expression in Acute HCV Infection Associated With Viral Persistence Lucy Golden-Mason,1 James R. Burton Jr,1 Nicole Castelblanco,1 Jared Klarquist,1 Salvador Benlloch,2 Chia Wang,3 and Hugo R. Rosen1 Interleukin-7 (IL-7) is required for the establishment and maintenance of memory CD4ⴙ and CD8ⴙ T lymphocytes, and cells lacking IL-7R␣ (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-7R␣ in the outcome of HCV infection, we used multiparameter flow cytometry to characterize patients with acute infection (n ⴝ 24), long-term chronic infection (12) and normal subjects (13). HCV infection per se resulted in downregulation of CD127 on total CD4ⴙ and CD8ⴙ 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 naı¨ve 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 CD4ⴙ and CD8ⴙ 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. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/ 0270-9139/suppmat/index.html). (HEPATOLOGY 2006;44:1098-1109.)

T

he 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 perforinAbbreviations: IL, interleukin; Bcl-2, B cell leukemia/lymphoma 2; CTL, cytotoxic T lymphocyte; CMV, cytomegalovirus; EBV, Epstein-Barr virus; HCV, hepatitis C virus. From the 1Division of Gastroenterology & Hepatology, Hepatitis C Center, and Integrated Program in Immunology, University of Colorado Health Sciences Center & National Jewish Hospital, Denver, CO; 2Hepatogastroenterology Department, Hospital Universitario La Fe, Valencia, Spain; and 3Harborview Medical Center/ University of Washington, Seattle, WA. Received May 12, 2006; accepted August 7, 2006. Research is supported by RO1 DK060590 (to H.R.R.). S.B. is supported in part by contract CM04/0217 (Instituto de Salud Carlos III). Address reprint requests to: Hugo R. Rosen, M.D., Division of Gastroenterology/ Hepatology, University of Colorado Health Sciences Center GI Division, 4200 East Ninth Ave. #B-158, Denver, CO 80262. E-mail: [email protected]; fax: 303-315-5711. Copyright © 2006 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hep.21365 Potential conflict of interest: Nothing to report. 1098

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 ␣-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-7R␣ survive for a long period and differentiate into central memory cells, whereas those lacking CD127 exhibit effector function

HEPATOLOGY, Vol. 44, No. 5, 2006

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 naı¨ve 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.

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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-naı¨ve 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 longterm chronic HCV (5 male and 7 female; mean age 43; all with HCV diagnosis more than 5 years) were used as controls. 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,

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Table 1. Demographic and Clinical Features of Patients With Acute HCV Infection C/R

Patient ID

Genotype

VL Baseline

C C C C C C C C C C C C C R R R R R R R R R R R

HS102 HS111 HS116 HS118 HS122 HS124 PD106 PD108 PD110 PD111 TN101 TN105 TN108 HS101 HS104 HS112 HS114 LA202 PA101 PA103 PD102 PD103 PD104 TN106

1b 1a 2b 1b 1a 1b 1 1a 1a 1a 1a/1b 1a 1a 1a 1b 1a 2 1* 1* 1* 1a 1a 1a 1a

619,756 52,816 1,483,424 ⬍615 893 52,159 1,009,178 5,180 ⬍615 ⬍615 25,107 323,466 651,105 1,073 5,318,925 199,414 ⬍615 ⬍615 ⬍615 ⬍615 ⬍615 ⬍615 ⬍615 5,185,599

VL 6 Month

104551‡ 2,526,131 ⬎7692316 705 2,498,935 137,089 2,537,333 166,863 152,545 1222‡ 1,746,654 6,147 1,445,738 ⬍615 ⬍615 ⬍615† ⬍615† ⬍615 ⬍615 ⬍615 ⬍615 ⬍615 ⬍615 ⬍615

Risk Factor

Age

Gender

ALT Baseline

IDU IDU IDU Unknown Unknown IDU IDU Sexual IDU IDU Sexual Unknown Unknown IDU Needle stick IDU IDU Unknown Surgery IDU Surgery IDU Needle stick IDU

45 20 24 46 40 19 45 46 42 24 38 30 48 41 53 24 17 48 36 25 52 27 27 48

M M M F M F M F M M F M M M F M F M F F F F F F

682 NA 603 21 84 248 (-1.5m) 187 589 1565 54 NA 208 68 222 3,190 197 22 675 (-1.5m) 37 15 19 214 18 14 (4m)

Abbreviations: C, chronic; R, resolved; VL, viral load (IU/mL); ALT, alanine aminotransferase; M, male; F, female. *HCV Serotype; †9 month; ‡12 month.

