of July 12, 2018.This information is current as

with Hepatitis C Virus InfectionHepatitis B Vaccine Failure in Individuals

Pathways: Role inkip1 and p27ink4ap16 T Cell Responses via+KLRG1 Impairs CD4

Guang Y. Li, Jonathan P. Moorman and Zhi Q. YaoYing, Xiao Y. Wu, Shu M. Lin, Jeddidiah W. D. Griffin, Lei Shi, Jia M. Wang, Jun P. Ren, Yong Q. Cheng, Ruo S.

http://www.jimmunol.org/content/192/2/649doi: 10.4049/jimmunol.1302069December 2013;

2014; 192:649-657; Prepublished online 13J Immunol 

Referenceshttp://www.jimmunol.org/content/192/2/649.full#ref-list-1

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The Journal of Immunology

KLRG1 Impairs CD4+ T Cell Responses via p16ink4a andp27kip1 Pathways: Role in Hepatitis B Vaccine Failure inIndividuals with Hepatitis C Virus Infection

Lei Shi,*,†,1 Jia M. Wang,*,‡,1 Jun P. Ren,* Yong Q. Cheng,*,x Ruo S. Ying,*,{

Xiao Y. Wu,* Shu M. Lin,† Jeddidiah W. D. Griffin,* Guang Y. Li,*

Jonathan P. Moorman,*,‖ and Zhi Q. Yao*,‖

Coinfection of hepatitis B virus (HBV) with hepatitis C virus (HCV) is quite common, leading to an increase in morbidity and

mortality. As such, HBV vaccination is recommended in HCV-infected individuals. However, HBV vaccine responses in HCV-

infected individuals are often blunted compared with uninfected populations. The mechanism for this failure of vaccine response

in HCV-infected subjects remains unclear. In this study, we investigated the expression and function of an inhibitory receptor, killer

cell lectin-like receptor subfamily Gmember 1 (KLRG1), in the regulation of CD4+ T cells and HBV vaccine responses during HCV

infection. We demonstrated that KLRG1 was overexpressed on CD4+ T cells from HCV-infected, HBV vaccine nonresponders

compared with HBV vaccine responders. The capacity of CD4+ T cells to proliferate and secrete IL-2 cytokine was inversely

associated with the level of KLRG1 expression. Importantly, blocking KLRG1 signaling resulted in a significant improvement in

CD4+ T cell proliferation and IL-2 production in HCV-infected, HBV vaccine nonresponders in response to TCR stimulation.

Moreover, blockade of KLRG1 increased the phosphorylation of Akt (Ser473) and decreased the expression of cell cycle inhibitors

p16ink4a and p27kip1, which subsequently enhanced the expression of cyclin-dependent kinase 2 and cyclin E. These results suggest

that the KLRG1 pathway impairs CD4+ T cell responses to neoantigen and induces a state of immune senescence in individuals

with HCV infection, raising the possibility that blocking this negative-signaling pathway might improve HBV vaccine responses in

the setting of chronic viral infection. The Journal of Immunology, 2014, 192: 649–657.

Hepatitis C virus (HCV) infection is a global public healthproblem, with ∼200 million people chronically infectedworldwide. HCV-mediated impairment of the host in-

nate to adaptive immune system is imperative for the developmentof persistent viral infection and poor vaccine responses, although

the underlying mechanisms for this failure require further study(1). It is well known that APCs and CD4+ T cells play a pivotal

role in the host immune responses to pathogenic infection and

vaccination (2, 3). We (2) reported previously that a differential

secretion of IL-12/IL-23 by APCs drives Th17 cell development

that may be involved in the vaccine failure observed during HCV

infection. In this study, we further explored the mechanisms of

CD4+ T cell dysfunction using a model of hepatitis B vaccine

failure in the setting of chronic HCV infection in humans.Given the shared risk factors for transmission, coinfection of

hepatitis B virus (HBV) with HCV is common and may lead to

a higher rate and more rapid progression to liver cirrhosis and liver

cancer (4, 5). Thus, HBV vaccine is recommended to prevent

HBV superinfection and its associated increases in morbidity and

mortality in HCV-infected individuals. However, vaccine response

in this setting is often blunted, with poor response rates to

a standard course of HBV vaccinations in chronically HCV-

infected patients, especially in those with advanced liver fibrosis

and cirrhosis compared with healthy populations (40–60% versus

90–95%) (6, 7). Our recent data suggest that, even in the setting of

relatively preserved hepatic function, seroconversion in HCV-

infected individuals is much lower than in age-matched healthy

subjects (HSs) (6). The reasons for vaccine nonresponse in 5–10%

of HSs and 40–60% of HCV-infected individuals remain poorly

understood, although several factors are known to play a role,

including age (HBV hyporesponsiveness is strongly correlated

with aging, with seroconversion rates showing declines as early as

35 years of age and waning markedly over the ensuing decades),

gender, smoking, obesity, and certain HLA alleles (8–10). Given

the fact that these HBV vaccine nonresponders (HBV-NRs) also

have poor recall responses to tetanus toxoid or Candida, it was

*Division of Infectious Diseases, Department of Internal Medicine, James H. QuillenCollege of Medicine, East Tennessee State University, Johnson City, TN 37614;†Department of Infectious Diseases, Xian Jiaotong University College of Medicine,Xian, China; ‡Department of Biochemistry and Molecular Biology, Soochow Uni-versity School of Medicine, Suzhou, China; xInternational Center for Diagnosis andTreatment of Liver Diseases, 302 Hospital, Beijing, China; {Department of Hepatol-ogy, Guangzhou Number 8 People’s Hospital, Guangzhou, China; and ‖Hepatitis(HCV/HIV) Program, James H. Quillen VA Medical Center, Department of VeteransAffairs, Johnson City, TN 37604

1L.S. and J.M.W. contributed equally to this work and share first authorship.

