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Cook, Laura, Munier, C. Mee Ling, Seddiki, Nabila, van Bockel, David, Ontiveros, Noé, Hardy,

Melinda Y., Gillies, Jana K., Levings, Megan K., Reid, Hugh H., Petersen, Jan, Rossjohn, Jamie,

Anderson, Robert P., Zaunders, John J., Tye-Din, Jason A. and Kelleher, Anthony D. 2017.

Circulating gluten-specific FOXP3 + CD39 + regulatory T cells have impaired suppressive function

in patients with celiac disease. Journal of Allergy and Clinical Immunology 140 (6) , pp. 1592-1603.

10.1016/j.jaci.2017.02.015 file

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Circulating gluten-specific forkhead box protein

3–positive CD391 regulatory T cells have impaired

suppressive function in patients with celiac disease

Q12 Laura Cook, PhD,a,b* C. Mee Ling Munier, PhD,a Nabila Seddiki, PhD,a,b� David van Bockel, PhD,a

No�e Ontiveros, MSc,c,d Melinda Y. Hardy, PhD,c,d Jana K. Gillies, MSc,e Megan K. Levings, PhD,e

Hugh Reid, PhD,f Jan Peterson, PhD,f Jamie Rossjohn, PhD,f,g,h Robert P. Anderson, PhD,c,d,i

John Zaunders, PhD,a,b Jason A. Tye-Din, PhD,c,d,j and Anthony D. Kelleher, PhDa,b Sydney, Parkville, Clayton, and

Brisbane, Australia; Vancouver, British Columbia, Canada; Cardiff, United Kingdom; and Cambridge, Mass

Background: Celiac disease is a chronic immune-mediated

inflammatory disorder of the gut triggered by dietary gluten.

Although the effector T-cell response in patients with celiac

disease has been well characterized, the role of regulatory

T (Treg) cells in the loss of tolerance to gluten remains poorly

understood.

Objective: We sought to define whether patients with celiac

disease have a dysfunction or lack of gluten-specific forkhead

box protein 3 (FOXP3)1 Treg cells.

Methods: Treated patients with celiac disease underwent oral

wheat challenge to stimulate recirculation of gluten-specific T

cells. Peripheral blood was collected before and after challenge.

To comprehensively measure the gluten-specific CD41 T-cell

response, we paired traditional IFN-g ELISpot with an assay to

detect antigen-specific CD41 T cells that does not rely on

tetramers, antigen-stimulated cytokine production, or

proliferation but rather on antigen-induced coexpression of

CD25 and OX40 (CD134).

Results: Numbers of circulating gluten-specific Treg cells and

effector T cells both increased significantly after oral wheat

challenge, peaking at day 6. Surprisingly, we found that

approximately 80% of the ex vivo circulating gluten-specific

CD41 T cells were FOXP31CD391 Treg cells, which reside

within the pool of memory CD41CD251CD127lowCD45RO1

Treg cells. Although we observed normal suppressive function

in peripheral polyclonal Treg cells from patients with celiac

disease, after a short in vitro expansion, the gluten-specific

FOXP31CD391 Treg cells exhibited significantly reduced

suppressive function compared with polyclonal Treg cells.

Conclusion: This study provides the first estimation of

FOXP31CD391 Treg cell frequency within circulating gluten-

specific CD41 T cells after oral gluten challenge of patients with

celiac disease. FOXP31CD391 Treg cells comprised a major

proportion of all circulating gluten-specific CD41 T cells but

had impaired suppressive function, indicating that Treg cell

dysfunction might be a key contributor to disease pathogenesis.

(J Allergy Clin Immunol 2017;nnn:nnn-nnn.)

Key words: Regulatory T cells, CD39, forkhead box protein 3, celiac

disease, gluten, OX40

Celiac disease is a chronic inflammatory disorder with featuresof autoimmune disease that results from a loss of glutentolerance.1 It is characterized by villous atrophy and the presenceof autoantibodies to tissue transglutaminase 2 (tTG), an enzyme

From athe Immunovirology and Pathogenesis Program, Kirby Institute, UNSWAustralia,

Sydney; bSt Vincent’s Centre for Applied Medical Research, St Vincent’s Hospital,

Sydney; cthe Immunology Division, Walter and Eliza Hall Institute, Parkville; dthe

Department of Medical Biology, University of Melbourne, Parkville; ethe Department

of Surgery, University of British Columbia, Vancouver; fthe Infection and Immunity

Program and Department of Biochemistry and Molecular Biology, Biomedicine Dis-

covery Institute, Monash University, Clayton; gthe ARC Centre of Excellence in

Advanced Molecular Imaging, University of Queensland, Brisbane; hthe Institute of

Infection and Immunity, Cardiff University School of Medicine, Heath Park, Cardiff;iImmusanT, Cambridge; and jthe Department of Gastroenterology, Royal Melbourne

Hospital, Parkville.

*Laura Cook, PhD, is currently affiliated with the Department of Medicine, University of

British Columbia, Vancouver, British Columbia, Canada.

�C. Mee Ling Munier, PhD, is currently affiliated with INSERM U955 and Universit�e

Paris-Est Cr�eteil (UPEC)/Vaccine Research Institute, Cr�eteil, France.

Supported by the Australian Government Department of Health and Ageing; the

NHMRC through a program (510448) grant, NHMRC project grant (1085875), an

NHMRC Australia Fellowship (to J.R.), and a Practitioner Fellowship (to A.D.K.); a

Coeliac Research Fund Grant (to N.S., R.P.A., J.T.-D., and A.D.K.); an Australian

Postgraduate Award; and a UNSW Research Excellence Scholarship (to L.C.).

Disclosure of potential conflict of interest: L. Cook has received a grant from the

Australian Postgraduate Award and the University of New South Wales Research

Excellence Award. N. Seddiki has received a grant from the Coeliac Research Fund

and is named inventor on a patent for the use of CD39 and the OX40 assay to identify

antigen-specific regulatory T cells held by St Vincent’s Hospital, Sydney, Australia. J.

Rossjohn has received payment from the National Health and Medical Research

Council Australia Fellowship. R. P. Anderson has received a grant from the Coeliac

Research Fund; is Chief Scientific Officer of ImmusanT; is a coinventor of patents

pertaining to the use of gluten peptides in therapeutics, diagnostics, and nontoxic

gluten; and is a shareholder of Nexpep and ImmusanT. J. Zaunders is named inventor

on a patent for the use of CD39 and the OX40 assay to identify antigen-specific

regulatory T cells held by St Vincent’s Hospital, Sydney, Australia. J. A. Tye-Din has

received a grant from the Coeliac Research Fund; has consultant arrangements with

ImmusanT; is coinventor of patents pertaining to the use of gluten peptides in

therapeutics, diagnostics, and nontoxic gluten; and is a shareholder in Nexpep. A. D.

Kelleher has received grants from the Australian Government Department of Health

and Ageing, the National Health and Medical Research Council (510448 and

1085875), and the Coeliac Research Fund; has received a Practitioner Fellowship

from the Australian Government Department of Health and Ageing; and is named

inventor on a patent for the use of CD39 and the OX40 assay to identify antigen-

specific regulatory T cells held by St Vincent’s Hospital, Sydney, Australia. The rest of

the authors declare that they have no relevant conflicts of interest.

Received for publication March 24, 2015; revised February 3, 2017; accepted for publi-

cation February 16, 2017.

Corresponding author: Laura Cook, PhD, Levings Lab, Child and Family Research

Institute, Room A4-102, 950 West 28th Ave, Vancouver, BC V5Z 4H4, Canada.

