TitleIncreased frequency of circulating follicular helper T cells inchildren with hand foot and mouth disease caused byenterovirus 71 infection

Author(s) Wu J Cui D Yang X Lou J Lin J Ye X Qin Z Huang LZhao D Huo Z Xie G Zheng S Yu F Lu L Chen Y

Citation Journal of Immunology Research 2014 v 2014 article no651872

Issued Date 2014

URL httphdlhandlenet10722212121

Rights Creative Commons Attribution 30 Hong Kong License

Research ArticleIncreased Frequency of Circulating Follicular HelperT Cells in Children with Hand Foot and Mouth DiseaseCaused by Enterovirus 71 Infection

Jianping Wu1 David Cui1 Xianzhi Yang1 Jianzhou Lou1 Jie Lin2 Xianfei Ye1

Zhimei Qin1 Li Huang1 Dejian Zhao1 Zhaoxia Huo1 Guoliang Xie1 Shufa Zheng1

Fei Yu1 Liwei Lu3 and Yu Chen1

1 Department of Clinical Laboratory The First Affiliated Hospital School of Medicine Zhejiang University79 Qingchun Road Hangzhou 310003 China

2Department of Clinical Laboratory Center of Community Health Service of Qingbo Street Hangzhou 310002 China3Department of Pathology and Center of Infection and Immunology The University of Hong Kong Hong Kong

Correspondence should be addressed to Liwei Lu liweiluhkuhk and Yu Chen chenyu zy163com

Received 8 March 2014 Revised 30 April 2014 Accepted 7 May 2014 Published 11 June 2014

Academic Editor Toshinori Nakayama

Copyright copy 2014 Jianping Wu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Enterovirus 71 (EV71) is amajor causative agent of hand foot andmouth disease (HFMD) in childrenThe role of T follicular helper(TFH) cells in EV71-infected children remains unclear in regulating humoral immunityThe frequency of circulating ICOShighPD-1highCXCR5+CD4+ TFH cells in the children with mild and severe EV71 infection and healthy controls (HC) was detected by flowcytometry respectively IL-21 and IL-6mRNA expression and their serum levels Bcl-6 mRNA expression and specific neutralizingantibodies against EV71 (NAb-EV71) were measured In the acute stage of patients with EV71 infection increased frequenciesof circulating TFH cells with ICOShigh and PD-1high expression in the mild and severe patients were observed and the positivecorrelations among the frequencies of circulating TFH cells and the serum levels of IL-21 IL-6 and NAb-EV71 titres were detectedrespectively Moreover the expressions of IL-6 and IL-21 mRNA in PBMCs from patients were also significantly higher than thoseof HC However further analysis did not reveal any significant differences between mild and severe patients These data indicate arole of TFH cells and associated cytokines in modulating the humoral response during the pathogenesis of EV71 infection

1 Introduction

Enterovirus 71 (EV71) is a positive-stranded RNA genomeand belongs to a member of the species A Enterovirus genusPicornaviridae family [1] EV71 is one of the major causativeagents of hand foot and mouth disease (HFMD) in youngchildren which has caused a series of outbreaks of HFMDthroughout the world with particular prevalence in Asian-Pacific region [2ndash6] Most HFMD cases are mild and self-limited disease is caused mainly by EV71 and CoxsackievirusA16 (CA16) but HFMD cases caused by EV71 have resultedin severe neurological complications pulmonary edema andfetal death EV71 has become a serious public health concern

[5ndash8] Currently there is no safe and effective vaccine avail-able for prevention and control of this disease There is alsono approved antiviral drug for treatment of EV71 infectionRecent studies suggest that neutralizing antibodies (NAb)against EV71 (NAb-EV71) are crucial for the protection fromEV71 infection in animals and young children [4ndash6]

In an adaptive immune response Th1 and Th2 subsetsof CD4+ helper T cells are considered to play a pivotal rolein helping B cells to class-switch Ig isotypes via secretingspecial cytokines such as IFN-120574 and IL-4 and Th1 andTh2 cytokines respectively [9] Recently T follicular helper(TFH) cells have been clearly described as a specializedsubset of CD4+ T cells which distinguish from Th1 Th2

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2014 Article ID 651872 11 pageshttpdxdoiorg1011552014651872

Research ArticleIncreased Frequency of Circulating Follicular HelperT Cells in Children with Hand Foot and Mouth DiseaseCaused by Enterovirus 71 Infection

Jianping Wu1 David Cui1 Xianzhi Yang1 Jianzhou Lou1 Jie Lin2 Xianfei Ye1

Zhimei Qin1 Li Huang1 Dejian Zhao1 Zhaoxia Huo1 Guoliang Xie1 Shufa Zheng1

Fei Yu1 Liwei Lu3 and Yu Chen1

1 Department of Clinical Laboratory The First Affiliated Hospital School of Medicine Zhejiang University79 Qingchun Road Hangzhou 310003 China

2Department of Clinical Laboratory Center of Community Health Service of Qingbo Street Hangzhou 310002 China3Department of Pathology and Center of Infection and Immunology The University of Hong Kong Hong Kong

Correspondence should be addressed to Liwei Lu liweiluhkuhk and Yu Chen chenyu zy163com

Received 8 March 2014 Revised 30 April 2014 Accepted 7 May 2014 Published 11 June 2014

Academic Editor Toshinori Nakayama

Copyright copy 2014 Jianping Wu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Enterovirus 71 (EV71) is amajor causative agent of hand foot andmouth disease (HFMD) in childrenThe role of T follicular helper(TFH) cells in EV71-infected children remains unclear in regulating humoral immunityThe frequency of circulating ICOShighPD-1highCXCR5+CD4+ TFH cells in the children with mild and severe EV71 infection and healthy controls (HC) was detected by flowcytometry respectively IL-21 and IL-6mRNA expression and their serum levels Bcl-6 mRNA expression and specific neutralizingantibodies against EV71 (NAb-EV71) were measured In the acute stage of patients with EV71 infection increased frequenciesof circulating TFH cells with ICOShigh and PD-1high expression in the mild and severe patients were observed and the positivecorrelations among the frequencies of circulating TFH cells and the serum levels of IL-21 IL-6 and NAb-EV71 titres were detectedrespectively Moreover the expressions of IL-6 and IL-21 mRNA in PBMCs from patients were also significantly higher than thoseof HC However further analysis did not reveal any significant differences between mild and severe patients These data indicate arole of TFH cells and associated cytokines in modulating the humoral response during the pathogenesis of EV71 infection

1 Introduction

Enterovirus 71 (EV71) is a positive-stranded RNA genomeand belongs to a member of the species A Enterovirus genusPicornaviridae family [1] EV71 is one of the major causativeagents of hand foot and mouth disease (HFMD) in youngchildren which has caused a series of outbreaks of HFMDthroughout the world with particular prevalence in Asian-Pacific region [2ndash6] Most HFMD cases are mild and self-limited disease is caused mainly by EV71 and CoxsackievirusA16 (CA16) but HFMD cases caused by EV71 have resultedin severe neurological complications pulmonary edema andfetal death EV71 has become a serious public health concern

[5ndash8] Currently there is no safe and effective vaccine avail-able for prevention and control of this disease There is alsono approved antiviral drug for treatment of EV71 infectionRecent studies suggest that neutralizing antibodies (NAb)against EV71 (NAb-EV71) are crucial for the protection fromEV71 infection in animals and young children [4ndash6]

In an adaptive immune response Th1 and Th2 subsetsof CD4+ helper T cells are considered to play a pivotal rolein helping B cells to class-switch Ig isotypes via secretingspecial cytokines such as IFN-120574 and IL-4 and Th1 andTh2 cytokines respectively [9] Recently T follicular helper(TFH) cells have been clearly described as a specializedsubset of CD4+ T cells which distinguish from Th1 Th2

Hindawi Publishing CorporationJournal of Immunology ResearchVolume 2014 Article ID 651872 11 pageshttpdxdoiorg1011552014651872

2 Journal of Immunology Research

Th17 and Treg and localize to B cell follicles of germinalcenter (GC) where they can regulate the humoral immuneresponses [10ndash14] Typical features of TFH cells are theexpression of chemokine (C-X-Cmotif) receptor 5 (CXCR5)inducible costimulator (ICOS) programmed death-1 (PD-1)interleukin- (IL-) 21 and B-cell lymphoma 6 (BCL-6) [14ndash16]Additionally TFH cells also express other surface moleculesincluding CD40 ligand (CD40L) OX40 IL-21 receptor (IL-21R) and IL-6 receptor (IL-6R) [14] TFH cells can migrateto C-X-C motif chemokine 13 (CXCL13) expressed on Bcells through CXCR5 which is carried out in GC whereTFH cells and relative cytokines regulate the developmentof B-cells responses as well as Ig isotype switching and theproduction of optimal antibodies [17ndash20] Studies indicatedthat ICOS and PD-1 of the CD28 family members are closelycorrelated with the functions of TFH cells some cytokinessuch as IL-6 IL-12 IL-21 and IL-23 can induce IL-21 secretionin human naıve CD4+ T cells but only IL-12 induces thesustained expression of CXCR5 and ICOS on these activatednaıve CD4+ T cells which promotes TFH cell developmentby upregulation of BCL-6 expression [20ndash25] Bcl-6 is anessential transcriptional factor that directly affects TFH cellsdifferentiation and represses transcriptional regulators ofother Th cells [17]

Recent studies have shown that circulating TFH cellswere dysregulated in patients with lymphoma autoimmunediseases and several infectious diseases [25ndash30] Increasingevidence indicates that Th1 Th2 Th17 and Treg subsetsand B cells are critically involved in the pathogenesis ofEV71 infection [31ndash35] However the role of circulating TFHcells is not yet characterized in regulating humoral immuneresponse in EV71-infected children In this study we reportedthat the frequency of circulating TFH cells and levels of NAb-EV71 IL-21 and IL-6 mRNA expression were significantlyincreased in patients at the acute stage of EV71 infectioncompared to HC Additionally the frequency of circulatingCXCR5+CD4+ TFH cells with ICOShigh and PD-1high waspositively correlatedwith levels of IL-21 IL-6 andNAb-EV71These data indicated that the TFH cell might play a crucialrole in the pathogenesis of HFMD at acute stage of EV71infection

2 Materials and Methods

21 Patients A total of 60 children below ten years withEV71 infections acquired during outbreaks betweenApril andSeptember 2013 in the First Affiliated Hospital of Collegeof Medicine Zhejiang University The diagnoses were estab-lished depending on clinical features and laboratory criteriaEV71-infected HFMD was defined by EV71-positive throatswabs stools rectal swabs vesicular swabs or cerebrospinalfluid (CSF) samples which were detected using specific EV71primers and probe from the enterovirus 71 nucleic aciddetection kit (Da An Gene Co Ltd Guangzhou China)Mild case was characterized by typical manifestations with orwithout fever which usually recovered spontaneously withinone week severe case was defined as a febrile exanthematousdisease with neurological signs of encephalitis meningi-tis andor cardiopulmonary complications as previously

described [3] Clinical samples and data were collected fromthe EV71-infected HFMD children who had not been treatedat the time the samples were collected in the first diagnosein the hospital and children with bacterial infections wereexcluded

43 healthy children with age and gender matched wereincluded in this study as controls from health checks requir-ing blood puncture on parentsrsquo request or physiciansrsquo adviceto test for HBV infection status liver function and bloodroutine examination at our hospital According to the Dec-laration of Helsinki (1964) informed consents were obtainedfrom the parents or guardians of the children and themedicalethical committee of our hospital approved this study Mainclinical data of these children are shown in Table 1

22 Cell Isolation Peripheral blood samples were obtainedfrom the healthy children and the patients in the acute stageof EV71 infection and peripheral blood mononuclear cells(PBMCs) were isolated by density-gradient centrifugationusing Ficoll-Hypaque solution

23 Flow Cytometric Analysis Human PBMCs were washedand stained with PerCPcy55-anti-CXCR5 and APC-anti-CD4 PE-anti-CD278 (ICOS) and FITC-anti-CD279 (PD-1) (Biolegend San Diego CA USA) Isotype-matched Abcontrols were used in all procedures All the staining was per-formed according to manufacturerrsquos protocol The frequencyof circulating TFH cells was determined by a FACSCaliburflow cytometry (Beckton Dickinson BD Bioscience USA)and FlowJo software version 765 (TreeStar San Carlos CA)

24 RNA Isolation and Real-Time PCR For detection ofIL-6 IL-21 and BCL-6 mRNA expression total RNA fromPBMCs was extracted with Trizol (Invitrogen USA) cDNAwas synthesized by reverse transcription reagent kits (TakaraDalian China) according to the manufacturerrsquos instructionReal-time PCR was performed in triplicate using TakaraSYBR super mix (Takara Dalian China) by ABI 7500analysis system (Applied Biosystems CA USA) Amplifiedconditions were as follows 5min at 95∘C for denaturationthen 40 cycles of 95∘C for 10 sec and 60∘C for 40 sec andcollecting for fluorescence at 60∘C Primer sequences wereas follows IL-6 sense 51015840-GGCCTTCCCTACTTCACAAG-31015840 antisense 51015840-ATTTCCACGATTTCCCAGAG-31015840 IL-21 sense 51015840-AGATCCAGTCCTGGCAACATG-31015840 antisense51015840-GGCAGAAATTCAGGGACCAAG-31015840 and BCL-6 sense51015840-TTGTGAGCCGTGAGCAGTTT-31015840 antisense 51015840-TGT-CTTGGGGCATCAGCAT-31015840 Each gene was normalizedusing 120573-actin gene with the following primers sense 51015840-TGGAATCCTGTGGCATCCATGAAAC-31015840 antisense 51015840-TAAAACGCAGCTCAGTAACAGTCCG-31015840 Data were ana-lyzed by ABI 7500 Software (Applied Biosystems CA USA)

25 Enzyme-Linked Immunosorbent Assay (ELISA) Serumsamples were stored at minus80∘C The levels of the sera IL-6and IL-21 in individual children and healthy controls weremeasured by enzyme-linked immunosorbent assay (ELISA)

Journal of Immunology Research 3

Table 1 Clinical characteristics of 60 recruited children with HFMD caused by EV71 infection

Group Number MF Age (Months) WBC (times109) CRP (mgL)Mild 29 1613 4641 plusmn 1801 790 plusmn 184 695 plusmn 480

Severe 31 2011 2619 plusmn 1298 1311 plusmn 416 1939 plusmn 1191

HC 43 2320 3744 plusmn 1114 634 plusmn 111 031 plusmn 010

Note data correspond to the arithmetic mean plusmn SD MF malefemale WBC white blood cell CRP C-reactive protein HC healthy controls60 Laboratory-confirmed HFMD children with EV71 infection were recruited and classified into two groups according to the clinical severity of disease anddegrees of neurological damage

using LEGEND MAX Human IL-6 ELISA Kit with Pre-coated Plates and LEGEND MAX Human IL-21 ELISA Kitwith Pre-coated PlatesELISA kits (Biolegend San DiegoCA USA) according to the manufacturerrsquos instructions Allsamples were run in triplicate

26 Neutralizing Antibody (NAb) against EV71 Assay(NAb-EV71) Neutralizing antibodies (NAb) against EV71(ZhejiangDTIDZJU-74) in sera was detected by a micro-neutralization test in 96-well plates as described previously[3] Briefly the sera inactivated at 56∘C for 30 minutes wereserially diluted from 1 8 to 1 1024 in DMEM containing2 fetal bovine serum Serum dilution (25120583L) and 25 120583Lviral cultures with 100 fifty percent tissue culture infectivedose (TCID

50) were mixed and incubated at 37∘C for 2

hours The serum-virus mixtures were then added into100 120583L of human rhabdomyosarcoma (RD) cell suspension(2 times 105 cellsmL) incubated at 37∘C for 3 days in 5 CO

2

and monitored for characteristic cytopathic effect (CPE)The neutralizing titer was defined as the reciprocal of thedilution of the serum that neutralized ge50 of the infectedcells If the serum neutralization titer was ge1 8 the specificneutralizing antibody (NAb) against EV71 was consideredto be positive An antibody-negative serum and uninfectedcells were defined as controls

27 Statistical Analysis One-way ANOVA analysis was per-formed to confirm whether there was an overall statisticallysignificant change among the groups Studentrsquos 119905-test was per-formed as appropriate Correlations between variables weredetermined using Spearmanrsquos correlation coefficient Datawere analyzed using GraphPad Prism 5 software (GraphPadSoftware Inc San Diego CA)

3 Results

31 Increased Frequency of Circulating CXCR5+CD4+ TFHCells in Children with EV71 Infection To identify the viral eti-ology of HFMD from each child in our recruited population60 children with EV71-mediated HFMD were identified byone-step real-time RT-PCR assay for at least one of clinicalsamples from throat swabs stools rectal swabs vesicularswabs or CSF 31 cases were considered to be severe HFMDwith CNS involvement and 29 cases were mild according toclinical symptoms (Table 1) We found that severe patientsoccurred below 3 years the number of peripheral bloodWBCand the concentration of CRP were notably different amongthe severe mild and HC groups

To explore the potential role of circulating TFH cells inEV71-mediated HFMD children the frequency of circulatingCXCR5+CD4+ TFH cells and the percentages of ICOShigh orPD-1highCXCR5+CD4+ TFH cells were analyzed using flowcytometry (Figures 1(a)ndash1(c)) The frequencies of circulatingCXCR5+CD4+ TFH cells in CD4+ T cells in bothmild (700plusmn369) and severe (715 plusmn 293) HFMD patients were signifi-cantly higher than those in HC (452 plusmn 065) (Figure 2(a))Moreover the frequencies of ICOShigh CXCR5+CD4+ TFHcells (HC 073 plusmn 033 mild 150 plusmn 138 severe 230 plusmn233) and PD-1highCXCR5+CD4+ TFH cells (HC 107plusmn028mild 196 plusmn 111 severe 244 plusmn 127) in CD4+ T cells weresimilar in both mild and severe cases they were notablyhigher than those of HC (Figures 2(c)-2(d)) respectivelyHowever the percentages of circulating CXCR5+CD4+ TFHcells ICOShigh or PD-1highCXCR5+CD4+ TFH cells werenot significantly different between mild and severe HFMDpatients (Figures 2(c)-2(d)) In addition a strongly positivecorrelation was observed between ICOShighCXCR5+CD4+TFH and PD-1highCXCR5+CD4+ TFH cells (119903 = 06762119875 lt 00001) in EV71-infected patients (Figure 2(b))

32 High Levels of Neutralizing Antibodies against EV71 (NAb-EV71) with Increased Circulating TFH Cells in Children withMild and Severe Infections The increased frequencies ofcirculating TFH cells in human autoimmune thyroid disease(AITD) including Graversquos disease (GD) and Hashimotorsquosthyroiditis (HT) rheumatoid arthritis (RA) and systemiclupus erythematosus (SLE) were significantly correlated withthe levels of autoantibodies and disease activity [13 14 27ndash30] Next we sought to examine whether the frequencyof circulating TFH cells was associated with the levels ofspecific NAb-EV71 in the mild and severe patients with EV71infection It was found that the levels of specific NAb-EV71 inboth mild and severe EV71-patients were significantly higherthan those of HC but no significant difference was observedbetween mild and severe patients (HC 1023 plusmn 747 mild11807 plusmn 10811 severe 11529 plusmn 10159) (Figure 3(a)) Thefrequencies of circulatingCXCR5+CD4+ TFHcells ICOShighor PD-1highCXCR5+CD4+ TFH cells were all positively sig-nificantly associated with the levels of specific NAb-EV71 inthe mild and severe patients but not with HC respectively(Figures 3(b)ndash3(d))

33 SerumLevels of Cytokines Related to Circulating TFHCellsin Children with Mild and Severe Infections Recent studiesshow that cytokines both interleukin-21 (IL-21) and IL-6

4 Journal of Immunology Research

MildHC Severe

844 688

Q7928

Q3000

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000

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000

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000

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950

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FSC-height SSC-height subset

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104

104

800

1 k

1 k

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400

200

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8006004002000

SSC-

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103

103

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104

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ICOS+ ICOS+ ICOS+

(c)

Figure 1 Detection of circulating CXCR5+CD4+ TFH cells with ICOShigh and PD-1high expression in peripheral blood of EV71-infectedpatients andHC Peripheral bloodmononuclear cells (PBMCs) frompatientswith EV71 infection andHCwere stainedwith labeled antibodiesand analyzed by flow cytometry as described in Section 2 The cells were gated initially on living lymphocytes (upper left) and then on CD4+

T cells (upper right) Representative plots of CXCR5+CD4+ TFH cells in the total CD4+ T cells (a) PD-1highCXCR5+CD4+ TFH cells (b) andCOShighCXCR5+CD4+ TFH cells (c)

Journal of Immunology Research 5

0

5

10

15

20

HC Mild Severe

nslowastlowastlowast

lowastlowastlowast

P = 00021 P = 05151P lt 00001

CXCR

5+

CD4+

in C

D4+

T ce

lls (

)

(a)

0

2

4

8

6

0 5 10 15

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ICOS+CXCR5+CD4+ TFH cells ()

r = 07220

PD-1

+CX

CR5+

CD4+

TFH

cells

()

(b)

0

2

4

6

8

HC Mild Severe

nslowastlowastlowast

lowastlowastlowastP = 00002 P = 01085P lt 00001

PD-1

+CX

CR5+

CD4+

in C

D4+

T ce

lls (

)

(c)

0HC Mild Severe

5

10

15

ns

lowastlowastlowastP = 00037 P = 01930P = 00005

lowastlowastlowast

ICO

S+CX

CR5+

CD4+

in C

D4+

T ce

lls (

)

(d)

Figure 2 Increased percentages of circulating CXCR5+CD4+ TFH cells with ICOShigh and PD-1high expression in peripheral blood of EV71-infected patients The percentages of CXCR5+CD4+ TFH cells in the total CD4+ T cells (a) the correlation between the percentages of PD-1+CXCR5+CD4+ TFH cells and ICOS+CXCR5+CD4+ TFH cells from EV71-infected patients (119899 = 60) (b) PD-1+CXCR5+CD4+ TFH cells (c)and ICOS+CXCR5+CD4+ TFH cells (d) Data shown were the mean plusmn SD (standard deviation SD) The horizontal lines show the medianlowast

119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 ns no significant difference

had critical roles in differentiation and function of follicularhelper CD4+ T cells [14 21 30] The levels of cytokinesboth IL-21 (HC 6665 plusmn 1588 mild 9234 plusmn 3326 severe10146plusmn2682) and IL-6 (HC 494plusmn243 mild 1768plusmn1003severe 2545 plusmn 2152) in sera from the patients with EV-71infection were notably higher than those of HC althoughno significant difference was found in both mild and severechildren respectively (Figure 4(a)) The levels of serum IL-21 were strongly positively correlated with the frequencies ofTFH cells amongHCmild and severe patients (Figure 4(b))Interestingly a significant positive correlation between IL-6levels andCXCR5+CD4+ TFH cells frequencies was observedbetween mild and severe patients respectively although itwas not found in HC (Figure 4(c)) Interestingly IL-6 levelshowed a significant positive correlation with IL-21 level inmild and severe-HFMD patients respectively (Figure 4(d))

34 Correlation between Sera IL-21 and IL-6 and Specific NAb-EV71 in Children with Mild and Severe Infections Recent

studies demonstrated that the levels of both IL-21 andIL-6 cytokines were significantly associated with antibodyexpression [14 21] In this study we found that the concen-trations of serum IL-21 were positively correlated with that ofspecific NAb-EV71 in mild or severe patients although thiscorrelation was not observed in HC (Figure 5(a)) Howeverno significant association was noted between serum IL-6levels and specific NAb-EV71 concentrations in any of thethree groups tested respectively (Figure 5(b))

35 Expression of Bcl-6 IL-21 and IL-6 mRNA in Childrenwith Mild and Severe Infections Previous studies indicatedthat Bcl-6 was a pivotal transcription factor for CD4+ T celldifferentiation into TFH cells [14 18 26] Both IL-21 and IL-6 share cooperative functions in TFH differentiation and Bcell immunity [21] Here we assessed the expression of Bcl-6IL-21 and IL-6 mRNA in HC (119899 = 15) mild (119899 = 10) andsevere (119899 = 12) EV71-patients The IL-6 mRNA expressionin severe patients or mild patients was significantly higher

6 Journal of Immunology Research

0

200

400

600

NAb

-EV

71 ti

tres ns

HC Mild Severe

lowastlowastlowast

lowastlowastlowast

(a)

00 5 10 15 20

200

400

600

NAb

-EV

71 ti

tres

MildP lt 00001

CXCR5+CD4+ in CD4

+ T cells ()

r = 08581

0

10

20

30

40

NAb

-EV

71 ti

tres

3 4 5 6

HCP = 02560

CXCR5+CD4+ in CD4

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r = 01771

0 5 10 15 200

200

400

600

NAb

-EV

71 ti

tres

SevereP = 00043

CXCR5+CD4+ in CD4

+ T cells ()

r = 04992

(b)

000 05 10 15 20 25

10

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NAb

-EV

71 ti

tres

P = 05120

PD-1+CXCR5+CD4+ TFH cells ()

r = 01028

0 2 4 60

200

400

600

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-EV

71 ti

tres

P lt 00001

PD-1+CXCR5+CD4+ TFH cells ()

r = 06637

0 2 4 860

200

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600

NAb

-EV

71 ti

tres

P = 00027

PD-1+CXCR5+CD4+ TFH cells ()

r = 05196

(c)

0 5 10 150

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NAb

-EV

71 ti

tres

P = 00103

ICOS+CXCR5+CD4+ TFH cells ()

r = 04542

0 2 4 860

200

400

600

NAb

-EV

71 ti

tres

P = 00039

ICOS+CXCR5+CD4+ TFH cells ()

r = 05190

00 05 10 15 200

10

20

30

40

NAb

-EV

71 ti

tres

P = 07634

ICOS+CXCR5+CD4+ TFH cells ()

r = 004728

(d)

Figure 3 Correlation of specific NAb-EV71 titres and circulating CXCR5+CD4+ TFH cells with PD-1high and ICOShigh expression in EV71-infected patients (a) Titres of specific NAb-EV71 in sera from HC and EV71-infected patients including mild and severe cases Relationshipbetween the titres of specific NAb-EV71 and the frequency of circulating CXCR5+CD4+ TFH cells (b) PD-1+CXCR5+CD4+ TFH cells (c)and ICOS+CXCR5+CD4+ TFH cells (d) Data shown were the mean plusmn SD The horizontal lines show the median lowast119875 lt 005 lowastlowast119875 lt 001lowastlowastlowast

119875 lt 0001 ns no significant difference

than that in HC but no significant difference of IL-6 mRNAexpression was observed between mild and severe patientsgroups (HC 1008 plusmn 416 mild 1802 plusmn 941 severe 2055 plusmn1138) (Figure 6(a)) Similarly the IL-21 mRNA expressions

in both mild and severe patients were significantly higherthan those inHC but no significant difference of IL-21mRNAwas found between mild patients and severe patients (HC117plusmn048 mild 221plusmn131 severe 269plusmn151) (Figure 6(b))

Journal of Immunology Research 7

0

50

100

150

200

IL-2

1 (p

gm

L)

ns

P lt 00001 P lt 00001 P = 03909

HC Mild Severe

lowastlowastlowast

lowastlowastlowast

0

20

40

60

80

100

IL-6

(pg

mL)

ns

HC Mild Severe

P lt 00001 P lt 00001 P = 03438

lowastlowastlowast

lowastlowastlowast

(a)

3 4 5 60

50

100

150

IL-2

1 (p

gm

L)

P = 00053

0 5 1510 200

50

100

150

200

IL-2

1 (p

gm

L)

P lt 00001

0 5 1510 200

50

100

150

200

IL-2

1 (p

gm

L)

P = 00005

CXCR5+CD4+ in CD4

+ T cells () CXCR5+CD4+ in CD4

+ T cells () CXCR5+CD4+ in CD4

+ T cells ()

r = 05919r = 08479r = 04177

HC Mild Severe

(b)

0 5 1510 200

10

20

30

40

50

IL-6

(pg

mL)

P = 00176

CXCR5+CD4+ in CD4

+ T cells ()

r = 04375

3 4 5 60

5

10

15

20

IL-6

(pg

mL)

P = 04986

CXCR5+CD4+ in CD4

+ T cells ()

r = minus01060

0 5 1510 200

20

40

60

80

100

IL-6

(pg

mL)

P = 00236

CXCR5+CD4+ in CD4

+ T cells ()

r = 04056

(c)

0

5

10

15

20

IL-6

(pg

mL)

0 50 150100IL-21 (pgmL)

P = 02594r = minus01758

0 50 150100 2000

10

20

30

40

50

IL-21 (pgmL)

IL-6

(pg

mL)

P = 00220r = 04237

0 50 150100 200IL-21 (pgmL)

0

20

40

60

80

100

IL-6

(pg

mL)

P = 00418r = 03677

(d)

Figure 4 Correlation of cytokines levels and circulating CXCR5+CD4+ TFH cells in EV71-infected patients including mild and severe cases(a) Cytokines levels of IL-21 and IL-6 in sera fromHC and EV71-infected patients (b) Relationship between the IL-21 levels and the frequencyof circulating CXCR5+CD4+ TFH cells (c) Relationship between the IL-6 levels and the frequency of circulating CXCR5+CD4+ TFH cells(d) Relationship between the IL-6 levels and IL-21 levels Data shown were the mean plusmn SDThe horizontal lines show the median lowast119875 lt 005lowastlowast

119875 lt 001 lowastlowastlowast119875 lt 0001 ns no significant difference

8 Journal of Immunology Research

0

10

20

30

40

IL-21 (pgmL)

NAb

-EV

71 ti

tres

HC

0 50 100 150

P = 01036

0

200

400

600

IL-21 (pgmL)

NAb

-EV

71 ti

tres

Severe

500 100 200150

P = 00182

minus200

0

200

400

600

IL-21 (pgmL)

NAb

-EV

71 ti

tres

Mild

50 100 200150

P lt 00001

r = 04216

r = 07174

r = 02516

(a)