Received for publication August 5, 2013. Accepted for publication November 15,2013.

This work was supported by National Institutes of Health National Institutes ofDiabetes and Digestive and Kidney Diseases Grant R01DK093526 (to Z.Q.Y. andJ.P.M.). L.S. is a visiting scholar who is supported in part by the Guanghua Foun-dation of Xian Jiaotong University. Y.Q.C. is a visiting scholar who is supported inpart by Beijing 302 Hospital. R.S.Y., a visiting scholar, holds a grant for viralhepatitis research from Guangzhou Municipal Health Bureau, China.

The content of this publication does not necessarily reflect the views of the Depart-ment of Veterans Affairs or the U.S. Government.

Address correspondence and reprint requests to Dr. Guangyu Li, Division of Infec-tious Diseases, Department of Internal Medicine, East Tennessee State University,Quillen College of Medicine, Johnson City, TN 37614. E-mail address: lig001@etsu.edu

Abbreviations used in this article: CDK, cyclin-dependent kinase; HAV, hepatitis Avirus; HBsAg, hepatitis B surface Ag; HBV, hepatitis B virus; HBV-NR, hepatitis Bvaccine nonresponder; HBV-R, hepatitis B vaccine responder; HCV, hepatitis C virus;HS, healthy subject; KLRG1, killer cell lectin-like receptor subfamily G member 1.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302069

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suggested that HBV vaccine failure may be due to a defect inCD4+ Th cells (11–14), regulatory T cells (15), or in APCs (16,17); however, this has remained controversial (18, 19). A numberof clinical studies (20–24) attempted to correct vaccine nonresponseby adding adjuvants, altering doses, and administering vaccinethrough different routes or strategies. These approaches led tovarying degrees of improvement in HSs but had limited success invirally infected individuals, in part because of our incompleteunderstanding of the mechanisms that inhibit vaccine responses inthis setting.Recently, it was reported that an inhibitory receptor and marker

for cell aging–killer cell lectin-like receptor subfamily G member1 (KLRG1)–increases substantially on T cells and NK cells duringpathogenic infections (25–35). KLRG1 is a transmembrane pro-tein with an ITIM in its cytoplasmic domain and a C-type lectin-like domain in the extracellular region. The expression and functionof KLRG1 during chronic viral infection remain elusive; thus,further defining its role in immune responses in a clinically relevantdisease model is significant and timely. In this article, we focus onexploring the role of KLRG1 in regulating CD4+ T cell responses toHBV vaccine in HCV-infected individuals. We found that the ex-pression of KLRG1 was significantly upregulated on CD4+ T cells,leading to an overexpression of cell cycle inhibitors (p16ink4a/p27kip1)and impaired cellular functions, which were more prominent in HBV-NRs compared with HBV-vaccine responders (HBV-Rs) duringchronic HCV infection.

Materials and MethodsSubjects

The study protocol was approved by a joint institutional review board atEast Tennessee State University and James H. Quillen VA Medical Center.A total of 48 HCV-infected subjects and 16 uninfected controls withoutserologic evidence of prior exposure to HBV or hepatitis A virus (HAV)were recruited into this study to receive either Engerix HBV (if HAV Abnegative) or Twinrix HAV/HBV combination vaccines, as appropriate. TheHCV-infected subjects comprised 24 HBV-Rs (defined as hepatitis Bsurface Ab titer. 10 IU/ml at 1–6 mo following a standard course of HBVvaccination) and 24 HBV-NRs (defined as hepatitis B surface Ab titer, 10IU/ml at 1–6 mo following a standard course of HBV vaccination). Allinfected subjects were virologically and serologically positive for HCV,prior to the antiviral treatment, with HCV genotype 1 (70%) and type 2 or3 (30%) and viral load ranging from 12,300 to 500,000 IU/ml. The subjectsnot infected with HCV comprised 1 spontaneously resolved individual,4 sustained virological responders following antiviral treatment, and 11HSs. Written informed consent was obtained from all participants. Themean age of HCV-infected HBV-NRs was comparable to HBV-Rs andcontrol subjects (p . 0.05).

Cell isolation and culture

Human PBMCswere isolated from the peripheral blood of study subjects byFicoll-density centrifugation with lympho-H (Atlanta Biological, Law-renceville, GA) and then viably cryopreserved in freezing medium in liquidnitrogen. If indicated, CD4+ T cells were further purified from isolatedPBMCs by negative selection with magnetic beads using a CD4+ T cellIsolation Kit (Miltenyi Biotec, Auburn, CA); cell purity was .95%. Cellswere cultured with RPMI 1640 containing 10% FBS (Life Technologies,Gaithersburg, MD), 100 mg/ml penicillin-streptomycin, and 2 mM L-glu-tamine (both from Thermo Scientific, Logan, UT) at 37˚C with 5% CO2

atmosphere for the subsequent experiments.