CD8, CD45RA, CD45RO, CD62L, HLA-DR, CCR5, and CXCR4 were obtained from BD Biosciences. AntiCD127-APC (IL-7 receptor ␣-chain) and anti-CCR7FITC 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 ␮L of 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO). Isotype-matched control antibodies were used to determine background levels of staining. Analysis of Antigen-Specific CD8ⴙ 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, CD8PerCP 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-␥, TNF-␣, 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 ␣-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 CD127ⴙ 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 HCVinfected 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 CD4ⴙ and CD8ⴙ 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, ex-

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pression 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). 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 naı¨ve 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⫹ naı¨ve T cells, 13.87%-77.09%, P ⫽ .0002) and CD8⫹ (11.93%, percentage of CD8⫹ naı¨ve T cells, 1.06%-44.41%, P ⫽ .0015) naı¨ve, 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⫹ naı¨ve; 33.55%, 12.25%-67.22%, % of CD8⫹ naı¨ve; 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⫹ naı¨ve cells (47.36%, 16.67%-70.13%, P ⫽ .0008) than uninfected controls and significantly more CD127⫹CD8⫹ naı¨ve cells (29.44%, 2.47%-

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Fig. 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) Naı¨ve 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 naı¨ve 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 naı¨ve compartment. * P ⬍ .05; ** P ⬍ .005.

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-7R␣ 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 naı¨ve 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 Comparison of CD127ⴙ and CD127ⴚ 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


Fig. 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 CD127⫺CD8⫹/CD4⫹ T cells for all subjects tested for both (B) and (C).

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

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differed significantly between CD8⫹CD127⫹ and CD8⫹CD127⫺ T cell populations (Fig. 3A, Supplemental Table 1). 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/ionomycinstimulated 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-␥ 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-␣ compared to the resolved group and normal controls. In contrast to CD8⫹ T cells, CD4⫹CD127⫹ T cells produce more TNF-␣ 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-tochronic 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-␣ production). Longitudinal Kinetics Analyses of CD127 Expression on HCV-Specific CTLs in Acute HCV. Next, we

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Fig. 3.


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

4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™ Fig. 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-␥ and TNF-␣ 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-␣ 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-␣ 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.

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Table 2. Levels of Tetramer-Positive Cells Patient ID

Time

CMV pp65

HCV 1073

HCV 1406

HCV 1992

HCV 2594

HS101 Acute resolved

M0 M2 M4 M6 M0 M2 M4 M6 M0 M4 M6 M12 M0 M2 M4 M6 M12 M0 M6 M12 M0 M2 M6 M12

0.36* 0.41 0.24 0.34 0.08 0.18 0.03 0.04 0.03 0.06 0.07 0.04 0.03 0.09 0.04 0.04 0.04 neg neg neg 0.25 0.21 0.23 0.22

1.36 0.74 0.49 0.46 0.06 0.06 0.04 0.16 0.05 0.06 0.03 0.03 0.03 0.06 0.07 0.04 0.06 0.06 0.01 0.03 3.15 2.10 3.29 2.42

0.72 0.25 0.15 0.12 neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg

0.10 0.22 0.09 0.06 0.17 0.12 0.11 0.09 0.10 0.05 0.10 0.12 neg neg neg neg neg 0.05 0.05 0.04 0.21 0.08 0.14 0.13

0.31 0.40 0.32 0.19 0.08 0.16 0.07 0.04 0.15 0.05 0.07 0.03 neg neg neg neg neg neg neg neg 0.10 0.04 0.08 0.10

TN106 Acute resolved

HS112 Acute resolved

HS118 Acute chronic

HS122 Acute chronic TN105 Acute chronic

Abbreviation: neg, negative. *Percent of CD8⫹ T cells (levels greater than .02 are considered positive).

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 HCVspecific CTLs fluctuate significantly over time, and there is not a direct contemporaneous correlation with viral level.

Discussion This study of HCV infection examined the expression of IL-7R␣ (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 naı¨ve/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-7R␣-neg-

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Table 3. CD127 Expression on Antigen-Specific Cells Patient

CMV

HCV

HCV

HCV

HCV

Mean†

Virus

ID

Time

pp65

1073

1406

1992

2594

TetⴙCD127ⴙ

IU/mL

HS101 Resolved

M0 M2 M4 M6 M0 M2 M4 M6 M0 M4 M6 M12 M0 M2 M4 M6 M12 M0 M6 M12 M0 M2 M6 M12

7.28* 10.6 7.39 7.56 68 78.12 25 50 66.67 82.35 80.85 56.52 21.74 77.78 25 61.11 46.88 neg neg neg 11.63 6.03 5.71 5.19