0

10

20

30

40

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 5 10 2015

P = 04936

0

200

400

600

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 10 20 30 40 50

P = 01039

0

200

400

600

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 20 40 60 80 100

P = 00741r = 03253r = 03082r = 01072

(b)

Figure 5 Correlation of specific NAb-EV71 titres and cytokines levels from HC and EV71-infected patients including mild and severe cases(a) Relationship between the titres of specific NAb-EV71 and the serum levels of IL-21 (b) Relationship between the titres of specific NAb-EV71 and the serum levels of IL-6

0

10

20

30

40

50

IL-6

mRA

N re

lativ

e exp

ress

ion

ns

HC Mild Severe

lowastlowast

lowast

P = 00116 P = 00078 P = 08175

(a)

0

2

4

6

IL-2

1 m

RAN

relat

ive e

xpre

ssio

n

ns

HC Mild Severe

lowastlowast

lowast

P = 00375 P = 00037 P = 04887

(b)

0

10

20

30

40

50

Bcl-6

mRA

N re

lativ

e exp

ress

ion

nsns ns

HC Mild Severe

P = 04212 P = 04495 P = 09737

(c)

Figure 6 Expression of IL-6 IL-21 and Bcl-6 mRNA in human PBMCs Human PBMCs from 10 mild and 12 severe patients with EV71infection and 15 HC were detected by real-time PCR assay as described in Section 2 (a)The levels of IL-6 mRNA in human PBMCs (b)Thelevels of IL-21 mRNA in human PBMCs (c) The levels of Bcl-6 mRNA in human PBMCs data shown were the mean plusmn SD The horizontallines show the median lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 ns no significant difference

Moreover there were no significant differences for Bcl-6mRNA expression among these groups tested (HC 2334 plusmn1292 mild 1845plusmn1028 severe 1954plusmn1332) (Figure 6(c))

4 Discussion

There aremore than onemillionHFMD cases and 200 deathseach year since 2008 in China [36] EV71 is one of thecommon causative factors of HFMD in young children less

than 5 years of age [3 5] Although mild diseases are the pre-dominant clinical features of EV71 infection severe diseaseswith neurological involvement and the fetal cases of HFMDare closely associated with EV71 infection identified by PCRandor virus isolation [6ndash8] In our study compared to mildpatients the age of severe patients wasmainly observed below3 years (severe versus mild cases 119875 = 00016) and evaluatedevidences with WBC numbers (severe versus mild cases 119875 lt00001 mild cases versus HC 119875 = 00035) and serum CRPlevels (severe versusmild cases119875 lt 00001 mild cases versus

Journal of Immunology Research 9

HC 119875 lt 00001) suggested that inappropriate inflammationmay be elicited in the progression of HFMD in the acutephase of EV71 infection whichwas consistentwith previouslyreported data [7 31 32]

Previous studies have demonstrated that both humoraland cellular immune responses such as antibodies B and Tcells including Th1Th2 and Th17 and other T cell lineagesand related cytokines play a critical role in protection fromEV71 infection in animal models [4 34 35] In humans EV71infection induced imbalance of TB cells and their relatedcytokines and even caused a systemic dysregulation of theimmune system [31ndash33] Recent studies show that follicularhelper T cells (TFH) a new T cell subpopulation play apivotal role in regulating immune responses and have beenassociated with many diseases [13 20 28] Simpson et alreported an increased frequency of circulatingCXCR5+CD4+TFH cells in patients with systemic lupus erythematosus(SLE) and Sjogrenrsquos syndrome [30] Feng et al demonstratedthat the frequency of circulating TFH cells was significantlyincreased in patients with HBV and these circulating TFHcells participated in the HBV-related immune responses[28] However the potential role of circulating TFH cells inpatients with EV71 infection is not yet known In the presentwork we firstly examined the frequency of circulating TFHcells in mild and severe patients with EV71 infection andhealthy controlsThe results demonstrated that the frequencyof circulating CXCR5+CD4+ TFH cells in mild and severepatients in the acute stage of EV71 infection was significantlyhigher than that of HC (severe or mild cases versus HC119875 lt 005) respectively However severe patients did notdiffer significantly from mild patients in the percentage ofperipheral blood CXCR5+CD4+ TFH cells (severe versusmild cases 119875 = 05151) These findings clearly indicatedthat TFH cells played an important role in the EV71-inducedimmune responses

Simpson et al detected that the high frequency of circu-lating TFH cells with high expression of ICOS andor PD-1 molecules was associated with the severity of end-organinvolvement in patients with systemic lupus erythematosus(SLE) and Sjogrenrsquos syndrome [30] Zhu et al demonstratedthe increased frequency of TFH cells with highly expressedICOS andor PD-1 in patients with autoimmune thyroiddisease and rheumatoid arthritis respectively and both ICOSand PD-1 molecules are closely positively correlated withthe function of TFH cells [13] We detected an enhancedfrequency of PD-1high and ICOShighCXCR5+CD4+ TFH cellsin the mild and severe EV71-induced patients comparedto HC (severe or mild cases versus HC 119875 lt 005) Inaddition a closely positive correlation was observed betweenthe frequencies of these two subpopulations of PD-1high andICOShighCXCR5+CD4+ T cells These data supported thedefinition of circulating TFH cells However the percentageof circulating TFH cells with ICOShigh and PD-1high expres-sion revealed no significant difference between the mild andsevere patients (severe versus mild cases PD-1high 119875 =01085 severe versus mild cases ICOShigh 119875 = 01930)In other words EV71 increased the frequency of circulatingTFH cells with ICOShigh and PD-1high expression that was not

notably associated with the severity of patients in the acutestage of EV71 infection

It is known that TFH cells are critical for promoting thedevelopment of antibody response by helper B cells in GC[10 11] Several seroepidemiologic investigations indicatedthat the neutralizing antibody can protect adults as well aschildren from EV71 infection once again although the titersof specific neutralizing antibody were not correlated withseverity of the disease with EV71 infection [3ndash6 34 35]Why is that To date that is not well understood Our dataalso indicated that the serum titers of specific NAb-EV71in patients with acute stage of EV71 infection were higherthan those in HC (severe or mild cases versus HC 119875 lt00001) but no significant difference was observed betweenmild and severe patients groups (severe versus mild cases119875 = 09693) which corresponded to previous studies [3]Additionally we found that enhanced frequency of circulat-ing CXCR5+CD4+ TFH PD-1highCXCR5+CD4+ TFH andICOShighCXCR5+CD4+ TFH cells was notably positively cor-related with the titres of specific NAb-EV71 in patients withEV71 infection and a significant associationwas not observedbetween themild and severe patients respectivelyThese dataalso indicated that TFH cells with ICOShigh and PD-1highexpression participated in regulating antibody responses inthe acute stage of EV71 infection at the same time whichmight give a good explanation to why there is no significantdifference between the levels of specific NAb-EV71 in bothmild and severe patients with EV71 infection

Recent studies demonstrated that not only did TFH cellssecrete IL-21 but IL-21 could also drive GC formation andTFH cell generation and function and promote terminalmature of B cell in GC Ig class-switching and antibodyresponses [10 11 20] IL-6 has also induced IL-21 productionand TFH cell generation and mice lacking both IL-6 andIL-21 are unable to generate TFH cell-dependent immuneresponses [20 21] Bcl-6 transcription factor is selectivelyexpressed in TFH cells which is required for programmingof TFH cell generation and Bcl-6 expression is regulated byIL-6 and IL-21 [14 21] In current study we found that thelevels of sera IL-21 and IL-6 concentrations were significantlyhigher in both mild and severe patients with EV71 infectionthan those in HC (severe ormild cases versus HC IL-21IL-6119875 lt 00001) and they were found to be positively correlatedwith the frequency of circulating CXCR5+CD4+ TFH cellsrespectively which suggested that IL-21 and IL-6 might playan important role in the generation of TFH cells In additiona strongly positive correlation was observed between sera IL-21 and NAb-EV71 as well as between sera IL-21 and IL-6indicating that IL-21 and IL-6 could promote the producingof specific NAb-EV71 and IL-6 might help in production ofIL-21 cytokines Furthermore PCR analysis indicated thatthe expression of IL-21 and IL-6 mRNA was significantlyincreased in both mild and severe patients compared to HC(severe or mild cases versus HC IL-21IL-6 mRNA 119875 lt005) However no difference of IL-21 and IL-6 cytokinesand mRNA expression was found between mild and severepatients (severe versus mild cases IL-21IL-6 cytokines 119875 =0390903438 IL-21IL-6 mRNA 119875 = 0488708175) these

10 Journal of Immunology Research

findings indicated that the expression of IL-21 or IL-6 wasnot notably related with severity of disease with acute stage ofEV71 infection and the IL-6 high expression was consistentwith pervious study [7] Additionally no significant differ-ence of peripheral blood Bcl-6mRNA expression amongHCmild and severe patients is consistent with pervious studywhich might be associated with formation of memory ofTFH cells [30 37] These data revealed that IL-21 and IL-6 participated in the generation of TFH cells and specificNAb-EV71 immune responses which were not significantlyassociated with the severity of patients in the acute stage ofEV71 infection

In conclusion our data indicated that increased freque-ncy of circulating TFH cells with ICOShigh and PD-1highexpression was significantly and positively associated withserum specific NAb-EV71 level and IL-21 and IL-6 con-centration in patients with EV71 infection suggesting thatTFH cells and associated cytokines might play a criticalrole in regulating humoral responses in the acute stage ofEV71 infection Further studies on the role of TFH cells inregulating B cell response and antibody production duringthe disease progression of EV71 infection will provide newinsight into understanding the immune pathogenesis of EV71infection in human

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

JianpingWu and David Cui contributed equally to this work

Acknowledgment

This study was supported by the National Key Programs forInfectious Diseases of China (Grant no 2012ZX10004-210)

References

[1] D Nasri L Bouslama S Pillet T Bourlet M Aouni and BPozzetto ldquoBasic rationale current methods and future direc-tions for molecular typing of human enterovirusrdquo ExpertReview of Molecular Diagnostics vol 7 no 4 pp 419ndash434 2007

[2] S-C Chen H-L Chang T-R Yan Y-T Cheng and K-T Chen ldquoAn eight-year study of epidemiologic features ofenterovirus 71 infection in Taiwanrdquo The American Journal ofTropical Medicine and Hygiene vol 77 no 1 pp 188ndash191 2007

[3] C Yang C Deng J Wan L Zhu and Q Leng ldquoNeutralizingantibody response in the patients with hand foot and mouthdisease to enterovirus 71 and its clinical implicationsrdquo VirologyJournal vol 8 article 306 2011

[4] QMao N Li X Yu et al ldquoAntigenicity animal protective effectand genetic characteristics of candidate vaccine strains ofenterovirus 71rdquo Archives of Virology vol 157 no 1 pp 37ndash412012

[5] M Zeng N F El Khatib S Tu et al ldquoSeroepidemiology ofEnterovirus 71 infection prior to the 2011 season in children in

Shanghairdquo Journal of Clinical Virology vol 53 no 4 pp 285ndash289 2012

[6] W-C Huang L-M Huang C-L Kao et al ldquoSeroprevalence ofenterovirus 71 and no evidence of crossprotection of enterovirus71 antibody against the other enteroviruses in kindergartenchildren in Taipei cityrdquo Journal of Microbiology Immunologyand Infection vol 45 no 2 pp 96ndash101 2012

[7] L-Y Chang C A Hsiung C-Y Lu et al ldquoStatus of cellularrather than humoral immunity is correlated with clinicaloutcome of enterovirus 71rdquo Pediatric Research vol 60 no 4 pp466ndash471 2006

[8] S-M Wang H-Y Lei K-J Huang et al ldquoPathogenesis ofenterovirus 71 brainstem encephalitis in pediatric patients rolesof cytokines and cellular immune activation in patients withpulmonary edemardquo Journal of Infectious Diseases vol 188 no4 pp 564ndash570 2003

[9] F D Finkelman J Holmes I M Katona et al ldquoLymphokinecontrol of in vivo immunoglobulin isotype selectionrdquo AnnualReview of Immunology vol 8 pp 303ndash330 1990

[10] C S Ma S Suryani D T Avery et al ldquoEarly commitment ofnave human CD4+ T cells to the T follicular helper (FH) celllineage is induced by IL-12rdquo Immunology and Cell Biology vol87 no 8 pp 590ndash600 2009

[11] T R Mosmann and R L Coffman ldquoTH1 and TH2 cells differ-ent patterns of lymphokine secretion lead to different functionalpropertiesrdquo Annual Review of Immunology vol 7 pp 145ndash1731989

[12] L E Harrington R D Hatton P R Mangan et al ldquoInterleukin17-producing CD4+ effector T cells develop via a lineage distinctfrom the T helper type 1 and 2 lineagesrdquo Nature Immunologyvol 6 no 11 pp 1123ndash1132 2005

[13] C Zhu M Jie Y Liu et al ldquoIncreased frequency of follicularhelper T cells in patients with autoimmune thyroid diseaserdquoJournal of Clinical Endocrinology and Metabolism vol 97 no3 pp 943ndash950 2012

[14] M A Kroenke D Eto M Locci et al ldquoBcl6 andMaf cooperateto instruct human follicular helper CD4 T cell differentiationrdquoJournal of Immunology vol 188 no 8 pp 3734ndash3744 2012

[15] D Breitfeld L Ohl E Kremmer et al ldquoFollicular B helper Tcells express CXC chemokine receptor 5 localize to B cellfollicles and support immunoglobulin productionrdquoThe Journalof Experimental Medicine vol 192 no 11 pp 1545ndash1551 2000

[16] P Schaerli K Willimann A B Lang M Lipp P Loetscherand B Moser ldquoCXC chemokine receptor 5 expression definesfollicular homing T cells with B cell helper functionrdquo Journal ofExperimental Medicine vol 192 no 11 pp 1553ndash1562 2000

[17] C N Arnold D J Campbell M Lipp and E C Butcher ldquoThegerminal center response is impaired in the absence of T cell-expressedCXCR5rdquoEuropean Journal of Immunology vol 37 no1 pp 100ndash109 2007

[18] N Fazilleau L Mark L J McHeyzer-Williams and M GMcHeyzer-Williams ldquoFollicular helper T cells lineage andlocationrdquo Immunity vol 30 no 3 pp 324ndash335 2009

[19] N M Haynes C D C Allen R Lesley K M Ansel N Killeenand J G Cyster ldquoRole of CXCR5 and CCR7 in follicular Thcell positioning and appearance of a programmed cell deathgene-1High germinal center-associated subpopulationrdquo Journalof Immunology vol 179 no 8 pp 5099ndash5108 2007

[20] R Spolski andW J Leonard ldquoIL-21 andT follicular helper cellsrdquoInternational Immunology vol 22 no 1 Article ID dxp112 pp7ndash12 2009

Journal of Immunology Research 11

[21] D Eto C Lao D DiToro et al ldquoIL-21 and IL-6 are critical fordifferent aspects of B cell immunity and redundantly induceoptimal follicular helper CD4 T cell (Tfh) differentiationrdquo PLoSONE vol 6 no 3 Article ID e17739 2011

[22] C Dong A E Juedes U-A Temann et al ldquoICOS co-stimu-linebreak latory receptor is essential for T-cell activation andfunctionrdquo Nature vol 409 no 6816 pp 97ndash101 2001

[23] T Okazaki and T Honjo ldquoThe PD-1-PD-L pathway in immuno-logical tolerancerdquo Trends in Immunology vol 27 no 4 pp 195ndash201 2006

[24] I Yusuf R Kageyama L Monticelli et al ldquoGerminal center Tfollicular helper cell IL-4 production is dependent on signalinglymphocytic activation molecule receptor (CD150)rdquo Journal ofImmunology vol 185 no 1 pp 190ndash202 2010

[25] D Yu and C G Vinuesa ldquoThe elusive identity of T follicularhelper cellsrdquo Trends in Immunology vol 31 no 10 pp 377ndash3832010

[26] M A Linterman R J Rigby RWong et al ldquoRoquin differenti-ates the specialized functions of duplicated T cell costimulatoryreceptor genes CD28 and ICOSrdquo Immunity vol 30 no 2 pp228ndash241 2009

[27] M Locci C Havenar-Daughton E Landais et al ldquoHuman cir-culating PD-1+1CXCR3minusCXCR5+ memory Tfh cells are highlyfunctional and correlate with broadly neutralizing HIV anti-body responsesrdquo Immunity vol 39 no 4 pp 758ndash769 2013

[28] J Feng L Lu C Hua et al ldquoHigh frequency of CD4+CXCR5+TFH cells in patients with immune-active chronic hepatitis BrdquoPLoS ONE vol 6 no 7 Article ID e21698 2011

[29] C Petrovas T Yamamoto M Y Gerner et al ldquoCD4 T follicularhelper cell dynamics during SIV infectionrdquo Journal of ClinicalInvestigation vol 122 no 9 pp 3281ndash3294 2012

[30] N Simpson P A Gatenby A Wilson et al ldquoExpansion ofcirculating T cells resembling follicular helper T cells is a fixedphenotype that identifies a subset of severe systemic lupuserythematosusrdquo Arthritis and Rheumatism vol 62 no 1 pp234ndash244 2010

[31] S Li C Cai J Feng et al ldquoPeripheral T lymphocyte subset imb-alances in children with enterovirus 71-induced hand foot andmouth diseaserdquo Virus Research vol 180 pp 84ndash91 2014

[32] J Chen J Tong H Liu et al ldquoIncreased frequency ofTh17 cellsin the peripheral blood of children infectedwith enterovirus 71rdquoJournal of Medical Virology vol 84 no 5 pp 763ndash767 2012

[33] J Xie Y Jiao Z Qiu Q Li and T Li ldquoSignificant elevation of Bcells at the acute stage in enterovirus 71-infected children withcentral nervous system involvementrdquo Scandinavian Journal ofInfectious Diseases vol 42 no 11-12 pp 931ndash935 2010

[34] Y Liang X Zhou E Yang et al ldquoAnalysis of the Th1Th2reaction in the immune response induced by EV71 inacti-vated vaccine in neonatal rhesus monkeysrdquo Journal of ClinicalImmunology vol 32 no 5 pp 1048ndash1058 2012

[35] Y-W Lin K-C Chang C-M Kao S-P Chang Y-Y Tungand S-H Chen ldquoLymphocyte and antibody responses reduceenterovirus 71 lethality in mice by decreasing tissue viral loadsrdquoJournal of Virology vol 83 no 13 pp 6477ndash6483 2009

[36] National notifiable diseases announced by National Health andFamily Planning Commission of the Peoplersquos Republic of Chinahttpwwwnhfpcgovcnzhuzhanyqxxlistsshtml

[37] R Morita N Schmitt S-E Bentebibel et al ldquoHuman bloodCXCR5+CD4+ T cells are counterparts of T follicular cellsand contain specific subsets that differentially support antibodysecretionrdquo Immunity vol 34 no 1 pp 108ndash121 2011

Journal of Immunology Research 3

Table 1 Clinical characteristics of 60 recruited children with HFMD caused by EV71 infection

Group Number MF Age (Months) WBC (times109) CRP (mgL)Mild 29 1613 4641 plusmn 1801 790 plusmn 184 695 plusmn 480

Severe 31 2011 2619 plusmn 1298 1311 plusmn 416 1939 plusmn 1191

HC 43 2320 3744 plusmn 1114 634 plusmn 111 031 plusmn 010

Note data correspond to the arithmetic mean plusmn SD MF malefemale WBC white blood cell CRP C-reactive protein HC healthy controls60 Laboratory-confirmed HFMD children with EV71 infection were recruited and classified into two groups according to the clinical severity of disease anddegrees of neurological damage

using LEGEND MAX Human IL-6 ELISA Kit with Pre-coated Plates and LEGEND MAX Human IL-21 ELISA Kitwith Pre-coated PlatesELISA kits (Biolegend San DiegoCA USA) according to the manufacturerrsquos instructions Allsamples were run in triplicate

26 Neutralizing Antibody (NAb) against EV71 Assay(NAb-EV71) Neutralizing antibodies (NAb) against EV71(ZhejiangDTIDZJU-74) in sera was detected by a micro-neutralization test in 96-well plates as described previously[3] Briefly the sera inactivated at 56∘C for 30 minutes wereserially diluted from 1 8 to 1 1024 in DMEM containing2 fetal bovine serum Serum dilution (25120583L) and 25 120583Lviral cultures with 100 fifty percent tissue culture infectivedose (TCID

50) were mixed and incubated at 37∘C for 2

hours The serum-virus mixtures were then added into100 120583L of human rhabdomyosarcoma (RD) cell suspension(2 times 105 cellsmL) incubated at 37∘C for 3 days in 5 CO

2

and monitored for characteristic cytopathic effect (CPE)The neutralizing titer was defined as the reciprocal of thedilution of the serum that neutralized ge50 of the infectedcells If the serum neutralization titer was ge1 8 the specificneutralizing antibody (NAb) against EV71 was consideredto be positive An antibody-negative serum and uninfectedcells were defined as controls

27 Statistical Analysis One-way ANOVA analysis was per-formed to confirm whether there was an overall statisticallysignificant change among the groups Studentrsquos 119905-test was per-formed as appropriate Correlations between variables weredetermined using Spearmanrsquos correlation coefficient Datawere analyzed using GraphPad Prism 5 software (GraphPadSoftware Inc San Diego CA)

3 Results

31 Increased Frequency of Circulating CXCR5+CD4+ TFHCells in Children with EV71 Infection To identify the viral eti-ology of HFMD from each child in our recruited population60 children with EV71-mediated HFMD were identified byone-step real-time RT-PCR assay for at least one of clinicalsamples from throat swabs stools rectal swabs vesicularswabs or CSF 31 cases were considered to be severe HFMDwith CNS involvement and 29 cases were mild according toclinical symptoms (Table 1) We found that severe patientsoccurred below 3 years the number of peripheral bloodWBCand the concentration of CRP were notably different amongthe severe mild and HC groups

To explore the potential role of circulating TFH cells inEV71-mediated HFMD children the frequency of circulatingCXCR5+CD4+ TFH cells and the percentages of ICOShigh orPD-1highCXCR5+CD4+ TFH cells were analyzed using flowcytometry (Figures 1(a)ndash1(c)) The frequencies of circulatingCXCR5+CD4+ TFH cells in CD4+ T cells in bothmild (700plusmn369) and severe (715 plusmn 293) HFMD patients were signifi-cantly higher than those in HC (452 plusmn 065) (Figure 2(a))Moreover the frequencies of ICOShigh CXCR5+CD4+ TFHcells (HC 073 plusmn 033 mild 150 plusmn 138 severe 230 plusmn233) and PD-1highCXCR5+CD4+ TFH cells (HC 107plusmn028mild 196 plusmn 111 severe 244 plusmn 127) in CD4+ T cells weresimilar in both mild and severe cases they were notablyhigher than those of HC (Figures 2(c)-2(d)) respectivelyHowever the percentages of circulating CXCR5+CD4+ TFHcells ICOShigh or PD-1highCXCR5+CD4+ TFH cells werenot significantly different between mild and severe HFMDpatients (Figures 2(c)-2(d)) In addition a strongly positivecorrelation was observed between ICOShighCXCR5+CD4+TFH and PD-1highCXCR5+CD4+ TFH cells (119903 = 06762119875 lt 00001) in EV71-infected patients (Figure 2(b))

32 High Levels of Neutralizing Antibodies against EV71 (NAb-EV71) with Increased Circulating TFH Cells in Children withMild and Severe Infections The increased frequencies ofcirculating TFH cells in human autoimmune thyroid disease(AITD) including Graversquos disease (GD) and Hashimotorsquosthyroiditis (HT) rheumatoid arthritis (RA) and systemiclupus erythematosus (SLE) were significantly correlated withthe levels of autoantibodies and disease activity [13 14 27ndash30] Next we sought to examine whether the frequencyof circulating TFH cells was associated with the levels ofspecific NAb-EV71 in the mild and severe patients with EV71infection It was found that the levels of specific NAb-EV71 inboth mild and severe EV71-patients were significantly higherthan those of HC but no significant difference was observedbetween mild and severe patients (HC 1023 plusmn 747 mild11807 plusmn 10811 severe 11529 plusmn 10159) (Figure 3(a)) Thefrequencies of circulatingCXCR5+CD4+ TFHcells ICOShighor PD-1highCXCR5+CD4+ TFH cells were all positively sig-nificantly associated with the levels of specific NAb-EV71 inthe mild and severe patients but not with HC respectively(Figures 3(b)ndash3(d))

33 SerumLevels of Cytokines Related to Circulating TFHCellsin Children with Mild and Severe Infections Recent studiesshow that cytokines both interleukin-21 (IL-21) and IL-6

4 Journal of Immunology Research

MildHC Severe

844 688

Q7928

Q3000

Q4

000

Q1

000

Q4

000

Q1720

Q2503

Q2

950

Q3 Q8

321

Q6556

349

CD4 FITC SSC-height subset728

FSC-height SSC-height subset

SSC-

H

SSC-

heig

ht

Q5

CD4+

104

104

800

1 k

1 k

600

400

200

0

8006004002000

SSC-

H

SSC-

heig

ht 800

1 k

600

400

200

0

103

103

102

102

101

101

100

100

CXCR

5+

CD4+

104

104

103

103

102

102

101

101

100

100

CD4+

104

104

103

103

102

102

101

101

100

100

104

103

102

101

100

(a)

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

104

103

102

101

100

100

104

103

102

101

100

925

Q8

359

Q5144

Q6

245

Q7859

Q8

388

Q5332

Q6

690 844

Q8 Q7

556

Q5321

Q6

688

Q7

PD-1+ PD-1+

CXCR

5+

PD-1+

(b)

817

Q12

488

Q11

Q9232

Q10

111820

Q12

583

Q11

Q9293

Q10

924857

Q12

329

Q11

Q9174

Q10

924

CXCR

5+

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

104

103

102

101

100

100

104

103

102

101

100

ICOS+ ICOS+ ICOS+

(c)

Figure 1 Detection of circulating CXCR5+CD4+ TFH cells with ICOShigh and PD-1high expression in peripheral blood of EV71-infectedpatients andHC Peripheral bloodmononuclear cells (PBMCs) frompatientswith EV71 infection andHCwere stainedwith labeled antibodiesand analyzed by flow cytometry as described in Section 2 The cells were gated initially on living lymphocytes (upper left) and then on CD4+

T cells (upper right) Representative plots of CXCR5+CD4+ TFH cells in the total CD4+ T cells (a) PD-1highCXCR5+CD4+ TFH cells (b) andCOShighCXCR5+CD4+ TFH cells (c)

Journal of Immunology Research 5

0

5

10

15

20

HC Mild Severe

nslowastlowastlowast

lowastlowastlowast

P = 00021 P = 05151P lt 00001

CXCR

5+

CD4+

in C

D4+

T ce

lls (

)

(a)

0

2

4

8

6

0 5 10 15

P lt 00001

ICOS+CXCR5+CD4+ TFH cells ()

r = 07220

PD-1

+CX

CR5+

CD4+

TFH

cells

()

(b)

0

2

4

6

8

HC Mild Severe

nslowastlowastlowast

lowastlowastlowastP = 00002 P = 01085P lt 00001

PD-1

+CX

CR5+

CD4+

in C

D4+

T ce

lls (

)

(c)

0HC Mild Severe

5

10

15

ns

lowastlowastlowastP = 00037 P = 01930P = 00005

lowastlowastlowast

ICO

S+CX

CR5+

CD4+

in C

D4+

T ce

lls (

)

(d)

Figure 2 Increased percentages of circulating CXCR5+CD4+ TFH cells with ICOShigh and PD-1high expression in peripheral blood of EV71-infected patients The percentages of CXCR5+CD4+ TFH cells in the total CD4+ T cells (a) the correlation between the percentages of PD-1+CXCR5+CD4+ TFH cells and ICOS+CXCR5+CD4+ TFH cells from EV71-infected patients (119899 = 60) (b) PD-1+CXCR5+CD4+ TFH cells (c)and ICOS+CXCR5+CD4+ TFH cells (d) Data shown were the mean plusmn SD (standard deviation SD) The horizontal lines show the medianlowast

119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 ns no significant difference

had critical roles in differentiation and function of follicularhelper CD4+ T cells [14 21 30] The levels of cytokinesboth IL-21 (HC 6665 plusmn 1588 mild 9234 plusmn 3326 severe10146plusmn2682) and IL-6 (HC 494plusmn243 mild 1768plusmn1003severe 2545 plusmn 2152) in sera from the patients with EV-71infection were notably higher than those of HC althoughno significant difference was found in both mild and severechildren respectively (Figure 4(a)) The levels of serum IL-21 were strongly positively correlated with the frequencies ofTFH cells amongHCmild and severe patients (Figure 4(b))Interestingly a significant positive correlation between IL-6levels andCXCR5+CD4+ TFH cells frequencies was observedbetween mild and severe patients respectively although itwas not found in HC (Figure 4(c)) Interestingly IL-6 levelshowed a significant positive correlation with IL-21 level inmild and severe-HFMD patients respectively (Figure 4(d))

34 Correlation between Sera IL-21 and IL-6 and Specific NAb-EV71 in Children with Mild and Severe Infections Recent

studies demonstrated that the levels of both IL-21 andIL-6 cytokines were significantly associated with antibodyexpression [14 21] In this study we found that the concen-trations of serum IL-21 were positively correlated with that ofspecific NAb-EV71 in mild or severe patients although thiscorrelation was not observed in HC (Figure 5(a)) Howeverno significant association was noted between serum IL-6levels and specific NAb-EV71 concentrations in any of thethree groups tested respectively (Figure 5(b))

35 Expression of Bcl-6 IL-21 and IL-6 mRNA in Childrenwith Mild and Severe Infections Previous studies indicatedthat Bcl-6 was a pivotal transcription factor for CD4+ T celldifferentiation into TFH cells [14 18 26] Both IL-21 and IL-6 share cooperative functions in TFH differentiation and Bcell immunity [21] Here we assessed the expression of Bcl-6IL-21 and IL-6 mRNA in HC (119899 = 15) mild (119899 = 10) andsevere (119899 = 12) EV71-patients The IL-6 mRNA expressionin severe patients or mild patients was significantly higher