Flow cytometry

Procedures for intracellular and cytokine staining were performed essentially asdescribed previously (2). Briefly, purified CD4+ T cells or PBMCs were in-cubated or not with anti-CD3/CD28 Abs (1 mg/ml; eBioscience, San Diego,CA) or hepatitis B surface Ag (HBsAg; 2.5 mg/ml; BiosPacific, Emeryville,CA) as indicated in the results. For IL-2 intracellular staining, 1 mg/ml bre-feldin A (BioLegend, San Diego, CA) was added 4 h prior to cell harvesting tohalt cytokine secretion. The cells were stained for surface marker expressionand then fixed and permeabilized using an Inside Stain kit (Miltenyi Biotec),according to the manufacturer’s instructions. Four-color flow cytometric

analysis was performed using the following Abs: Alexa Fluor 488–conjugatedKLRG1 (13F12; a gift from Dr. Hanspeter Pircher, Institute of Medical Mi-crobiology and Hygiene, Department of Immunology, University of Freiburg,Freiburg, Germany), allophycocyanin-CD4/PE–IL-2 (eBioscience), and AlexaFluor 488–Phospho-Akt (Ser473) (193H12; Cell Signaling, Danvers, MA). ForAkt phosphocytometry staining, purified CD4+ T cells were incubated withKLRG1-blocking or control-IgG Abs in the presence of 1 mg/ml anti-CD3/CD28 for 72 h. The cells were restimulated with 3 mg/ml anti-CD3/CD28and incubated on ice for 15 min, fixed in 4% paraformaldehyde for 10 min,and permeabilized with 90% methanol on ice for 30 min. The cells weresubsequently incubated with p-Akt (Ser473) (D9E) or rabbit isotype control(DA1E; both from Cell Signaling) for 1 h at room temperature. Fluorescenceminus one strategy was used to determine background levels of staining andadjust multicolor compensation for cell gating. The cell analysis was per-formed on a FACSCalibur or Accuri C6 flow cytometer (BD, Franklin Lakes,NJ) using CellQuest or FlowJo software (TreeStar, Ashland, OR).

KLRG1 blockade

Purified CD4+ T cells or PBMCs from HCV patients were incubated withanti-human KLRG1 Ab (4 mg/ml; gift from Dr. Hanspeter Pircher) orisotype-control IgG in the presence of various concentrations of anti-CD3/CD28 for different times, as indicated in the Results, and then subjected toflow cytometric analysis of intracellular IL-2 and pAkt expression, CFSEassay, and Western blot.

Proliferation assay

PBMCs were labeled with CFSE (2.5 mM; Invitrogen) for 10 min at 37˚C,per the manufacturer’s instructions, washed with complete medium, andcultured (5 3 104/well) in a 96-well plate in the presence of anti-CD3(1 mg/ml), anti-CD28 (1 mg/ml), and recombinant human IL-2 (100 U/ml;R&D Systems). After culture for 5 d, the cells were immunostained withPE-CD4 and Alexa Fluor 488–KLRG1 and analyzed with a FACSCaliburflow cytometer and FlowJo software.

Western blot

Purified CD4+ T cells from HBV-NRs of HCV patients were incubated withanti-human KLRG1 or control IgG Ab (4 mg/ml) in the presence of anti-CD3/CD28 (1 mg/ml) for 3 d. The expression of P16ink4a, P27kip1, cyclin-dependent kinase (CDK)2, and cyclin E in CD4+ T cell was measuredby Western blot. Briefly, the cells were lysed in 13 RIPA lysis buffer(Boston BioProducts, Ashland, MA) and supplied with protease inhibitors/phosphorylase inhibitors (Thermo Scientific) and EDTA on ice. Cell lysateswere centrifuged for 15 min at 4˚C, and the protein concentrations weremeasured. Thereafter, protein samples were combined with 43 Laemmlisample buffer (Boston BioProducts), denatured, and separated by SDS-PAGE. Following transfer to an Amersham Hybond-P membrane (GEHealthcare, Piscataway, NJ), the membrane was blocked by 3% BSA-TBSTand probed with P16ink4a (Bethyl Laboratories, Montgomery, TX), P27kip1

(BioLegend), cyclin E (BioLegend), CDK2 (BioLegend), or b-actin (SantaCruz Biotechnology, Santa Cruz, CA) at 4˚C overnight. Finally, the mem-brane was incubated with an HRP-conjugated secondary Ab (Millipore,Temecula, CA) and developed by Amersham ECL Prime Western blottingDetection Reagents (GE Healthcare Biosciences, Pittsburgh, PA) on KodakX-OMAT-LS X-ray film (Sigma-Aldrich, St. Louis, MO). Specific bandswere quantified by AlphaEaseFC software (Alpha Innotech).

RT-PCR

Purified CD4+ T cells from HSs and HCV-infected HBV-NRs and HBV-Rswere subject to RT-PCR assay to determine the mRNA level of p16ink4a.Total RNA was isolated using an RNeasy Mini Kit (QIAGEN, Valencia,CA). A total of 1 mg/ml RNA was reverse transcribed (Ambion, Austin,TX), and the cDNAwas amplified by PCR using the following conditions:95˚C for 10 min, followed by 95˚C for 45 s, 60˚C for 45 s, and 72˚C for45 s for 30 cycles and then 72˚C for 5 min. The primers for p16ink4a geneamplifications were sense: 59-CCA TCA TCA TGA CCT GGA TCG-39antisense: 59-AGC ATG GAG CCT TCG GCT GA-39 (Integrated DNATechnologies, Coralville, IA). b-actin gene served as a control for nor-malization. The amplified products were analyzed by electrophoresis on2% agarose gels. OD values of the DNA products were determined usinga Gal-Pro Analyzer (Version 4.0; Media Cybernetics).