E-mail: lcook@cfri.ca.

0091-6749/$36.00

� 2017 American Academy of Allergy, Asthma & Immunology

http://dx.doi.org/10.1016/j.jaci.2017.02.015

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Abbreviations used

APC: Antigen-presenting cell

CTV: CellTrace Violet

FOXP3: Forkhead box protein 3

PE: Phycoerythrin

SEB: Staphylococcal enterotoxin B

Tconv: Conventional T

Treg: Regulatory T

TSDR: Regulatory T cell–specific demethylated region

tTG: Tissue transglutaminase 2

that deamidates gluten. Intestinal damage is caused by CD41

T cells, which recognize deamidated gluten peptides presentedin complex with HLA-DQ2.5, HLA-DQ2.2, and/or HLA-DQ8,2,3

and the immunodominant hierarchy of wheat gliadin T-cellepitopes in HLA-DQ2.51 patients with celiac disease has beencomprehensively mapped.4 Although HLA susceptibilityhaplotypes are expressed by 30% to 40% of the generalpopulation, celiac disease affects only approximately 1%,indicating that immune tolerance to gluten is the norm. However,the mechanisms that underpin maintenance of gluten toleranceremain poorly described.

Gluten-responsive effector CD41 T cells can be detected in theperipheral blood of patients with celiac disease on a gluten-freediet 6 to 8 days after a 3-day oral gluten challenge.5 Onactivation, these cells secrete high levels of IFN-g,6,7 support Bcell–mediated production of antibodies to tTG and modifiedgluten peptides, and enhance lysis of stressed epithelial cells byCD81 T cells.4,6 Studies of total regulatory T (Treg) cells inpatients with celiac disease have provided evidence for bothnormal suppressive function8-10 and impaired function,11,12 aswell as suggesting that effector T cells have become resistant toTreg cell suppression.10,13,14 However, although forkhead boxprotein 3 (FOXP3)1 Treg cells have an important role inmaintaining peripheral tolerance, until now, the frequency andfunction of gluten-specific FOXP31 Treg cells in patients withceliac disease have not been studied.

For the first time, this study exploited acute in vivo glutenchallenge in patients with celiac disease to interrogate botheffector and regulatory components of the recall response togluten. Specifically, we aimed to estimate the frequency of Tregcells within gluten-specific CD41T-cell recall responses; identifychanges in the frequency of peripheral gut-homing memoryCD41 T-cell populations after gluten challenge; andphenotypically and functionally characterize gluten-specificTreg cells.

METHODS

Subjects and samplesPatients with celiac disease were recruited after provision of informed

consent (Human Research Ethics Committees: Royal Melbourne Hospital ID

2003.009; Walter and Eliza Hall Institute of Medical Research ID 03/04).

Enrollment criteria were biopsy-proved disease conforming to European

Society for Paediatric Gastroenterology Hepatology and Nutrition

guidelines,15HLA-DQ21, and compliancewith a gluten-free diet for 6months

or more. Healthy donor blood was obtained from the Australian Red Cross

Blood Service and volunteers (St Vincent’s Hospital Human Research Ethics

Committee ID HREC/13/SVH/145). Peripheral blood was collected into

lithium heparin vacutainers (BD, San Jose, Calif), transported at ambient

temperature, and processed within 8 hours of collection. Mononuclear cells

were obtained by means of centrifugation over Ficoll-Paque (GE Healthcare,

Fairfield, Conn).

Serology and HLA typingSerum titers of tTG IgA and deamidated gliadin peptide IgGwere evaluated

with commercial kits (INV 708760 and 704525; INOVA Diagnostics, San

Diego, Calif) by a diagnostic laboratory (Gribbles-Healthscope, nnn,

Australia) Q1. The presence of alleles encoding HLA-DQ2.5, HLA-DQ2.2, and

HLA_DQ8 was determined by detecting 5 single nucleotide polymorphisms

(rs2187668, rs2395182, rs4713586, rs7454108, and rs7775228), as previously

described.16,17 HLA-DQB1 and HLA-DQA1 alleles were determined by

using PCR sequence–specific oligonucleotide hybridization (Victorian

Transplantation and Immunogenetics Service, Victoria, Australia).

Oral gluten challengeAll participants undertook a gluten challenge5 from days 1 to 3 by

consuming 4 slices of commercial white bread daily (approximately 10 g/d

wheat gluten) and recorded symptoms daily to day 6, grading them as mild,

moderate, or severe.18

ReagentsWe used 2 HLA-DQ2.5–restricted 15mers that encompass the

immunodominant deamidated wheat gliadin T-cell epitopes DQ2.5-glia-a1/

a2 (LQPFPQPELPYPQPQ) andDQ2.5-glia-v1/v2 (QPFPQPEQPFPWQP).4

Gluten peptide mix contained an equimolar mixture of these two 15mers. An

HLA-DQ2.5–restricted 15mer that encompasses the immunodominant barley

hordein T-cell epitope DQ2.5-hor-3 (PEQPIPEQPQPYPQQ) acted as a

specificity control.4 Peptides were synthesized to 95% purity or greater, as

confirmed by means of high-performance liquid chromatography (Pepscan,

Lelystad, The Netherlands); dissolved in dimethyl sulfoxide (Sigma-Aldrich,

St Louis, Mo); and stored at2808C until use.5,18 Chymotrypsin digestion and

deamidation of gliadin (#101778; ICN Biomedicals, Cost Mesa, Calif) were

performed, as previously described.5,18 Tetanus toxoid was from CSL (nnn,

Australia) Q2, and staphylococcal enterotoxin B (SEB) and PHA were from

Sigma-Aldrich.

IFN-g secretion assaysAntigen-stimulated IFN-g secretion from PBMCs was assessed by using

either ELISpot (Mabtech, Nacka Strand, Sweden) or ELISA (Mabtech),

assays that are equivalent in their ability to detect gluten-specific responses.18

Gluten peptide mix and deamidated gliadin were used at 100 mg/mL and

tetanus toxoid was used at 10 LfU/mL, and assays were performed in

triplicate, as previously described.5,18,19

OX40 assayThe OX40 assay was performed, as previously described,20,21 with either

fresh whole blood (diluted 1:1 with RPMI 1640 media, Invitrogen) or PBMCs

at 2 3 106 cells/mL and incubation with antigen for 44 hours at 378C (5%

CO2). Antigen concentrations were as follows: SEB, 1mg/mL; tetanus toxoid,

2 LfU/mL; deamidated gliadin, 100 mg/mL; and DQ2.5-hor-3m 50 mg/mL.

Optimal concentrations of gluten antigens were determined in a pilot study

(see Fig E1, B, in this article’s Online Repository at www.jacionline.org).

DQ2.5-glia-a1/a2 and DQ2.5-glia-v1/v2 were used separately at

50 mg/mL and in an equimolar gluten peptide mix. Assay cutoffs were as

follows: greater than 0.02% of CD41 T cells (mean1 3 SDs Q3of unstimulated

wells) and greater than 20 cells.

Population tracking within the OX40 assayPostchallenge PBMCs were fluorescence-activated cell sorting purified

into 4 CD41 T-cell populations (Fig 3, A) Q4: (1) CD45RO2 T cells and

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(2) CD45RO1CD127highCD25low conventional T (Tconv) cells, with the

CD45RO1CD127lowCD25high Treg cells subdivided into (3) CD391 and

(4) CD392 cells. Portions of the 3 CD45RO1 populations were stained with

CellTrace Violet (CTV) to enable cell tracking. In some experiments

PBMCs were stained with DQ2.5-glia a21/a2 tetramer and sorted into

tetramer-negative and tetramer-positive CD41 T cells that were then labeled

with CTV. PBMCs were reconstituted, maintaining ex vivo cell ratios, and

supplemented with CD3-depleted autologous PBMCs (Dynabeads CD3,

Invitrogen) to one sixth of the total cell number. Each well contained a single

CTV-labeled population, and the OX40 assay was performed and analyzed, as

described above.