6 Journal of Immunology Research

0

200

400

600

NAb

-EV

71 ti

tres ns

HC Mild Severe

lowastlowastlowast

lowastlowastlowast

(a)

00 5 10 15 20

200

400

600

NAb

-EV

71 ti

tres

MildP lt 00001

CXCR5+CD4+ in CD4

+ T cells ()

r = 08581

0

10

20

30

40

NAb

-EV

71 ti

tres

3 4 5 6

HCP = 02560

CXCR5+CD4+ in CD4

+ T cells ()

r = 01771

0 5 10 15 200

200

400

600

NAb

-EV

71 ti

tres

SevereP = 00043

CXCR5+CD4+ in CD4

+ T cells ()

r = 04992

(b)

000 05 10 15 20 25

10

20

30

40

NAb

-EV

71 ti

tres

P = 05120

PD-1+CXCR5+CD4+ TFH cells ()

r = 01028

0 2 4 60

200

400

600

NAb

-EV

71 ti

tres

P lt 00001

PD-1+CXCR5+CD4+ TFH cells ()

r = 06637

0 2 4 860

200

400

600

NAb

-EV

71 ti

tres

P = 00027

PD-1+CXCR5+CD4+ TFH cells ()

r = 05196

(c)

0 5 10 150

200

400

600

NAb

-EV

71 ti

tres

P = 00103

ICOS+CXCR5+CD4+ TFH cells ()

r = 04542

0 2 4 860

200

400

600

NAb

-EV

71 ti

tres

P = 00039

ICOS+CXCR5+CD4+ TFH cells ()

r = 05190

00 05 10 15 200

10

20

30

40

NAb

-EV

71 ti

tres

P = 07634

ICOS+CXCR5+CD4+ TFH cells ()

r = 004728

(d)

Figure 3 Correlation of specific NAb-EV71 titres and circulating CXCR5+CD4+ TFH cells with PD-1high and ICOShigh expression in EV71-infected patients (a) Titres of specific NAb-EV71 in sera from HC and EV71-infected patients including mild and severe cases Relationshipbetween the titres of specific NAb-EV71 and the frequency of circulating CXCR5+CD4+ TFH cells (b) PD-1+CXCR5+CD4+ TFH cells (c)and ICOS+CXCR5+CD4+ TFH cells (d) Data shown were the mean plusmn SD The horizontal lines show the median lowast119875 lt 005 lowastlowast119875 lt 001lowastlowastlowast

119875 lt 0001 ns no significant difference

than that in HC but no significant difference of IL-6 mRNAexpression was observed between mild and severe patientsgroups (HC 1008 plusmn 416 mild 1802 plusmn 941 severe 2055 plusmn1138) (Figure 6(a)) Similarly the IL-21 mRNA expressions

in both mild and severe patients were significantly higherthan those inHC but no significant difference of IL-21mRNAwas found between mild patients and severe patients (HC117plusmn048 mild 221plusmn131 severe 269plusmn151) (Figure 6(b))

Journal of Immunology Research 7

0

50

100

150

200

IL-2

1 (p

gm

L)

ns

P lt 00001 P lt 00001 P = 03909

HC Mild Severe

lowastlowastlowast

lowastlowastlowast

0

20

40

60

80

100

IL-6

(pg

mL)

ns

HC Mild Severe

P lt 00001 P lt 00001 P = 03438

lowastlowastlowast

lowastlowastlowast

(a)

3 4 5 60

50

100

150

IL-2

1 (p

gm

L)

P = 00053

0 5 1510 200

50

100

150

200

IL-2

1 (p

gm

L)

P lt 00001

0 5 1510 200

50

100

150

200

IL-2

1 (p

gm

L)

P = 00005

CXCR5+CD4+ in CD4

+ T cells () CXCR5+CD4+ in CD4

+ T cells () CXCR5+CD4+ in CD4

+ T cells ()

r = 05919r = 08479r = 04177

HC Mild Severe

(b)

0 5 1510 200

10

20

30

40

50

IL-6

(pg

mL)

P = 00176

CXCR5+CD4+ in CD4

+ T cells ()

r = 04375

3 4 5 60

5

10

15

20

IL-6

(pg

mL)

P = 04986

CXCR5+CD4+ in CD4

+ T cells ()

r = minus01060

0 5 1510 200

20

40

60

80

100

IL-6

(pg

mL)

P = 00236

CXCR5+CD4+ in CD4

+ T cells ()

r = 04056

(c)

0

5

10

15

20

IL-6

(pg

mL)

0 50 150100IL-21 (pgmL)

P = 02594r = minus01758

0 50 150100 2000

10

20

30

40

50

IL-21 (pgmL)

IL-6

(pg

mL)

P = 00220r = 04237

0 50 150100 200IL-21 (pgmL)

0

20

40

60

80

100

IL-6

(pg

mL)

P = 00418r = 03677

(d)

Figure 4 Correlation of cytokines levels and circulating CXCR5+CD4+ TFH cells in EV71-infected patients including mild and severe cases(a) Cytokines levels of IL-21 and IL-6 in sera fromHC and EV71-infected patients (b) Relationship between the IL-21 levels and the frequencyof circulating CXCR5+CD4+ TFH cells (c) Relationship between the IL-6 levels and the frequency of circulating CXCR5+CD4+ TFH cells(d) Relationship between the IL-6 levels and IL-21 levels Data shown were the mean plusmn SDThe horizontal lines show the median lowast119875 lt 005lowastlowast

119875 lt 001 lowastlowastlowast119875 lt 0001 ns no significant difference

8 Journal of Immunology Research

0

10

20

30

40

IL-21 (pgmL)

NAb

-EV

71 ti

tres

HC

0 50 100 150

P = 01036

0

200

400

600

IL-21 (pgmL)

NAb

-EV

71 ti

tres

Severe

500 100 200150

P = 00182

minus200

0

200

400

600

IL-21 (pgmL)

NAb

-EV

71 ti

tres

Mild

50 100 200150

P lt 00001

r = 04216

r = 07174

r = 02516

(a)

0

10

20

30

40

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 5 10 2015

P = 04936

0

200

400

600

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 10 20 30 40 50

P = 01039

0

200

400

600

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 20 40 60 80 100

P = 00741r = 03253r = 03082r = 01072

(b)

Figure 5 Correlation of specific NAb-EV71 titres and cytokines levels from HC and EV71-infected patients including mild and severe cases(a) Relationship between the titres of specific NAb-EV71 and the serum levels of IL-21 (b) Relationship between the titres of specific NAb-EV71 and the serum levels of IL-6

0

10

20

30

40

50

IL-6

mRA

N re

lativ

e exp

ress

ion

ns

HC Mild Severe

lowastlowast

lowast

P = 00116 P = 00078 P = 08175

(a)

0

2

4

6

IL-2

1 m

RAN

relat

ive e

xpre

ssio

n

ns

HC Mild Severe

lowastlowast

lowast

P = 00375 P = 00037 P = 04887

(b)

0

10

20

30

40

50

Bcl-6

mRA

N re

lativ

e exp

ress

ion

nsns ns

HC Mild Severe

P = 04212 P = 04495 P = 09737

(c)

Figure 6 Expression of IL-6 IL-21 and Bcl-6 mRNA in human PBMCs Human PBMCs from 10 mild and 12 severe patients with EV71infection and 15 HC were detected by real-time PCR assay as described in Section 2 (a)The levels of IL-6 mRNA in human PBMCs (b)Thelevels of IL-21 mRNA in human PBMCs (c) The levels of Bcl-6 mRNA in human PBMCs data shown were the mean plusmn SD The horizontallines show the median lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 ns no significant difference

Moreover there were no significant differences for Bcl-6mRNA expression among these groups tested (HC 2334 plusmn1292 mild 1845plusmn1028 severe 1954plusmn1332) (Figure 6(c))

4 Discussion

There aremore than onemillionHFMD cases and 200 deathseach year since 2008 in China [36] EV71 is one of thecommon causative factors of HFMD in young children less

than 5 years of age [3 5] Although mild diseases are the pre-dominant clinical features of EV71 infection severe diseaseswith neurological involvement and the fetal cases of HFMDare closely associated with EV71 infection identified by PCRandor virus isolation [6ndash8] In our study compared to mildpatients the age of severe patients wasmainly observed below3 years (severe versus mild cases 119875 = 00016) and evaluatedevidences with WBC numbers (severe versus mild cases 119875 lt00001 mild cases versus HC 119875 = 00035) and serum CRPlevels (severe versusmild cases119875 lt 00001 mild cases versus

Journal of Immunology Research 9

HC 119875 lt 00001) suggested that inappropriate inflammationmay be elicited in the progression of HFMD in the acutephase of EV71 infection whichwas consistentwith previouslyreported data [7 31 32]

Previous studies have demonstrated that both humoraland cellular immune responses such as antibodies B and Tcells including Th1Th2 and Th17 and other T cell lineagesand related cytokines play a critical role in protection fromEV71 infection in animal models [4 34 35] In humans EV71infection induced imbalance of TB cells and their relatedcytokines and even caused a systemic dysregulation of theimmune system [31ndash33] Recent studies show that follicularhelper T cells (TFH) a new T cell subpopulation play apivotal role in regulating immune responses and have beenassociated with many diseases [13 20 28] Simpson et alreported an increased frequency of circulatingCXCR5+CD4+TFH cells in patients with systemic lupus erythematosus(SLE) and Sjogrenrsquos syndrome [30] Feng et al demonstratedthat the frequency of circulating TFH cells was significantlyincreased in patients with HBV and these circulating TFHcells participated in the HBV-related immune responses[28] However the potential role of circulating TFH cells inpatients with EV71 infection is not yet known In the presentwork we firstly examined the frequency of circulating TFHcells in mild and severe patients with EV71 infection andhealthy controlsThe results demonstrated that the frequencyof circulating CXCR5+CD4+ TFH cells in mild and severepatients in the acute stage of EV71 infection was significantlyhigher than that of HC (severe or mild cases versus HC119875 lt 005) respectively However severe patients did notdiffer significantly from mild patients in the percentage ofperipheral blood CXCR5+CD4+ TFH cells (severe versusmild cases 119875 = 05151) These findings clearly indicatedthat TFH cells played an important role in the EV71-inducedimmune responses

Simpson et al detected that the high frequency of circu-lating TFH cells with high expression of ICOS andor PD-1 molecules was associated with the severity of end-organinvolvement in patients with systemic lupus erythematosus(SLE) and Sjogrenrsquos syndrome [30] Zhu et al demonstratedthe increased frequency of TFH cells with highly expressedICOS andor PD-1 in patients with autoimmune thyroiddisease and rheumatoid arthritis respectively and both ICOSand PD-1 molecules are closely positively correlated withthe function of TFH cells [13] We detected an enhancedfrequency of PD-1high and ICOShighCXCR5+CD4+ TFH cellsin the mild and severe EV71-induced patients comparedto HC (severe or mild cases versus HC 119875 lt 005) Inaddition a closely positive correlation was observed betweenthe frequencies of these two subpopulations of PD-1high andICOShighCXCR5+CD4+ T cells These data supported thedefinition of circulating TFH cells However the percentageof circulating TFH cells with ICOShigh and PD-1high expres-sion revealed no significant difference between the mild andsevere patients (severe versus mild cases PD-1high 119875 =01085 severe versus mild cases ICOShigh 119875 = 01930)In other words EV71 increased the frequency of circulatingTFH cells with ICOShigh and PD-1high expression that was not

notably associated with the severity of patients in the acutestage of EV71 infection

It is known that TFH cells are critical for promoting thedevelopment of antibody response by helper B cells in GC[10 11] Several seroepidemiologic investigations indicatedthat the neutralizing antibody can protect adults as well aschildren from EV71 infection once again although the titersof specific neutralizing antibody were not correlated withseverity of the disease with EV71 infection [3ndash6 34 35]Why is that To date that is not well understood Our dataalso indicated that the serum titers of specific NAb-EV71in patients with acute stage of EV71 infection were higherthan those in HC (severe or mild cases versus HC 119875 lt00001) but no significant difference was observed betweenmild and severe patients groups (severe versus mild cases119875 = 09693) which corresponded to previous studies [3]Additionally we found that enhanced frequency of circulat-ing CXCR5+CD4+ TFH PD-1highCXCR5+CD4+ TFH andICOShighCXCR5+CD4+ TFH cells was notably positively cor-related with the titres of specific NAb-EV71 in patients withEV71 infection and a significant associationwas not observedbetween themild and severe patients respectivelyThese dataalso indicated that TFH cells with ICOShigh and PD-1highexpression participated in regulating antibody responses inthe acute stage of EV71 infection at the same time whichmight give a good explanation to why there is no significantdifference between the levels of specific NAb-EV71 in bothmild and severe patients with EV71 infection

Recent studies demonstrated that not only did TFH cellssecrete IL-21 but IL-21 could also drive GC formation andTFH cell generation and function and promote terminalmature of B cell in GC Ig class-switching and antibodyresponses [10 11 20] IL-6 has also induced IL-21 productionand TFH cell generation and mice lacking both IL-6 andIL-21 are unable to generate TFH cell-dependent immuneresponses [20 21] Bcl-6 transcription factor is selectivelyexpressed in TFH cells which is required for programmingof TFH cell generation and Bcl-6 expression is regulated byIL-6 and IL-21 [14 21] In current study we found that thelevels of sera IL-21 and IL-6 concentrations were significantlyhigher in both mild and severe patients with EV71 infectionthan those in HC (severe ormild cases versus HC IL-21IL-6119875 lt 00001) and they were found to be positively correlatedwith the frequency of circulating CXCR5+CD4+ TFH cellsrespectively which suggested that IL-21 and IL-6 might playan important role in the generation of TFH cells In additiona strongly positive correlation was observed between sera IL-21 and NAb-EV71 as well as between sera IL-21 and IL-6indicating that IL-21 and IL-6 could promote the producingof specific NAb-EV71 and IL-6 might help in production ofIL-21 cytokines Furthermore PCR analysis indicated thatthe expression of IL-21 and IL-6 mRNA was significantlyincreased in both mild and severe patients compared to HC(severe or mild cases versus HC IL-21IL-6 mRNA 119875 lt005) However no difference of IL-21 and IL-6 cytokinesand mRNA expression was found between mild and severepatients (severe versus mild cases IL-21IL-6 cytokines 119875 =0390903438 IL-21IL-6 mRNA 119875 = 0488708175) these

10 Journal of Immunology Research

findings indicated that the expression of IL-21 or IL-6 wasnot notably related with severity of disease with acute stage ofEV71 infection and the IL-6 high expression was consistentwith pervious study [7] Additionally no significant differ-ence of peripheral blood Bcl-6mRNA expression amongHCmild and severe patients is consistent with pervious studywhich might be associated with formation of memory ofTFH cells [30 37] These data revealed that IL-21 and IL-6 participated in the generation of TFH cells and specificNAb-EV71 immune responses which were not significantlyassociated with the severity of patients in the acute stage ofEV71 infection

In conclusion our data indicated that increased freque-ncy of circulating TFH cells with ICOShigh and PD-1highexpression was significantly and positively associated withserum specific NAb-EV71 level and IL-21 and IL-6 con-centration in patients with EV71 infection suggesting thatTFH cells and associated cytokines might play a criticalrole in regulating humoral responses in the acute stage ofEV71 infection Further studies on the role of TFH cells inregulating B cell response and antibody production duringthe disease progression of EV71 infection will provide newinsight into understanding the immune pathogenesis of EV71infection in human

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

JianpingWu and David Cui contributed equally to this work

Acknowledgment

This study was supported by the National Key Programs forInfectious Diseases of China (Grant no 2012ZX10004-210)

References

[1] D Nasri L Bouslama S Pillet T Bourlet M Aouni and BPozzetto ldquoBasic rationale current methods and future direc-tions for molecular typing of human enterovirusrdquo ExpertReview of Molecular Diagnostics vol 7 no 4 pp 419ndash434 2007

[2] S-C Chen H-L Chang T-R Yan Y-T Cheng and K-T Chen ldquoAn eight-year study of epidemiologic features ofenterovirus 71 infection in Taiwanrdquo The American Journal ofTropical Medicine and Hygiene vol 77 no 1 pp 188ndash191 2007

[3] C Yang C Deng J Wan L Zhu and Q Leng ldquoNeutralizingantibody response in the patients with hand foot and mouthdisease to enterovirus 71 and its clinical implicationsrdquo VirologyJournal vol 8 article 306 2011

[4] QMao N Li X Yu et al ldquoAntigenicity animal protective effectand genetic characteristics of candidate vaccine strains ofenterovirus 71rdquo Archives of Virology vol 157 no 1 pp 37ndash412012

[5] M Zeng N F El Khatib S Tu et al ldquoSeroepidemiology ofEnterovirus 71 infection prior to the 2011 season in children in

Shanghairdquo Journal of Clinical Virology vol 53 no 4 pp 285ndash289 2012

[6] W-C Huang L-M Huang C-L Kao et al ldquoSeroprevalence ofenterovirus 71 and no evidence of crossprotection of enterovirus71 antibody against the other enteroviruses in kindergartenchildren in Taipei cityrdquo Journal of Microbiology Immunologyand Infection vol 45 no 2 pp 96ndash101 2012

[7] L-Y Chang C A Hsiung C-Y Lu et al ldquoStatus of cellularrather than humoral immunity is correlated with clinicaloutcome of enterovirus 71rdquo Pediatric Research vol 60 no 4 pp466ndash471 2006

[8] S-M Wang H-Y Lei K-J Huang et al ldquoPathogenesis ofenterovirus 71 brainstem encephalitis in pediatric patients rolesof cytokines and cellular immune activation in patients withpulmonary edemardquo Journal of Infectious Diseases vol 188 no4 pp 564ndash570 2003

[9] F D Finkelman J Holmes I M Katona et al ldquoLymphokinecontrol of in vivo immunoglobulin isotype selectionrdquo AnnualReview of Immunology vol 8 pp 303ndash330 1990

[10] C S Ma S Suryani D T Avery et al ldquoEarly commitment ofnave human CD4+ T cells to the T follicular helper (FH) celllineage is induced by IL-12rdquo Immunology and Cell Biology vol87 no 8 pp 590ndash600 2009

[11] T R Mosmann and R L Coffman ldquoTH1 and TH2 cells differ-ent patterns of lymphokine secretion lead to different functionalpropertiesrdquo Annual Review of Immunology vol 7 pp 145ndash1731989

[12] L E Harrington R D Hatton P R Mangan et al ldquoInterleukin17-producing CD4+ effector T cells develop via a lineage distinctfrom the T helper type 1 and 2 lineagesrdquo Nature Immunologyvol 6 no 11 pp 1123ndash1132 2005

[13] C Zhu M Jie Y Liu et al ldquoIncreased frequency of follicularhelper T cells in patients with autoimmune thyroid diseaserdquoJournal of Clinical Endocrinology and Metabolism vol 97 no3 pp 943ndash950 2012

[14] M A Kroenke D Eto M Locci et al ldquoBcl6 andMaf cooperateto instruct human follicular helper CD4 T cell differentiationrdquoJournal of Immunology vol 188 no 8 pp 3734ndash3744 2012

[15] D Breitfeld L Ohl E Kremmer et al ldquoFollicular B helper Tcells express CXC chemokine receptor 5 localize to B cellfollicles and support immunoglobulin productionrdquoThe Journalof Experimental Medicine vol 192 no 11 pp 1545ndash1551 2000

[16] P Schaerli K Willimann A B Lang M Lipp P Loetscherand B Moser ldquoCXC chemokine receptor 5 expression definesfollicular homing T cells with B cell helper functionrdquo Journal ofExperimental Medicine vol 192 no 11 pp 1553ndash1562 2000

[17] C N Arnold D J Campbell M Lipp and E C Butcher ldquoThegerminal center response is impaired in the absence of T cell-expressedCXCR5rdquoEuropean Journal of Immunology vol 37 no1 pp 100ndash109 2007

[18] N Fazilleau L Mark L J McHeyzer-Williams and M GMcHeyzer-Williams ldquoFollicular helper T cells lineage andlocationrdquo Immunity vol 30 no 3 pp 324ndash335 2009

[19] N M Haynes C D C Allen R Lesley K M Ansel N Killeenand J G Cyster ldquoRole of CXCR5 and CCR7 in follicular Thcell positioning and appearance of a programmed cell deathgene-1High germinal center-associated subpopulationrdquo Journalof Immunology vol 179 no 8 pp 5099ndash5108 2007

[20] R Spolski andW J Leonard ldquoIL-21 andT follicular helper cellsrdquoInternational Immunology vol 22 no 1 Article ID dxp112 pp7ndash12 2009

Journal of Immunology Research 11

[21] D Eto C Lao D DiToro et al ldquoIL-21 and IL-6 are critical fordifferent aspects of B cell immunity and redundantly induceoptimal follicular helper CD4 T cell (Tfh) differentiationrdquo PLoSONE vol 6 no 3 Article ID e17739 2011

[22] C Dong A E Juedes U-A Temann et al ldquoICOS co-stimu-linebreak latory receptor is essential for T-cell activation andfunctionrdquo Nature vol 409 no 6816 pp 97ndash101 2001

[23] T Okazaki and T Honjo ldquoThe PD-1-PD-L pathway in immuno-logical tolerancerdquo Trends in Immunology vol 27 no 4 pp 195ndash201 2006

[24] I Yusuf R Kageyama L Monticelli et al ldquoGerminal center Tfollicular helper cell IL-4 production is dependent on signalinglymphocytic activation molecule receptor (CD150)rdquo Journal ofImmunology vol 185 no 1 pp 190ndash202 2010

[25] D Yu and C G Vinuesa ldquoThe elusive identity of T follicularhelper cellsrdquo Trends in Immunology vol 31 no 10 pp 377ndash3832010

[26] M A Linterman R J Rigby RWong et al ldquoRoquin differenti-ates the specialized functions of duplicated T cell costimulatoryreceptor genes CD28 and ICOSrdquo Immunity vol 30 no 2 pp228ndash241 2009

[27] M Locci C Havenar-Daughton E Landais et al ldquoHuman cir-culating PD-1+1CXCR3minusCXCR5+ memory Tfh cells are highlyfunctional and correlate with broadly neutralizing HIV anti-body responsesrdquo Immunity vol 39 no 4 pp 758ndash769 2013

[28] J Feng L Lu C Hua et al ldquoHigh frequency of CD4+CXCR5+TFH cells in patients with immune-active chronic hepatitis BrdquoPLoS ONE vol 6 no 7 Article ID e21698 2011

[29] C Petrovas T Yamamoto M Y Gerner et al ldquoCD4 T follicularhelper cell dynamics during SIV infectionrdquo Journal of ClinicalInvestigation vol 122 no 9 pp 3281ndash3294 2012

[30] N Simpson P A Gatenby A Wilson et al ldquoExpansion ofcirculating T cells resembling follicular helper T cells is a fixedphenotype that identifies a subset of severe systemic lupuserythematosusrdquo Arthritis and Rheumatism vol 62 no 1 pp234ndash244 2010

[31] S Li C Cai J Feng et al ldquoPeripheral T lymphocyte subset imb-alances in children with enterovirus 71-induced hand foot andmouth diseaserdquo Virus Research vol 180 pp 84ndash91 2014

[32] J Chen J Tong H Liu et al ldquoIncreased frequency ofTh17 cellsin the peripheral blood of children infectedwith enterovirus 71rdquoJournal of Medical Virology vol 84 no 5 pp 763ndash767 2012

[33] J Xie Y Jiao Z Qiu Q Li and T Li ldquoSignificant elevation of Bcells at the acute stage in enterovirus 71-infected children withcentral nervous system involvementrdquo Scandinavian Journal ofInfectious Diseases vol 42 no 11-12 pp 931ndash935 2010

[34] Y Liang X Zhou E Yang et al ldquoAnalysis of the Th1Th2reaction in the immune response induced by EV71 inacti-vated vaccine in neonatal rhesus monkeysrdquo Journal of ClinicalImmunology vol 32 no 5 pp 1048ndash1058 2012

[35] Y-W Lin K-C Chang C-M Kao S-P Chang Y-Y Tungand S-H Chen ldquoLymphocyte and antibody responses reduceenterovirus 71 lethality in mice by decreasing tissue viral loadsrdquoJournal of Virology vol 83 no 13 pp 6477ndash6483 2009

[36] National notifiable diseases announced by National Health andFamily Planning Commission of the Peoplersquos Republic of Chinahttpwwwnhfpcgovcnzhuzhanyqxxlistsshtml

[37] R Morita N Schmitt S-E Bentebibel et al ldquoHuman bloodCXCR5+CD4+ T cells are counterparts of T follicular cellsand contain specific subsets that differentially support antibodysecretionrdquo Immunity vol 34 no 1 pp 108ndash121 2011

4 Journal of Immunology Research

MildHC Severe

844 688

Q7928

Q3000

Q4

000

Q1

000

Q4

000

Q1720

Q2503

Q2

950

Q3 Q8

321

Q6556

349

CD4 FITC SSC-height subset728

FSC-height SSC-height subset

SSC-

H

SSC-

heig

ht

Q5

CD4+

104

104

800

1 k

1 k

600

400

200

0

8006004002000

SSC-

H

SSC-

heig

ht 800

1 k

600

400

200

0

103

103

102

102

101

101

100

100

CXCR

5+

CD4+

104

104

103

103

102

102

101

101

100

100

CD4+

104

104

103

103

102

102

101

101

100

100

104

103

102

101

100

(a)

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

104

103

102

101

100

100

104

103

102

101

100

925

Q8

359

Q5144

Q6

245

Q7859

Q8

388

Q5332

Q6

690 844

Q8 Q7

556

Q5321

Q6

688

Q7

PD-1+ PD-1+

CXCR

5+

PD-1+

(b)

817

Q12

488

Q11

Q9232

Q10

111820

Q12

583

Q11

Q9293

Q10

924857

Q12

329

Q11

Q9174

Q10

924

CXCR

5+

104

104

103

103

102

102

101

101

100

100

104

104

103

103

102

102

101

101

100

104

103

102

101

100

100

104

103

102

101

100

ICOS+ ICOS+ ICOS+

(c)

Figure 1 Detection of circulating CXCR5+CD4+ TFH cells with ICOShigh and PD-1high expression in peripheral blood of EV71-infectedpatients andHC Peripheral bloodmononuclear cells (PBMCs) frompatientswith EV71 infection andHCwere stainedwith labeled antibodiesand analyzed by flow cytometry as described in Section 2 The cells were gated initially on living lymphocytes (upper left) and then on CD4+

T cells (upper right) Representative plots of CXCR5+CD4+ TFH cells in the total CD4+ T cells (a) PD-1highCXCR5+CD4+ TFH cells (b) andCOShighCXCR5+CD4+ TFH cells (c)

Journal of Immunology Research 5

0

5

10

15

20

HC Mild Severe

nslowastlowastlowast

lowastlowastlowast

P = 00021 P = 05151P lt 00001

CXCR

5+

CD4+

in C

D4+

T ce

lls (

)

(a)

0

2

4

8

6

0 5 10 15

P lt 00001

ICOS+CXCR5+CD4+ TFH cells ()

r = 07220

PD-1

+CX

CR5+

CD4+

TFH

cells

()

(b)

0

2

4

6

8

HC Mild Severe

nslowastlowastlowast

lowastlowastlowastP = 00002 P = 01085P lt 00001

PD-1

+CX

CR5+

CD4+

in C

D4+

T ce

lls (

)

(c)

0HC Mild Severe

5

10

15

ns

lowastlowastlowastP = 00037 P = 01930P = 00005

lowastlowastlowast

ICO

S+CX

CR5+

CD4+

in C

D4+

T ce

lls (

)

(d)

Figure 2 Increased percentages of circulating CXCR5+CD4+ TFH cells with ICOShigh and PD-1high expression in peripheral blood of EV71-infected patients The percentages of CXCR5+CD4+ TFH cells in the total CD4+ T cells (a) the correlation between the percentages of PD-1+CXCR5+CD4+ TFH cells and ICOS+CXCR5+CD4+ TFH cells from EV71-infected patients (119899 = 60) (b) PD-1+CXCR5+CD4+ TFH cells (c)and ICOS+CXCR5+CD4+ TFH cells (d) Data shown were the mean plusmn SD (standard deviation SD) The horizontal lines show the medianlowast

119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 ns no significant difference

had critical roles in differentiation and function of follicularhelper CD4+ T cells [14 21 30] The levels of cytokinesboth IL-21 (HC 6665 plusmn 1588 mild 9234 plusmn 3326 severe10146plusmn2682) and IL-6 (HC 494plusmn243 mild 1768plusmn1003severe 2545 plusmn 2152) in sera from the patients with EV-71infection were notably higher than those of HC althoughno significant difference was found in both mild and severechildren respectively (Figure 4(a)) The levels of serum IL-21 were strongly positively correlated with the frequencies ofTFH cells amongHCmild and severe patients (Figure 4(b))Interestingly a significant positive correlation between IL-6levels andCXCR5+CD4+ TFH cells frequencies was observedbetween mild and severe patients respectively although itwas not found in HC (Figure 4(c)) Interestingly IL-6 levelshowed a significant positive correlation with IL-21 level inmild and severe-HFMD patients respectively (Figure 4(d))

34 Correlation between Sera IL-21 and IL-6 and Specific NAb-EV71 in Children with Mild and Severe Infections Recent

studies demonstrated that the levels of both IL-21 andIL-6 cytokines were significantly associated with antibodyexpression [14 21] In this study we found that the concen-trations of serum IL-21 were positively correlated with that ofspecific NAb-EV71 in mild or severe patients although thiscorrelation was not observed in HC (Figure 5(a)) Howeverno significant association was noted between serum IL-6levels and specific NAb-EV71 concentrations in any of thethree groups tested respectively (Figure 5(b))