Statistical analysis

Study results were summarized for each group, and the results are expressedas mean 6 SD. Comparison between indicated groups in the Results wasperformed using a variety of tests to demonstrate the least significant

650 KLRG1 IMPAIRS CD4+ T CELL RESPONSES VIA p16 AND p27 PATHWAYS

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difference, including the Tukey procedure, depending on the ANOVA Ftest Prism software (version 4; GraphPad Software), or a nonparametricMann–Whitney U test. A pairwise t test was used to compare the signif-icance of changes in KLRG1-blockage experiments. Correlations betweenKLRG1 expression on CD4+ T cells and IL-2 expression were analyzedusing a Pearson Correlation program. The p values were considered sig-nificant (*p , 0.05) or very significant (**p , 0.01 and ***p , 0.001).

ResultsKLRG1 is overexpressed on CD4+ T cells in HCV-infectedHBV-NRs versus HBV-Rs

We demonstrated previously that impaired APC and T cell func-tions might play a role in blunting the HBV vaccine response inindividuals with HCV infection, although the underlying mecha-nisms for these cell defects is unclear (2, 6). KLRG1 is an in-hibitory receptor expressed on T cells and NK cells and is knownmarker for cell aging or immune senescence (28–30). As an initialapproach to study the role of KLRG1 on CD4+ T cell function andHBV vaccine responses during HCV infection, we first examinedKLRG1 expression on CD4+ T cells from HCV-infected versusHCV-uninfected subjects, with or without anti-CD3/CD28 stim-ulation ex vivo. As shown in Fig. 1A, KLRG1 expression on CD4+

T cells derived from HCV-infected patients was much higher thanon cells from HCV-uninfected individuals, but no difference inKLRG1 expression was observed between HCV-resolved subjectsand HSs. Also, as we and other investigators (1, 36) reportedpreviously for the relationship between viral load and clinicaldisease progression or immunological changes, no apparent cor-relation was found between KLRG1 expression and HCV RNAlevel. We then examined KLRG1 expression on CD4+ T cells fromHCV-infected HBV-NRs and HBV-Rs. As shown in Fig. 1B, aftercostimulation of TCR with anti-CD3 and anti-CD28, KLRG1expression on CD4+ T cells increased in both HBV-NRs and HBV-Rs; however, KLRG1 expression on CD4+ T cells in HBV-NRswas significantly higher than that observed in HBV-Rs with HCVinfection, either with or without TCR stimulation. This hold truein terms of the percentage of KLRG1+ CD4+ T cell populations

(Fig. 1C, p , 0.05), as well as the mean fluorescence intensity ofKLRG1 expression levels on CD4+ T cells from HBV-NRscompared with HBV-Rs, regardless of TCR stimulation (Fig.1D, p , 0.01). These results suggest that HCV infection mayinduce T cell aging or senescence by upregulation of KLRG1expression on CD4+ T cells, which may contribute to HBV vac-cine nonresponsiveness.

KLRG1 expression is inversely associated with IL-2 expressionby CD4+ T cells in HCV-infected HBV-Rs versus HBV-NRs

Previous work revealed that a higher level of KLRG1 expression onCD4+ or CD8+ T cells led to an anergic or senescent status thatwas characterized by a decrease in IL-2 production or cell pro-liferation (25, 35). To better understand the effect of KLRG1expression on human CD4+ T cell function and its role in vaccineresponses in the setting of persistent viral infection, we examinedIL-2 expression by CD4+ T cells from HBV-NRs and HBV-Rswith chronic HCV infection. As shown in Fig. 2A, HCV-infectedHBV-NRs exhibited significantly less IL-2 production compared withHBV-Rs. We then analyzed the relationship between KLRG1 ex-pression and IL-2 production by purified CD4+ T cells in response toTCR stimulation. As shown in Fig. 2B, almost all IL-2–producingcells were KLRG12 T cells, whereas the majority of KLRG1+ Thcells failed to produce IL-2. To determine whether IL-2 was producedby Ag-specific CD4+ T cells, we stimulated PBMCs from HCV-infected HBV-Rs ex vivo with HBsAg for 20 h, followed by FACSstaining and gating on CD4+KLRG12 cells, and then analyzed IL-2expression by CD45RA (naive) and CD45RO (memory) T cells. Asshown in Fig. 2C, IL-2 was expressed primarily by memory, ratherthan naıve, CD4+KLRG12 T cells from HBV-Rs stimulated withHBsAg ex vivo, and the data were reproducible in repeated experi-ments. Notably, KLRG1 expression was inversely associated withIL-2 production by CD4+ T cells during HCV infection, with HBV-NR CD4+ T cells expressing more KLRG1 and producing less IL-2compared with those from HBV-Rs (Fig. 2D). Importantly, blockadeof KLRG1 signaling with specific anti-KLRG1 significantly in-creased IL-2 production by CD4+ T cells from HBV-NRs among

FIGURE 1. Higher levels of KLRG1 ex-

pression on CD4+ T cells in HBV-NRs versus

HBV-Rs among chronically HCV-infected in-

dividuals. PBMCs from HCV-infected HBV-

NRs and HBV-Rs and HCV-uninfected sub-

jects were stimulated or not with anti-CD3/

CD28 for 18 h and immunostained with CD4

and KLRG1, followed by flow cytometric

analysis. (A) KLRG1+ cell frequency in gated

CD4+ T cells from HCV-infected and unin-

fected individuals, including HCV-resolved

subjects and HSs, with or without ex vivo

TCR stimulation. (B) Representative dot plots

of KLRG1 expression on CD4+ T cells from

HCV-infected HBV-NRs and HBV-Rs, with-

out or with ex vivo TCR stimulation. (C)

KLRG1+ cell frequency in gated CD4+ T cells

from each group. (D) Mean fluorescence in-

tensity of KLRG1 expression level in CD4+

T cells from each group. *p , 0.05, **p ,0.01, ***p , 0.001.