Flow cytometryStaining was performed, as previously described,22 with anti-CD3–

peridinin-chlorophyll-protein complex–Cy5.5 (SK7), CD4–Alexa Fluor 700

(RPA-T4), CD25–allophycocyanin (2A3), OX40 (CD134)–phycoerythrin

(PE; L106), cytotoxic T lymphocyte–associated antigen 4–PE (BNI3),

GARP-BV711 (7B11), integrin b7–PE (FIB504; BD); CD45RO-ECD

(UCHL1; Beckman Coulter, Fullerton, Calif); CD127-eFluor450

(eBioRDR5), CD39-PECy7 (A1); LAP-PECy7Q5 (FNLAP; eBioscience),

Helios–Alex Fluor 488 (22F6), and anti-FOXP3–Alexa Fluor 488 (259D;

BioLegend, San Diego, Calif). FOXP3 staining was performed with the

FOXP3 buffer kit (BD), and the IgG1k-FITC antibody (BD) was used to set

analysis gates. All mAbswere used at manufacturers’ recommended dilutions.

Cell labeling was performed with the CTV Cell Proliferation Kit (Invitrogen)

and PKH26 Red Fluorescent Cell Linker Kit for General Cell Membrane

Labelling (Sigma-Aldrich) per the manufacturers’ recommendations by using

5 mmol/L dye concentrations. HLA-DQ2 proteins were synthesized with

bound DQ2.5-glia-a1, DQ2.5-glia-a2, or the HLA class II invariant peptide

by using previously described constructs and methods.23 Tetramers were

produced by means of addition of either NeutrAvidin R-allophycocyanin or

R-PE conjugate (Invitrogen) to biotinylated protein, as previously described.24

Staining was performed with 50 mg/mL tetramer in complete media at 378C

for 1 hour. A 4-laser LSRII flow cytometer (BD) was used, and analysis

was performed with FlowJo software (v8.8.7; TreeStar, Ashland, Ore).

In vitro expansion of T-cell populationsThe protocol used to expand Treg cells, Tconv cells, and DQ2.5-glia-a1/

a2–specific CD391 T cells was adapted from Gregori et al,25 as previously

described20; T-cell cloning was performed by plating cells at 1 cell/well.

Irradiated feeder cell mix consisted of 5 3 105 cells/mL of mixed PBMCs

(equal mix of autologous PBMCs and PBMCs from 2 allogeneic healthy

donors) and 5 3 104 cells/mL of an autologous EBV-transformed B-cell

line generated and maintained, as previously described.26

Quantitative RT-PCR and T-cell receptor clonotype

analysis by 59 Rapid Amplification of cDNA EndsQuantitative RT-PCR was performed on resting T-cell populations, and

relative expression levels to b-actin were calculated, as previously

described20: FOXP3 forward, 59-TCACCTACGCCACGCTCAT-39;

FOXP3 reverse, 59-TCATTGAGTGTCCGCTGCTT-39; TGF-b forward,

59-CCCTGGACACCAACTATTGC-39; and TGF-b reverse, 59-CAGA

AGTTGGCATGGTAGCC-39. T-cell receptor clonotypes were analyzed by

using 59RapidAmplification of cDNAEnds (Clontech,MountainView, Calif)

PCR, as previously described.27 Sequences were analyzed by using the

ImmunoGenetics V-quest database.28

Suppression assaySuppression assays were performed, as previously described.20

CD41CD1271CD252 responder T cells were labeled with CTV (Invitrogen),

and suppressor cells were labeled with PKH26 (Sigma-Aldrich). Wells

contained 50,000 irradiated autologous antigen-presenting cells (APCs) and

20,000 responder T cells, with suppressor T cells added at the ratios indicated.

Assays were stimulated for 4 days with 0.25 mg/mL soluble anti-CD3

(Invitrogen). CD39 enzyme activity was blocked in some assays with

250 mmol/L ARL67156 (Sigma-Aldrich). The division index was used to

calculate the percentage of suppression.29

Treg cell–specific demethylated region analysisGenomic DNAwas isolated, and bisulfite conversion was performed with

the EZ DNAMethylation-Direct kit (Zymo Research, Irvine, Calif). PCR was

performedwith the PyroMark PCR kit (Qiagen, Hilden, Germany) andHuman

FoxP3 Methylation Assay ADS783FS2 (EpigenDx, Ashland, Mass),

which reports the methylation of 8 representative CpG sites in the regulatory

T cell–specific demethylated region (TSDR). Pyrosequencing was performed

on a PyroMark Q96 ID (Qiagen) with PyroMark Gold Q96 reagents (Qiagen)

and Streptavidin Sepharose (GE Healthcare). All kits/reagents were used,

according to the manufacturer’s instructions. Analysis was performed on

female subjects, and the levels of methylation have not been adjusted to

account for X-inactivation.

StatisticsMann-Whitney U tests or 1-way ANOVAwere used unless samples were

matched, and then Wilcoxon signed-rank tests were performed. Correlation

analyses used Spearman rho (rs). P values were considered significant at

less than .05. Prism 6.0 software (GraphPad Software, La Jolla, Calif) was

used for all statistical analyses.

RESULTS

Numbers of circulating gluten-specific

FOXP31CD391 Treg cells are significantly increased

after gluten challengeTo investigate CD41 T-cell recall responses to gluten, we

recruited a cohort of 17 treated patients with celiac disease(see Tables E1 and E2 in this article’s Online Repository atwww.jacionline.org). We used our previously developed OX40assay, which detects antigen-specific CD41 T cells throughantigen-induced coexpression of CD25 and OX40,21 to measurechanges in the frequency of circulating gluten-specific CD41

T cells in patients with celiac disease after gluten challenge(Fig 1 [F1-4/C], A). In a pilot study we found the optimal time for detectingresponses was 6 to 8 days after gluten challenge (termed days 6and 8; see Fig E1, A). At day 6, we observed significant increasesin deamidated gliadin levels (n5 15,P5.007) and gluten peptideresponses (n5 9,P5.008; Fig 1,B). The overall peak response togluten antigens occurred at day 6 (median response 0.27% ofCD41 T cells). There were no detectable responses to the barleyhordein peptide DQ2.5-hor-3 (Fig 1, A), indicating that the wheatpeptide responses are specifically induced by oral wheat glutenchallenge. Patients with detectable gluten peptide responsesalso had a significant increase in numbers of both total andgut-homing CD391FOXP31 Treg cells at day 6 (Fig 1,C), a trendnot seen in gluten peptide nonresponders (see Fig E2, A, in thisarticle’s Online Repository at www.jacionline.org). Q6There wereno significant changes observed within numbers of total orgut-homing CD41 Tconv cells (data not shown).

We assessed the presence of gluten-specific CD41 T cells in 6healthy volunteers (HLA-DQ genotypes were known for 4, andonly 1 carried HLA-DQ2.5). The median deamidatedgliadin-specific response for the non-HLA-DQ2.5 subjects was0.09% of CD41 T cells (range, 0% to 0.15%), and the responsefor all 6 subjects was 0.19% (range, 0% to 0.77%; see Fig E1,C). No deamidated gluten peptide responses were detected in

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these subjects (data not shown), indicating that CD41 T-cellresponses to deamidated gluten peptides are a more specificmarker of celiac disease.