35 Expression of Bcl-6 IL-21 and IL-6 mRNA in Childrenwith Mild and Severe Infections Previous studies indicatedthat Bcl-6 was a pivotal transcription factor for CD4+ T celldifferentiation into TFH cells [14 18 26] Both IL-21 and IL-6 share cooperative functions in TFH differentiation and Bcell immunity [21] Here we assessed the expression of Bcl-6IL-21 and IL-6 mRNA in HC (119899 = 15) mild (119899 = 10) andsevere (119899 = 12) EV71-patients The IL-6 mRNA expressionin severe patients or mild patients was significantly higher

6 Journal of Immunology Research

0

200

400

600

NAb

-EV

71 ti

tres ns

HC Mild Severe

lowastlowastlowast

lowastlowastlowast

(a)

00 5 10 15 20

200

400

600

NAb

-EV

71 ti

tres

MildP lt 00001

CXCR5+CD4+ in CD4

+ T cells ()

r = 08581

0

10

20

30

40

NAb

-EV

71 ti

tres

3 4 5 6

HCP = 02560

CXCR5+CD4+ in CD4

+ T cells ()

r = 01771

0 5 10 15 200

200

400

600

NAb

-EV

71 ti

tres

SevereP = 00043

CXCR5+CD4+ in CD4

+ T cells ()

r = 04992

(b)

000 05 10 15 20 25

10

20

30

40

NAb

-EV

71 ti

tres

P = 05120

PD-1+CXCR5+CD4+ TFH cells ()

r = 01028

0 2 4 60

200

400

600

NAb

-EV

71 ti

tres

P lt 00001

PD-1+CXCR5+CD4+ TFH cells ()

r = 06637

0 2 4 860

200

400

600

NAb

-EV

71 ti

tres

P = 00027

PD-1+CXCR5+CD4+ TFH cells ()

r = 05196

(c)

0 5 10 150

200

400

600

NAb

-EV

71 ti

tres

P = 00103

ICOS+CXCR5+CD4+ TFH cells ()

r = 04542

0 2 4 860

200

400

600

NAb

-EV

71 ti

tres

P = 00039

ICOS+CXCR5+CD4+ TFH cells ()

r = 05190

00 05 10 15 200

10

20

30

40

NAb

-EV

71 ti

tres

P = 07634

ICOS+CXCR5+CD4+ TFH cells ()

r = 004728

(d)

Figure 3 Correlation of specific NAb-EV71 titres and circulating CXCR5+CD4+ TFH cells with PD-1high and ICOShigh expression in EV71-infected patients (a) Titres of specific NAb-EV71 in sera from HC and EV71-infected patients including mild and severe cases Relationshipbetween the titres of specific NAb-EV71 and the frequency of circulating CXCR5+CD4+ TFH cells (b) PD-1+CXCR5+CD4+ TFH cells (c)and ICOS+CXCR5+CD4+ TFH cells (d) Data shown were the mean plusmn SD The horizontal lines show the median lowast119875 lt 005 lowastlowast119875 lt 001lowastlowastlowast

119875 lt 0001 ns no significant difference

than that in HC but no significant difference of IL-6 mRNAexpression was observed between mild and severe patientsgroups (HC 1008 plusmn 416 mild 1802 plusmn 941 severe 2055 plusmn1138) (Figure 6(a)) Similarly the IL-21 mRNA expressions

in both mild and severe patients were significantly higherthan those inHC but no significant difference of IL-21mRNAwas found between mild patients and severe patients (HC117plusmn048 mild 221plusmn131 severe 269plusmn151) (Figure 6(b))

Journal of Immunology Research 7

0

50

100

150

200

IL-2

1 (p

gm

L)

ns

P lt 00001 P lt 00001 P = 03909

HC Mild Severe

lowastlowastlowast

lowastlowastlowast

0

20

40

60

80

100

IL-6

(pg

mL)

ns

HC Mild Severe

P lt 00001 P lt 00001 P = 03438

lowastlowastlowast

lowastlowastlowast

(a)

3 4 5 60

50

100

150

IL-2

1 (p

gm

L)

P = 00053

0 5 1510 200

50

100

150

200

IL-2

1 (p

gm

L)

P lt 00001

0 5 1510 200

50

100

150

200

IL-2

1 (p

gm

L)

P = 00005

CXCR5+CD4+ in CD4

+ T cells () CXCR5+CD4+ in CD4

+ T cells () CXCR5+CD4+ in CD4

+ T cells ()

r = 05919r = 08479r = 04177

HC Mild Severe

(b)

0 5 1510 200

10

20

30

40

50

IL-6

(pg

mL)

P = 00176

CXCR5+CD4+ in CD4

+ T cells ()

r = 04375

3 4 5 60

5

10

15

20

IL-6

(pg

mL)

P = 04986

CXCR5+CD4+ in CD4

+ T cells ()

r = minus01060

0 5 1510 200

20

40

60

80

100

IL-6

(pg

mL)

P = 00236

CXCR5+CD4+ in CD4

+ T cells ()

r = 04056

(c)

0

5

10

15

20

IL-6

(pg

mL)

0 50 150100IL-21 (pgmL)

P = 02594r = minus01758

0 50 150100 2000

10

20

30

40

50

IL-21 (pgmL)

IL-6

(pg

mL)

P = 00220r = 04237

0 50 150100 200IL-21 (pgmL)

0

20

40

60

80

100

IL-6

(pg

mL)

P = 00418r = 03677

(d)

Figure 4 Correlation of cytokines levels and circulating CXCR5+CD4+ TFH cells in EV71-infected patients including mild and severe cases(a) Cytokines levels of IL-21 and IL-6 in sera fromHC and EV71-infected patients (b) Relationship between the IL-21 levels and the frequencyof circulating CXCR5+CD4+ TFH cells (c) Relationship between the IL-6 levels and the frequency of circulating CXCR5+CD4+ TFH cells(d) Relationship between the IL-6 levels and IL-21 levels Data shown were the mean plusmn SDThe horizontal lines show the median lowast119875 lt 005lowastlowast

119875 lt 001 lowastlowastlowast119875 lt 0001 ns no significant difference

8 Journal of Immunology Research

0

10

20

30

40

IL-21 (pgmL)

NAb

-EV

71 ti

tres

HC

0 50 100 150

P = 01036

0

200

400

600

IL-21 (pgmL)

NAb

-EV

71 ti

tres

Severe

500 100 200150

P = 00182

minus200

0

200

400

600

IL-21 (pgmL)

NAb

-EV

71 ti

tres

Mild

50 100 200150

P lt 00001

r = 04216

r = 07174

r = 02516

(a)

0

10

20

30

40

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 5 10 2015

P = 04936

0

200

400

600

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 10 20 30 40 50

P = 01039

0

200

400

600

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 20 40 60 80 100

P = 00741r = 03253r = 03082r = 01072

(b)

Figure 5 Correlation of specific NAb-EV71 titres and cytokines levels from HC and EV71-infected patients including mild and severe cases(a) Relationship between the titres of specific NAb-EV71 and the serum levels of IL-21 (b) Relationship between the titres of specific NAb-EV71 and the serum levels of IL-6

0

10

20

30

40

50

IL-6

mRA

N re

lativ

e exp

ress

ion

ns

HC Mild Severe

lowastlowast

lowast

P = 00116 P = 00078 P = 08175

(a)

0

2

4

6

IL-2

1 m

RAN

relat

ive e

xpre

ssio

n

ns

HC Mild Severe

lowastlowast

lowast

P = 00375 P = 00037 P = 04887

(b)

0

10

20

30

40

50

Bcl-6

mRA

N re

lativ

e exp

ress

ion

nsns ns

HC Mild Severe

P = 04212 P = 04495 P = 09737

(c)

Figure 6 Expression of IL-6 IL-21 and Bcl-6 mRNA in human PBMCs Human PBMCs from 10 mild and 12 severe patients with EV71infection and 15 HC were detected by real-time PCR assay as described in Section 2 (a)The levels of IL-6 mRNA in human PBMCs (b)Thelevels of IL-21 mRNA in human PBMCs (c) The levels of Bcl-6 mRNA in human PBMCs data shown were the mean plusmn SD The horizontallines show the median lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 ns no significant difference

Moreover there were no significant differences for Bcl-6mRNA expression among these groups tested (HC 2334 plusmn1292 mild 1845plusmn1028 severe 1954plusmn1332) (Figure 6(c))

4 Discussion

There aremore than onemillionHFMD cases and 200 deathseach year since 2008 in China [36] EV71 is one of thecommon causative factors of HFMD in young children less

than 5 years of age [3 5] Although mild diseases are the pre-dominant clinical features of EV71 infection severe diseaseswith neurological involvement and the fetal cases of HFMDare closely associated with EV71 infection identified by PCRandor virus isolation [6ndash8] In our study compared to mildpatients the age of severe patients wasmainly observed below3 years (severe versus mild cases 119875 = 00016) and evaluatedevidences with WBC numbers (severe versus mild cases 119875 lt00001 mild cases versus HC 119875 = 00035) and serum CRPlevels (severe versusmild cases119875 lt 00001 mild cases versus

Journal of Immunology Research 9

HC 119875 lt 00001) suggested that inappropriate inflammationmay be elicited in the progression of HFMD in the acutephase of EV71 infection whichwas consistentwith previouslyreported data [7 31 32]

Previous studies have demonstrated that both humoraland cellular immune responses such as antibodies B and Tcells including Th1Th2 and Th17 and other T cell lineagesand related cytokines play a critical role in protection fromEV71 infection in animal models [4 34 35] In humans EV71infection induced imbalance of TB cells and their relatedcytokines and even caused a systemic dysregulation of theimmune system [31ndash33] Recent studies show that follicularhelper T cells (TFH) a new T cell subpopulation play apivotal role in regulating immune responses and have beenassociated with many diseases [13 20 28] Simpson et alreported an increased frequency of circulatingCXCR5+CD4+TFH cells in patients with systemic lupus erythematosus(SLE) and Sjogrenrsquos syndrome [30] Feng et al demonstratedthat the frequency of circulating TFH cells was significantlyincreased in patients with HBV and these circulating TFHcells participated in the HBV-related immune responses[28] However the potential role of circulating TFH cells inpatients with EV71 infection is not yet known In the presentwork we firstly examined the frequency of circulating TFHcells in mild and severe patients with EV71 infection andhealthy controlsThe results demonstrated that the frequencyof circulating CXCR5+CD4+ TFH cells in mild and severepatients in the acute stage of EV71 infection was significantlyhigher than that of HC (severe or mild cases versus HC119875 lt 005) respectively However severe patients did notdiffer significantly from mild patients in the percentage ofperipheral blood CXCR5+CD4+ TFH cells (severe versusmild cases 119875 = 05151) These findings clearly indicatedthat TFH cells played an important role in the EV71-inducedimmune responses

Simpson et al detected that the high frequency of circu-lating TFH cells with high expression of ICOS andor PD-1 molecules was associated with the severity of end-organinvolvement in patients with systemic lupus erythematosus(SLE) and Sjogrenrsquos syndrome [30] Zhu et al demonstratedthe increased frequency of TFH cells with highly expressedICOS andor PD-1 in patients with autoimmune thyroiddisease and rheumatoid arthritis respectively and both ICOSand PD-1 molecules are closely positively correlated withthe function of TFH cells [13] We detected an enhancedfrequency of PD-1high and ICOShighCXCR5+CD4+ TFH cellsin the mild and severe EV71-induced patients comparedto HC (severe or mild cases versus HC 119875 lt 005) Inaddition a closely positive correlation was observed betweenthe frequencies of these two subpopulations of PD-1high andICOShighCXCR5+CD4+ T cells These data supported thedefinition of circulating TFH cells However the percentageof circulating TFH cells with ICOShigh and PD-1high expres-sion revealed no significant difference between the mild andsevere patients (severe versus mild cases PD-1high 119875 =01085 severe versus mild cases ICOShigh 119875 = 01930)In other words EV71 increased the frequency of circulatingTFH cells with ICOShigh and PD-1high expression that was not

notably associated with the severity of patients in the acutestage of EV71 infection

It is known that TFH cells are critical for promoting thedevelopment of antibody response by helper B cells in GC[10 11] Several seroepidemiologic investigations indicatedthat the neutralizing antibody can protect adults as well aschildren from EV71 infection once again although the titersof specific neutralizing antibody were not correlated withseverity of the disease with EV71 infection [3ndash6 34 35]Why is that To date that is not well understood Our dataalso indicated that the serum titers of specific NAb-EV71in patients with acute stage of EV71 infection were higherthan those in HC (severe or mild cases versus HC 119875 lt00001) but no significant difference was observed betweenmild and severe patients groups (severe versus mild cases119875 = 09693) which corresponded to previous studies [3]Additionally we found that enhanced frequency of circulat-ing CXCR5+CD4+ TFH PD-1highCXCR5+CD4+ TFH andICOShighCXCR5+CD4+ TFH cells was notably positively cor-related with the titres of specific NAb-EV71 in patients withEV71 infection and a significant associationwas not observedbetween themild and severe patients respectivelyThese dataalso indicated that TFH cells with ICOShigh and PD-1highexpression participated in regulating antibody responses inthe acute stage of EV71 infection at the same time whichmight give a good explanation to why there is no significantdifference between the levels of specific NAb-EV71 in bothmild and severe patients with EV71 infection

Recent studies demonstrated that not only did TFH cellssecrete IL-21 but IL-21 could also drive GC formation andTFH cell generation and function and promote terminalmature of B cell in GC Ig class-switching and antibodyresponses [10 11 20] IL-6 has also induced IL-21 productionand TFH cell generation and mice lacking both IL-6 andIL-21 are unable to generate TFH cell-dependent immuneresponses [20 21] Bcl-6 transcription factor is selectivelyexpressed in TFH cells which is required for programmingof TFH cell generation and Bcl-6 expression is regulated byIL-6 and IL-21 [14 21] In current study we found that thelevels of sera IL-21 and IL-6 concentrations were significantlyhigher in both mild and severe patients with EV71 infectionthan those in HC (severe ormild cases versus HC IL-21IL-6119875 lt 00001) and they were found to be positively correlatedwith the frequency of circulating CXCR5+CD4+ TFH cellsrespectively which suggested that IL-21 and IL-6 might playan important role in the generation of TFH cells In additiona strongly positive correlation was observed between sera IL-21 and NAb-EV71 as well as between sera IL-21 and IL-6indicating that IL-21 and IL-6 could promote the producingof specific NAb-EV71 and IL-6 might help in production ofIL-21 cytokines Furthermore PCR analysis indicated thatthe expression of IL-21 and IL-6 mRNA was significantlyincreased in both mild and severe patients compared to HC(severe or mild cases versus HC IL-21IL-6 mRNA 119875 lt005) However no difference of IL-21 and IL-6 cytokinesand mRNA expression was found between mild and severepatients (severe versus mild cases IL-21IL-6 cytokines 119875 =0390903438 IL-21IL-6 mRNA 119875 = 0488708175) these

10 Journal of Immunology Research

findings indicated that the expression of IL-21 or IL-6 wasnot notably related with severity of disease with acute stage ofEV71 infection and the IL-6 high expression was consistentwith pervious study [7] Additionally no significant differ-ence of peripheral blood Bcl-6mRNA expression amongHCmild and severe patients is consistent with pervious studywhich might be associated with formation of memory ofTFH cells [30 37] These data revealed that IL-21 and IL-6 participated in the generation of TFH cells and specificNAb-EV71 immune responses which were not significantlyassociated with the severity of patients in the acute stage ofEV71 infection

In conclusion our data indicated that increased freque-ncy of circulating TFH cells with ICOShigh and PD-1highexpression was significantly and positively associated withserum specific NAb-EV71 level and IL-21 and IL-6 con-centration in patients with EV71 infection suggesting thatTFH cells and associated cytokines might play a criticalrole in regulating humoral responses in the acute stage ofEV71 infection Further studies on the role of TFH cells inregulating B cell response and antibody production duringthe disease progression of EV71 infection will provide newinsight into understanding the immune pathogenesis of EV71infection in human

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

JianpingWu and David Cui contributed equally to this work

Acknowledgment

This study was supported by the National Key Programs forInfectious Diseases of China (Grant no 2012ZX10004-210)

References

[1] D Nasri L Bouslama S Pillet T Bourlet M Aouni and BPozzetto ldquoBasic rationale current methods and future direc-tions for molecular typing of human enterovirusrdquo ExpertReview of Molecular Diagnostics vol 7 no 4 pp 419ndash434 2007

[2] S-C Chen H-L Chang T-R Yan Y-T Cheng and K-T Chen ldquoAn eight-year study of epidemiologic features ofenterovirus 71 infection in Taiwanrdquo The American Journal ofTropical Medicine and Hygiene vol 77 no 1 pp 188ndash191 2007

[3] C Yang C Deng J Wan L Zhu and Q Leng ldquoNeutralizingantibody response in the patients with hand foot and mouthdisease to enterovirus 71 and its clinical implicationsrdquo VirologyJournal vol 8 article 306 2011

[4] QMao N Li X Yu et al ldquoAntigenicity animal protective effectand genetic characteristics of candidate vaccine strains ofenterovirus 71rdquo Archives of Virology vol 157 no 1 pp 37ndash412012

[5] M Zeng N F El Khatib S Tu et al ldquoSeroepidemiology ofEnterovirus 71 infection prior to the 2011 season in children in

Shanghairdquo Journal of Clinical Virology vol 53 no 4 pp 285ndash289 2012

[6] W-C Huang L-M Huang C-L Kao et al ldquoSeroprevalence ofenterovirus 71 and no evidence of crossprotection of enterovirus71 antibody against the other enteroviruses in kindergartenchildren in Taipei cityrdquo Journal of Microbiology Immunologyand Infection vol 45 no 2 pp 96ndash101 2012

[7] L-Y Chang C A Hsiung C-Y Lu et al ldquoStatus of cellularrather than humoral immunity is correlated with clinicaloutcome of enterovirus 71rdquo Pediatric Research vol 60 no 4 pp466ndash471 2006

[8] S-M Wang H-Y Lei K-J Huang et al ldquoPathogenesis ofenterovirus 71 brainstem encephalitis in pediatric patients rolesof cytokines and cellular immune activation in patients withpulmonary edemardquo Journal of Infectious Diseases vol 188 no4 pp 564ndash570 2003

[9] F D Finkelman J Holmes I M Katona et al ldquoLymphokinecontrol of in vivo immunoglobulin isotype selectionrdquo AnnualReview of Immunology vol 8 pp 303ndash330 1990

[10] C S Ma S Suryani D T Avery et al ldquoEarly commitment ofnave human CD4+ T cells to the T follicular helper (FH) celllineage is induced by IL-12rdquo Immunology and Cell Biology vol87 no 8 pp 590ndash600 2009

[11] T R Mosmann and R L Coffman ldquoTH1 and TH2 cells differ-ent patterns of lymphokine secretion lead to different functionalpropertiesrdquo Annual Review of Immunology vol 7 pp 145ndash1731989

[12] L E Harrington R D Hatton P R Mangan et al ldquoInterleukin17-producing CD4+ effector T cells develop via a lineage distinctfrom the T helper type 1 and 2 lineagesrdquo Nature Immunologyvol 6 no 11 pp 1123ndash1132 2005

[13] C Zhu M Jie Y Liu et al ldquoIncreased frequency of follicularhelper T cells in patients with autoimmune thyroid diseaserdquoJournal of Clinical Endocrinology and Metabolism vol 97 no3 pp 943ndash950 2012

[14] M A Kroenke D Eto M Locci et al ldquoBcl6 andMaf cooperateto instruct human follicular helper CD4 T cell differentiationrdquoJournal of Immunology vol 188 no 8 pp 3734ndash3744 2012

[15] D Breitfeld L Ohl E Kremmer et al ldquoFollicular B helper Tcells express CXC chemokine receptor 5 localize to B cellfollicles and support immunoglobulin productionrdquoThe Journalof Experimental Medicine vol 192 no 11 pp 1545ndash1551 2000

[16] P Schaerli K Willimann A B Lang M Lipp P Loetscherand B Moser ldquoCXC chemokine receptor 5 expression definesfollicular homing T cells with B cell helper functionrdquo Journal ofExperimental Medicine vol 192 no 11 pp 1553ndash1562 2000

[17] C N Arnold D J Campbell M Lipp and E C Butcher ldquoThegerminal center response is impaired in the absence of T cell-expressedCXCR5rdquoEuropean Journal of Immunology vol 37 no1 pp 100ndash109 2007

[18] N Fazilleau L Mark L J McHeyzer-Williams and M GMcHeyzer-Williams ldquoFollicular helper T cells lineage andlocationrdquo Immunity vol 30 no 3 pp 324ndash335 2009

[19] N M Haynes C D C Allen R Lesley K M Ansel N Killeenand J G Cyster ldquoRole of CXCR5 and CCR7 in follicular Thcell positioning and appearance of a programmed cell deathgene-1High germinal center-associated subpopulationrdquo Journalof Immunology vol 179 no 8 pp 5099ndash5108 2007

[20] R Spolski andW J Leonard ldquoIL-21 andT follicular helper cellsrdquoInternational Immunology vol 22 no 1 Article ID dxp112 pp7ndash12 2009

Journal of Immunology Research 11

[21] D Eto C Lao D DiToro et al ldquoIL-21 and IL-6 are critical fordifferent aspects of B cell immunity and redundantly induceoptimal follicular helper CD4 T cell (Tfh) differentiationrdquo PLoSONE vol 6 no 3 Article ID e17739 2011

[22] C Dong A E Juedes U-A Temann et al ldquoICOS co-stimu-linebreak latory receptor is essential for T-cell activation andfunctionrdquo Nature vol 409 no 6816 pp 97ndash101 2001

[23] T Okazaki and T Honjo ldquoThe PD-1-PD-L pathway in immuno-logical tolerancerdquo Trends in Immunology vol 27 no 4 pp 195ndash201 2006

[24] I Yusuf R Kageyama L Monticelli et al ldquoGerminal center Tfollicular helper cell IL-4 production is dependent on signalinglymphocytic activation molecule receptor (CD150)rdquo Journal ofImmunology vol 185 no 1 pp 190ndash202 2010

[25] D Yu and C G Vinuesa ldquoThe elusive identity of T follicularhelper cellsrdquo Trends in Immunology vol 31 no 10 pp 377ndash3832010

[26] M A Linterman R J Rigby RWong et al ldquoRoquin differenti-ates the specialized functions of duplicated T cell costimulatoryreceptor genes CD28 and ICOSrdquo Immunity vol 30 no 2 pp228ndash241 2009

[27] M Locci C Havenar-Daughton E Landais et al ldquoHuman cir-culating PD-1+1CXCR3minusCXCR5+ memory Tfh cells are highlyfunctional and correlate with broadly neutralizing HIV anti-body responsesrdquo Immunity vol 39 no 4 pp 758ndash769 2013

[28] J Feng L Lu C Hua et al ldquoHigh frequency of CD4+CXCR5+TFH cells in patients with immune-active chronic hepatitis BrdquoPLoS ONE vol 6 no 7 Article ID e21698 2011

[29] C Petrovas T Yamamoto M Y Gerner et al ldquoCD4 T follicularhelper cell dynamics during SIV infectionrdquo Journal of ClinicalInvestigation vol 122 no 9 pp 3281ndash3294 2012

[30] N Simpson P A Gatenby A Wilson et al ldquoExpansion ofcirculating T cells resembling follicular helper T cells is a fixedphenotype that identifies a subset of severe systemic lupuserythematosusrdquo Arthritis and Rheumatism vol 62 no 1 pp234ndash244 2010

[31] S Li C Cai J Feng et al ldquoPeripheral T lymphocyte subset imb-alances in children with enterovirus 71-induced hand foot andmouth diseaserdquo Virus Research vol 180 pp 84ndash91 2014

[32] J Chen J Tong H Liu et al ldquoIncreased frequency ofTh17 cellsin the peripheral blood of children infectedwith enterovirus 71rdquoJournal of Medical Virology vol 84 no 5 pp 763ndash767 2012

[33] J Xie Y Jiao Z Qiu Q Li and T Li ldquoSignificant elevation of Bcells at the acute stage in enterovirus 71-infected children withcentral nervous system involvementrdquo Scandinavian Journal ofInfectious Diseases vol 42 no 11-12 pp 931ndash935 2010

[34] Y Liang X Zhou E Yang et al ldquoAnalysis of the Th1Th2reaction in the immune response induced by EV71 inacti-vated vaccine in neonatal rhesus monkeysrdquo Journal of ClinicalImmunology vol 32 no 5 pp 1048ndash1058 2012

[35] Y-W Lin K-C Chang C-M Kao S-P Chang Y-Y Tungand S-H Chen ldquoLymphocyte and antibody responses reduceenterovirus 71 lethality in mice by decreasing tissue viral loadsrdquoJournal of Virology vol 83 no 13 pp 6477ndash6483 2009

[36] National notifiable diseases announced by National Health andFamily Planning Commission of the Peoplersquos Republic of Chinahttpwwwnhfpcgovcnzhuzhanyqxxlistsshtml

[37] R Morita N Schmitt S-E Bentebibel et al ldquoHuman bloodCXCR5+CD4+ T cells are counterparts of T follicular cellsand contain specific subsets that differentially support antibodysecretionrdquo Immunity vol 34 no 1 pp 108ndash121 2011

Journal of Immunology Research 5

0

5

10

15

20

HC Mild Severe

nslowastlowastlowast

lowastlowastlowast

P = 00021 P = 05151P lt 00001

CXCR

5+

CD4+

in C

D4+

T ce

lls (

)

(a)

0

2

4

8

6

0 5 10 15

P lt 00001

ICOS+CXCR5+CD4+ TFH cells ()

r = 07220

PD-1

+CX

CR5+

CD4+

TFH

cells

()

(b)

0

2

4

6

8

HC Mild Severe

nslowastlowastlowast

lowastlowastlowastP = 00002 P = 01085P lt 00001

PD-1

+CX

CR5+

CD4+

in C

D4+

T ce

lls (

)

(c)

0HC Mild Severe

5

10

15

ns

lowastlowastlowastP = 00037 P = 01930P = 00005

lowastlowastlowast

ICO

S+CX

CR5+

CD4+

in C

D4+

T ce

lls (

)

(d)

Figure 2 Increased percentages of circulating CXCR5+CD4+ TFH cells with ICOShigh and PD-1high expression in peripheral blood of EV71-infected patients The percentages of CXCR5+CD4+ TFH cells in the total CD4+ T cells (a) the correlation between the percentages of PD-1+CXCR5+CD4+ TFH cells and ICOS+CXCR5+CD4+ TFH cells from EV71-infected patients (119899 = 60) (b) PD-1+CXCR5+CD4+ TFH cells (c)and ICOS+CXCR5+CD4+ TFH cells (d) Data shown were the mean plusmn SD (standard deviation SD) The horizontal lines show the medianlowast

119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 ns no significant difference

had critical roles in differentiation and function of follicularhelper CD4+ T cells [14 21 30] The levels of cytokinesboth IL-21 (HC 6665 plusmn 1588 mild 9234 plusmn 3326 severe10146plusmn2682) and IL-6 (HC 494plusmn243 mild 1768plusmn1003severe 2545 plusmn 2152) in sera from the patients with EV-71infection were notably higher than those of HC althoughno significant difference was found in both mild and severechildren respectively (Figure 4(a)) The levels of serum IL-21 were strongly positively correlated with the frequencies ofTFH cells amongHCmild and severe patients (Figure 4(b))Interestingly a significant positive correlation between IL-6levels andCXCR5+CD4+ TFH cells frequencies was observedbetween mild and severe patients respectively although itwas not found in HC (Figure 4(c)) Interestingly IL-6 levelshowed a significant positive correlation with IL-21 level inmild and severe-HFMD patients respectively (Figure 4(d))

34 Correlation between Sera IL-21 and IL-6 and Specific NAb-EV71 in Children with Mild and Severe Infections Recent

studies demonstrated that the levels of both IL-21 andIL-6 cytokines were significantly associated with antibodyexpression [14 21] In this study we found that the concen-trations of serum IL-21 were positively correlated with that ofspecific NAb-EV71 in mild or severe patients although thiscorrelation was not observed in HC (Figure 5(a)) Howeverno significant association was noted between serum IL-6levels and specific NAb-EV71 concentrations in any of thethree groups tested respectively (Figure 5(b))

35 Expression of Bcl-6 IL-21 and IL-6 mRNA in Childrenwith Mild and Severe Infections Previous studies indicatedthat Bcl-6 was a pivotal transcription factor for CD4+ T celldifferentiation into TFH cells [14 18 26] Both IL-21 and IL-6 share cooperative functions in TFH differentiation and Bcell immunity [21] Here we assessed the expression of Bcl-6IL-21 and IL-6 mRNA in HC (119899 = 15) mild (119899 = 10) andsevere (119899 = 12) EV71-patients The IL-6 mRNA expressionin severe patients or mild patients was significantly higher

6 Journal of Immunology Research

0

200

400

600

NAb

-EV

71 ti

tres ns

HC Mild Severe

lowastlowastlowast

lowastlowastlowast

(a)

00 5 10 15 20

200

400

600

NAb

-EV

71 ti

tres

MildP lt 00001

CXCR5+CD4+ in CD4

+ T cells ()

r = 08581

0

10

20

30

40

NAb

-EV

71 ti

tres

3 4 5 6

HCP = 02560

CXCR5+CD4+ in CD4

+ T cells ()

r = 01771

0 5 10 15 200

200

400

600

NAb

-EV

71 ti

tres

SevereP = 00043

CXCR5+CD4+ in CD4

+ T cells ()

r = 04992

(b)

000 05 10 15 20 25

10

20

30

40

NAb

-EV

71 ti

tres

P = 05120

PD-1+CXCR5+CD4+ TFH cells ()

r = 01028

0 2 4 60

200

400

600

NAb

-EV

71 ti

tres

P lt 00001

PD-1+CXCR5+CD4+ TFH cells ()

r = 06637

0 2 4 860

200

400

600

NAb

-EV

71 ti

tres

P = 00027

PD-1+CXCR5+CD4+ TFH cells ()

r = 05196

(c)