5.26 9.05 9.95 8.24 77.78 72.23 50 61.66 71.43 72.73 80 43.75 25 55.56 94.44 64.29 44.23 16.67 55.56 46.67 1.56 1.58 0.67 0.65

6.5 7.69 9.86 3.39 neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg neg

48.75 40.78 44.26 22.92 42.86 31.58 47.34 47.06 23.08 35.29 21.43 48.28 neg neg neg neg neg 19.44 25 45.45 42.42 44.44 37.5 29.03

13.6 17.08 22.54 32.5 45.16 80.95 36.36 25 46.43 63.64 61.29 41.67 neg neg neg neg neg neg neg neg 36.67 28.57 46.67 34.78

18.53 18.65 21.65 16.76 55.27 61.59 44.57 44.57 46.98 57.22 54.24 44.57 25.00 55.56 94.44 64.29 44.23 18.06 40.28 46.06 26.88 24.86 28.28 21.49

1,073 ⬍615 689 ⬍615 5,185,599 60,774 ⬍615 ⬍615 199,414 ⬍615 ⬍615 ⬍615 ⬍615 ⬍615 ⬍615 705 54,852 893 2,498,935 901,266 323,466 4,226 6,147 34,025

TN106 Resolved

HS112 Resolved

HS118 Chronic

HS122 Chronic TN105 Chronic

*Percentage of specific tetramer-positive cells expressing CD127. †Mean of all HCV-specific tetramer-positive cells expressing CD127.

ative cells differentiated from the pool of IL-7R␣⫹ 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 naı¨ve 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 naı¨ve or memory phenotype,24 we found that in patients who subsequently develop chronic infection, the proportion of CD127expressing cells is lower in the different lymphocytes subsets, i.e., naı¨ve 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-7R␣ 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-7R␣negative cells. We recognize that one of the inherent limitations of our study is that the time interval between ™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™3 Fig. 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 HCVspecific 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 tetramerpositive 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.


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Fig. 4.

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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-␥ 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-␥ 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, subclin-


ical 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.

References 1. Kaech SM, Whery EJ, Ahmed R. Effector and memory T-cell differentiation: implications for vaccine development. Nat Rev Immunol 2002;2: 251-262. 2. Williams MA, Bevan MJ. Exhausted T cells perk up. Nature 2006;439: 669-670. 3. Harty JT, Badovinac VP. Influence of effector molecules on the CD8⫹ T cell response to infection. Curr Opin Immunol 2002;14:360-365. 4. Lang KS, Recher M, Navarini AA, Harris NL, Lohning M, Junt T, et al. Inverse correlation between IL-7 receptor expression and CD8 T cell exhaustion during persistent antigen stimulation. Eur J Immunol 2005;35: 738-745. 5. Prlic M, Lefrancois L, Jameson SC. Multiple choices: regulation of memory CD8 T cell generation and homeostasis by interleukin (IL)-7 and IL-15. J Exp Med 2002;195:F49-F52 6. Schluns KS, Kieper WC, Jameson SC, Lefrancois L. Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo. Nat Immunol 2000;1:426-432. 7. Fuller MJ, Hildeman DA, Sabbaj S, Gaddis DE, Tebo AE, Shang L, et al. Cutting edge: emergence of CD127 high functionally competent memory T cells is compromised by high viral loads and inadequate T cell help. J Immunol 2005;174:5926-5930. 8. van Leeuwen EM, de Bree GJ, Remmerswaal EB, Yong SL, Tesselaar K, ten Berge IJ, et al. IL-7 receptor alpha chain expression distinguishes functional subsets of virus-specific human CD8⫹ T cells. Blood 2005;106: 2091-2098. 9. Paiardini M, Cervasi B, Albrecht H, Muthukumar A, Dunham R, Gordon S, et al. Loss of CD127 expression defines an expansion of effector CD8⫹ T cells in HIV-infected individuals. J Immunol 2005;174:2900-2909. 10. MacPherson PA, Fex C, Sanchez-Dardon J, Hawley-Foss N, Angel JB. Interleukin-7 receptor expression on CD8(⫹) T cells is reduced in HIV infection and partially restored with effective antiretroviral therapy. J Acquir Immune Defic Syndr 2001;28:454-457 11. Smyk-Pearson S, Tester I, Lezotte D, Sasaki A, Lewinsohn DM, Rosen HR. Differential antigenic hierarchy associated with spontaneous recovery from HCV: implications for vaccine design. J Infect Dis 2006;194:454463. 12. Shoukry NH, Grakoui A, Houghton M, Chien DY, Ghrayeb J, Reimann KA, et al. Memory CD8⫹ T cells are required for protection from persistent hepatitis C virus infection. J Exp Med 2003;197:1645-1655. 13. Thimme R, Oldach D, Chang KM, Steiger C, Ray SC, Chisari FV. Determinants of viral clearance and persistence during acute hepatitis C virus infection. J Exp Med 2004;194:1395-1406. 14. Lechner F, Wong DKH, Dunbar PR, Chapman R, Chung RT, Dohrenwend P, et al. Analysis of successful immune responses in persons infected with hepatitis C virus. J Exp Med 2000;191:1499-1512. 15. Gru¨ner NH, Gerlach TJ, Jung MC, Diepolder HM, Schirren CA, Schraut WW, et al. Association of hepatitis C virus-specific CD8⫹ T cells with viral clearance in acute hepatitis C. J Infect Dis 2000;181:1528-1536.