The majority of gluten peptide–specific

CD41CD251OX401 T cells express CD39We have previously shown that within antigen-responsive

CD251OX401 T cells, a subset of Treg cells can beidentified on the basis of CD39 expression, providing asensitive and specific way to measure and isolate viableantigen-specific FOXP31 Treg cells.20 We used this approach todetermine the contribution of CD391FOXP31 Treg cells togluten-specific OX40 assay responses (Fig 2[F2-4/C]

[F3-4/C], A). On average,

72% of deamidated gliadin-specific T cells were CD391

(75% coexpressed FOXP31), and 89% of gluten peptide-specific T cells were CD391, with 82% of these cells expressingFOXP3 (Fig 2, B).

We found similar CpG methylation patterns within the TSDRin DQ2.5-glia-a1/a2–specific CD391 T cells sorted directly aftera 44-hour OX40 assay compared with ex vivo Treg cellsisolated from both healthy subjects and patients with celiacdisease (Fig 2, C). These data are consistent with gluten-specific CD251OX401CD391 T cells being highly enriched forTreg cells.

We confirmed OX40 assay specificity through severalexperiments using DQ2.5-glia-a1/a2 tetramer reagents. Wecostained DQ2.5-glia-a1/a2 peptide–stimulated OX40 assayswith DQ2.5-glia-a1/a2 tetramer and observed that of allquadrants in the CD25 versus OX40 plot, the CD251OX401

quadrant had the highest proportion of tetramer-positive cells(Fig 2, D). We confirmed that, similar to OX40 responses,tetramer staining was only observed in day 6 postchallengePBMCs (not prechallenge PBMCs) and that the majority of theCD41 tetramer-positive cells were CD391 (median, 70.3%;n 5 7; Fig 2, E). Finally, we sorted CD41CD251OX401CD391

FIG 1. OX40 assay responses to gluten antigen peak at day 6 after gluten challenge. A, Representative OX40

assay responses from patients with celiac disease at day 6. B, For 15 patients with celiac disease, the

percentage of CD41 T cells responding to deamidated gliadin, gluten peptide mix, SEB, or tetanus toxoid

(Tet Tox) are shown. Dotted line, Assay cutoff. C, Flow cytometric analysis of total and integrin

b71CD391FOXP31 Treg cell (CD45RO1CD127lowCD25high) frequencies in ex vivo peripheral blood at days

0, 6, and 8 for patients with celiac disease and detectable gluten peptide responses (n 5 7). Red lines,

Patients with gluten peptide mix responses (n 5 9); black lines, patients with undetectable gluten peptide

responses (n 5 7). Statistical analyses used Wilcoxon signed-rank tests.

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T cells and labeled with cell proliferation dye beforerestimulating with a panel of antigens in the presence ofautologous APCs. We observed that stimulation with cognateantigen generated substantially more proliferation (87%)than DQ2.5-glia-v1/v2 (29%), DQ2.5-hor-3 (29%), or noantigen (21.7%). DQ2.5-glia-a1/a2 restimulation of matchedCD252OX402 T cells did not stimulate cell proliferation(see Fig E3, D, in this article’s Online Repository atwww.jacionline.org).

Importantly, although CD39 expression varies betweensubjects, in each subject the proportion of CD391 cells withinCD41T cells did not vary within the timeframe of theOX40 assay

(see Fig E4, C, in this article’s Online Repository atwww.jacionline.org). The proportion of CD391 cells withinrecall responses also did not significantly vary from beforechallenge through day 8 after challenge (see Fig E2, C, OnlineRepository). Responses to the mitogen SEBwere similar betweenthe celiac cohort (n 5 15; mean response, 5.8% CD41 T-cells)and healthy subjects (n 5 15; mean age, 37; 60% female; meanresponse, 7.2% of CD41 T cells) and consisted of less than30% CD391 T cells (20.7% in healthy subjects vs 29.1% inpatients with celiac disease; see Fig E2, E). This suggests that ahigh proportion of CD391 cells within antigen-specific responsesis not an inherent feature of the OX40 assay but is instead a unique

FIG 2. The majority of circulating gluten peptide–specific CD41 T cells are CD391FOXP31. A and B, CD39

and FOXP3 expression within OX40 assay responses: representative data (Fig 2, A) and within responses

to deamidated gliadin (n 5 14) and gluten peptide mix (n 5 9; mean 6 SEM; Fig 2, B). C, Percentage

methylation at 8 CpG sites in the TSDR of FOXP3 within ex vivo Treg and Tconv cells (n 5 3 healthy female

subjects and n 5 3 female patients with celiac disease) and DQ2.5-glia-a1/a2–specific CD391 cells (n 5 3;

median 6 interquartile range). ns, Not significant. D, The frequency of DQ2.5-glia-a1/a2 tetramer-positive

cells is shown within each quadrant of the CD25/OX40 plot for DQ2.5-glia-a1/a2–stimulated OX40 assays

(n 5 3; mean 6 SEM). E, Representative DQ2.5-glia-a1/a2 tetramer and CD39 staining of unstimulated

PBMCs at days 0 and 6 after gluten challenge. For 3 patients, tetramer staining is shown at both day

0 and day 6. The proportion of tetramer-positive cells that are CD391 is shown for 7 patients with celiac

disease at day 6 after gluten challenge (mean 6 SEM). Statistical analyses in Fig 2, B and D, used

Mann-Whitney U tests, and those in Fig 2, E, used Wilcoxon signed-rank tests.

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feature of the recall response to gluten antigen in patients withceliac disease.

To confirm that gluten-specific CD251OX401CD391 T cellsoriginated from the pool of circulating Treg cells, we used ourpreviously described method for population tracking within theOX40 assay (Fig 3, A and B).20 We found that CD391 memoryTreg cells comprised an average of 88% of DQ2.5-glia-a1/a2-specific CD251OX401CD391 T cells, with CD392 Treg cellsalso the dominant population (average, 76%; n 5 4) within theCD251OX401CD392 T cells (Fig 3, E). Again, we observed

that during a 44-hour OX40 assay, CD39 expression was notaffected by cell activation (Fig 3, C). For 2 subjects, we trackedsorted, CTV-labeled CD41DQ2.5-glia-a1/a2 tetramer-positivecells within OX40 assays stimulated with DQ2.5-glia-a1/a2peptide. We observed that approximately 60% of theCD251OX401 cells were composed of CD391CTV1 cells(Fig 3, F). These data support our findings that peripheralCD41CD45RO1CD25highCD127low Treg cells constitute morethan 80% of the total DQ2.5-glia-a1/a2–specific CD251OX401

T-cell response after gluten challenge.

FIG 3. DQ2.5-glia-a1/a2–specific CD251OX401CD391 T cells originate from peripheral CD391 Treg cells.

A and B, Overview of the method and gating strategy used to conduct population tracking within an

OX40 assay performed as described in the Methods section with peripheral blood after gluten challenge.

C, Changes in CD39 expression between ex vivo isolation and after a 44-hour OX40 assay for Tconv cells,

CD392 Treg cells, and CD391 Treg cells (mean 6 SEM). D, Representative plots for unstimulated wells

and DQ2.5-glia-a1/a2–stimulated wells that contained CTV-labeled CD391 Treg cells. E, Proportion of Tconv

cells (gray), CD392 Treg cells (blue), and CD391 Treg cells (red) within the total DQ2.5-glia-a1/a2–specific

CD251OX401 T-cell response and the CD392 and CD391 fractions of this response. Data in Fig 3, C and E,

are the mean of 5 independent experiments with 4 patients with celiac disease. F, Tetramer-positive cells

were sorted from day 6 PBMCs, labeled with CTV, and either left unstimulated or stimulated for 44 hours

with DQ2.5-glia-a1/a2 peptide in the presence of autologous APCs. The proportion of CD391CTV1 cells

within the CD251OX401 quadrant is representative of 2.