0 5 10 150

200

400

600

NAb

-EV

71 ti

tres

P = 00103

ICOS+CXCR5+CD4+ TFH cells ()

r = 04542

0 2 4 860

200

400

600

NAb

-EV

71 ti

tres

P = 00039

ICOS+CXCR5+CD4+ TFH cells ()

r = 05190

00 05 10 15 200

10

20

30

40

NAb

-EV

71 ti

tres

P = 07634

ICOS+CXCR5+CD4+ TFH cells ()

r = 004728

(d)

Figure 3 Correlation of specific NAb-EV71 titres and circulating CXCR5+CD4+ TFH cells with PD-1high and ICOShigh expression in EV71-infected patients (a) Titres of specific NAb-EV71 in sera from HC and EV71-infected patients including mild and severe cases Relationshipbetween the titres of specific NAb-EV71 and the frequency of circulating CXCR5+CD4+ TFH cells (b) PD-1+CXCR5+CD4+ TFH cells (c)and ICOS+CXCR5+CD4+ TFH cells (d) Data shown were the mean plusmn SD The horizontal lines show the median lowast119875 lt 005 lowastlowast119875 lt 001lowastlowastlowast

119875 lt 0001 ns no significant difference

than that in HC but no significant difference of IL-6 mRNAexpression was observed between mild and severe patientsgroups (HC 1008 plusmn 416 mild 1802 plusmn 941 severe 2055 plusmn1138) (Figure 6(a)) Similarly the IL-21 mRNA expressions

in both mild and severe patients were significantly higherthan those inHC but no significant difference of IL-21mRNAwas found between mild patients and severe patients (HC117plusmn048 mild 221plusmn131 severe 269plusmn151) (Figure 6(b))

Journal of Immunology Research 7

0

50

100

150

200

IL-2

1 (p

gm

L)

ns

P lt 00001 P lt 00001 P = 03909

HC Mild Severe

lowastlowastlowast

lowastlowastlowast

0

20

40

60

80

100

IL-6

(pg

mL)

ns

HC Mild Severe

P lt 00001 P lt 00001 P = 03438

lowastlowastlowast

lowastlowastlowast

(a)

3 4 5 60

50

100

150

IL-2

1 (p

gm

L)

P = 00053

0 5 1510 200

50

100

150

200

IL-2

1 (p

gm

L)

P lt 00001

0 5 1510 200

50

100

150

200

IL-2

1 (p

gm

L)

P = 00005

CXCR5+CD4+ in CD4

+ T cells () CXCR5+CD4+ in CD4

+ T cells () CXCR5+CD4+ in CD4

+ T cells ()

r = 05919r = 08479r = 04177

HC Mild Severe

(b)

0 5 1510 200

10

20

30

40

50

IL-6

(pg

mL)

P = 00176

CXCR5+CD4+ in CD4

+ T cells ()

r = 04375

3 4 5 60

5

10

15

20

IL-6

(pg

mL)

P = 04986

CXCR5+CD4+ in CD4

+ T cells ()

r = minus01060

0 5 1510 200

20

40

60

80

100

IL-6

(pg

mL)

P = 00236

CXCR5+CD4+ in CD4

+ T cells ()

r = 04056

(c)

0

5

10

15

20

IL-6

(pg

mL)

0 50 150100IL-21 (pgmL)

P = 02594r = minus01758

0 50 150100 2000

10

20

30

40

50

IL-21 (pgmL)

IL-6

(pg

mL)

P = 00220r = 04237

0 50 150100 200IL-21 (pgmL)

0

20

40

60

80

100

IL-6

(pg

mL)

P = 00418r = 03677

(d)

Figure 4 Correlation of cytokines levels and circulating CXCR5+CD4+ TFH cells in EV71-infected patients including mild and severe cases(a) Cytokines levels of IL-21 and IL-6 in sera fromHC and EV71-infected patients (b) Relationship between the IL-21 levels and the frequencyof circulating CXCR5+CD4+ TFH cells (c) Relationship between the IL-6 levels and the frequency of circulating CXCR5+CD4+ TFH cells(d) Relationship between the IL-6 levels and IL-21 levels Data shown were the mean plusmn SDThe horizontal lines show the median lowast119875 lt 005lowastlowast

119875 lt 001 lowastlowastlowast119875 lt 0001 ns no significant difference

8 Journal of Immunology Research

0

10

20

30

40

IL-21 (pgmL)

NAb

-EV

71 ti

tres

HC

0 50 100 150

P = 01036

0

200

400

600

IL-21 (pgmL)

NAb

-EV

71 ti

tres

Severe

500 100 200150

P = 00182

minus200

0

200

400

600

IL-21 (pgmL)

NAb

-EV

71 ti

tres

Mild

50 100 200150

P lt 00001

r = 04216

r = 07174

r = 02516

(a)

0

10

20

30

40

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 5 10 2015

P = 04936

0

200

400

600

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 10 20 30 40 50

P = 01039

0

200

400

600

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 20 40 60 80 100

P = 00741r = 03253r = 03082r = 01072

(b)

Figure 5 Correlation of specific NAb-EV71 titres and cytokines levels from HC and EV71-infected patients including mild and severe cases(a) Relationship between the titres of specific NAb-EV71 and the serum levels of IL-21 (b) Relationship between the titres of specific NAb-EV71 and the serum levels of IL-6

0

10

20

30

40

50

IL-6

mRA

N re

lativ

e exp

ress

ion

ns

HC Mild Severe

lowastlowast

lowast

P = 00116 P = 00078 P = 08175

(a)

0

2

4

6

IL-2

1 m

RAN

relat

ive e

xpre

ssio

n

ns

HC Mild Severe

lowastlowast

lowast

P = 00375 P = 00037 P = 04887

(b)

0

10

20

30

40

50

Bcl-6

mRA

N re

lativ

e exp

ress

ion

nsns ns

HC Mild Severe

P = 04212 P = 04495 P = 09737

(c)

Figure 6 Expression of IL-6 IL-21 and Bcl-6 mRNA in human PBMCs Human PBMCs from 10 mild and 12 severe patients with EV71infection and 15 HC were detected by real-time PCR assay as described in Section 2 (a)The levels of IL-6 mRNA in human PBMCs (b)Thelevels of IL-21 mRNA in human PBMCs (c) The levels of Bcl-6 mRNA in human PBMCs data shown were the mean plusmn SD The horizontallines show the median lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 ns no significant difference

Moreover there were no significant differences for Bcl-6mRNA expression among these groups tested (HC 2334 plusmn1292 mild 1845plusmn1028 severe 1954plusmn1332) (Figure 6(c))

4 Discussion

There aremore than onemillionHFMD cases and 200 deathseach year since 2008 in China [36] EV71 is one of thecommon causative factors of HFMD in young children less

than 5 years of age [3 5] Although mild diseases are the pre-dominant clinical features of EV71 infection severe diseaseswith neurological involvement and the fetal cases of HFMDare closely associated with EV71 infection identified by PCRandor virus isolation [6ndash8] In our study compared to mildpatients the age of severe patients wasmainly observed below3 years (severe versus mild cases 119875 = 00016) and evaluatedevidences with WBC numbers (severe versus mild cases 119875 lt00001 mild cases versus HC 119875 = 00035) and serum CRPlevels (severe versusmild cases119875 lt 00001 mild cases versus

Journal of Immunology Research 9

HC 119875 lt 00001) suggested that inappropriate inflammationmay be elicited in the progression of HFMD in the acutephase of EV71 infection whichwas consistentwith previouslyreported data [7 31 32]

Previous studies have demonstrated that both humoraland cellular immune responses such as antibodies B and Tcells including Th1Th2 and Th17 and other T cell lineagesand related cytokines play a critical role in protection fromEV71 infection in animal models [4 34 35] In humans EV71infection induced imbalance of TB cells and their relatedcytokines and even caused a systemic dysregulation of theimmune system [31ndash33] Recent studies show that follicularhelper T cells (TFH) a new T cell subpopulation play apivotal role in regulating immune responses and have beenassociated with many diseases [13 20 28] Simpson et alreported an increased frequency of circulatingCXCR5+CD4+TFH cells in patients with systemic lupus erythematosus(SLE) and Sjogrenrsquos syndrome [30] Feng et al demonstratedthat the frequency of circulating TFH cells was significantlyincreased in patients with HBV and these circulating TFHcells participated in the HBV-related immune responses[28] However the potential role of circulating TFH cells inpatients with EV71 infection is not yet known In the presentwork we firstly examined the frequency of circulating TFHcells in mild and severe patients with EV71 infection andhealthy controlsThe results demonstrated that the frequencyof circulating CXCR5+CD4+ TFH cells in mild and severepatients in the acute stage of EV71 infection was significantlyhigher than that of HC (severe or mild cases versus HC119875 lt 005) respectively However severe patients did notdiffer significantly from mild patients in the percentage ofperipheral blood CXCR5+CD4+ TFH cells (severe versusmild cases 119875 = 05151) These findings clearly indicatedthat TFH cells played an important role in the EV71-inducedimmune responses

Simpson et al detected that the high frequency of circu-lating TFH cells with high expression of ICOS andor PD-1 molecules was associated with the severity of end-organinvolvement in patients with systemic lupus erythematosus(SLE) and Sjogrenrsquos syndrome [30] Zhu et al demonstratedthe increased frequency of TFH cells with highly expressedICOS andor PD-1 in patients with autoimmune thyroiddisease and rheumatoid arthritis respectively and both ICOSand PD-1 molecules are closely positively correlated withthe function of TFH cells [13] We detected an enhancedfrequency of PD-1high and ICOShighCXCR5+CD4+ TFH cellsin the mild and severe EV71-induced patients comparedto HC (severe or mild cases versus HC 119875 lt 005) Inaddition a closely positive correlation was observed betweenthe frequencies of these two subpopulations of PD-1high andICOShighCXCR5+CD4+ T cells These data supported thedefinition of circulating TFH cells However the percentageof circulating TFH cells with ICOShigh and PD-1high expres-sion revealed no significant difference between the mild andsevere patients (severe versus mild cases PD-1high 119875 =01085 severe versus mild cases ICOShigh 119875 = 01930)In other words EV71 increased the frequency of circulatingTFH cells with ICOShigh and PD-1high expression that was not

notably associated with the severity of patients in the acutestage of EV71 infection

It is known that TFH cells are critical for promoting thedevelopment of antibody response by helper B cells in GC[10 11] Several seroepidemiologic investigations indicatedthat the neutralizing antibody can protect adults as well aschildren from EV71 infection once again although the titersof specific neutralizing antibody were not correlated withseverity of the disease with EV71 infection [3ndash6 34 35]Why is that To date that is not well understood Our dataalso indicated that the serum titers of specific NAb-EV71in patients with acute stage of EV71 infection were higherthan those in HC (severe or mild cases versus HC 119875 lt00001) but no significant difference was observed betweenmild and severe patients groups (severe versus mild cases119875 = 09693) which corresponded to previous studies [3]Additionally we found that enhanced frequency of circulat-ing CXCR5+CD4+ TFH PD-1highCXCR5+CD4+ TFH andICOShighCXCR5+CD4+ TFH cells was notably positively cor-related with the titres of specific NAb-EV71 in patients withEV71 infection and a significant associationwas not observedbetween themild and severe patients respectivelyThese dataalso indicated that TFH cells with ICOShigh and PD-1highexpression participated in regulating antibody responses inthe acute stage of EV71 infection at the same time whichmight give a good explanation to why there is no significantdifference between the levels of specific NAb-EV71 in bothmild and severe patients with EV71 infection

Recent studies demonstrated that not only did TFH cellssecrete IL-21 but IL-21 could also drive GC formation andTFH cell generation and function and promote terminalmature of B cell in GC Ig class-switching and antibodyresponses [10 11 20] IL-6 has also induced IL-21 productionand TFH cell generation and mice lacking both IL-6 andIL-21 are unable to generate TFH cell-dependent immuneresponses [20 21] Bcl-6 transcription factor is selectivelyexpressed in TFH cells which is required for programmingof TFH cell generation and Bcl-6 expression is regulated byIL-6 and IL-21 [14 21] In current study we found that thelevels of sera IL-21 and IL-6 concentrations were significantlyhigher in both mild and severe patients with EV71 infectionthan those in HC (severe ormild cases versus HC IL-21IL-6119875 lt 00001) and they were found to be positively correlatedwith the frequency of circulating CXCR5+CD4+ TFH cellsrespectively which suggested that IL-21 and IL-6 might playan important role in the generation of TFH cells In additiona strongly positive correlation was observed between sera IL-21 and NAb-EV71 as well as between sera IL-21 and IL-6indicating that IL-21 and IL-6 could promote the producingof specific NAb-EV71 and IL-6 might help in production ofIL-21 cytokines Furthermore PCR analysis indicated thatthe expression of IL-21 and IL-6 mRNA was significantlyincreased in both mild and severe patients compared to HC(severe or mild cases versus HC IL-21IL-6 mRNA 119875 lt005) However no difference of IL-21 and IL-6 cytokinesand mRNA expression was found between mild and severepatients (severe versus mild cases IL-21IL-6 cytokines 119875 =0390903438 IL-21IL-6 mRNA 119875 = 0488708175) these

10 Journal of Immunology Research

findings indicated that the expression of IL-21 or IL-6 wasnot notably related with severity of disease with acute stage ofEV71 infection and the IL-6 high expression was consistentwith pervious study [7] Additionally no significant differ-ence of peripheral blood Bcl-6mRNA expression amongHCmild and severe patients is consistent with pervious studywhich might be associated with formation of memory ofTFH cells [30 37] These data revealed that IL-21 and IL-6 participated in the generation of TFH cells and specificNAb-EV71 immune responses which were not significantlyassociated with the severity of patients in the acute stage ofEV71 infection

In conclusion our data indicated that increased freque-ncy of circulating TFH cells with ICOShigh and PD-1highexpression was significantly and positively associated withserum specific NAb-EV71 level and IL-21 and IL-6 con-centration in patients with EV71 infection suggesting thatTFH cells and associated cytokines might play a criticalrole in regulating humoral responses in the acute stage ofEV71 infection Further studies on the role of TFH cells inregulating B cell response and antibody production duringthe disease progression of EV71 infection will provide newinsight into understanding the immune pathogenesis of EV71infection in human

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

JianpingWu and David Cui contributed equally to this work

Acknowledgment

This study was supported by the National Key Programs forInfectious Diseases of China (Grant no 2012ZX10004-210)

References

[1] D Nasri L Bouslama S Pillet T Bourlet M Aouni and BPozzetto ldquoBasic rationale current methods and future direc-tions for molecular typing of human enterovirusrdquo ExpertReview of Molecular Diagnostics vol 7 no 4 pp 419ndash434 2007

[2] S-C Chen H-L Chang T-R Yan Y-T Cheng and K-T Chen ldquoAn eight-year study of epidemiologic features ofenterovirus 71 infection in Taiwanrdquo The American Journal ofTropical Medicine and Hygiene vol 77 no 1 pp 188ndash191 2007

[3] C Yang C Deng J Wan L Zhu and Q Leng ldquoNeutralizingantibody response in the patients with hand foot and mouthdisease to enterovirus 71 and its clinical implicationsrdquo VirologyJournal vol 8 article 306 2011

[4] QMao N Li X Yu et al ldquoAntigenicity animal protective effectand genetic characteristics of candidate vaccine strains ofenterovirus 71rdquo Archives of Virology vol 157 no 1 pp 37ndash412012

[5] M Zeng N F El Khatib S Tu et al ldquoSeroepidemiology ofEnterovirus 71 infection prior to the 2011 season in children in

Shanghairdquo Journal of Clinical Virology vol 53 no 4 pp 285ndash289 2012

[6] W-C Huang L-M Huang C-L Kao et al ldquoSeroprevalence ofenterovirus 71 and no evidence of crossprotection of enterovirus71 antibody against the other enteroviruses in kindergartenchildren in Taipei cityrdquo Journal of Microbiology Immunologyand Infection vol 45 no 2 pp 96ndash101 2012

[7] L-Y Chang C A Hsiung C-Y Lu et al ldquoStatus of cellularrather than humoral immunity is correlated with clinicaloutcome of enterovirus 71rdquo Pediatric Research vol 60 no 4 pp466ndash471 2006

[8] S-M Wang H-Y Lei K-J Huang et al ldquoPathogenesis ofenterovirus 71 brainstem encephalitis in pediatric patients rolesof cytokines and cellular immune activation in patients withpulmonary edemardquo Journal of Infectious Diseases vol 188 no4 pp 564ndash570 2003

[9] F D Finkelman J Holmes I M Katona et al ldquoLymphokinecontrol of in vivo immunoglobulin isotype selectionrdquo AnnualReview of Immunology vol 8 pp 303ndash330 1990

[10] C S Ma S Suryani D T Avery et al ldquoEarly commitment ofnave human CD4+ T cells to the T follicular helper (FH) celllineage is induced by IL-12rdquo Immunology and Cell Biology vol87 no 8 pp 590ndash600 2009

[11] T R Mosmann and R L Coffman ldquoTH1 and TH2 cells differ-ent patterns of lymphokine secretion lead to different functionalpropertiesrdquo Annual Review of Immunology vol 7 pp 145ndash1731989

[12] L E Harrington R D Hatton P R Mangan et al ldquoInterleukin17-producing CD4+ effector T cells develop via a lineage distinctfrom the T helper type 1 and 2 lineagesrdquo Nature Immunologyvol 6 no 11 pp 1123ndash1132 2005

[13] C Zhu M Jie Y Liu et al ldquoIncreased frequency of follicularhelper T cells in patients with autoimmune thyroid diseaserdquoJournal of Clinical Endocrinology and Metabolism vol 97 no3 pp 943ndash950 2012

[14] M A Kroenke D Eto M Locci et al ldquoBcl6 andMaf cooperateto instruct human follicular helper CD4 T cell differentiationrdquoJournal of Immunology vol 188 no 8 pp 3734ndash3744 2012

[15] D Breitfeld L Ohl E Kremmer et al ldquoFollicular B helper Tcells express CXC chemokine receptor 5 localize to B cellfollicles and support immunoglobulin productionrdquoThe Journalof Experimental Medicine vol 192 no 11 pp 1545ndash1551 2000

[16] P Schaerli K Willimann A B Lang M Lipp P Loetscherand B Moser ldquoCXC chemokine receptor 5 expression definesfollicular homing T cells with B cell helper functionrdquo Journal ofExperimental Medicine vol 192 no 11 pp 1553ndash1562 2000

[17] C N Arnold D J Campbell M Lipp and E C Butcher ldquoThegerminal center response is impaired in the absence of T cell-expressedCXCR5rdquoEuropean Journal of Immunology vol 37 no1 pp 100ndash109 2007

[18] N Fazilleau L Mark L J McHeyzer-Williams and M GMcHeyzer-Williams ldquoFollicular helper T cells lineage andlocationrdquo Immunity vol 30 no 3 pp 324ndash335 2009

[19] N M Haynes C D C Allen R Lesley K M Ansel N Killeenand J G Cyster ldquoRole of CXCR5 and CCR7 in follicular Thcell positioning and appearance of a programmed cell deathgene-1High germinal center-associated subpopulationrdquo Journalof Immunology vol 179 no 8 pp 5099ndash5108 2007

[20] R Spolski andW J Leonard ldquoIL-21 andT follicular helper cellsrdquoInternational Immunology vol 22 no 1 Article ID dxp112 pp7ndash12 2009

Journal of Immunology Research 11

[21] D Eto C Lao D DiToro et al ldquoIL-21 and IL-6 are critical fordifferent aspects of B cell immunity and redundantly induceoptimal follicular helper CD4 T cell (Tfh) differentiationrdquo PLoSONE vol 6 no 3 Article ID e17739 2011

[22] C Dong A E Juedes U-A Temann et al ldquoICOS co-stimu-linebreak latory receptor is essential for T-cell activation andfunctionrdquo Nature vol 409 no 6816 pp 97ndash101 2001

[23] T Okazaki and T Honjo ldquoThe PD-1-PD-L pathway in immuno-logical tolerancerdquo Trends in Immunology vol 27 no 4 pp 195ndash201 2006

[24] I Yusuf R Kageyama L Monticelli et al ldquoGerminal center Tfollicular helper cell IL-4 production is dependent on signalinglymphocytic activation molecule receptor (CD150)rdquo Journal ofImmunology vol 185 no 1 pp 190ndash202 2010

[25] D Yu and C G Vinuesa ldquoThe elusive identity of T follicularhelper cellsrdquo Trends in Immunology vol 31 no 10 pp 377ndash3832010

[26] M A Linterman R J Rigby RWong et al ldquoRoquin differenti-ates the specialized functions of duplicated T cell costimulatoryreceptor genes CD28 and ICOSrdquo Immunity vol 30 no 2 pp228ndash241 2009

[27] M Locci C Havenar-Daughton E Landais et al ldquoHuman cir-culating PD-1+1CXCR3minusCXCR5+ memory Tfh cells are highlyfunctional and correlate with broadly neutralizing HIV anti-body responsesrdquo Immunity vol 39 no 4 pp 758ndash769 2013

[28] J Feng L Lu C Hua et al ldquoHigh frequency of CD4+CXCR5+TFH cells in patients with immune-active chronic hepatitis BrdquoPLoS ONE vol 6 no 7 Article ID e21698 2011

[29] C Petrovas T Yamamoto M Y Gerner et al ldquoCD4 T follicularhelper cell dynamics during SIV infectionrdquo Journal of ClinicalInvestigation vol 122 no 9 pp 3281ndash3294 2012

[30] N Simpson P A Gatenby A Wilson et al ldquoExpansion ofcirculating T cells resembling follicular helper T cells is a fixedphenotype that identifies a subset of severe systemic lupuserythematosusrdquo Arthritis and Rheumatism vol 62 no 1 pp234ndash244 2010

[31] S Li C Cai J Feng et al ldquoPeripheral T lymphocyte subset imb-alances in children with enterovirus 71-induced hand foot andmouth diseaserdquo Virus Research vol 180 pp 84ndash91 2014

[32] J Chen J Tong H Liu et al ldquoIncreased frequency ofTh17 cellsin the peripheral blood of children infectedwith enterovirus 71rdquoJournal of Medical Virology vol 84 no 5 pp 763ndash767 2012

[33] J Xie Y Jiao Z Qiu Q Li and T Li ldquoSignificant elevation of Bcells at the acute stage in enterovirus 71-infected children withcentral nervous system involvementrdquo Scandinavian Journal ofInfectious Diseases vol 42 no 11-12 pp 931ndash935 2010

[34] Y Liang X Zhou E Yang et al ldquoAnalysis of the Th1Th2reaction in the immune response induced by EV71 inacti-vated vaccine in neonatal rhesus monkeysrdquo Journal of ClinicalImmunology vol 32 no 5 pp 1048ndash1058 2012

[35] Y-W Lin K-C Chang C-M Kao S-P Chang Y-Y Tungand S-H Chen ldquoLymphocyte and antibody responses reduceenterovirus 71 lethality in mice by decreasing tissue viral loadsrdquoJournal of Virology vol 83 no 13 pp 6477ndash6483 2009

[36] National notifiable diseases announced by National Health andFamily Planning Commission of the Peoplersquos Republic of Chinahttpwwwnhfpcgovcnzhuzhanyqxxlistsshtml

[37] R Morita N Schmitt S-E Bentebibel et al ldquoHuman bloodCXCR5+CD4+ T cells are counterparts of T follicular cellsand contain specific subsets that differentially support antibodysecretionrdquo Immunity vol 34 no 1 pp 108ndash121 2011

6 Journal of Immunology Research

0

200

400

600

NAb

-EV

71 ti

tres ns

HC Mild Severe

lowastlowastlowast

lowastlowastlowast

(a)

00 5 10 15 20

200

400

600

NAb

-EV

71 ti

tres

MildP lt 00001

CXCR5+CD4+ in CD4

+ T cells ()

r = 08581

0

10

20

30

40

NAb

-EV

71 ti

tres

3 4 5 6

HCP = 02560

CXCR5+CD4+ in CD4

+ T cells ()

r = 01771

0 5 10 15 200

200

400

600

NAb

-EV

71 ti

tres

SevereP = 00043

CXCR5+CD4+ in CD4

+ T cells ()

r = 04992

(b)

000 05 10 15 20 25

10

20

30

40

NAb

-EV

71 ti

tres

P = 05120

PD-1+CXCR5+CD4+ TFH cells ()

r = 01028

0 2 4 60

200

400

600

NAb

-EV

71 ti

tres

P lt 00001

PD-1+CXCR5+CD4+ TFH cells ()

r = 06637

0 2 4 860

200

400

600

NAb

-EV

71 ti

tres

P = 00027

PD-1+CXCR5+CD4+ TFH cells ()

r = 05196

(c)

0 5 10 150

200

400

600

NAb

-EV

71 ti

tres

P = 00103

ICOS+CXCR5+CD4+ TFH cells ()

r = 04542

0 2 4 860

200

400

600

NAb

-EV

71 ti

tres

P = 00039

ICOS+CXCR5+CD4+ TFH cells ()

r = 05190

00 05 10 15 200

10

20

30

40

NAb

-EV

71 ti

tres

P = 07634

ICOS+CXCR5+CD4+ TFH cells ()

r = 004728

(d)

Figure 3 Correlation of specific NAb-EV71 titres and circulating CXCR5+CD4+ TFH cells with PD-1high and ICOShigh expression in EV71-infected patients (a) Titres of specific NAb-EV71 in sera from HC and EV71-infected patients including mild and severe cases Relationshipbetween the titres of specific NAb-EV71 and the frequency of circulating CXCR5+CD4+ TFH cells (b) PD-1+CXCR5+CD4+ TFH cells (c)and ICOS+CXCR5+CD4+ TFH cells (d) Data shown were the mean plusmn SD The horizontal lines show the median lowast119875 lt 005 lowastlowast119875 lt 001lowastlowastlowast

119875 lt 0001 ns no significant difference

than that in HC but no significant difference of IL-6 mRNAexpression was observed between mild and severe patientsgroups (HC 1008 plusmn 416 mild 1802 plusmn 941 severe 2055 plusmn1138) (Figure 6(a)) Similarly the IL-21 mRNA expressions

in both mild and severe patients were significantly higherthan those inHC but no significant difference of IL-21mRNAwas found between mild patients and severe patients (HC117plusmn048 mild 221plusmn131 severe 269plusmn151) (Figure 6(b))

Journal of Immunology Research 7

0

50

100

150

200

IL-2

1 (p

gm

L)

ns

P lt 00001 P lt 00001 P = 03909

HC Mild Severe

lowastlowastlowast

lowastlowastlowast

0

20

40

60

80

100

IL-6

(pg

mL)

ns

HC Mild Severe

P lt 00001 P lt 00001 P = 03438

lowastlowastlowast

lowastlowastlowast

(a)

3 4 5 60

50

100

150

IL-2

1 (p

gm

L)

P = 00053

0 5 1510 200

50

100

150

200

IL-2

1 (p

gm

L)

P lt 00001

0 5 1510 200

50

100

150

200

IL-2

1 (p

gm

L)

P = 00005

CXCR5+CD4+ in CD4

+ T cells () CXCR5+CD4+ in CD4

+ T cells () CXCR5+CD4+ in CD4

+ T cells ()

r = 05919r = 08479r = 04177

HC Mild Severe

(b)

0 5 1510 200

10

20

30

40

50

IL-6

(pg

mL)

P = 00176

CXCR5+CD4+ in CD4

+ T cells ()

r = 04375

3 4 5 60

5

10

15

20

IL-6

(pg

mL)

P = 04986

CXCR5+CD4+ in CD4

+ T cells ()

r = minus01060

0 5 1510 200

20

40

60

80

100

IL-6

(pg

mL)

P = 00236

CXCR5+CD4+ in CD4

+ T cells ()

r = 04056

(c)

0

5

10

15

20

IL-6

(pg

mL)

0 50 150100IL-21 (pgmL)

P = 02594r = minus01758

0 50 150100 2000

10

20

30

40

50

IL-21 (pgmL)

IL-6

(pg

mL)

P = 00220r = 04237

0 50 150100 200IL-21 (pgmL)

0

20

40

60

80

100

IL-6

(pg

mL)

P = 00418r = 03677

(d)

Figure 4 Correlation of cytokines levels and circulating CXCR5+CD4+ TFH cells in EV71-infected patients including mild and severe cases(a) Cytokines levels of IL-21 and IL-6 in sera fromHC and EV71-infected patients (b) Relationship between the IL-21 levels and the frequencyof circulating CXCR5+CD4+ TFH cells (c) Relationship between the IL-6 levels and the frequency of circulating CXCR5+CD4+ TFH cells(d) Relationship between the IL-6 levels and IL-21 levels Data shown were the mean plusmn SDThe horizontal lines show the median lowast119875 lt 005lowastlowast

119875 lt 001 lowastlowastlowast119875 lt 0001 ns no significant difference

8 Journal of Immunology Research

0

10

20

30

40

IL-21 (pgmL)

NAb

-EV

71 ti

tres

HC

0 50 100 150

P = 01036

0

200

400

600

IL-21 (pgmL)

NAb

-EV

71 ti

tres

Severe

500 100 200150

P = 00182

minus200

0

200

400

600

IL-21 (pgmL)

NAb

-EV

71 ti

tres

Mild

50 100 200150

P lt 00001

r = 04216

r = 07174

r = 02516

(a)

0

10

20

30

40

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 5 10 2015

P = 04936

0

200

400

600

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 10 20 30 40 50

P = 01039

0

200

400

600

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 20 40 60 80 100

P = 00741r = 03253r = 03082r = 01072

(b)

Figure 5 Correlation of specific NAb-EV71 titres and cytokines levels from HC and EV71-infected patients including mild and severe cases(a) Relationship between the titres of specific NAb-EV71 and the serum levels of IL-21 (b) Relationship between the titres of specific NAb-EV71 and the serum levels of IL-6

0

10

20

30

40

50

IL-6

mRA

N re

lativ

e exp

ress

ion

ns

HC Mild Severe

lowastlowast

lowast

P = 00116 P = 00078 P = 08175

(a)

0

2

4

6

IL-2

1 m

RAN

relat

ive e

xpre

ssio

n

ns

HC Mild Severe

lowastlowast

lowast

P = 00375 P = 00037 P = 04887

(b)