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HCV-infected individuals compared with subjects treated withIgG control (Fig. 2E). These results indicate that HBV-NRs ex-hibit poor IL-2 production by their CD4+ T cells compared withHBV-Rs among HCV-infected individuals; KLRG1 expression isnegatively associated with IL-2 production in HCV-infected patients;and KLRG1 blockade can recover the impaired IL-2 production byCD4+ T cells from HCV-infected HBV-NRs.

KLRG1 negatively regulates the proliferative capacity of CD4+

T cells that are more significantly suppressed in HCV-infectedHBV-NRs than HBV-Rs

The ability of KLRG1 to inhibit human T cell proliferative capacityis crucial for T cell aging and immune senescence. AlthoughKLRG1 expression on CD8+ T cells was shown to correlate in-

versely with their proliferative capacity (25, 35), a role forKLRG1 in the regulation of CD4+ T cell proliferation has not beendemonstrated in the setting of HCV infection. In this study, wecompared the relationship of KLRG1 expression and proliferationof CD4+ T cells from HCV-infected HBV-NRs and HBV-Rs inresponse to TCR stimulation using CFSE dilution to track celldivision. To this end, we labeled PBMCs with CFSE, stimulatedthem with anti-CD3/CD28 in the presence of IL-2 for 5 d, andthen double stained them with anti-human CD4 and KLRG1conjugates. CFSE dilution in KLRG1+/2 populations in gatedCD4+ T cells is shown in Fig. 3A. Consistent with their ability toproduce IL-2, the proliferative capacity of CD4+ T cells fromHBV-NRs was much lower than that seen in HBV-Rs with HCVinfection (p , 0.001). Interestingly, most of the proliferative

FIGURE 2. KLRG1 expression is inversely associated with IL-2 expression by CD4+ T cells in HCV-infected HBV-NRs and HBV-Rs. (A) PBMCs from

chronically HCV-infected HBV-NRs (n = 12) and HBV-Rs (n = 12) were stimulated or not with CD3/CD28 for 18 h and immunostained with conjugated

Abs to human IL-2 and CD4, followed by flow cytometric analysis. Representative dot plots of IL-2 expression in CD4+ T cells from HCV-infected HBV-

NRs and HBV-Rs (upper panels). Percentage of IL-2+ cell frequency in gated CD4+ T cells from HCV-infected HBV-NRs and HBV-Rs (lower panel). Each

symbol represents an individual subject, and the horizontal lines represent median values. (B) Representative dot plots of isotype staining and IL-2 versus

KLRG1 staining in purified CD4+ T cells from a patient with HCV infection. (C) Representative dot plots of IL-2 expression by naive versus memory CD4+

KLRG12 T cells from HCV-infected HBV-Rs stimulated with HBsAg ex vivo. PBMCs from HCV-infected HBV-Rs were stimulated with HBsAg ex vivo

for 20 h, followed by FACS staining and gating on CD4+KLRG12 cells, and were analyzed for IL-2 expression by CD45RA (naive) versus CD45RO

(memory) T cells. (D) The relationship between KLRG1 expression and IL-2 production by CD4+ T cells from HBV-NRs (s) and HBV-Rs (d) among

HCV-infected individuals. Data were analyzed by Pearson Correlation with two-tailed significance. (E) Purified CD4+ T cells from chronically HCV-

infected HBV-NRs (n = 12) were incubated with anti-KLRG1 or control IgG in the presence of TCRs and stimulated for 72 h, immunostained with

conjugated Abs to human IL-2, and analyzed by flow cytometry. Representative graph of IL-2 expression by CD4+ T cells treated with anti-KLRG1 versus

isotype-IgG control (left panel). Percentages of IL-2–expressing CD4+ T cells treated with IgG and anti-KLRG1 (right panel). Each symbol represents an

individual subject, and the horizontal lines represent median values. *p , 0.05.

652 KLRG1 IMPAIRS CD4+ T CELL RESPONSES VIA p16 AND p27 PATHWAYS

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CD4+ T cells were KLRG12 populations, whereas KLRG1+

T cells proliferated with less cycle following 5 d of TCR stimulationcompared with KLRG12 T cells; this was particularly true forHBV-NRs compared with HBV-Rs.To further investigate the role of KLRG1 in the control of T cell

proliferation, we carried out the T cell proliferation assay withCFSE dilution concomitant with KLRG1 blockade in CD4+ T cellsfrom HBV-NRs with HCV infection. In this case, purified CD4+

T cells were used for blocking experiments to avoid the secondaryeffects from accessory cells in bulk PBMCs. Notably, T cellstreated with anti-KLRG1 alone (without TCR stimulation) failedto proliferate, suggesting that the blocking Ab inhibits negativesignaling by KLRG1, rather than by directly activating T cells,and its effect requires TCR stimulation to drive cell proliferation.

As shown in Fig. 3B, inhibition of KLRG1 signaling in con-junction with TCR stimulation significantly enhanced the pro-liferative capacity of purified CD4+ T cells, although the cellcycle progression using purified CD4+ T cells was much lessthan that observed using bulk PBMCs (Fig. 3A), likely as a re-sult of the lack of other cytokine stimulation from accessorycells. Nevertheless, the cell division events were markedly in-creased in purified CD4+ T cells from six HBV-NRs with anti-KLRG1 blockade compared with those treated with isotype-control IgG (p , 0.001). These results indicate that HBV-NRCD4+ T cells proliferate inadequately, and KLRG1 negativelycontrols CD4+ T cell proliferation; as such, blocking this in-hibitory pathway can rescue T cell proliferation in HCV-infectedHBV-NRs.