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DQ2.5-glia-a1/a2–specific CD391 T cells have

impaired suppressive functionDQ2.5-glia-a1/a2–specificCD41CD251OX401CD391Tcells

were expanded in vitro from post–gluten challenge PBMCs (sortpurities, >90%) to further investigate the phenotype and functionof gluten-specific FOXP31CD391 Treg cells. For femalepatients with celiac disease and healthy donors, we also expandednon–gluten-specific CD252OX402 T cells and, from unstimulatedPBMCs, CD25highCD127lowTreg cells and CD252CD1271Tconvcells. We generated 3 T-cell clones, 2 from patient #0174 (C1and C2) and 1 from patient #0251 (C3). Clonality wasconfirmed through sequencing the T-cell receptor b chain variableregion. Both T-cell clones from patient #0174 expressed thesame TRBV7-2 clonotype (CASSLRYTDTQYF), which mightbe a public clonotype, as previously identified in another celiaccohort.30

Suppression assays used soluble anti-CD3 stimulus for 4 daysin the presence of autologous APCs with a 1:1 ratio of suppressorto responder cells (Fig 4[F4-4/C] , A). Suppressive function of ex vivo

celiac Treg cells (CD25highCD127low; median, 73.4%; n 5 5)before oral gluten challenge was comparable with that of healthyTreg cells (median, 70.5%; n 5 6) but significantly greater thanthat of Tconv cells (CD252CD1271; median, 12%; n 5 5), asexpected (P 5 .016; Fig 4, B). Interestingly, the subset ofex vivo CD25highCD127low Treg cells that was isolated asCD391 cells before challenge had reduced suppressive function(median, 46.3%; n 5 5; P 5 .016) that was not further affectedby the CD39 inhibitor ARL67156 (median, 45.5%). Similarly,56-day expanded DQ2.5-glia-a1/a2–specific CD391 T cellshad slightly reduced suppression comparedwith ex vivoTreg cellsfrom patients with celiac disease (mean, 55.8%; range, 30.1% to74.8%), as did the CD391 T-cell clones C1 and C2 (62% and 52%suppression, respectively; Fig 4, C). Addition of the CD39inhibitor ARL67156 had a minimal effect (mean reduction insuppression, 11%; Fig 4, C).

We assessed in vitro suppressive function with suppressor/responder cell ratios of 1:1 to 1:32 for 14 days expandedQ7 :DQ2.5-glia-a1/a2–specific CD391 T cells (n5 4), a T-cell clone(C3), and, for 4 healthy subjects and patients with celiac disease,Treg cells (CD25highCD127low) and Tconv cells(CD252CD1271). The celiac Treg cells exerted suppressionacross all cell ratios comparable with that of Treg cells fromhealthy subjects, whereas the gluten-specific CD391 T-cell cloneC3 began to exhibit markedly lower suppressive function at a1:8 cell ratio (Fig 4, D). The expanded gluten-specific CD391

T cells had significantly reduced suppressive function comparedwith that of polyclonal Treg cells from both healthysubjects and patients with celiac disease across all ratios tested(Fig 4, D). These data indicate that in patients with celiac diseaseafter gluten challenge, the expanded subset of peripheralgluten-specific CD391 Treg cells, but not polyclonal Treg cells,has impaired suppressive function.

Approximately 50% of expanded gluten-specificCD251OX401CD391 T cells stained positive for DQ2.5-glia-a1/a2 tetramer compared with greater than 2%tetramer-positive cells within non–gluten-specific CD252OX402

T cells (see Fig E3, B). These cells had substantial expression ofCD39, CD25, cytotoxic T lymphocyte–associated antigen 4, andintegrin b7, but FOXP3 expression was low or absent (see TableE3 in this article’s Online Repository at www.jacionline.org).

Interestingly, gluten-specific CD391 T cells were Heliosnegative, suggesting they originate from a peripherally derivedTreg cell population (see Fig E3, C). Quantitative RT-PCRconfirmed that expanded gluten-specific CD391 T cells hadvery low levels of FOXP3 and moderate-to-high levels ofTGF-b expression that corresponded to increased surfaceexpression of GARP and LAP, which tether latent TGF-b to thecell membrane (see Fig E3, A and E). Loss of FOXP3protein expression corresponded to increased CpG methylationin the TSDR of expanded cells. CD391 T cells had anaverage 2.8-fold increase in methylation, whereas Tregcells from healthy subjects and patients with celiac diseasehad 1.5- and 1.4-fold increases in methylation, respectively (seeFig E3, F).

Antigen-stimulated expression of CD25 and OX40

detects significantly more gluten-specific T cells

than conventional IFN-g secretion assaysWe performed correlation analyses to compare the sensitivity

of the OX40 assay with that of conventional IFN-g ELISpotassays. We observed a positive correlation between the IFN-gELISpot assay at day 6 and peak OX40 assay responses to glutenpeptide stimulus (n 5 13, rs 5 0.876, P 5 .0002; Fig 5 ½F5�, A, andTable I ½T1�). For the 12 HLA-DQ2.5 patients with celiac diseaseand detectable IFN-g responses to gluten peptide antigen, 10(83%) also had detectable OX40 assay responses. A linearregression analysis of responses to gluten peptide antigendetected by using each assay generated a line of best fit with aslope (m) of 4.806. This indicates the OX40 assay detectsapproximately 5 times the number of gluten peptide–specificCD41 T cells than the IFN-g ELISpot (Fig 5, B).

A high proportion of memory Treg cells from

patients with celiac disease express CD39Flow cytometry was used to measure the frequency of

peripheral lymphocyte populations (see gating in Fig E4, B) inhealthy volunteers (n 5 13; mean age, 47; 69% female) andpatients before gluten challenge (n 5 13; mean age, 58;69% female). The proportion of CD391 cells within memoryTreg cells was significantly higher in patients (mean,74.65%; range, 59.7% to 83.7%) than healthy control subjects(mean, 48.07%; range, 11.10% to 73.30%; P < .0001; Fig 5,C). Patients with celiac disease also had significantly reducedtotal memory Treg cell numbers at day 0 (mean, 3.14% ofCD41 T cells) compared with healthy control subjects(mean, 7.97%; P < .0001; Fig 5, D), which persisted at days6 and 8 after challenge (data not shown), and significantlymore CD391 memory Treg cells within CD41 T cells at day0 (P 5 .037; Fig 5, E).

Symptom severity associated with stronger gluten

peptide recall responses in the OX40 assayPatients with celiac disease were split into 2 groups based on

their symptom severity to identify associations betweenimmunologic variables and clinical symptoms (Table I). Nosignificant differences were observed for the frequency of totalCD45RO1CD391 Treg cells at day 0 or for ELISpot responses

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to gluten peptide mix (see Fig E5, A and C, in this article’s OnlineRepository at www.jacionline.org). However, patients with celiacdisease andmore severe symptoms had significantlymore CD391

cells within deamidated gliadin-specific CD251OX401 T cells(P 5 .014) and significantly larger OX40 assay responses togluten peptides (P 5 .011; see Fig E5, B and D).