0

10

20

30

40

50

Bcl-6

mRA

N re

lativ

e exp

ress

ion

nsns ns

HC Mild Severe

P = 04212 P = 04495 P = 09737

(c)

Figure 6 Expression of IL-6 IL-21 and Bcl-6 mRNA in human PBMCs Human PBMCs from 10 mild and 12 severe patients with EV71infection and 15 HC were detected by real-time PCR assay as described in Section 2 (a)The levels of IL-6 mRNA in human PBMCs (b)Thelevels of IL-21 mRNA in human PBMCs (c) The levels of Bcl-6 mRNA in human PBMCs data shown were the mean plusmn SD The horizontallines show the median lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 ns no significant difference

Moreover there were no significant differences for Bcl-6mRNA expression among these groups tested (HC 2334 plusmn1292 mild 1845plusmn1028 severe 1954plusmn1332) (Figure 6(c))

4 Discussion

There aremore than onemillionHFMD cases and 200 deathseach year since 2008 in China [36] EV71 is one of thecommon causative factors of HFMD in young children less

than 5 years of age [3 5] Although mild diseases are the pre-dominant clinical features of EV71 infection severe diseaseswith neurological involvement and the fetal cases of HFMDare closely associated with EV71 infection identified by PCRandor virus isolation [6ndash8] In our study compared to mildpatients the age of severe patients wasmainly observed below3 years (severe versus mild cases 119875 = 00016) and evaluatedevidences with WBC numbers (severe versus mild cases 119875 lt00001 mild cases versus HC 119875 = 00035) and serum CRPlevels (severe versusmild cases119875 lt 00001 mild cases versus

Journal of Immunology Research 9

HC 119875 lt 00001) suggested that inappropriate inflammationmay be elicited in the progression of HFMD in the acutephase of EV71 infection whichwas consistentwith previouslyreported data [7 31 32]

Previous studies have demonstrated that both humoraland cellular immune responses such as antibodies B and Tcells including Th1Th2 and Th17 and other T cell lineagesand related cytokines play a critical role in protection fromEV71 infection in animal models [4 34 35] In humans EV71infection induced imbalance of TB cells and their relatedcytokines and even caused a systemic dysregulation of theimmune system [31ndash33] Recent studies show that follicularhelper T cells (TFH) a new T cell subpopulation play apivotal role in regulating immune responses and have beenassociated with many diseases [13 20 28] Simpson et alreported an increased frequency of circulatingCXCR5+CD4+TFH cells in patients with systemic lupus erythematosus(SLE) and Sjogrenrsquos syndrome [30] Feng et al demonstratedthat the frequency of circulating TFH cells was significantlyincreased in patients with HBV and these circulating TFHcells participated in the HBV-related immune responses[28] However the potential role of circulating TFH cells inpatients with EV71 infection is not yet known In the presentwork we firstly examined the frequency of circulating TFHcells in mild and severe patients with EV71 infection andhealthy controlsThe results demonstrated that the frequencyof circulating CXCR5+CD4+ TFH cells in mild and severepatients in the acute stage of EV71 infection was significantlyhigher than that of HC (severe or mild cases versus HC119875 lt 005) respectively However severe patients did notdiffer significantly from mild patients in the percentage ofperipheral blood CXCR5+CD4+ TFH cells (severe versusmild cases 119875 = 05151) These findings clearly indicatedthat TFH cells played an important role in the EV71-inducedimmune responses

Simpson et al detected that the high frequency of circu-lating TFH cells with high expression of ICOS andor PD-1 molecules was associated with the severity of end-organinvolvement in patients with systemic lupus erythematosus(SLE) and Sjogrenrsquos syndrome [30] Zhu et al demonstratedthe increased frequency of TFH cells with highly expressedICOS andor PD-1 in patients with autoimmune thyroiddisease and rheumatoid arthritis respectively and both ICOSand PD-1 molecules are closely positively correlated withthe function of TFH cells [13] We detected an enhancedfrequency of PD-1high and ICOShighCXCR5+CD4+ TFH cellsin the mild and severe EV71-induced patients comparedto HC (severe or mild cases versus HC 119875 lt 005) Inaddition a closely positive correlation was observed betweenthe frequencies of these two subpopulations of PD-1high andICOShighCXCR5+CD4+ T cells These data supported thedefinition of circulating TFH cells However the percentageof circulating TFH cells with ICOShigh and PD-1high expres-sion revealed no significant difference between the mild andsevere patients (severe versus mild cases PD-1high 119875 =01085 severe versus mild cases ICOShigh 119875 = 01930)In other words EV71 increased the frequency of circulatingTFH cells with ICOShigh and PD-1high expression that was not

notably associated with the severity of patients in the acutestage of EV71 infection

It is known that TFH cells are critical for promoting thedevelopment of antibody response by helper B cells in GC[10 11] Several seroepidemiologic investigations indicatedthat the neutralizing antibody can protect adults as well aschildren from EV71 infection once again although the titersof specific neutralizing antibody were not correlated withseverity of the disease with EV71 infection [3ndash6 34 35]Why is that To date that is not well understood Our dataalso indicated that the serum titers of specific NAb-EV71in patients with acute stage of EV71 infection were higherthan those in HC (severe or mild cases versus HC 119875 lt00001) but no significant difference was observed betweenmild and severe patients groups (severe versus mild cases119875 = 09693) which corresponded to previous studies [3]Additionally we found that enhanced frequency of circulat-ing CXCR5+CD4+ TFH PD-1highCXCR5+CD4+ TFH andICOShighCXCR5+CD4+ TFH cells was notably positively cor-related with the titres of specific NAb-EV71 in patients withEV71 infection and a significant associationwas not observedbetween themild and severe patients respectivelyThese dataalso indicated that TFH cells with ICOShigh and PD-1highexpression participated in regulating antibody responses inthe acute stage of EV71 infection at the same time whichmight give a good explanation to why there is no significantdifference between the levels of specific NAb-EV71 in bothmild and severe patients with EV71 infection

Recent studies demonstrated that not only did TFH cellssecrete IL-21 but IL-21 could also drive GC formation andTFH cell generation and function and promote terminalmature of B cell in GC Ig class-switching and antibodyresponses [10 11 20] IL-6 has also induced IL-21 productionand TFH cell generation and mice lacking both IL-6 andIL-21 are unable to generate TFH cell-dependent immuneresponses [20 21] Bcl-6 transcription factor is selectivelyexpressed in TFH cells which is required for programmingof TFH cell generation and Bcl-6 expression is regulated byIL-6 and IL-21 [14 21] In current study we found that thelevels of sera IL-21 and IL-6 concentrations were significantlyhigher in both mild and severe patients with EV71 infectionthan those in HC (severe ormild cases versus HC IL-21IL-6119875 lt 00001) and they were found to be positively correlatedwith the frequency of circulating CXCR5+CD4+ TFH cellsrespectively which suggested that IL-21 and IL-6 might playan important role in the generation of TFH cells In additiona strongly positive correlation was observed between sera IL-21 and NAb-EV71 as well as between sera IL-21 and IL-6indicating that IL-21 and IL-6 could promote the producingof specific NAb-EV71 and IL-6 might help in production ofIL-21 cytokines Furthermore PCR analysis indicated thatthe expression of IL-21 and IL-6 mRNA was significantlyincreased in both mild and severe patients compared to HC(severe or mild cases versus HC IL-21IL-6 mRNA 119875 lt005) However no difference of IL-21 and IL-6 cytokinesand mRNA expression was found between mild and severepatients (severe versus mild cases IL-21IL-6 cytokines 119875 =0390903438 IL-21IL-6 mRNA 119875 = 0488708175) these

10 Journal of Immunology Research

findings indicated that the expression of IL-21 or IL-6 wasnot notably related with severity of disease with acute stage ofEV71 infection and the IL-6 high expression was consistentwith pervious study [7] Additionally no significant differ-ence of peripheral blood Bcl-6mRNA expression amongHCmild and severe patients is consistent with pervious studywhich might be associated with formation of memory ofTFH cells [30 37] These data revealed that IL-21 and IL-6 participated in the generation of TFH cells and specificNAb-EV71 immune responses which were not significantlyassociated with the severity of patients in the acute stage ofEV71 infection

In conclusion our data indicated that increased freque-ncy of circulating TFH cells with ICOShigh and PD-1highexpression was significantly and positively associated withserum specific NAb-EV71 level and IL-21 and IL-6 con-centration in patients with EV71 infection suggesting thatTFH cells and associated cytokines might play a criticalrole in regulating humoral responses in the acute stage ofEV71 infection Further studies on the role of TFH cells inregulating B cell response and antibody production duringthe disease progression of EV71 infection will provide newinsight into understanding the immune pathogenesis of EV71infection in human

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

JianpingWu and David Cui contributed equally to this work

Acknowledgment

This study was supported by the National Key Programs forInfectious Diseases of China (Grant no 2012ZX10004-210)

References

[1] D Nasri L Bouslama S Pillet T Bourlet M Aouni and BPozzetto ldquoBasic rationale current methods and future direc-tions for molecular typing of human enterovirusrdquo ExpertReview of Molecular Diagnostics vol 7 no 4 pp 419ndash434 2007

[2] S-C Chen H-L Chang T-R Yan Y-T Cheng and K-T Chen ldquoAn eight-year study of epidemiologic features ofenterovirus 71 infection in Taiwanrdquo The American Journal ofTropical Medicine and Hygiene vol 77 no 1 pp 188ndash191 2007

[3] C Yang C Deng J Wan L Zhu and Q Leng ldquoNeutralizingantibody response in the patients with hand foot and mouthdisease to enterovirus 71 and its clinical implicationsrdquo VirologyJournal vol 8 article 306 2011

[4] QMao N Li X Yu et al ldquoAntigenicity animal protective effectand genetic characteristics of candidate vaccine strains ofenterovirus 71rdquo Archives of Virology vol 157 no 1 pp 37ndash412012

[5] M Zeng N F El Khatib S Tu et al ldquoSeroepidemiology ofEnterovirus 71 infection prior to the 2011 season in children in

Shanghairdquo Journal of Clinical Virology vol 53 no 4 pp 285ndash289 2012

[6] W-C Huang L-M Huang C-L Kao et al ldquoSeroprevalence ofenterovirus 71 and no evidence of crossprotection of enterovirus71 antibody against the other enteroviruses in kindergartenchildren in Taipei cityrdquo Journal of Microbiology Immunologyand Infection vol 45 no 2 pp 96ndash101 2012

[7] L-Y Chang C A Hsiung C-Y Lu et al ldquoStatus of cellularrather than humoral immunity is correlated with clinicaloutcome of enterovirus 71rdquo Pediatric Research vol 60 no 4 pp466ndash471 2006

[8] S-M Wang H-Y Lei K-J Huang et al ldquoPathogenesis ofenterovirus 71 brainstem encephalitis in pediatric patients rolesof cytokines and cellular immune activation in patients withpulmonary edemardquo Journal of Infectious Diseases vol 188 no4 pp 564ndash570 2003

[9] F D Finkelman J Holmes I M Katona et al ldquoLymphokinecontrol of in vivo immunoglobulin isotype selectionrdquo AnnualReview of Immunology vol 8 pp 303ndash330 1990

[10] C S Ma S Suryani D T Avery et al ldquoEarly commitment ofnave human CD4+ T cells to the T follicular helper (FH) celllineage is induced by IL-12rdquo Immunology and Cell Biology vol87 no 8 pp 590ndash600 2009

[11] T R Mosmann and R L Coffman ldquoTH1 and TH2 cells differ-ent patterns of lymphokine secretion lead to different functionalpropertiesrdquo Annual Review of Immunology vol 7 pp 145ndash1731989

[12] L E Harrington R D Hatton P R Mangan et al ldquoInterleukin17-producing CD4+ effector T cells develop via a lineage distinctfrom the T helper type 1 and 2 lineagesrdquo Nature Immunologyvol 6 no 11 pp 1123ndash1132 2005

[13] C Zhu M Jie Y Liu et al ldquoIncreased frequency of follicularhelper T cells in patients with autoimmune thyroid diseaserdquoJournal of Clinical Endocrinology and Metabolism vol 97 no3 pp 943ndash950 2012

[14] M A Kroenke D Eto M Locci et al ldquoBcl6 andMaf cooperateto instruct human follicular helper CD4 T cell differentiationrdquoJournal of Immunology vol 188 no 8 pp 3734ndash3744 2012

[15] D Breitfeld L Ohl E Kremmer et al ldquoFollicular B helper Tcells express CXC chemokine receptor 5 localize to B cellfollicles and support immunoglobulin productionrdquoThe Journalof Experimental Medicine vol 192 no 11 pp 1545ndash1551 2000

[16] P Schaerli K Willimann A B Lang M Lipp P Loetscherand B Moser ldquoCXC chemokine receptor 5 expression definesfollicular homing T cells with B cell helper functionrdquo Journal ofExperimental Medicine vol 192 no 11 pp 1553ndash1562 2000

[17] C N Arnold D J Campbell M Lipp and E C Butcher ldquoThegerminal center response is impaired in the absence of T cell-expressedCXCR5rdquoEuropean Journal of Immunology vol 37 no1 pp 100ndash109 2007

[18] N Fazilleau L Mark L J McHeyzer-Williams and M GMcHeyzer-Williams ldquoFollicular helper T cells lineage andlocationrdquo Immunity vol 30 no 3 pp 324ndash335 2009

[19] N M Haynes C D C Allen R Lesley K M Ansel N Killeenand J G Cyster ldquoRole of CXCR5 and CCR7 in follicular Thcell positioning and appearance of a programmed cell deathgene-1High germinal center-associated subpopulationrdquo Journalof Immunology vol 179 no 8 pp 5099ndash5108 2007

[20] R Spolski andW J Leonard ldquoIL-21 andT follicular helper cellsrdquoInternational Immunology vol 22 no 1 Article ID dxp112 pp7ndash12 2009

Journal of Immunology Research 11

[21] D Eto C Lao D DiToro et al ldquoIL-21 and IL-6 are critical fordifferent aspects of B cell immunity and redundantly induceoptimal follicular helper CD4 T cell (Tfh) differentiationrdquo PLoSONE vol 6 no 3 Article ID e17739 2011

[22] C Dong A E Juedes U-A Temann et al ldquoICOS co-stimu-linebreak latory receptor is essential for T-cell activation andfunctionrdquo Nature vol 409 no 6816 pp 97ndash101 2001

[23] T Okazaki and T Honjo ldquoThe PD-1-PD-L pathway in immuno-logical tolerancerdquo Trends in Immunology vol 27 no 4 pp 195ndash201 2006

[24] I Yusuf R Kageyama L Monticelli et al ldquoGerminal center Tfollicular helper cell IL-4 production is dependent on signalinglymphocytic activation molecule receptor (CD150)rdquo Journal ofImmunology vol 185 no 1 pp 190ndash202 2010

[25] D Yu and C G Vinuesa ldquoThe elusive identity of T follicularhelper cellsrdquo Trends in Immunology vol 31 no 10 pp 377ndash3832010

[26] M A Linterman R J Rigby RWong et al ldquoRoquin differenti-ates the specialized functions of duplicated T cell costimulatoryreceptor genes CD28 and ICOSrdquo Immunity vol 30 no 2 pp228ndash241 2009

[27] M Locci C Havenar-Daughton E Landais et al ldquoHuman cir-culating PD-1+1CXCR3minusCXCR5+ memory Tfh cells are highlyfunctional and correlate with broadly neutralizing HIV anti-body responsesrdquo Immunity vol 39 no 4 pp 758ndash769 2013

[28] J Feng L Lu C Hua et al ldquoHigh frequency of CD4+CXCR5+TFH cells in patients with immune-active chronic hepatitis BrdquoPLoS ONE vol 6 no 7 Article ID e21698 2011

[29] C Petrovas T Yamamoto M Y Gerner et al ldquoCD4 T follicularhelper cell dynamics during SIV infectionrdquo Journal of ClinicalInvestigation vol 122 no 9 pp 3281ndash3294 2012

[30] N Simpson P A Gatenby A Wilson et al ldquoExpansion ofcirculating T cells resembling follicular helper T cells is a fixedphenotype that identifies a subset of severe systemic lupuserythematosusrdquo Arthritis and Rheumatism vol 62 no 1 pp234ndash244 2010

[31] S Li C Cai J Feng et al ldquoPeripheral T lymphocyte subset imb-alances in children with enterovirus 71-induced hand foot andmouth diseaserdquo Virus Research vol 180 pp 84ndash91 2014

[32] J Chen J Tong H Liu et al ldquoIncreased frequency ofTh17 cellsin the peripheral blood of children infectedwith enterovirus 71rdquoJournal of Medical Virology vol 84 no 5 pp 763ndash767 2012

[33] J Xie Y Jiao Z Qiu Q Li and T Li ldquoSignificant elevation of Bcells at the acute stage in enterovirus 71-infected children withcentral nervous system involvementrdquo Scandinavian Journal ofInfectious Diseases vol 42 no 11-12 pp 931ndash935 2010

[34] Y Liang X Zhou E Yang et al ldquoAnalysis of the Th1Th2reaction in the immune response induced by EV71 inacti-vated vaccine in neonatal rhesus monkeysrdquo Journal of ClinicalImmunology vol 32 no 5 pp 1048ndash1058 2012

[35] Y-W Lin K-C Chang C-M Kao S-P Chang Y-Y Tungand S-H Chen ldquoLymphocyte and antibody responses reduceenterovirus 71 lethality in mice by decreasing tissue viral loadsrdquoJournal of Virology vol 83 no 13 pp 6477ndash6483 2009

[36] National notifiable diseases announced by National Health andFamily Planning Commission of the Peoplersquos Republic of Chinahttpwwwnhfpcgovcnzhuzhanyqxxlistsshtml

[37] R Morita N Schmitt S-E Bentebibel et al ldquoHuman bloodCXCR5+CD4+ T cells are counterparts of T follicular cellsand contain specific subsets that differentially support antibodysecretionrdquo Immunity vol 34 no 1 pp 108ndash121 2011

Journal of Immunology Research 7

0

50

100

150

200

IL-2

1 (p

gm

L)

ns

P lt 00001 P lt 00001 P = 03909

HC Mild Severe

lowastlowastlowast

lowastlowastlowast

0

20

40

60

80

100

IL-6

(pg

mL)

ns

HC Mild Severe

P lt 00001 P lt 00001 P = 03438

lowastlowastlowast

lowastlowastlowast

(a)

3 4 5 60

50

100

150

IL-2

1 (p

gm

L)

P = 00053

0 5 1510 200

50

100

150

200

IL-2

1 (p

gm

L)

P lt 00001

0 5 1510 200

50

100

150

200

IL-2

1 (p

gm

L)

P = 00005

CXCR5+CD4+ in CD4

+ T cells () CXCR5+CD4+ in CD4

+ T cells () CXCR5+CD4+ in CD4

+ T cells ()

r = 05919r = 08479r = 04177

HC Mild Severe

(b)

0 5 1510 200

10

20

30

40

50

IL-6

(pg

mL)

P = 00176

CXCR5+CD4+ in CD4

+ T cells ()

r = 04375

3 4 5 60

5

10

15

20

IL-6

(pg

mL)

P = 04986

CXCR5+CD4+ in CD4

+ T cells ()

r = minus01060

0 5 1510 200

20

40

60

80

100

IL-6

(pg

mL)

P = 00236

CXCR5+CD4+ in CD4

+ T cells ()

r = 04056

(c)

0

5

10

15

20

IL-6

(pg

mL)

0 50 150100IL-21 (pgmL)

P = 02594r = minus01758

0 50 150100 2000

10

20

30

40

50

IL-21 (pgmL)

IL-6

(pg

mL)

P = 00220r = 04237

0 50 150100 200IL-21 (pgmL)

0

20

40

60

80

100

IL-6

(pg

mL)

P = 00418r = 03677

(d)

Figure 4 Correlation of cytokines levels and circulating CXCR5+CD4+ TFH cells in EV71-infected patients including mild and severe cases(a) Cytokines levels of IL-21 and IL-6 in sera fromHC and EV71-infected patients (b) Relationship between the IL-21 levels and the frequencyof circulating CXCR5+CD4+ TFH cells (c) Relationship between the IL-6 levels and the frequency of circulating CXCR5+CD4+ TFH cells(d) Relationship between the IL-6 levels and IL-21 levels Data shown were the mean plusmn SDThe horizontal lines show the median lowast119875 lt 005lowastlowast

119875 lt 001 lowastlowastlowast119875 lt 0001 ns no significant difference

8 Journal of Immunology Research

0

10

20

30

40

IL-21 (pgmL)

NAb

-EV

71 ti

tres

HC

0 50 100 150

P = 01036

0

200

400

600

IL-21 (pgmL)

NAb

-EV

71 ti

tres

Severe

500 100 200150

P = 00182

minus200

0

200

400

600

IL-21 (pgmL)

NAb

-EV

71 ti

tres

Mild

50 100 200150

P lt 00001

r = 04216

r = 07174

r = 02516

(a)

0

10

20

30

40

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 5 10 2015

P = 04936

0

200

400

600

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 10 20 30 40 50

P = 01039

0

200

400

600

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 20 40 60 80 100

P = 00741r = 03253r = 03082r = 01072

(b)

Figure 5 Correlation of specific NAb-EV71 titres and cytokines levels from HC and EV71-infected patients including mild and severe cases(a) Relationship between the titres of specific NAb-EV71 and the serum levels of IL-21 (b) Relationship between the titres of specific NAb-EV71 and the serum levels of IL-6

0

10

20

30

40

50

IL-6

mRA

N re

lativ

e exp

ress

ion

ns

HC Mild Severe

lowastlowast

lowast

P = 00116 P = 00078 P = 08175

(a)

0

2

4

6

IL-2

1 m

RAN

relat

ive e

xpre

ssio

n

ns

HC Mild Severe

lowastlowast

lowast

P = 00375 P = 00037 P = 04887

(b)

0

10

20

30

40

50

Bcl-6

mRA

N re

lativ

e exp

ress

ion

nsns ns

HC Mild Severe

P = 04212 P = 04495 P = 09737

(c)

Figure 6 Expression of IL-6 IL-21 and Bcl-6 mRNA in human PBMCs Human PBMCs from 10 mild and 12 severe patients with EV71infection and 15 HC were detected by real-time PCR assay as described in Section 2 (a)The levels of IL-6 mRNA in human PBMCs (b)Thelevels of IL-21 mRNA in human PBMCs (c) The levels of Bcl-6 mRNA in human PBMCs data shown were the mean plusmn SD The horizontallines show the median lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 ns no significant difference

Moreover there were no significant differences for Bcl-6mRNA expression among these groups tested (HC 2334 plusmn1292 mild 1845plusmn1028 severe 1954plusmn1332) (Figure 6(c))

4 Discussion

There aremore than onemillionHFMD cases and 200 deathseach year since 2008 in China [36] EV71 is one of thecommon causative factors of HFMD in young children less

than 5 years of age [3 5] Although mild diseases are the pre-dominant clinical features of EV71 infection severe diseaseswith neurological involvement and the fetal cases of HFMDare closely associated with EV71 infection identified by PCRandor virus isolation [6ndash8] In our study compared to mildpatients the age of severe patients wasmainly observed below3 years (severe versus mild cases 119875 = 00016) and evaluatedevidences with WBC numbers (severe versus mild cases 119875 lt00001 mild cases versus HC 119875 = 00035) and serum CRPlevels (severe versusmild cases119875 lt 00001 mild cases versus

Journal of Immunology Research 9

HC 119875 lt 00001) suggested that inappropriate inflammationmay be elicited in the progression of HFMD in the acutephase of EV71 infection whichwas consistentwith previouslyreported data [7 31 32]

Previous studies have demonstrated that both humoraland cellular immune responses such as antibodies B and Tcells including Th1Th2 and Th17 and other T cell lineagesand related cytokines play a critical role in protection fromEV71 infection in animal models [4 34 35] In humans EV71infection induced imbalance of TB cells and their relatedcytokines and even caused a systemic dysregulation of theimmune system [31ndash33] Recent studies show that follicularhelper T cells (TFH) a new T cell subpopulation play apivotal role in regulating immune responses and have beenassociated with many diseases [13 20 28] Simpson et alreported an increased frequency of circulatingCXCR5+CD4+TFH cells in patients with systemic lupus erythematosus(SLE) and Sjogrenrsquos syndrome [30] Feng et al demonstratedthat the frequency of circulating TFH cells was significantlyincreased in patients with HBV and these circulating TFHcells participated in the HBV-related immune responses[28] However the potential role of circulating TFH cells inpatients with EV71 infection is not yet known In the presentwork we firstly examined the frequency of circulating TFHcells in mild and severe patients with EV71 infection andhealthy controlsThe results demonstrated that the frequencyof circulating CXCR5+CD4+ TFH cells in mild and severepatients in the acute stage of EV71 infection was significantlyhigher than that of HC (severe or mild cases versus HC119875 lt 005) respectively However severe patients did notdiffer significantly from mild patients in the percentage ofperipheral blood CXCR5+CD4+ TFH cells (severe versusmild cases 119875 = 05151) These findings clearly indicatedthat TFH cells played an important role in the EV71-inducedimmune responses

Simpson et al detected that the high frequency of circu-lating TFH cells with high expression of ICOS andor PD-1 molecules was associated with the severity of end-organinvolvement in patients with systemic lupus erythematosus(SLE) and Sjogrenrsquos syndrome [30] Zhu et al demonstratedthe increased frequency of TFH cells with highly expressedICOS andor PD-1 in patients with autoimmune thyroiddisease and rheumatoid arthritis respectively and both ICOSand PD-1 molecules are closely positively correlated withthe function of TFH cells [13] We detected an enhancedfrequency of PD-1high and ICOShighCXCR5+CD4+ TFH cellsin the mild and severe EV71-induced patients comparedto HC (severe or mild cases versus HC 119875 lt 005) Inaddition a closely positive correlation was observed betweenthe frequencies of these two subpopulations of PD-1high andICOShighCXCR5+CD4+ T cells These data supported thedefinition of circulating TFH cells However the percentageof circulating TFH cells with ICOShigh and PD-1high expres-sion revealed no significant difference between the mild andsevere patients (severe versus mild cases PD-1high 119875 =01085 severe versus mild cases ICOShigh 119875 = 01930)In other words EV71 increased the frequency of circulatingTFH cells with ICOShigh and PD-1high expression that was not

notably associated with the severity of patients in the acutestage of EV71 infection

It is known that TFH cells are critical for promoting thedevelopment of antibody response by helper B cells in GC[10 11] Several seroepidemiologic investigations indicatedthat the neutralizing antibody can protect adults as well aschildren from EV71 infection once again although the titersof specific neutralizing antibody were not correlated withseverity of the disease with EV71 infection [3ndash6 34 35]Why is that To date that is not well understood Our dataalso indicated that the serum titers of specific NAb-EV71in patients with acute stage of EV71 infection were higherthan those in HC (severe or mild cases versus HC 119875 lt00001) but no significant difference was observed betweenmild and severe patients groups (severe versus mild cases119875 = 09693) which corresponded to previous studies [3]Additionally we found that enhanced frequency of circulat-ing CXCR5+CD4+ TFH PD-1highCXCR5+CD4+ TFH andICOShighCXCR5+CD4+ TFH cells was notably positively cor-related with the titres of specific NAb-EV71 in patients withEV71 infection and a significant associationwas not observedbetween themild and severe patients respectivelyThese dataalso indicated that TFH cells with ICOShigh and PD-1highexpression participated in regulating antibody responses inthe acute stage of EV71 infection at the same time whichmight give a good explanation to why there is no significantdifference between the levels of specific NAb-EV71 in bothmild and severe patients with EV71 infection

Recent studies demonstrated that not only did TFH cellssecrete IL-21 but IL-21 could also drive GC formation andTFH cell generation and function and promote terminalmature of B cell in GC Ig class-switching and antibodyresponses [10 11 20] IL-6 has also induced IL-21 productionand TFH cell generation and mice lacking both IL-6 andIL-21 are unable to generate TFH cell-dependent immuneresponses [20 21] Bcl-6 transcription factor is selectivelyexpressed in TFH cells which is required for programmingof TFH cell generation and Bcl-6 expression is regulated byIL-6 and IL-21 [14 21] In current study we found that thelevels of sera IL-21 and IL-6 concentrations were significantlyhigher in both mild and severe patients with EV71 infectionthan those in HC (severe ormild cases versus HC IL-21IL-6119875 lt 00001) and they were found to be positively correlatedwith the frequency of circulating CXCR5+CD4+ TFH cellsrespectively which suggested that IL-21 and IL-6 might playan important role in the generation of TFH cells In additiona strongly positive correlation was observed between sera IL-21 and NAb-EV71 as well as between sera IL-21 and IL-6indicating that IL-21 and IL-6 could promote the producingof specific NAb-EV71 and IL-6 might help in production ofIL-21 cytokines Furthermore PCR analysis indicated thatthe expression of IL-21 and IL-6 mRNA was significantlyincreased in both mild and severe patients compared to HC(severe or mild cases versus HC IL-21IL-6 mRNA 119875 lt005) However no difference of IL-21 and IL-6 cytokinesand mRNA expression was found between mild and severepatients (severe versus mild cases IL-21IL-6 cytokines 119875 =0390903438 IL-21IL-6 mRNA 119875 = 0488708175) these

10 Journal of Immunology Research

findings indicated that the expression of IL-21 or IL-6 wasnot notably related with severity of disease with acute stage ofEV71 infection and the IL-6 high expression was consistentwith pervious study [7] Additionally no significant differ-ence of peripheral blood Bcl-6mRNA expression amongHCmild and severe patients is consistent with pervious studywhich might be associated with formation of memory ofTFH cells [30 37] These data revealed that IL-21 and IL-6 participated in the generation of TFH cells and specificNAb-EV71 immune responses which were not significantlyassociated with the severity of patients in the acute stage ofEV71 infection