FIGURE 3. KLRG1 negatively regulates the proliferative

capacity of CD4+ T cells that are repressed more in HBV-NRs

than in HBV-Rs among HCV-infected individuals. (A) PBMCs

isolated from HCV-infected HBV-NRs and HBV-Rs were la-

beled with CFSE, stimulated with anti-CD3/CD28 in the

presence of IL-2 ex vivo for 5 d, double stained with conjugated

Abs to CD4 and KLRG1, and analyzed by flow cytometry.

Representative dot plots of cell division as CFSE dilution in

KLRG1+/2 populations in the gated CD4+ T cells from HBV-

NRs and HBV-Rs (left panel). KLRG1+ CD4+ T cell CFSE

dilution (right panel). Each symbol represents an individual

subject, and the horizontal lines represent median values. (B)

Overlaid graphs showing the proliferation of purified CD4+

T cells from HBV-NRs treated with isotype-control IgG1 and

anti-KLRG1 mAb (left panel). Percentage of CFSE dilution in

CD4+ T cells treated with control IgG1 and anti-KLRG1 from

six HBV-NRs (right panel). Each symbol represents an indi-

vidual subject, and the horizontal lines represent median values.

***p , 0.001.

FIGURE 4. KLRG1 impairs T cell responses via p16ink4a pathway in HBV-Rs and HBV-NRs among HCV-infected individuals. Purified CD4+ T cells

from HCV-infected HBV-NRs and HBV-Rs or HSs were incubated with anti-CD3/CD28 for 3 d and total protein and mRNAwere extracted from the cell

lysates, followed by RT-PCR analysis of p16ink4a mRNA expression (A) and Western blot for p16ink4a protein expression (B). (C) Purified CD4+ T cells from

HCV-infected HBV-NRs incubated with anti-CD3/CD28 in the presence of anti-KLRG1 or control IgG for 3 d were subjected to Western blot analysis of

p16ink4a protein expression. b-actin served as loading control. Representative images (upper panel). Densitometry data of p16ink4a expression, corrected by

b-actin level, from three independent experiments (lower panel). *p , 0.05, **p , 0.01.

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KLRG1 impairs T cell responses via the p16ink4a pathway inHBV-Rs and HBV-NRs with HCV infection

To determine how KLRG1 inhibits CD4+ T cell proliferation, wefurther examined the downstream signaling molecules that controlT cell cycle progression. p16ink4a is a well-known cell cycle in-hibitor and marker of cell aging (37–39). The INK4a pathwayregulates cell cycle progression by blocking the CDK4/6–cyclin Dcomplex. This complex increases the phosphorylation of RB,causing it to release the transcription factor E2F. E2F mediates thetranscription of several cellular genes that are involved in G1/Sprogression (39). Given its key role in T cell proliferation, weexamined p16ink4a mRNA by RT-PCR and protein expression byWestern blot using purified CD4+ T cells from HCV-infectedHBV-Rs and HBV-NRs and compared them with HSs. Asshown in Fig. 4A, the level of p16ink4a mRNA expression in CD4+

T cells from HBV-NRs was significantly higher than that fromHBV-Rs (p , 0.05) and HSs (p , 0.01). Although a relativelyhigher level of p16ink4a mRNAwas detected in CD4+ T cells fromHCV-infected HBV-Rs versus HSs, it was not significantly higher.This difference in mRNA expression from three groups of subjectswas reproducible in repeated experiments by RT-PCR and wasconfirmed by its protein-expression levels detected by Westernblot (Fig. 4B). Because both KLRG1 and p16ink4a are regarded asmarkers for cell aging and immune senescence (28–30, 37–39),we next determined whether these two molecules are functionallylinked by blocking KLRG1 signaling in CD4+ T cells and sub-sequently detecting p16ink4a level by Western blot. As shown inFig. 4C, p16ink4a expression was inhibited in T cells following

TCR stimulation in the presence of anti-KLRG1 compared withthose treated with IgG-control Ab. These results indicate that in-tracellular p16ink4a is involved in the KLRG1-mediated T celldysfunction and HBV vaccine nonresponsiveness during chronicHCV infection.

KLRG1 inhibits TCR-mediated Akt (Ser473) phosphorylationand downstream signaling pathways in CD4+ T cells duringHCV infection

Akt (Thr308 or Ser473) phosphorylation is the initial event of T cellactivation upon TCR stimulation (35, 37). Recent evidence sug-gests an enhancement of Akt (Ser473) phosphorylation in CD8+

CD282CD272 senescent T cells when PBMCs were stimulatedwith anti-CD3 in the presence of E-cadherin–blocking Ab (35). Tofurther define the underlying mechanisms involved in the im-provement of senescent CD4+ T cell activation and proliferationfollowing blockade of KLRG1 signaling, we assessed the phos-phorylation of Akt in CD4+ T cells from HCV-infected HBV-NRsby flow cytometry following TCR stimulation in conjunction withKLRG1 blockade. As shown in Fig. 5A, compared with cellstreated with control IgG, blockade of KLRG1 signaling signifi-cantly enhanced the phosphorylation of p-Akt (Ser473) in purifiedCD4+ T cells from HBV-NRs with chronic HCV infection. Thedata were reproducible using purified CD4+ T cells from eightHCV-infected HBV-NRs and stimulated ex vivo with anti-CD3/CD28 in the presence of anti-KLRG1 versus control IgG(p , 0.01).