DISCUSSIONThese data are the first report of the contribution of

FOXP31CD391 Treg cells to gluten-specific CD41 T-cellresponses in patients with celiac disease after in vivo glutenchallenge. Surprisingly, we observed that FOXP31CD391

Treg cells comprised more than 80% of circulating gluten

FIG 4. In vitro–expanded DQ2.5-glia-a1/a2–specific T cells retain suppressive function. A, Representative

responder cell proliferation showing division index (DI). B, Percentage of suppression of ex vivo Treg cells

from healthy subjects (n 5 6), Treg cells from patients with celiac disease (n 5 5), CD391 Treg cells from

patients with celiac disease (n 5 5), and Tconv cells from patients with celiac disease (n 5 5) at a 1:1 ratio

with responder T cells. ns, Not significant. C, Percentage suppression of 56-day expanded DQ2.5-glia-a1/a2–

specific CD391 T-cell populations (n5 2) and CD391 T-cell clones (C1 and C2). The CD39 inhibitor ARL67156

was added as indicated. D, Percentage suppression of 14-day expanded Treg cells (n 5 4), Tconv cells

(n 5 4), and DQ2.5-glia-a1/a2–specific CD391 T cells (n 5 4) from healthy subjects and patients with celiac

disease and T-cell clone C3 for 1:1 to 1:32 suppressor/responder cell ratios. Data in Fig 4, B-D, are

medians 1 interquartile ranges Q8of 1 to 3 independent experiments, and statistical analyses used

Mann-Whitney U tests.

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peptide–specific CD41T cells in patients with celiac disease aftergluten challenge. We confirmed that more than 85% of the glutenpeptide–specific FOXP31CD391 T cells originated from theperipheral pool of CD391 Treg cells and that the extent of CpGmethylation in the TSDR within the FOXP3 loci of these cellsis similar to that seen in CD127lowCD25high Treg cells fromhealthy subjects.

Because the majority of FOXP31 Treg cells do not secreteIFN-g, our data indicate that IFN-g–based methods only detectapproximately 20% of the total CD41 T-cell response to glutenantigen (supported by our linear regression analysis). TheOX40 assay correlated with the IFN-g ELISpot for the detectionof gluten-specific CD41 T-cell responses. This concurs withprevious studies that found the OX40 assay has strong agreementwith IFN-g release assays for Mycobacterium tuberculosis31,32

and concordance with serology, proliferation, and cytokineresponses to HIV-1,21 hepatitis C virus,33 human papilloma-virus,34 Mycobacterium avium complex, varicella zostervirus, EBV, CMV, Candida albicans, and Streptococcus

pneumonia.21,35 For the first time, we also show that the OX40

assay corresponds with class II tetramer staining for responsesto the DQ2.5-glia-a1/a2 epitopes in patients with celiac diseaseafter gluten challenge.

These data complement our previous study showing thatCD391 Treg cells comprise a substantial proportion of CD41

T-cell recall responses to viral and bacterial antigens.20 Previousstudies of FOXP31 Treg cells within in vivo recall responses tovaricella zoster virus in human subjects36,37 and within secondaryimmune responses to influenza virus in mice38,39 identified a keyrole for pathogen-specific Treg cells in controlling the cellular im-mune response. Of particular interest is the recent discovery thatparticle-associated antigens drive a Treg cell response, whereasdistinct soluble antigens instead drive an effector T-cellresponse.40 In addition to the type of antigen, the balance betweenTreg and Tconv cells within antigen-specific responses is alsoinfluenced by chronicity of antigen exposure.41 This study is alarge contribution to the relatively underexplored area ofantigen-specific human Treg cells and shows that, for patientswith celiac disease on a gluten-free diet, the CD41 T-cellresponse to acute dietary gluten re-exposure is skewed toward

FIG 5. Analysis of assay correlation and peripheral Treg cell frequency and phenotype. A and B, Gluten

peptide mix IFN-g ELISpot responses (SFU/106 PBMCs; n 5 13) are correlated with OX40 assay responses

(n 5 13) expressed as either CD251OX401 cells as a percentage of CD41 T cells (Fig 5, A) or CD251OX401

cells/106 PBMCs (Fig 5, B). In Fig 5, A, the calculated Spearman rho (rs) and P values are shown, and in Fig 5,

B, the line of best fit (solid line) and 95% CIs (dashed line) are shown. C-E, Flow cytometric phenotyping data

from 13 patients with celiac disease were compared with those in healthy subjects (n 5 13) for the

proportion of CD41CD45RO1CD127lowCD25high Treg cells that expressed CD39 (Fig 5, C), the proportion

of CD45RO1 Treg cells within CD41 T cells (Fig 5, D), and the proportion of CD41 T cells that were

CD45RO1CD391 Treg cells (Fig 5, E). Error bars represent medians 6 interquartile ranges, and statistical

analyses used Mann-Whitney U tests.

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Treg cells. Furthermore, we demonstrate that functionaldefects can be unmasked by studying the relevant diseaseantigen-specific population rather than polyclonal Treg cells.

On average, maximal OX40 responses to gluten antigensoccurred at day 6 after gluten challenge, a similar time courseto that previously noted for IFN-g responses.7,18 These dynamicchanges were restricted to the gluten-derived antigens becausethere was little change in responses to mitogen or controlantigens. Importantly, CD391 proportions within all OX40 assayresponses were not significantly altered after gluten challenge,indicating the high CD391 proportions observed within glutenpeptide responses occur independently of immune activation.For those patients with celiac disease who did not respond tothe gluten peptides in this study, there is no evidence of durableimmune tolerance because the gluten challenge still causedclinical symptoms for these patients. It is more likely that theimmune response is present but either less than the assay’s limitof detection or not detectable in peripheral blood. The severityof gastrointestinal symptoms also corresponded to larger immuneresponses to gluten and a higher frequency of CD391 cells withinthese responses. This is similar to a previous observation that thedensity of intestinal FOXP31Treg cells positively correlated withthe severity of histologic damage.10

Both expanded DQ2.5-glia-a1/a2–specific CD391 T-cell linesand clones and total ex vivo CD391 Treg cells from patients withceliac disease had reduced suppressive function in response to apolyclonal stimulus that was not dependent on CD39 functionbut corresponded to a loss of stable FOXP3 expression, indicatingthat gluten-specific CD391 Treg cells from patients with celiac

disease might have an inherent functional defect. These datacontrast with a previous observation that found expandedIL-10–secreting gluten-specific Treg type 1 cell clones hadnormal in vitro suppressive function.8 Our in vitro–expandedDQ2.5-glia-a1/a2–specific CD391 T cells had aCD251FOXP32TGF-b1GARP1LAP1 phenotype that mostclosely resembles that of human regulatory TH3 cells.42 Incontrast to the previously observed stable FOXP3 expressionseen in expanded CMV-P1–specific T-cell clones,20 expandedgluten-specific CD391 T-cell populations lost FOXP3 expressionin vitro within 14 days, which corresponded to increased CpGmethylation in the TSDR. This might indicate thatgluten-specific CD391 Treg cells retain a high degree ofplasticity,43 although expanded healthy Treg cells also acquiredmethylation in vitro to a lesser extent.