In conclusion our data indicated that increased freque-ncy of circulating TFH cells with ICOShigh and PD-1highexpression was significantly and positively associated withserum specific NAb-EV71 level and IL-21 and IL-6 con-centration in patients with EV71 infection suggesting thatTFH cells and associated cytokines might play a criticalrole in regulating humoral responses in the acute stage ofEV71 infection Further studies on the role of TFH cells inregulating B cell response and antibody production duringthe disease progression of EV71 infection will provide newinsight into understanding the immune pathogenesis of EV71infection in human

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

JianpingWu and David Cui contributed equally to this work

Acknowledgment

This study was supported by the National Key Programs forInfectious Diseases of China (Grant no 2012ZX10004-210)

References

[1] D Nasri L Bouslama S Pillet T Bourlet M Aouni and BPozzetto ldquoBasic rationale current methods and future direc-tions for molecular typing of human enterovirusrdquo ExpertReview of Molecular Diagnostics vol 7 no 4 pp 419ndash434 2007

[2] S-C Chen H-L Chang T-R Yan Y-T Cheng and K-T Chen ldquoAn eight-year study of epidemiologic features ofenterovirus 71 infection in Taiwanrdquo The American Journal ofTropical Medicine and Hygiene vol 77 no 1 pp 188ndash191 2007

[3] C Yang C Deng J Wan L Zhu and Q Leng ldquoNeutralizingantibody response in the patients with hand foot and mouthdisease to enterovirus 71 and its clinical implicationsrdquo VirologyJournal vol 8 article 306 2011

[4] QMao N Li X Yu et al ldquoAntigenicity animal protective effectand genetic characteristics of candidate vaccine strains ofenterovirus 71rdquo Archives of Virology vol 157 no 1 pp 37ndash412012

[5] M Zeng N F El Khatib S Tu et al ldquoSeroepidemiology ofEnterovirus 71 infection prior to the 2011 season in children in

Shanghairdquo Journal of Clinical Virology vol 53 no 4 pp 285ndash289 2012

[6] W-C Huang L-M Huang C-L Kao et al ldquoSeroprevalence ofenterovirus 71 and no evidence of crossprotection of enterovirus71 antibody against the other enteroviruses in kindergartenchildren in Taipei cityrdquo Journal of Microbiology Immunologyand Infection vol 45 no 2 pp 96ndash101 2012

[7] L-Y Chang C A Hsiung C-Y Lu et al ldquoStatus of cellularrather than humoral immunity is correlated with clinicaloutcome of enterovirus 71rdquo Pediatric Research vol 60 no 4 pp466ndash471 2006

[8] S-M Wang H-Y Lei K-J Huang et al ldquoPathogenesis ofenterovirus 71 brainstem encephalitis in pediatric patients rolesof cytokines and cellular immune activation in patients withpulmonary edemardquo Journal of Infectious Diseases vol 188 no4 pp 564ndash570 2003

[9] F D Finkelman J Holmes I M Katona et al ldquoLymphokinecontrol of in vivo immunoglobulin isotype selectionrdquo AnnualReview of Immunology vol 8 pp 303ndash330 1990

[10] C S Ma S Suryani D T Avery et al ldquoEarly commitment ofnave human CD4+ T cells to the T follicular helper (FH) celllineage is induced by IL-12rdquo Immunology and Cell Biology vol87 no 8 pp 590ndash600 2009

[11] T R Mosmann and R L Coffman ldquoTH1 and TH2 cells differ-ent patterns of lymphokine secretion lead to different functionalpropertiesrdquo Annual Review of Immunology vol 7 pp 145ndash1731989

[12] L E Harrington R D Hatton P R Mangan et al ldquoInterleukin17-producing CD4+ effector T cells develop via a lineage distinctfrom the T helper type 1 and 2 lineagesrdquo Nature Immunologyvol 6 no 11 pp 1123ndash1132 2005

[13] C Zhu M Jie Y Liu et al ldquoIncreased frequency of follicularhelper T cells in patients with autoimmune thyroid diseaserdquoJournal of Clinical Endocrinology and Metabolism vol 97 no3 pp 943ndash950 2012

[14] M A Kroenke D Eto M Locci et al ldquoBcl6 andMaf cooperateto instruct human follicular helper CD4 T cell differentiationrdquoJournal of Immunology vol 188 no 8 pp 3734ndash3744 2012

[15] D Breitfeld L Ohl E Kremmer et al ldquoFollicular B helper Tcells express CXC chemokine receptor 5 localize to B cellfollicles and support immunoglobulin productionrdquoThe Journalof Experimental Medicine vol 192 no 11 pp 1545ndash1551 2000

[16] P Schaerli K Willimann A B Lang M Lipp P Loetscherand B Moser ldquoCXC chemokine receptor 5 expression definesfollicular homing T cells with B cell helper functionrdquo Journal ofExperimental Medicine vol 192 no 11 pp 1553ndash1562 2000

[17] C N Arnold D J Campbell M Lipp and E C Butcher ldquoThegerminal center response is impaired in the absence of T cell-expressedCXCR5rdquoEuropean Journal of Immunology vol 37 no1 pp 100ndash109 2007

[18] N Fazilleau L Mark L J McHeyzer-Williams and M GMcHeyzer-Williams ldquoFollicular helper T cells lineage andlocationrdquo Immunity vol 30 no 3 pp 324ndash335 2009

[19] N M Haynes C D C Allen R Lesley K M Ansel N Killeenand J G Cyster ldquoRole of CXCR5 and CCR7 in follicular Thcell positioning and appearance of a programmed cell deathgene-1High germinal center-associated subpopulationrdquo Journalof Immunology vol 179 no 8 pp 5099ndash5108 2007

[20] R Spolski andW J Leonard ldquoIL-21 andT follicular helper cellsrdquoInternational Immunology vol 22 no 1 Article ID dxp112 pp7ndash12 2009

Journal of Immunology Research 11

[21] D Eto C Lao D DiToro et al ldquoIL-21 and IL-6 are critical fordifferent aspects of B cell immunity and redundantly induceoptimal follicular helper CD4 T cell (Tfh) differentiationrdquo PLoSONE vol 6 no 3 Article ID e17739 2011

[22] C Dong A E Juedes U-A Temann et al ldquoICOS co-stimu-linebreak latory receptor is essential for T-cell activation andfunctionrdquo Nature vol 409 no 6816 pp 97ndash101 2001

[23] T Okazaki and T Honjo ldquoThe PD-1-PD-L pathway in immuno-logical tolerancerdquo Trends in Immunology vol 27 no 4 pp 195ndash201 2006

[24] I Yusuf R Kageyama L Monticelli et al ldquoGerminal center Tfollicular helper cell IL-4 production is dependent on signalinglymphocytic activation molecule receptor (CD150)rdquo Journal ofImmunology vol 185 no 1 pp 190ndash202 2010

[25] D Yu and C G Vinuesa ldquoThe elusive identity of T follicularhelper cellsrdquo Trends in Immunology vol 31 no 10 pp 377ndash3832010

[26] M A Linterman R J Rigby RWong et al ldquoRoquin differenti-ates the specialized functions of duplicated T cell costimulatoryreceptor genes CD28 and ICOSrdquo Immunity vol 30 no 2 pp228ndash241 2009

[27] M Locci C Havenar-Daughton E Landais et al ldquoHuman cir-culating PD-1+1CXCR3minusCXCR5+ memory Tfh cells are highlyfunctional and correlate with broadly neutralizing HIV anti-body responsesrdquo Immunity vol 39 no 4 pp 758ndash769 2013

[28] J Feng L Lu C Hua et al ldquoHigh frequency of CD4+CXCR5+TFH cells in patients with immune-active chronic hepatitis BrdquoPLoS ONE vol 6 no 7 Article ID e21698 2011

[29] C Petrovas T Yamamoto M Y Gerner et al ldquoCD4 T follicularhelper cell dynamics during SIV infectionrdquo Journal of ClinicalInvestigation vol 122 no 9 pp 3281ndash3294 2012

[30] N Simpson P A Gatenby A Wilson et al ldquoExpansion ofcirculating T cells resembling follicular helper T cells is a fixedphenotype that identifies a subset of severe systemic lupuserythematosusrdquo Arthritis and Rheumatism vol 62 no 1 pp234ndash244 2010

[31] S Li C Cai J Feng et al ldquoPeripheral T lymphocyte subset imb-alances in children with enterovirus 71-induced hand foot andmouth diseaserdquo Virus Research vol 180 pp 84ndash91 2014

[32] J Chen J Tong H Liu et al ldquoIncreased frequency ofTh17 cellsin the peripheral blood of children infectedwith enterovirus 71rdquoJournal of Medical Virology vol 84 no 5 pp 763ndash767 2012

[33] J Xie Y Jiao Z Qiu Q Li and T Li ldquoSignificant elevation of Bcells at the acute stage in enterovirus 71-infected children withcentral nervous system involvementrdquo Scandinavian Journal ofInfectious Diseases vol 42 no 11-12 pp 931ndash935 2010

[34] Y Liang X Zhou E Yang et al ldquoAnalysis of the Th1Th2reaction in the immune response induced by EV71 inacti-vated vaccine in neonatal rhesus monkeysrdquo Journal of ClinicalImmunology vol 32 no 5 pp 1048ndash1058 2012

[35] Y-W Lin K-C Chang C-M Kao S-P Chang Y-Y Tungand S-H Chen ldquoLymphocyte and antibody responses reduceenterovirus 71 lethality in mice by decreasing tissue viral loadsrdquoJournal of Virology vol 83 no 13 pp 6477ndash6483 2009

[36] National notifiable diseases announced by National Health andFamily Planning Commission of the Peoplersquos Republic of Chinahttpwwwnhfpcgovcnzhuzhanyqxxlistsshtml

[37] R Morita N Schmitt S-E Bentebibel et al ldquoHuman bloodCXCR5+CD4+ T cells are counterparts of T follicular cellsand contain specific subsets that differentially support antibodysecretionrdquo Immunity vol 34 no 1 pp 108ndash121 2011

8 Journal of Immunology Research

0

10

20

30

40

IL-21 (pgmL)

NAb

-EV

71 ti

tres

HC

0 50 100 150

P = 01036

0

200

400

600

IL-21 (pgmL)

NAb

-EV

71 ti

tres

Severe

500 100 200150

P = 00182

minus200

0

200

400

600

IL-21 (pgmL)

NAb

-EV

71 ti

tres

Mild

50 100 200150

P lt 00001

r = 04216

r = 07174

r = 02516

(a)

0

10

20

30

40

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 5 10 2015

P = 04936

0

200

400

600

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 10 20 30 40 50

P = 01039

0

200

400

600

IL-6 (pgmL)

NAb

-EV

71 ti

tres

0 20 40 60 80 100

P = 00741r = 03253r = 03082r = 01072

(b)

Figure 5 Correlation of specific NAb-EV71 titres and cytokines levels from HC and EV71-infected patients including mild and severe cases(a) Relationship between the titres of specific NAb-EV71 and the serum levels of IL-21 (b) Relationship between the titres of specific NAb-EV71 and the serum levels of IL-6

0

10

20

30

40

50

IL-6

mRA

N re

lativ

e exp

ress

ion

ns

HC Mild Severe

lowastlowast

lowast

P = 00116 P = 00078 P = 08175

(a)

0

2

4

6

IL-2

1 m

RAN

relat

ive e

xpre

ssio

n

ns

HC Mild Severe

lowastlowast

lowast

P = 00375 P = 00037 P = 04887

(b)

0

10

20

30

40

50

Bcl-6

mRA

N re

lativ

e exp

ress

ion

nsns ns

HC Mild Severe

P = 04212 P = 04495 P = 09737

(c)

Figure 6 Expression of IL-6 IL-21 and Bcl-6 mRNA in human PBMCs Human PBMCs from 10 mild and 12 severe patients with EV71infection and 15 HC were detected by real-time PCR assay as described in Section 2 (a)The levels of IL-6 mRNA in human PBMCs (b)Thelevels of IL-21 mRNA in human PBMCs (c) The levels of Bcl-6 mRNA in human PBMCs data shown were the mean plusmn SD The horizontallines show the median lowast119875 lt 005 lowastlowast119875 lt 001 lowastlowastlowast119875 lt 0001 ns no significant difference

Moreover there were no significant differences for Bcl-6mRNA expression among these groups tested (HC 2334 plusmn1292 mild 1845plusmn1028 severe 1954plusmn1332) (Figure 6(c))

4 Discussion

There aremore than onemillionHFMD cases and 200 deathseach year since 2008 in China [36] EV71 is one of thecommon causative factors of HFMD in young children less

than 5 years of age [3 5] Although mild diseases are the pre-dominant clinical features of EV71 infection severe diseaseswith neurological involvement and the fetal cases of HFMDare closely associated with EV71 infection identified by PCRandor virus isolation [6ndash8] In our study compared to mildpatients the age of severe patients wasmainly observed below3 years (severe versus mild cases 119875 = 00016) and evaluatedevidences with WBC numbers (severe versus mild cases 119875 lt00001 mild cases versus HC 119875 = 00035) and serum CRPlevels (severe versusmild cases119875 lt 00001 mild cases versus

Journal of Immunology Research 9

HC 119875 lt 00001) suggested that inappropriate inflammationmay be elicited in the progression of HFMD in the acutephase of EV71 infection whichwas consistentwith previouslyreported data [7 31 32]

Previous studies have demonstrated that both humoraland cellular immune responses such as antibodies B and Tcells including Th1Th2 and Th17 and other T cell lineagesand related cytokines play a critical role in protection fromEV71 infection in animal models [4 34 35] In humans EV71infection induced imbalance of TB cells and their relatedcytokines and even caused a systemic dysregulation of theimmune system [31ndash33] Recent studies show that follicularhelper T cells (TFH) a new T cell subpopulation play apivotal role in regulating immune responses and have beenassociated with many diseases [13 20 28] Simpson et alreported an increased frequency of circulatingCXCR5+CD4+TFH cells in patients with systemic lupus erythematosus(SLE) and Sjogrenrsquos syndrome [30] Feng et al demonstratedthat the frequency of circulating TFH cells was significantlyincreased in patients with HBV and these circulating TFHcells participated in the HBV-related immune responses[28] However the potential role of circulating TFH cells inpatients with EV71 infection is not yet known In the presentwork we firstly examined the frequency of circulating TFHcells in mild and severe patients with EV71 infection andhealthy controlsThe results demonstrated that the frequencyof circulating CXCR5+CD4+ TFH cells in mild and severepatients in the acute stage of EV71 infection was significantlyhigher than that of HC (severe or mild cases versus HC119875 lt 005) respectively However severe patients did notdiffer significantly from mild patients in the percentage ofperipheral blood CXCR5+CD4+ TFH cells (severe versusmild cases 119875 = 05151) These findings clearly indicatedthat TFH cells played an important role in the EV71-inducedimmune responses

Simpson et al detected that the high frequency of circu-lating TFH cells with high expression of ICOS andor PD-1 molecules was associated with the severity of end-organinvolvement in patients with systemic lupus erythematosus(SLE) and Sjogrenrsquos syndrome [30] Zhu et al demonstratedthe increased frequency of TFH cells with highly expressedICOS andor PD-1 in patients with autoimmune thyroiddisease and rheumatoid arthritis respectively and both ICOSand PD-1 molecules are closely positively correlated withthe function of TFH cells [13] We detected an enhancedfrequency of PD-1high and ICOShighCXCR5+CD4+ TFH cellsin the mild and severe EV71-induced patients comparedto HC (severe or mild cases versus HC 119875 lt 005) Inaddition a closely positive correlation was observed betweenthe frequencies of these two subpopulations of PD-1high andICOShighCXCR5+CD4+ T cells These data supported thedefinition of circulating TFH cells However the percentageof circulating TFH cells with ICOShigh and PD-1high expres-sion revealed no significant difference between the mild andsevere patients (severe versus mild cases PD-1high 119875 =01085 severe versus mild cases ICOShigh 119875 = 01930)In other words EV71 increased the frequency of circulatingTFH cells with ICOShigh and PD-1high expression that was not

notably associated with the severity of patients in the acutestage of EV71 infection

It is known that TFH cells are critical for promoting thedevelopment of antibody response by helper B cells in GC[10 11] Several seroepidemiologic investigations indicatedthat the neutralizing antibody can protect adults as well aschildren from EV71 infection once again although the titersof specific neutralizing antibody were not correlated withseverity of the disease with EV71 infection [3ndash6 34 35]Why is that To date that is not well understood Our dataalso indicated that the serum titers of specific NAb-EV71in patients with acute stage of EV71 infection were higherthan those in HC (severe or mild cases versus HC 119875 lt00001) but no significant difference was observed betweenmild and severe patients groups (severe versus mild cases119875 = 09693) which corresponded to previous studies [3]Additionally we found that enhanced frequency of circulat-ing CXCR5+CD4+ TFH PD-1highCXCR5+CD4+ TFH andICOShighCXCR5+CD4+ TFH cells was notably positively cor-related with the titres of specific NAb-EV71 in patients withEV71 infection and a significant associationwas not observedbetween themild and severe patients respectivelyThese dataalso indicated that TFH cells with ICOShigh and PD-1highexpression participated in regulating antibody responses inthe acute stage of EV71 infection at the same time whichmight give a good explanation to why there is no significantdifference between the levels of specific NAb-EV71 in bothmild and severe patients with EV71 infection

Recent studies demonstrated that not only did TFH cellssecrete IL-21 but IL-21 could also drive GC formation andTFH cell generation and function and promote terminalmature of B cell in GC Ig class-switching and antibodyresponses [10 11 20] IL-6 has also induced IL-21 productionand TFH cell generation and mice lacking both IL-6 andIL-21 are unable to generate TFH cell-dependent immuneresponses [20 21] Bcl-6 transcription factor is selectivelyexpressed in TFH cells which is required for programmingof TFH cell generation and Bcl-6 expression is regulated byIL-6 and IL-21 [14 21] In current study we found that thelevels of sera IL-21 and IL-6 concentrations were significantlyhigher in both mild and severe patients with EV71 infectionthan those in HC (severe ormild cases versus HC IL-21IL-6119875 lt 00001) and they were found to be positively correlatedwith the frequency of circulating CXCR5+CD4+ TFH cellsrespectively which suggested that IL-21 and IL-6 might playan important role in the generation of TFH cells In additiona strongly positive correlation was observed between sera IL-21 and NAb-EV71 as well as between sera IL-21 and IL-6indicating that IL-21 and IL-6 could promote the producingof specific NAb-EV71 and IL-6 might help in production ofIL-21 cytokines Furthermore PCR analysis indicated thatthe expression of IL-21 and IL-6 mRNA was significantlyincreased in both mild and severe patients compared to HC(severe or mild cases versus HC IL-21IL-6 mRNA 119875 lt005) However no difference of IL-21 and IL-6 cytokinesand mRNA expression was found between mild and severepatients (severe versus mild cases IL-21IL-6 cytokines 119875 =0390903438 IL-21IL-6 mRNA 119875 = 0488708175) these

10 Journal of Immunology Research

findings indicated that the expression of IL-21 or IL-6 wasnot notably related with severity of disease with acute stage ofEV71 infection and the IL-6 high expression was consistentwith pervious study [7] Additionally no significant differ-ence of peripheral blood Bcl-6mRNA expression amongHCmild and severe patients is consistent with pervious studywhich might be associated with formation of memory ofTFH cells [30 37] These data revealed that IL-21 and IL-6 participated in the generation of TFH cells and specificNAb-EV71 immune responses which were not significantlyassociated with the severity of patients in the acute stage ofEV71 infection

In conclusion our data indicated that increased freque-ncy of circulating TFH cells with ICOShigh and PD-1highexpression was significantly and positively associated withserum specific NAb-EV71 level and IL-21 and IL-6 con-centration in patients with EV71 infection suggesting thatTFH cells and associated cytokines might play a criticalrole in regulating humoral responses in the acute stage ofEV71 infection Further studies on the role of TFH cells inregulating B cell response and antibody production duringthe disease progression of EV71 infection will provide newinsight into understanding the immune pathogenesis of EV71infection in human

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

JianpingWu and David Cui contributed equally to this work

Acknowledgment

This study was supported by the National Key Programs forInfectious Diseases of China (Grant no 2012ZX10004-210)

References

[1] D Nasri L Bouslama S Pillet T Bourlet M Aouni and BPozzetto ldquoBasic rationale current methods and future direc-tions for molecular typing of human enterovirusrdquo ExpertReview of Molecular Diagnostics vol 7 no 4 pp 419ndash434 2007

[2] S-C Chen H-L Chang T-R Yan Y-T Cheng and K-T Chen ldquoAn eight-year study of epidemiologic features ofenterovirus 71 infection in Taiwanrdquo The American Journal ofTropical Medicine and Hygiene vol 77 no 1 pp 188ndash191 2007

[3] C Yang C Deng J Wan L Zhu and Q Leng ldquoNeutralizingantibody response in the patients with hand foot and mouthdisease to enterovirus 71 and its clinical implicationsrdquo VirologyJournal vol 8 article 306 2011

[4] QMao N Li X Yu et al ldquoAntigenicity animal protective effectand genetic characteristics of candidate vaccine strains ofenterovirus 71rdquo Archives of Virology vol 157 no 1 pp 37ndash412012

[5] M Zeng N F El Khatib S Tu et al ldquoSeroepidemiology ofEnterovirus 71 infection prior to the 2011 season in children in

Shanghairdquo Journal of Clinical Virology vol 53 no 4 pp 285ndash289 2012

[6] W-C Huang L-M Huang C-L Kao et al ldquoSeroprevalence ofenterovirus 71 and no evidence of crossprotection of enterovirus71 antibody against the other enteroviruses in kindergartenchildren in Taipei cityrdquo Journal of Microbiology Immunologyand Infection vol 45 no 2 pp 96ndash101 2012

[7] L-Y Chang C A Hsiung C-Y Lu et al ldquoStatus of cellularrather than humoral immunity is correlated with clinicaloutcome of enterovirus 71rdquo Pediatric Research vol 60 no 4 pp466ndash471 2006

[8] S-M Wang H-Y Lei K-J Huang et al ldquoPathogenesis ofenterovirus 71 brainstem encephalitis in pediatric patients rolesof cytokines and cellular immune activation in patients withpulmonary edemardquo Journal of Infectious Diseases vol 188 no4 pp 564ndash570 2003

[9] F D Finkelman J Holmes I M Katona et al ldquoLymphokinecontrol of in vivo immunoglobulin isotype selectionrdquo AnnualReview of Immunology vol 8 pp 303ndash330 1990

[10] C S Ma S Suryani D T Avery et al ldquoEarly commitment ofnave human CD4+ T cells to the T follicular helper (FH) celllineage is induced by IL-12rdquo Immunology and Cell Biology vol87 no 8 pp 590ndash600 2009

[11] T R Mosmann and R L Coffman ldquoTH1 and TH2 cells differ-ent patterns of lymphokine secretion lead to different functionalpropertiesrdquo Annual Review of Immunology vol 7 pp 145ndash1731989

[12] L E Harrington R D Hatton P R Mangan et al ldquoInterleukin17-producing CD4+ effector T cells develop via a lineage distinctfrom the T helper type 1 and 2 lineagesrdquo Nature Immunologyvol 6 no 11 pp 1123ndash1132 2005

[13] C Zhu M Jie Y Liu et al ldquoIncreased frequency of follicularhelper T cells in patients with autoimmune thyroid diseaserdquoJournal of Clinical Endocrinology and Metabolism vol 97 no3 pp 943ndash950 2012

[14] M A Kroenke D Eto M Locci et al ldquoBcl6 andMaf cooperateto instruct human follicular helper CD4 T cell differentiationrdquoJournal of Immunology vol 188 no 8 pp 3734ndash3744 2012

[15] D Breitfeld L Ohl E Kremmer et al ldquoFollicular B helper Tcells express CXC chemokine receptor 5 localize to B cellfollicles and support immunoglobulin productionrdquoThe Journalof Experimental Medicine vol 192 no 11 pp 1545ndash1551 2000

[16] P Schaerli K Willimann A B Lang M Lipp P Loetscherand B Moser ldquoCXC chemokine receptor 5 expression definesfollicular homing T cells with B cell helper functionrdquo Journal ofExperimental Medicine vol 192 no 11 pp 1553ndash1562 2000

[17] C N Arnold D J Campbell M Lipp and E C Butcher ldquoThegerminal center response is impaired in the absence of T cell-expressedCXCR5rdquoEuropean Journal of Immunology vol 37 no1 pp 100ndash109 2007

[18] N Fazilleau L Mark L J McHeyzer-Williams and M GMcHeyzer-Williams ldquoFollicular helper T cells lineage andlocationrdquo Immunity vol 30 no 3 pp 324ndash335 2009

[19] N M Haynes C D C Allen R Lesley K M Ansel N Killeenand J G Cyster ldquoRole of CXCR5 and CCR7 in follicular Thcell positioning and appearance of a programmed cell deathgene-1High germinal center-associated subpopulationrdquo Journalof Immunology vol 179 no 8 pp 5099ndash5108 2007

[20] R Spolski andW J Leonard ldquoIL-21 andT follicular helper cellsrdquoInternational Immunology vol 22 no 1 Article ID dxp112 pp7ndash12 2009

Journal of Immunology Research 11

[21] D Eto C Lao D DiToro et al ldquoIL-21 and IL-6 are critical fordifferent aspects of B cell immunity and redundantly induceoptimal follicular helper CD4 T cell (Tfh) differentiationrdquo PLoSONE vol 6 no 3 Article ID e17739 2011

[22] C Dong A E Juedes U-A Temann et al ldquoICOS co-stimu-linebreak latory receptor is essential for T-cell activation andfunctionrdquo Nature vol 409 no 6816 pp 97ndash101 2001

[23] T Okazaki and T Honjo ldquoThe PD-1-PD-L pathway in immuno-logical tolerancerdquo Trends in Immunology vol 27 no 4 pp 195ndash201 2006

[24] I Yusuf R Kageyama L Monticelli et al ldquoGerminal center Tfollicular helper cell IL-4 production is dependent on signalinglymphocytic activation molecule receptor (CD150)rdquo Journal ofImmunology vol 185 no 1 pp 190ndash202 2010

[25] D Yu and C G Vinuesa ldquoThe elusive identity of T follicularhelper cellsrdquo Trends in Immunology vol 31 no 10 pp 377ndash3832010

[26] M A Linterman R J Rigby RWong et al ldquoRoquin differenti-ates the specialized functions of duplicated T cell costimulatoryreceptor genes CD28 and ICOSrdquo Immunity vol 30 no 2 pp228ndash241 2009

[27] M Locci C Havenar-Daughton E Landais et al ldquoHuman cir-culating PD-1+1CXCR3minusCXCR5+ memory Tfh cells are highlyfunctional and correlate with broadly neutralizing HIV anti-body responsesrdquo Immunity vol 39 no 4 pp 758ndash769 2013

[28] J Feng L Lu C Hua et al ldquoHigh frequency of CD4+CXCR5+TFH cells in patients with immune-active chronic hepatitis BrdquoPLoS ONE vol 6 no 7 Article ID e21698 2011

[29] C Petrovas T Yamamoto M Y Gerner et al ldquoCD4 T follicularhelper cell dynamics during SIV infectionrdquo Journal of ClinicalInvestigation vol 122 no 9 pp 3281ndash3294 2012

[30] N Simpson P A Gatenby A Wilson et al ldquoExpansion ofcirculating T cells resembling follicular helper T cells is a fixedphenotype that identifies a subset of severe systemic lupuserythematosusrdquo Arthritis and Rheumatism vol 62 no 1 pp234ndash244 2010

[31] S Li C Cai J Feng et al ldquoPeripheral T lymphocyte subset imb-alances in children with enterovirus 71-induced hand foot andmouth diseaserdquo Virus Research vol 180 pp 84ndash91 2014

[32] J Chen J Tong H Liu et al ldquoIncreased frequency ofTh17 cellsin the peripheral blood of children infectedwith enterovirus 71rdquoJournal of Medical Virology vol 84 no 5 pp 763ndash767 2012

[33] J Xie Y Jiao Z Qiu Q Li and T Li ldquoSignificant elevation of Bcells at the acute stage in enterovirus 71-infected children withcentral nervous system involvementrdquo Scandinavian Journal ofInfectious Diseases vol 42 no 11-12 pp 931ndash935 2010

[34] Y Liang X Zhou E Yang et al ldquoAnalysis of the Th1Th2reaction in the immune response induced by EV71 inacti-vated vaccine in neonatal rhesus monkeysrdquo Journal of ClinicalImmunology vol 32 no 5 pp 1048ndash1058 2012

[35] Y-W Lin K-C Chang C-M Kao S-P Chang Y-Y Tungand S-H Chen ldquoLymphocyte and antibody responses reduceenterovirus 71 lethality in mice by decreasing tissue viral loadsrdquoJournal of Virology vol 83 no 13 pp 6477ndash6483 2009

[36] National notifiable diseases announced by National Health andFamily Planning Commission of the Peoplersquos Republic of Chinahttpwwwnhfpcgovcnzhuzhanyqxxlistsshtml

[37] R Morita N Schmitt S-E Bentebibel et al ldquoHuman bloodCXCR5+CD4+ T cells are counterparts of T follicular cellsand contain specific subsets that differentially support antibodysecretionrdquo Immunity vol 34 no 1 pp 108ndash121 2011

Journal of Immunology Research 9

HC 119875 lt 00001) suggested that inappropriate inflammationmay be elicited in the progression of HFMD in the acutephase of EV71 infection whichwas consistentwith previouslyreported data [7 31 32]