FIGURE 5. KLRG1 inhibits Akt (Ser473) phosphorylation and downstream signaling pathways in CD4+ T cells during HCV infection. (A) KLRG1

blockade increases p-Akt (Ser473) phosphorylation in purified CD4+ T cells from HCV-infected HBV-NRs. Representative graphs of p-Akt (Ser473) ex-

pression in the CD4+ T cells treated with control IgG1 or anti-KLRG1 (left panel). Gray-filled graph represents isotype-control staining. Percentages of

p-Akt (Ser473) phosphorylation in CD4+ T cells from HBV-NRs following treatment with anti-KLRG1 or control IgG (right panel). The horizontal lines

indicate the median values for eight HCV-infected HBV-NRs. (B) T cells of PBMCs from HCV-infected HBV-NRs were stimulated by anti-CD3/CD28 in

the presence of anti-KLRG1 or control IgG for 3 d. Total protein was extracted from the cell lysates, followed by Western blot analysis of CDK2, cyclin E,

and p27kip1 expression. b-actin served as loading control. Representative images (left panel). Densitometry data of CDK2, cyclin E, and p27kip1 expression,

corrected by b-actin level, from three independent experiments (right panel). *p , 0.05, **p , 0.01, ***p , 0.001.

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We showed previously that HCV core protein inhibits T cellcycle progression through Akt/p27kip1 pathway (40, 41). Thus,the immune senescence mediated by inhibitory receptors, such asKLRG1, may prevent TCR-mediated PI3K/Akt phosphorylation(25, 35). This, in turn, lifts the block on forkhead box O tran-scription factors and activates p27kip1, causing G1/S phase growtharrest by blocking the activation of cyclins and CDKs. Therefore,we expect that improved Akt phosphorylation by blockingKLRG1 signaling subsequently will decrease p27kip1 expressionand enhance cyclin and CDK activation. To test this hypothesis,T cells from HCV-infected HBV-NRs were stimulated with anti-CD3/CD28 in the presence of anti-KLRG1 or control IgG. p27kip1,as well as cyclin E and CDK2, were detected by Western blot. Asshown in Fig. 5B, the expression level of p27kip1 was decreased,whereas cyclin E and CDK2 were increased, in cells treated withanti-KLRG1 versus IgG. The results were reproducible in threeindependent experiments using purified cells from different HBV-NRs with HCV infection. These results indicate that KLRG1negatively regulates CD4+ T cell functions by affecting multipleintrinsic regulators, including Akt/p27kip1-related cell cycle pro-teins (Fig. 6). Thus, manipulating these signaling molecules mayprovide an alternative approach to improve HBV vaccine re-sponsiveness in HCV-infected individuals.

DiscussionHCV infection is a world-wide infectious disease that can leadto chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma.After decades of studies on this immunomodulatory virus, it hasbecome evident that HCV-mediated host immune dysfunctionplays a major role in viral persistence, as well as disease pro-gression. Notably, like HIV infection, individuals with HCV in-fection often do not respond well to HBV vaccinations, and effortsto boost vaccine response have proven to be futile, in part becauseof our poor understanding of the mechanisms that inhibit vaccineresponse in this setting. In this study, we used the model of HBVvaccine failure in HCV-infected individuals to explore the role ofKLRG1 in regulating CD4+ T cell functions and to examinewhether blocking the KLRG1 pathway affects immune responsesin HCV patients who have failed HBV vaccinations. Our datashow that KLRG1 is overexpressed on CD4+ T cells from HBV-NRs compared with HBV-Rs among HCV-infected individuals.Moreover, HCV-infected HBV-NRs exhibit a more profounddysfunction of CD4+ T cell proliferation and secretion of IL-2cytokine compared with HBV-Rs, which is inversely associatedwith the level of KLRG1 expression. Importantly, blocking KLRG1signaling leads to a significant improvement in CD4+ T cell pro-liferation and IL-2 production in HCV-infected HBV-NRs in re-sponse to TCR stimulation. Additionally, blockade of KLRG1increases the phosphorylation of Akt (Ser473) and decreases theexpression of cell cycle inhibitors p16ink4a and p27kip1, whichsubsequently enhances the expression of CDK2 and cyclin E. Theseresults suggest that KLRG1 impairs CD4+ T cell responses viap16ink4a and p27kip1 pathways, and the blunted HBV vaccine re-sponse during HCV infection might be a result, at least in part, ofvirus-mediated premature cell aging through the KLRG1-signalingpathway. Based on this study, we propose a model, as depicted inFig. 6, to illustrate the role of KLRG1 in impairing CD4+ T cellfunction and HBV vaccine responses in the setting of chronic HCVinfection.Although the role of KLRG1 in immune senescence has been

emerging, its link to chronic infection and vaccine response israther novel. Recently, Lindenstrøm et al. (42) used a bacillusCalmette-Guerin vaccine model to demonstrate that CD4+