Compared with healthy control subjects, patients with celiacdisease before challenge had significantly more CD391 cellswithin CD45RO1 Treg cells yet lower absolute numbers ofCD45RO1 Treg cells. This is likely due to a single nucleotidepolymorphism in the CD39 gene that determines CD39expression levels in Treg cells, and futurework should investigatethe association of such CD39 single nucleotide polymorphismswith celiac disease.44 CD391 Treg cells have been shown to bepotent suppressors of IFN-g and IL-17 and to be increased inthe synovia of patients with juvenile arthritis.44 ThereforeCD391 Treg cells might be preferentially expanded, yet theirnumbers are insufficient to control inflammation after glutenexposure in patients with celiac disease. CD39 expression onTreg cells might also be useful in predicting clinical outcomes

TABLE I. Celiac disease cohort symptoms and CD41 T-cell responses to gluten antigens Q9

ID Symptoms after gluten challenge

Post–gluten challenge

gluten peptide mix response,

OX40 assayz

Post–gluten challenge

gluten peptide mix response,

IFN-g secretion

0062* Mild depressed mood and lethargy (days 4-6) Detected Detected

0077* Asymptomatic Not detected§ Detected

0080* Mild nausea (days 1-3) Not detected Not detected

0152� Severe vomiting, lethargy, and diarrhea (days 1-3); moderate nausea, bloating,

and abdominal pain (days 1-3)

Detected Detected

0159* Mild bloating, abdominal pain, and lethargy (days 2-3) Detected Detected

0174� Severe nausea, vomiting, abdominal pain, and diarrhea (days 1-3); moderate

lethargy, hot flushes, cold sweats, and flatulence (days 2-4)

Detected Detected

0196� Moderate-to-severe abdominal pain, nausea, and lethargy and mild diarrhea

(days 1-3)

Detected Detected (ELISA)

0230* Mild nausea and diarrhea (days 1-3) Detected Detected

0239* Asymptomatic Not detected Detected (at assay

limit of detection)

0250� Severe vomiting, nausea, abdominal pain, lethargy, and cold sweats

(days 1-3); mild bloating and diarrhea (days 2-4)

Detected Detected

0251� Moderate-to-severe abdominal pain, bloating, nausea, and lethargy Detected Detected (ELISA)

0505* Severe lethargy and mild diarrhea (days 1-3) Not detected Not detected

0506* Mild constipation (days 1-3) Not detected Not detected

0509* Mild bloating, abdominal pain, and diarrhea (day 1) Not detected Not detected

0510� Severe bloating, abdominal pain, and constipation (days 203) Not detected Not detected

0512* Asymptomatic Detected Detected

0072� Moderate-to-severe nausea, bloating, and vomiting (day 1);

moderate abdominal pain and lethargy (day 1)

Detected Detected

*Nonsevere group: reported mild or no symptoms.

�Severe group: reported moderate-to-severe symptoms.

�Post–gluten challenge OX40 assay responses were only listed as detected if they were greater than the baseline response.

§Cohort analyses were performed with 15 patients: patient #0077 was not included because day 8 OX40 assays used cryopreserved PBMCs, and patient #0072 was not included

because day 8 analysis was not performed.

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because low CD39 expression has been associated with betterCD41 T-cell recovery after antiretroviral therapy in HIV1

patients20 and methotrexate resistance in patients withrheumatoid arthritis.45

Together, the data presented here indicate that in patients withceliac disease after gluten challenge, FOXP31CD391 Treg cellsdominate peripheral recall responses to gluten and can be readilyexpanded on in vivo antigen challenge yet exhibit impairedin vitro suppressive function. Therefore one interpretation ofthese data is that, in response to gluten challenge,FOXP31CD391 Treg cells are induced in vivo in an attempt torestore homeostasis. However, the generated cells have impairedsuppressive function, possibly as a result of generation underinflammatory conditions in vivo. A key area for furtherinvestigation is whether in vivo challenge conditions could bemanipulated to drive expansion of functional Treg cells.

We thank Dr Anne Pesenacker and Dr Kate MacDonald for helpful

discussions; Ms Cathy Pizzey for her assistance with patient visit scheduling

and data and sample collection; andMs Lisa Xu,MsYinXu,MsAnnett Howe,

andMsMichelle Bailey for fluorescence-activated cell sorting isolation of cell

populations. All healthy subject and patients with celiac disease are thanked

for their participation in the study.

Key messages

d In patients with celiac disease, 6 days after gluten

challenge in vivo, a surprisingly large proportion of

circulating gluten-specific CD41 T cells are FOXP31

CD391 Treg cells.

d In patients with celiac disease after gluten challenge,

gluten-specific Treg cells exhibit impaired polyclonal sup-

pressive function in vitro, suggesting that an intrinsic

dysfunction of expanded CD391 Treg cells might

contribute to the loss of tolerance to gluten.

d Detection of gluten-specific CD41 T cells based on

antigen-induced coexpression of CD25 and OX40 is

more sensitive than traditional methods relying on

antigen-induced cytokine production and, for the first

time, allows detailed characterization of antigen-specific

Treg cells in patients with this disease.

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FIG E1. Optimization of OX40 assay time course and gluten antigen concentration. A, In a pilot study OX40

assays were performed with peripheral blood from 3 patients (#0080, #0152, and #0250) and 1 HLA-DQ21

subject without celiac disease (#0458). Blood was collected before gluten challenge (day 0) and at days 4,

6, 8, and 10 after challenge. OX40 assay responses (CD251OX401) are shown for gluten peptide mix,

DQ2.5-glia-a1/a2, DQ2.5-glia-v1/v2 deamidated gliadin, SEB, or tetanus toxoid (Tet Tox). B, OX40 assay

responses are shown at days 6 and 8 to 10, 50, and 100 mg/mL deamidated gliadin; 50 and 125 mg/mL

DQ2.5-glia-a1/a2; or 50 and 125 mg/mL DQ2.5-glia-v1/v2. C, OX40 assays were performed with peripheral

blood of 6 healthy subjects. The deamidated gliadin responses are shown for a single experiment with

each subject. Error bars represent means 6 SEMs.

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FIG E2. Changes in peripheral Treg cell frequencies and OX40 assay CD391 proportions after gluten

challenge. A and B, Frequencies of CD41 T cells that were memory CD391FOXP31 Treg cells (Fig E3, A)

and integrin b71CD391 memory Treg cells (Fig E3, B) are shown for patients who did not have detectable

gluten peptide responses (n 5 6). C, The proportion of CD39 expression within OX40 assay responses is

plotted for assays stimulated with SEB (n 5 15), tetanus toxoid (Tet Tox; n 5 15), deamidated gliadin

(n 5 10), and gluten peptide mix (n 5 7). Subjects were only included in analyses if they had detectable

responses at all 3 time points or, for gluten peptide mix, if they had detectable responses at both day 6

and day 8. Statistical analyses were performed with Wilcoxon signed-rank tests. ND, Not detected; ns,

not significant. Each subject is represented by a different symbol, and red connecting lines indicate those

subjects with gluten peptide responses. D and E, Total CD41CD251OX401 T-cell responses to SEB and

the proportion of CD391 cells within these responses are shown for healthy subjects (n 5 15; Fig E3, D)

and patients with celiac disease (n 5 15; Fig E3, E). Means 6 SEMs are shown.