Previous studies have demonstrated that both humoraland cellular immune responses such as antibodies B and Tcells including Th1Th2 and Th17 and other T cell lineagesand related cytokines play a critical role in protection fromEV71 infection in animal models [4 34 35] In humans EV71infection induced imbalance of TB cells and their relatedcytokines and even caused a systemic dysregulation of theimmune system [31ndash33] Recent studies show that follicularhelper T cells (TFH) a new T cell subpopulation play apivotal role in regulating immune responses and have beenassociated with many diseases [13 20 28] Simpson et alreported an increased frequency of circulatingCXCR5+CD4+TFH cells in patients with systemic lupus erythematosus(SLE) and Sjogrenrsquos syndrome [30] Feng et al demonstratedthat the frequency of circulating TFH cells was significantlyincreased in patients with HBV and these circulating TFHcells participated in the HBV-related immune responses[28] However the potential role of circulating TFH cells inpatients with EV71 infection is not yet known In the presentwork we firstly examined the frequency of circulating TFHcells in mild and severe patients with EV71 infection andhealthy controlsThe results demonstrated that the frequencyof circulating CXCR5+CD4+ TFH cells in mild and severepatients in the acute stage of EV71 infection was significantlyhigher than that of HC (severe or mild cases versus HC119875 lt 005) respectively However severe patients did notdiffer significantly from mild patients in the percentage ofperipheral blood CXCR5+CD4+ TFH cells (severe versusmild cases 119875 = 05151) These findings clearly indicatedthat TFH cells played an important role in the EV71-inducedimmune responses

Simpson et al detected that the high frequency of circu-lating TFH cells with high expression of ICOS andor PD-1 molecules was associated with the severity of end-organinvolvement in patients with systemic lupus erythematosus(SLE) and Sjogrenrsquos syndrome [30] Zhu et al demonstratedthe increased frequency of TFH cells with highly expressedICOS andor PD-1 in patients with autoimmune thyroiddisease and rheumatoid arthritis respectively and both ICOSand PD-1 molecules are closely positively correlated withthe function of TFH cells [13] We detected an enhancedfrequency of PD-1high and ICOShighCXCR5+CD4+ TFH cellsin the mild and severe EV71-induced patients comparedto HC (severe or mild cases versus HC 119875 lt 005) Inaddition a closely positive correlation was observed betweenthe frequencies of these two subpopulations of PD-1high andICOShighCXCR5+CD4+ T cells These data supported thedefinition of circulating TFH cells However the percentageof circulating TFH cells with ICOShigh and PD-1high expres-sion revealed no significant difference between the mild andsevere patients (severe versus mild cases PD-1high 119875 =01085 severe versus mild cases ICOShigh 119875 = 01930)In other words EV71 increased the frequency of circulatingTFH cells with ICOShigh and PD-1high expression that was not

notably associated with the severity of patients in the acutestage of EV71 infection

It is known that TFH cells are critical for promoting thedevelopment of antibody response by helper B cells in GC[10 11] Several seroepidemiologic investigations indicatedthat the neutralizing antibody can protect adults as well aschildren from EV71 infection once again although the titersof specific neutralizing antibody were not correlated withseverity of the disease with EV71 infection [3ndash6 34 35]Why is that To date that is not well understood Our dataalso indicated that the serum titers of specific NAb-EV71in patients with acute stage of EV71 infection were higherthan those in HC (severe or mild cases versus HC 119875 lt00001) but no significant difference was observed betweenmild and severe patients groups (severe versus mild cases119875 = 09693) which corresponded to previous studies [3]Additionally we found that enhanced frequency of circulat-ing CXCR5+CD4+ TFH PD-1highCXCR5+CD4+ TFH andICOShighCXCR5+CD4+ TFH cells was notably positively cor-related with the titres of specific NAb-EV71 in patients withEV71 infection and a significant associationwas not observedbetween themild and severe patients respectivelyThese dataalso indicated that TFH cells with ICOShigh and PD-1highexpression participated in regulating antibody responses inthe acute stage of EV71 infection at the same time whichmight give a good explanation to why there is no significantdifference between the levels of specific NAb-EV71 in bothmild and severe patients with EV71 infection

Recent studies demonstrated that not only did TFH cellssecrete IL-21 but IL-21 could also drive GC formation andTFH cell generation and function and promote terminalmature of B cell in GC Ig class-switching and antibodyresponses [10 11 20] IL-6 has also induced IL-21 productionand TFH cell generation and mice lacking both IL-6 andIL-21 are unable to generate TFH cell-dependent immuneresponses [20 21] Bcl-6 transcription factor is selectivelyexpressed in TFH cells which is required for programmingof TFH cell generation and Bcl-6 expression is regulated byIL-6 and IL-21 [14 21] In current study we found that thelevels of sera IL-21 and IL-6 concentrations were significantlyhigher in both mild and severe patients with EV71 infectionthan those in HC (severe ormild cases versus HC IL-21IL-6119875 lt 00001) and they were found to be positively correlatedwith the frequency of circulating CXCR5+CD4+ TFH cellsrespectively which suggested that IL-21 and IL-6 might playan important role in the generation of TFH cells In additiona strongly positive correlation was observed between sera IL-21 and NAb-EV71 as well as between sera IL-21 and IL-6indicating that IL-21 and IL-6 could promote the producingof specific NAb-EV71 and IL-6 might help in production ofIL-21 cytokines Furthermore PCR analysis indicated thatthe expression of IL-21 and IL-6 mRNA was significantlyincreased in both mild and severe patients compared to HC(severe or mild cases versus HC IL-21IL-6 mRNA 119875 lt005) However no difference of IL-21 and IL-6 cytokinesand mRNA expression was found between mild and severepatients (severe versus mild cases IL-21IL-6 cytokines 119875 =0390903438 IL-21IL-6 mRNA 119875 = 0488708175) these

10 Journal of Immunology Research

findings indicated that the expression of IL-21 or IL-6 wasnot notably related with severity of disease with acute stage ofEV71 infection and the IL-6 high expression was consistentwith pervious study [7] Additionally no significant differ-ence of peripheral blood Bcl-6mRNA expression amongHCmild and severe patients is consistent with pervious studywhich might be associated with formation of memory ofTFH cells [30 37] These data revealed that IL-21 and IL-6 participated in the generation of TFH cells and specificNAb-EV71 immune responses which were not significantlyassociated with the severity of patients in the acute stage ofEV71 infection

In conclusion our data indicated that increased freque-ncy of circulating TFH cells with ICOShigh and PD-1highexpression was significantly and positively associated withserum specific NAb-EV71 level and IL-21 and IL-6 con-centration in patients with EV71 infection suggesting thatTFH cells and associated cytokines might play a criticalrole in regulating humoral responses in the acute stage ofEV71 infection Further studies on the role of TFH cells inregulating B cell response and antibody production duringthe disease progression of EV71 infection will provide newinsight into understanding the immune pathogenesis of EV71infection in human

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

JianpingWu and David Cui contributed equally to this work

Acknowledgment

This study was supported by the National Key Programs forInfectious Diseases of China (Grant no 2012ZX10004-210)

References

[1] D Nasri L Bouslama S Pillet T Bourlet M Aouni and BPozzetto ldquoBasic rationale current methods and future direc-tions for molecular typing of human enterovirusrdquo ExpertReview of Molecular Diagnostics vol 7 no 4 pp 419ndash434 2007

[2] S-C Chen H-L Chang T-R Yan Y-T Cheng and K-T Chen ldquoAn eight-year study of epidemiologic features ofenterovirus 71 infection in Taiwanrdquo The American Journal ofTropical Medicine and Hygiene vol 77 no 1 pp 188ndash191 2007

[3] C Yang C Deng J Wan L Zhu and Q Leng ldquoNeutralizingantibody response in the patients with hand foot and mouthdisease to enterovirus 71 and its clinical implicationsrdquo VirologyJournal vol 8 article 306 2011

[4] QMao N Li X Yu et al ldquoAntigenicity animal protective effectand genetic characteristics of candidate vaccine strains ofenterovirus 71rdquo Archives of Virology vol 157 no 1 pp 37ndash412012

[5] M Zeng N F El Khatib S Tu et al ldquoSeroepidemiology ofEnterovirus 71 infection prior to the 2011 season in children in

Shanghairdquo Journal of Clinical Virology vol 53 no 4 pp 285ndash289 2012

[6] W-C Huang L-M Huang C-L Kao et al ldquoSeroprevalence ofenterovirus 71 and no evidence of crossprotection of enterovirus71 antibody against the other enteroviruses in kindergartenchildren in Taipei cityrdquo Journal of Microbiology Immunologyand Infection vol 45 no 2 pp 96ndash101 2012

[7] L-Y Chang C A Hsiung C-Y Lu et al ldquoStatus of cellularrather than humoral immunity is correlated with clinicaloutcome of enterovirus 71rdquo Pediatric Research vol 60 no 4 pp466ndash471 2006

[8] S-M Wang H-Y Lei K-J Huang et al ldquoPathogenesis ofenterovirus 71 brainstem encephalitis in pediatric patients rolesof cytokines and cellular immune activation in patients withpulmonary edemardquo Journal of Infectious Diseases vol 188 no4 pp 564ndash570 2003

[9] F D Finkelman J Holmes I M Katona et al ldquoLymphokinecontrol of in vivo immunoglobulin isotype selectionrdquo AnnualReview of Immunology vol 8 pp 303ndash330 1990

[10] C S Ma S Suryani D T Avery et al ldquoEarly commitment ofnave human CD4+ T cells to the T follicular helper (FH) celllineage is induced by IL-12rdquo Immunology and Cell Biology vol87 no 8 pp 590ndash600 2009

[11] T R Mosmann and R L Coffman ldquoTH1 and TH2 cells differ-ent patterns of lymphokine secretion lead to different functionalpropertiesrdquo Annual Review of Immunology vol 7 pp 145ndash1731989

[12] L E Harrington R D Hatton P R Mangan et al ldquoInterleukin17-producing CD4+ effector T cells develop via a lineage distinctfrom the T helper type 1 and 2 lineagesrdquo Nature Immunologyvol 6 no 11 pp 1123ndash1132 2005

[13] C Zhu M Jie Y Liu et al ldquoIncreased frequency of follicularhelper T cells in patients with autoimmune thyroid diseaserdquoJournal of Clinical Endocrinology and Metabolism vol 97 no3 pp 943ndash950 2012

[14] M A Kroenke D Eto M Locci et al ldquoBcl6 andMaf cooperateto instruct human follicular helper CD4 T cell differentiationrdquoJournal of Immunology vol 188 no 8 pp 3734ndash3744 2012

[15] D Breitfeld L Ohl E Kremmer et al ldquoFollicular B helper Tcells express CXC chemokine receptor 5 localize to B cellfollicles and support immunoglobulin productionrdquoThe Journalof Experimental Medicine vol 192 no 11 pp 1545ndash1551 2000

[16] P Schaerli K Willimann A B Lang M Lipp P Loetscherand B Moser ldquoCXC chemokine receptor 5 expression definesfollicular homing T cells with B cell helper functionrdquo Journal ofExperimental Medicine vol 192 no 11 pp 1553ndash1562 2000

[17] C N Arnold D J Campbell M Lipp and E C Butcher ldquoThegerminal center response is impaired in the absence of T cell-expressedCXCR5rdquoEuropean Journal of Immunology vol 37 no1 pp 100ndash109 2007

[18] N Fazilleau L Mark L J McHeyzer-Williams and M GMcHeyzer-Williams ldquoFollicular helper T cells lineage andlocationrdquo Immunity vol 30 no 3 pp 324ndash335 2009

[19] N M Haynes C D C Allen R Lesley K M Ansel N Killeenand J G Cyster ldquoRole of CXCR5 and CCR7 in follicular Thcell positioning and appearance of a programmed cell deathgene-1High germinal center-associated subpopulationrdquo Journalof Immunology vol 179 no 8 pp 5099ndash5108 2007

[20] R Spolski andW J Leonard ldquoIL-21 andT follicular helper cellsrdquoInternational Immunology vol 22 no 1 Article ID dxp112 pp7ndash12 2009

Journal of Immunology Research 11

[21] D Eto C Lao D DiToro et al ldquoIL-21 and IL-6 are critical fordifferent aspects of B cell immunity and redundantly induceoptimal follicular helper CD4 T cell (Tfh) differentiationrdquo PLoSONE vol 6 no 3 Article ID e17739 2011

[22] C Dong A E Juedes U-A Temann et al ldquoICOS co-stimu-linebreak latory receptor is essential for T-cell activation andfunctionrdquo Nature vol 409 no 6816 pp 97ndash101 2001

[23] T Okazaki and T Honjo ldquoThe PD-1-PD-L pathway in immuno-logical tolerancerdquo Trends in Immunology vol 27 no 4 pp 195ndash201 2006

[24] I Yusuf R Kageyama L Monticelli et al ldquoGerminal center Tfollicular helper cell IL-4 production is dependent on signalinglymphocytic activation molecule receptor (CD150)rdquo Journal ofImmunology vol 185 no 1 pp 190ndash202 2010

[25] D Yu and C G Vinuesa ldquoThe elusive identity of T follicularhelper cellsrdquo Trends in Immunology vol 31 no 10 pp 377ndash3832010

[26] M A Linterman R J Rigby RWong et al ldquoRoquin differenti-ates the specialized functions of duplicated T cell costimulatoryreceptor genes CD28 and ICOSrdquo Immunity vol 30 no 2 pp228ndash241 2009

[27] M Locci C Havenar-Daughton E Landais et al ldquoHuman cir-culating PD-1+1CXCR3minusCXCR5+ memory Tfh cells are highlyfunctional and correlate with broadly neutralizing HIV anti-body responsesrdquo Immunity vol 39 no 4 pp 758ndash769 2013

[28] J Feng L Lu C Hua et al ldquoHigh frequency of CD4+CXCR5+TFH cells in patients with immune-active chronic hepatitis BrdquoPLoS ONE vol 6 no 7 Article ID e21698 2011

[29] C Petrovas T Yamamoto M Y Gerner et al ldquoCD4 T follicularhelper cell dynamics during SIV infectionrdquo Journal of ClinicalInvestigation vol 122 no 9 pp 3281ndash3294 2012

[30] N Simpson P A Gatenby A Wilson et al ldquoExpansion ofcirculating T cells resembling follicular helper T cells is a fixedphenotype that identifies a subset of severe systemic lupuserythematosusrdquo Arthritis and Rheumatism vol 62 no 1 pp234ndash244 2010

[31] S Li C Cai J Feng et al ldquoPeripheral T lymphocyte subset imb-alances in children with enterovirus 71-induced hand foot andmouth diseaserdquo Virus Research vol 180 pp 84ndash91 2014

[32] J Chen J Tong H Liu et al ldquoIncreased frequency ofTh17 cellsin the peripheral blood of children infectedwith enterovirus 71rdquoJournal of Medical Virology vol 84 no 5 pp 763ndash767 2012

[33] J Xie Y Jiao Z Qiu Q Li and T Li ldquoSignificant elevation of Bcells at the acute stage in enterovirus 71-infected children withcentral nervous system involvementrdquo Scandinavian Journal ofInfectious Diseases vol 42 no 11-12 pp 931ndash935 2010

[34] Y Liang X Zhou E Yang et al ldquoAnalysis of the Th1Th2reaction in the immune response induced by EV71 inacti-vated vaccine in neonatal rhesus monkeysrdquo Journal of ClinicalImmunology vol 32 no 5 pp 1048ndash1058 2012

[35] Y-W Lin K-C Chang C-M Kao S-P Chang Y-Y Tungand S-H Chen ldquoLymphocyte and antibody responses reduceenterovirus 71 lethality in mice by decreasing tissue viral loadsrdquoJournal of Virology vol 83 no 13 pp 6477ndash6483 2009

[36] National notifiable diseases announced by National Health andFamily Planning Commission of the Peoplersquos Republic of Chinahttpwwwnhfpcgovcnzhuzhanyqxxlistsshtml

[37] R Morita N Schmitt S-E Bentebibel et al ldquoHuman bloodCXCR5+CD4+ T cells are counterparts of T follicular cellsand contain specific subsets that differentially support antibodysecretionrdquo Immunity vol 34 no 1 pp 108ndash121 2011

10 Journal of Immunology Research

findings indicated that the expression of IL-21 or IL-6 wasnot notably related with severity of disease with acute stage ofEV71 infection and the IL-6 high expression was consistentwith pervious study [7] Additionally no significant differ-ence of peripheral blood Bcl-6mRNA expression amongHCmild and severe patients is consistent with pervious studywhich might be associated with formation of memory ofTFH cells [30 37] These data revealed that IL-21 and IL-6 participated in the generation of TFH cells and specificNAb-EV71 immune responses which were not significantlyassociated with the severity of patients in the acute stage ofEV71 infection

In conclusion our data indicated that increased freque-ncy of circulating TFH cells with ICOShigh and PD-1highexpression was significantly and positively associated withserum specific NAb-EV71 level and IL-21 and IL-6 con-centration in patients with EV71 infection suggesting thatTFH cells and associated cytokines might play a criticalrole in regulating humoral responses in the acute stage ofEV71 infection Further studies on the role of TFH cells inregulating B cell response and antibody production duringthe disease progression of EV71 infection will provide newinsight into understanding the immune pathogenesis of EV71infection in human

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

JianpingWu and David Cui contributed equally to this work

Acknowledgment

This study was supported by the National Key Programs forInfectious Diseases of China (Grant no 2012ZX10004-210)

References

[1] D Nasri L Bouslama S Pillet T Bourlet M Aouni and BPozzetto ldquoBasic rationale current methods and future direc-tions for molecular typing of human enterovirusrdquo ExpertReview of Molecular Diagnostics vol 7 no 4 pp 419ndash434 2007

[2] S-C Chen H-L Chang T-R Yan Y-T Cheng and K-T Chen ldquoAn eight-year study of epidemiologic features ofenterovirus 71 infection in Taiwanrdquo The American Journal ofTropical Medicine and Hygiene vol 77 no 1 pp 188ndash191 2007

[3] C Yang C Deng J Wan L Zhu and Q Leng ldquoNeutralizingantibody response in the patients with hand foot and mouthdisease to enterovirus 71 and its clinical implicationsrdquo VirologyJournal vol 8 article 306 2011

[4] QMao N Li X Yu et al ldquoAntigenicity animal protective effectand genetic characteristics of candidate vaccine strains ofenterovirus 71rdquo Archives of Virology vol 157 no 1 pp 37ndash412012

[5] M Zeng N F El Khatib S Tu et al ldquoSeroepidemiology ofEnterovirus 71 infection prior to the 2011 season in children in

Shanghairdquo Journal of Clinical Virology vol 53 no 4 pp 285ndash289 2012

[6] W-C Huang L-M Huang C-L Kao et al ldquoSeroprevalence ofenterovirus 71 and no evidence of crossprotection of enterovirus71 antibody against the other enteroviruses in kindergartenchildren in Taipei cityrdquo Journal of Microbiology Immunologyand Infection vol 45 no 2 pp 96ndash101 2012

[7] L-Y Chang C A Hsiung C-Y Lu et al ldquoStatus of cellularrather than humoral immunity is correlated with clinicaloutcome of enterovirus 71rdquo Pediatric Research vol 60 no 4 pp466ndash471 2006

[8] S-M Wang H-Y Lei K-J Huang et al ldquoPathogenesis ofenterovirus 71 brainstem encephalitis in pediatric patients rolesof cytokines and cellular immune activation in patients withpulmonary edemardquo Journal of Infectious Diseases vol 188 no4 pp 564ndash570 2003

[9] F D Finkelman J Holmes I M Katona et al ldquoLymphokinecontrol of in vivo immunoglobulin isotype selectionrdquo AnnualReview of Immunology vol 8 pp 303ndash330 1990

[10] C S Ma S Suryani D T Avery et al ldquoEarly commitment ofnave human CD4+ T cells to the T follicular helper (FH) celllineage is induced by IL-12rdquo Immunology and Cell Biology vol87 no 8 pp 590ndash600 2009

[11] T R Mosmann and R L Coffman ldquoTH1 and TH2 cells differ-ent patterns of lymphokine secretion lead to different functionalpropertiesrdquo Annual Review of Immunology vol 7 pp 145ndash1731989

[12] L E Harrington R D Hatton P R Mangan et al ldquoInterleukin17-producing CD4+ effector T cells develop via a lineage distinctfrom the T helper type 1 and 2 lineagesrdquo Nature Immunologyvol 6 no 11 pp 1123ndash1132 2005

[13] C Zhu M Jie Y Liu et al ldquoIncreased frequency of follicularhelper T cells in patients with autoimmune thyroid diseaserdquoJournal of Clinical Endocrinology and Metabolism vol 97 no3 pp 943ndash950 2012

[14] M A Kroenke D Eto M Locci et al ldquoBcl6 andMaf cooperateto instruct human follicular helper CD4 T cell differentiationrdquoJournal of Immunology vol 188 no 8 pp 3734ndash3744 2012

[15] D Breitfeld L Ohl E Kremmer et al ldquoFollicular B helper Tcells express CXC chemokine receptor 5 localize to B cellfollicles and support immunoglobulin productionrdquoThe Journalof Experimental Medicine vol 192 no 11 pp 1545ndash1551 2000

[16] P Schaerli K Willimann A B Lang M Lipp P Loetscherand B Moser ldquoCXC chemokine receptor 5 expression definesfollicular homing T cells with B cell helper functionrdquo Journal ofExperimental Medicine vol 192 no 11 pp 1553ndash1562 2000

[17] C N Arnold D J Campbell M Lipp and E C Butcher ldquoThegerminal center response is impaired in the absence of T cell-expressedCXCR5rdquoEuropean Journal of Immunology vol 37 no1 pp 100ndash109 2007

[18] N Fazilleau L Mark L J McHeyzer-Williams and M GMcHeyzer-Williams ldquoFollicular helper T cells lineage andlocationrdquo Immunity vol 30 no 3 pp 324ndash335 2009

[19] N M Haynes C D C Allen R Lesley K M Ansel N Killeenand J G Cyster ldquoRole of CXCR5 and CCR7 in follicular Thcell positioning and appearance of a programmed cell deathgene-1High germinal center-associated subpopulationrdquo Journalof Immunology vol 179 no 8 pp 5099ndash5108 2007

[20] R Spolski andW J Leonard ldquoIL-21 andT follicular helper cellsrdquoInternational Immunology vol 22 no 1 Article ID dxp112 pp7ndash12 2009

Journal of Immunology Research 11

[21] D Eto C Lao D DiToro et al ldquoIL-21 and IL-6 are critical fordifferent aspects of B cell immunity and redundantly induceoptimal follicular helper CD4 T cell (Tfh) differentiationrdquo PLoSONE vol 6 no 3 Article ID e17739 2011

[22] C Dong A E Juedes U-A Temann et al ldquoICOS co-stimu-linebreak latory receptor is essential for T-cell activation andfunctionrdquo Nature vol 409 no 6816 pp 97ndash101 2001

[23] T Okazaki and T Honjo ldquoThe PD-1-PD-L pathway in immuno-logical tolerancerdquo Trends in Immunology vol 27 no 4 pp 195ndash201 2006

[24] I Yusuf R Kageyama L Monticelli et al ldquoGerminal center Tfollicular helper cell IL-4 production is dependent on signalinglymphocytic activation molecule receptor (CD150)rdquo Journal ofImmunology vol 185 no 1 pp 190ndash202 2010

[25] D Yu and C G Vinuesa ldquoThe elusive identity of T follicularhelper cellsrdquo Trends in Immunology vol 31 no 10 pp 377ndash3832010

[26] M A Linterman R J Rigby RWong et al ldquoRoquin differenti-ates the specialized functions of duplicated T cell costimulatoryreceptor genes CD28 and ICOSrdquo Immunity vol 30 no 2 pp228ndash241 2009

[27] M Locci C Havenar-Daughton E Landais et al ldquoHuman cir-culating PD-1+1CXCR3minusCXCR5+ memory Tfh cells are highlyfunctional and correlate with broadly neutralizing HIV anti-body responsesrdquo Immunity vol 39 no 4 pp 758ndash769 2013

[28] J Feng L Lu C Hua et al ldquoHigh frequency of CD4+CXCR5+TFH cells in patients with immune-active chronic hepatitis BrdquoPLoS ONE vol 6 no 7 Article ID e21698 2011

[29] C Petrovas T Yamamoto M Y Gerner et al ldquoCD4 T follicularhelper cell dynamics during SIV infectionrdquo Journal of ClinicalInvestigation vol 122 no 9 pp 3281ndash3294 2012

[30] N Simpson P A Gatenby A Wilson et al ldquoExpansion ofcirculating T cells resembling follicular helper T cells is a fixedphenotype that identifies a subset of severe systemic lupuserythematosusrdquo Arthritis and Rheumatism vol 62 no 1 pp234ndash244 2010

[31] S Li C Cai J Feng et al ldquoPeripheral T lymphocyte subset imb-alances in children with enterovirus 71-induced hand foot andmouth diseaserdquo Virus Research vol 180 pp 84ndash91 2014

[32] J Chen J Tong H Liu et al ldquoIncreased frequency ofTh17 cellsin the peripheral blood of children infectedwith enterovirus 71rdquoJournal of Medical Virology vol 84 no 5 pp 763ndash767 2012

[33] J Xie Y Jiao Z Qiu Q Li and T Li ldquoSignificant elevation of Bcells at the acute stage in enterovirus 71-infected children withcentral nervous system involvementrdquo Scandinavian Journal ofInfectious Diseases vol 42 no 11-12 pp 931ndash935 2010

[34] Y Liang X Zhou E Yang et al ldquoAnalysis of the Th1Th2reaction in the immune response induced by EV71 inacti-vated vaccine in neonatal rhesus monkeysrdquo Journal of ClinicalImmunology vol 32 no 5 pp 1048ndash1058 2012

[35] Y-W Lin K-C Chang C-M Kao S-P Chang Y-Y Tungand S-H Chen ldquoLymphocyte and antibody responses reduceenterovirus 71 lethality in mice by decreasing tissue viral loadsrdquoJournal of Virology vol 83 no 13 pp 6477ndash6483 2009

[36] National notifiable diseases announced by National Health andFamily Planning Commission of the Peoplersquos Republic of Chinahttpwwwnhfpcgovcnzhuzhanyqxxlistsshtml

[37] R Morita N Schmitt S-E Bentebibel et al ldquoHuman bloodCXCR5+CD4+ T cells are counterparts of T follicular cellsand contain specific subsets that differentially support antibodysecretionrdquo Immunity vol 34 no 1 pp 108ndash121 2011

Journal of Immunology Research 11

[21] D Eto C Lao D DiToro et al ldquoIL-21 and IL-6 are critical fordifferent aspects of B cell immunity and redundantly induceoptimal follicular helper CD4 T cell (Tfh) differentiationrdquo PLoSONE vol 6 no 3 Article ID e17739 2011

[22] C Dong A E Juedes U-A Temann et al ldquoICOS co-stimu-linebreak latory receptor is essential for T-cell activation andfunctionrdquo Nature vol 409 no 6816 pp 97ndash101 2001

[23] T Okazaki and T Honjo ldquoThe PD-1-PD-L pathway in immuno-logical tolerancerdquo Trends in Immunology vol 27 no 4 pp 195ndash201 2006

[24] I Yusuf R Kageyama L Monticelli et al ldquoGerminal center Tfollicular helper cell IL-4 production is dependent on signalinglymphocytic activation molecule receptor (CD150)rdquo Journal ofImmunology vol 185 no 1 pp 190ndash202 2010

[25] D Yu and C G Vinuesa ldquoThe elusive identity of T follicularhelper cellsrdquo Trends in Immunology vol 31 no 10 pp 377ndash3832010

[26] M A Linterman R J Rigby RWong et al ldquoRoquin differenti-ates the specialized functions of duplicated T cell costimulatoryreceptor genes CD28 and ICOSrdquo Immunity vol 30 no 2 pp228ndash241 2009

[27] M Locci C Havenar-Daughton E Landais et al ldquoHuman cir-culating PD-1+1CXCR3minusCXCR5+ memory Tfh cells are highlyfunctional and correlate with broadly neutralizing HIV anti-body responsesrdquo Immunity vol 39 no 4 pp 758ndash769 2013

[28] J Feng L Lu C Hua et al ldquoHigh frequency of CD4+CXCR5+TFH cells in patients with immune-active chronic hepatitis BrdquoPLoS ONE vol 6 no 7 Article ID e21698 2011

[29] C Petrovas T Yamamoto M Y Gerner et al ldquoCD4 T follicularhelper cell dynamics during SIV infectionrdquo Journal of ClinicalInvestigation vol 122 no 9 pp 3281ndash3294 2012

[30] N Simpson P A Gatenby A Wilson et al ldquoExpansion ofcirculating T cells resembling follicular helper T cells is a fixedphenotype that identifies a subset of severe systemic lupuserythematosusrdquo Arthritis and Rheumatism vol 62 no 1 pp234ndash244 2010

[31] S Li C Cai J Feng et al ldquoPeripheral T lymphocyte subset imb-alances in children with enterovirus 71-induced hand foot andmouth diseaserdquo Virus Research vol 180 pp 84ndash91 2014

[32] J Chen J Tong H Liu et al ldquoIncreased frequency ofTh17 cellsin the peripheral blood of children infectedwith enterovirus 71rdquoJournal of Medical Virology vol 84 no 5 pp 763ndash767 2012

[33] J Xie Y Jiao Z Qiu Q Li and T Li ldquoSignificant elevation of Bcells at the acute stage in enterovirus 71-infected children withcentral nervous system involvementrdquo Scandinavian Journal ofInfectious Diseases vol 42 no 11-12 pp 931ndash935 2010

[34] Y Liang X Zhou E Yang et al ldquoAnalysis of the Th1Th2reaction in the immune response induced by EV71 inacti-vated vaccine in neonatal rhesus monkeysrdquo Journal of ClinicalImmunology vol 32 no 5 pp 1048ndash1058 2012

[35] Y-W Lin K-C Chang C-M Kao S-P Chang Y-Y Tungand S-H Chen ldquoLymphocyte and antibody responses reduceenterovirus 71 lethality in mice by decreasing tissue viral loadsrdquoJournal of Virology vol 83 no 13 pp 6477ndash6483 2009

[36] National notifiable diseases announced by National Health andFamily Planning Commission of the Peoplersquos Republic of Chinahttpwwwnhfpcgovcnzhuzhanyqxxlistsshtml

[37] R Morita N Schmitt S-E Bentebibel et al ldquoHuman bloodCXCR5+CD4+ T cells are counterparts of T follicular cellsand contain specific subsets that differentially support antibodysecretionrdquo Immunity vol 34 no 1 pp 108ndash121 2011