KLRG12 IL-2–secreting subsets were central to increased pro-tective efficacy. Their data suggested that the waning of memoryimmunity that occurred as tuberculous infections became chronicwas associated with a loss of IL-2–producing CD4 cells and anincrease in KLGR1+ anergic T cells. These findings could be re-versed by vaccine boosting, leading to selection induction, ex-pansion, and maintenance of CD4+KLRG12 memory T cells. Ourstudy supports this concept, in that chronic HCV infection leads toblunted vaccine responses characterized by high KLRG1 expres-sion on CD4+ T cells with impaired ability to proliferate and toproduce IL-2.It is well-recognized that elderly individuals are more suscep-

tible to infections and have decreased responses to vaccinations(43). In general, the immune responses in the elderly are signifi-cantly less robust than responses by younger individuals in mag-nitude, duration, and quality of response, leading to generally poorefficacy in terms of vaccine responses (44–46). Remarkably, vac-cine responses, such as HBV, influenza, and pneumococcal vaccine,are also impaired in individuals with chronic viral infection, notedin the setting of HIV, HCV, and CMV infections (47, 48). It is im-perative to characterize the mechanisms underlying vaccine non-responsiveness in chronically viral-infected individuals, because they

FIGURE 6. A putative model for HCV-induced, KLRG1-mediated

CD4+ T cell dysfunction via p16ink4a and p27kip1 pathways. HCV-driven

upregulation of KLRG1 expression on CD4+ T cells inhibits TCR-induced

Akt phosphorylation. This, in turn, lifts the block on forkhead box O

transcription factors and activates p27kip1, causing G1 growth arrest by

blocking the activation of cyclins and CDKs (40, 41). HCV infection also

upregulates p16ink4a expression in CD4+ T cells, which is linked to KLRG1

signaling. This, in turn, blocks the activation of cyclins and CDKs, causing

G1 growth arrest (1). This KLRG1-mediated p16ink4a/p27kip1 alteration

inhibits CD4+ T cell responses that are likely involved in the vaccine

responses during chronic HCV infection. Therefore, blocking KLRG1 and

its downstream-signaling molecules may provide a novel approach to boost

vaccine responses in virally infected individuals.

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are the individuals most susceptible to superinfection-mediated in-creases in morbidity and mortality. Most vaccines aim to developgood neutralizing Ab, which involves the activation of APCs, theinteraction of the activated CD4+ T cells with their cognate B cells toform germinal centers, and maturation of the B cells to producespecific Abs. Each of these steps can be affected by viral infectionand/or cell aging. The mechanisms for the virus-mediated impairmentof CD4+ T cell responses, including T cell proliferation and IL-2production, is a fascinating, yet unclear, research theme because itbridges the innate and adaptive immune (vaccine) responses (46).To elucidate the mechanisms by which persistent viral infection

mediates host immune dysfunction and ultimately leads to bluntedvaccine responses, we previously explored the role of HCV-mediated immune exhaustion in HBV vaccine responses duringHCV infection. We showed that PD-1 and Tim-3, markers for cellexhaustion, are overexpressed on APCs and T cells of HBV-NRsversus HBV-Rs among HCV-infected individuals (2, 6). In additionto inducing immune exhaustion that impairs essential functionalactivity, persistent viral infections can lead to immune senescence,with accelerated premature cell aging due to telomere erosion orunrepaired DNA damage (26, 49). In this study, we further dem-onstrate that KLRG1 and p16ink4a, markers for cell aging, areupregulated in CD4+ T cells in HBV-NRs compared with HBV-Rswith HCV infection. Thus, we believe that HCV may use twocritical cell regulatory mechanisms, cell exhaustion and cell aging,through upregulation of two inhibitory pathways, PD-1/Tim-3 andKLRG1/p16ink4a, to dampen the functions of immune cells torespond appropriately to vaccines during chronic infection.It is possible that modulating these inhibitory receptors, like

Tim-3 and KLRG1, which are preferentially expressed in highlydifferentiated T cells during viral infection, may boost immuneresponses. Although there has been substantial progress in iden-tifying the mechanisms that regulate both processes separately, it isunclear how these processes interrelate and whether blockingpathways that maintain either the exhausted or the senescent state,or both, can boost vaccine responses, especially in virally infectedindividuals. A recent study (49) demonstrated that young HIV-infected patients, with a duration of infection , 4 y, have earlyimmune exhaustion leading to premature aging and senescencecomparable to the elderly, suggesting that virus induces prematureimmune senescence associated with high rates of immune ex-haustion following short-term infection. We are also exploring themechanisms underlying how HCV infection induces immune ex-haustion and immune senescence that are essential for developingspecific strategies to improve vaccine responses in the setting ofchronic viral infection. These studies have led to an intersectingfield of virus-mediated immune exhaustion and immune senes-cence with regard to vaccine responses.In summary, this study delineates the mechanisms of persistent

infection-induced immune senescence or cell aging by measuringT cell responses to neoantigen stimulation. Our findings suggestthat KLRG1 expression is upregulated and associated with CD4+

T cell dysfunctions that are more prominent in HBV-NRs com-pared with HBV-Rs among HCV-infected individuals. HCV-induced KLRG1 expression impairs CD4+ T cell responses viap16ink4a and p27kip1 pathways (Fig. 6); thus, inhibition of theKLRG1 pathway and downstream signaling molecules in CD4+

T cells can be therapeutically exploited for improving humanimmune (vaccine) responses. Our data presented in this articlesuggest that KLRG1 may be used as a potential predictor forvaccine responses and progression of immune status in chronicHCV infection. Further characterization of KLRG1 expressionand functional changes in CD4+ T cells will help us to betterunderstand the pathogenesis of chronic HCV infection, and

it may reveal a potential therapeutic target for managing thisdisease.

AcknowledgmentsWe thank Dr. Hanspeter Pircher for providing anti-KLRG1 Abs. This pub-

lication is the result of work supported with resources and the use of facil-

ities at the James H. Quillen Veterans Affairs Medical Center.

DisclosuresThe authors have no financial conflicts of interest.

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