16. Tester I, Smyk-Pearson S, Wang P, Wertheimer A, Yao E, Lewinsohn DM, et al. Immune evasion versus recovery after acute hepatitis C virus infection from a shared source. J Exp Med 2005;201:1725-1731. 17. Cox AL, Mosbruger T, Lauer GM, Pardoll D, Thomas DL, Ray SC. Comprehensive analyses of CD8⫹ T cell responses during longitudinal study of acute human hepatitis C. HEPATOLOGY 2005;42:104-112 18. Wedemeyer H, He XS, Nascimbeni M, Davis AR, Greenberg HB, Hoofnagle JH, et al. Impaired effector function of hepatitis C virus-specific CD8⫹ T cells in chronic hepatitis C virus infection. J Immunol 2002;169: 3447-3458. 19. Gruener NH, Lechner F, Jung MC, Diepolder H, Gerlach T, Lauer G, et al. Sustained dysfunction of antiviral CD8⫹ T lymphocytes after infection with hepatitis C virus. J Virol 2001;75:5550-5558. 20. Appay V, Dunbar PR, Callan M, Klenerman P, Gillespie GMA, Papagno L, et al. Memory CD8⫹ T cells vary in differentiation phenotype in different persistent virus infections. Nature Med 2002;8:379-385. 21. Lucas M, Vargas-Cuero AL, Lauer GM, Barnes E, Willberg CB, Semmo N, et al. Pervasive influence of hepatitis C virus on the phenotype of antiviral CD8⫹ T cells. J Immunol 2004;172:1744-1753. 22. Park JH, Yu Q, Erman B, Appelbaum JS, Montoya-Durango D, Grimes HL, et al. Suppression of IL7Ralpha transcription by IL-7 and other prosurvival cytokines: a novel mechanism for maximizing IL-7-dependent T cell survival. Immunity 2004;21:289-302. 23. Lang KS, Recher M, Navarini AA, Harris NL, Lohning M, Junt T, et al. Inverse correlation between IL-7 receptor expression and CD8 T cell exhaustion during persistent antigen stimulation. Eur J Immunol 2005;35: 738-745.

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24. Colle JH, Moreau JL, Fontanet A, Lambotte O, Joussemet M, Delfraissy JF, et al. CD127 expression and regulation are altered in the memory CD8 T cells of HIV-infected patients–reversal by highly active anti-retroviral therapy (HAART). Clin Exp Immunol 2006;143:398-403. 25. Sasson SC, Zaunders JJ, Zanetti G, King EM, Merlin KM, Smith DE, et al. Increased plasma interleukin-7 level correlates with decreased CD127 and Increased CD132 extracellular expression on T cell subsets in patients with HIV-1 infection. J Infect Dis 2006;193:505-514. 26. Zhang M, Drenkow J, Lankford CS, Frucht DM, Rabin RL, Gingeras TR, et al. HIV regulation of the IL-7R: a viral mechanism for enhancing HIV-1 replication in human macrophages in vitro. J Leukoc Biol 2006;79:13281338. 27. Heller T, Rehermann B. Acute hepatitis C: a multifaceted disease. Semin Liver Dis 2005;25:1-17. 28. Semmo N, Day CL, Ward SM, Lucas M, Harcourt G, Loughry A, et al. Preferential loss of IL-2-secreting CD4⫹ T helper cells in chronic HCV infection. HEPATOLOGY 2005;41:1019-1028. 29. Klenerman P, Hill A. T cells and viral persistence: lessons from diverse infections. Nat Immunol 2005;6:873-879. 30. Boettler T, Panther E, Bengsch B, Nazarova N, Spangenberg HC, Blum HE, et al. 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. J Virol 2006;80:3532-3540. 31. Pham TN, MacParland SA, Mulrooney PM, Cooksley H, Naoumov NV, Michalak TI. Hepatitis C virus persistence after spontaneous or treatmentinduced resolution of hepatitis C. J Virol 2004;78:5867-5874.