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FIG E3. Characterization of expanded DQ2.5-glia-a1/a2–specific CD391 T cells. A, Quantitative RT-PCR was

used to compare FOXP3 and TGF-b mRNA expression levels in 14-day expanded gluten-specific CD391 T

cells to levels in expanded CD127lowCD25high Treg cells from healthy subjects (n 5 3) and expanded Treg

and CD1271CD252 Tconv cells from patients with celiac disease (n 5 2). Relative expression is shown after

normalization to b-actin. Means 6 SEMs are shown for 2 independent experiments. B, DQ2.5

tetramer (loaded with HLA class II invariant peptide or DQ2.5-glia-a1/a2) staining of 14-day expanded

DQ2.5-glia-a1/a2–specific CD251OX401 and non–gluten-specific CD252OX402 T cells. Frequencies of

tetramer-positive cells are representative of expanded CD391 cells and clones from 2 subjects. C, CD39

and Helios staining (representative of n 5 3) of expanded CD127lowCD25high Treg cells and

gluten-specific CD391 T cells from healthy subjects and patients with celiac disease. D, DQ2.5-glia-a1/a2–

specific CD251OX401CD391 (red) and non–gluten specific CD252OX402 (black) T cells were

fluorescence-activated cell sorting isolated directly after an OX40 assay and restimulated for 5 days with

the antigens indicated. Percentage proliferation shown is representative of 3 subjects. E, Histograms

showing expression of GARP and LAP in cells expanded from patient #0251 with celiac disease and a

healthy control subject. Median fluorescence intensity (MFI) Q10is shown in the legend. F, Percentage

methylation in the TSDR of FOXP3 is shown for 14-day expanded CD127lowCD25high Treg cells from healthy

subjects (n 5 3) and patients with celiac disease (n 5 3) and gluten-specific CD391 T cells (n 5 3;

median 6 interquartile range; all donors were female). ns, Not significant.

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FIG E4. Flow cytometric gating strategies. A, Gating strategy for OX40 assay analysis. Lymphocytes were

gated based on their forward- and side-scatter profiles, followed by gating CD31CD41 T cells. The

crosshatch gates used to define the antigen-specific CD251OX401 T cells were set initially on unstimulated

wells and refined by using responses in SEB-stimulated positive control wells. B,Gating strategy shown for

isolation and phenotypic analysis of peripheral CD41 memory T-cell populations. After gating on

lymphocytes and CD31CD41 T cells, the CD45RO1 memory cells were divided into Treg cells

(CD127lowCD25high) and the remaining non-Treg cells, with the Treg cells separated further into CD391

and CD392 populations. Expression of phenotypic markers, such as the gut-homing marker integrin b7,

was analyzed within each gated T-cell population. C, For 12 patients with celiac disease, the percentage

of CD391 cells within CD41 T cells is shown for PBMCs sourced directly ex vivo and from OX40 assay wells

after 44 hours of stimulation with no antigen, SEB, or gluten peptide mix.

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FIG E5. Symptom severity associated with greater responses to gluten peptide antigen. Patients with celiac

disease were divided into groups based on symptom severity: severe, n 5 7; nonsevere, n 5 10. Groups

were compared by identifying differences in the proportion of CD391 cells within ex vivomemory Treg cells

(A), the proportion of CD39 expression within deamidated gliadin-specific CD251OX401 T cells (B), the

magnitude of day 6 postchallenge IFN-g ELISpot response to gluten peptide mix (n 5 5 in severe group

because n 5 2 only had ELISA data; (C), and the maximal OX40 assay response to gluten peptide mix

(D). Error bars represent means 6 SEMs. Mann-Whitney U tests were performed. ns, Not significant.

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TABLE E1. Celiac disease cohort characteristics

ID Age (y) Sex Time since diagnosis tTG IgA (U/mL)* DGP IgA (U/mL)* HLA-DQ Haplotype

0062 66 F 5 y, 9 mo <20 <20 HLA-DQ2.5

0077 48 M 21 y <20 <20 HLA-DQ2.5

0080� 68 F 31 y <20 <20 HLA-DQ2.5

0152� 54 M 1 y, 9 mo <20 <20 HLA-DQ2.5

0159 67 F 14 y, 9 mo <20 <20 HLA-DQ2.5 (homozygous)

0174 51 F 15 y, 7 mo <20 <20 HLA-DQ2.5

0196 60 F 9 y, 3 mo <20 <20 HLA-DQ2.5

0230 53 F 12 y, 9 mo <20 <20 HLA-DQ2.5

0239 71 F 39 y, 3 mo <20 <20 HLA-DQ2.5 (homozygous)

0250 62 M 8 y, 5 mo <20 <20 HLA-DQ2.5

0251� 53 F 8 y, 2 mo <20 <20 HLA-DQ2.5

0505 35 F 34 y, 11 mo <20 <20 HLA-DQ2.2

0506 69 M 2 y, 1 mo <20 <20 HLA-DQ2.5 (heterozygous)

0509 29 F 7 y, 11 mo <20 <20 HLA-DQ2.5 (heterozygous)

0510 38 M 4 y, 11 mo 62 (strongly positive) 27 (weakly positive) HLA-DQ2.5 and HLA-DQ2.2

0512 52 F 1 y, 4 mo <20 29 (weakly positive) HLA-DQ2.5 (heterozygous)

0072 57 F 20 y, 2 mo <20 <20 HLA-DQ2.5 (heterozygous)

Mean 55 71% F

29% M

14 y, 1 mo <20 <20

All data were collected at the time of gluten challenge. All subjects have been on a gluten-free diet since diagnosis.

DGP, Deamidated gliadin peptide; F, female; M, male.

*Normal serology is less than 20 U/mL.

�Indicates subjects analyzed in the pilot study.

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TABLE E2. HLA-DQ alleles detected in the celiac disease

cohort

ID HLA-DQA HLA-DQB

0062 DQA1*05 DQB1*02

DQB1*03

0077 DQA1*01

DQA1*05

DQB1*02

DQB1*05

0080* DQA1*05:01 DQB1*02:01

DQB1*02:02

0152* DQA1*01

DQA1*05

DQB1*02

DQB1*06

0159 DQA1*05:01 DQB1*02:01

0174 DQA1*05:01 DQB1*02:01/02

DQB1*05:02

0196 DQA1*05:01 DQB1*02:01/02

DQB1*06:02

0230 DQA1*05:01 DQB1*02:01

DQB1*05:02

0239 DQA1*05:01 DQB1*02:01

0250* DQA1*02

DQA1*05

DQB1*02

0251 DQA1*01

DQA1*05

DQB1*02:01

DQB1*05:01

0505 DQA1*02

DQA1*04/06

DQB1*02

DQB1*04

0506 N/A N/A

0509 N/A N/A

0510 DQA1*02:01

DQA1*05:01

DQB1*02:01

DQB1*02:02

0512 NA NA

0072 NA NA

HLA-DQ2.5 refers to the DQA1*0501:DQB1*0201 haplotype, and HLA-DQ2.2

refers to the DQA1*0201:DQB1*0202 haplotype.

NA, HLA-DQB1 and HLA-DQA1 alleles were not assessed, and instead only the

presence of alleles encoding HLA-DQ2.5, HLA-DQ2.2, and HLA-DQ8 were detected,

as described in section 2.4.Q11

*Indicates subjects analyzed in the pilot study.

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TABLE E3. Phenotype of in vitro–expanded DQ2.5-glia-a1/a2–specific CD41CD251CD1341CD391 T cells

Molecule Clone C1 (#0174) Clone C2 (#0174) Clone C3 (#0251) CD391 cells (#0174) CD391 cells (#0230) CD391 cells (#0196) CD391 cells (#0251)

CD25 111 111 111 111 111 111 111

CD39 111 111 111 111 111 111 111

FOXP3 1 2 11 1 2 1 1

CTLA-4 111 111 11 111 111 11 111

CD45RO 111 111 111 111 111 111 111

Integrin b7 11 11 11 11 111 1 1

LAP NA NA 111 NA NA 1 1

GARP NA NA 11 NA NA 11 11

Helios NA NA 2 NA NA 2 2

CTLA-4, Cytotoxic T lymphocyte–associated antigen 4; NA, not assessed; 111, expression on greater than 90% of cells; 11, expression on 50% to 90% of cells; 1, expression

on 10% to 50% of cells; 2, expression on less than 10% of cells.

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