of July 25, 2022.This information is current as

Addison's Disease Patients T Cells in Autoimmune+Specific CD8−

High Frequency of Cytolytic 21-Hydroxylase

Vincenzo CerundoloSimon H. Pearce, Klaus Badenhoop, Olle Kämpe andGan, Eirik Bratland, Sophie Bensing, Eystein S. Husebye, Penna-Martinez, Gesine Meyer, Anna L. Mitchell, Earn H.Andrea Tarlton, Mohammad Alimohammadi, Marissa Amina Dawoodji, Ji-Li Chen, Dawn Shepherd, Frida Dalin,

http://www.jimmunol.org/content/193/5/2118doi: 10.4049/jimmunol.1400056July 2014;

2014; 193:2118-2126; Prepublished online 25J Immunol 

MaterialSupplementary

6.DCSupplementalhttp://www.jimmunol.org/content/suppl/2014/07/25/jimmunol.140005

Referenceshttp://www.jimmunol.org/content/193/5/2118.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2014 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

High Frequency of Cytolytic 21-Hydroxylase–Specific CD8+

T Cells in Autoimmune Addison’s Disease Patients

Amina Dawoodji,*,1 Ji-Li Chen,*,1 Dawn Shepherd,*,1 Frida Dalin,†,1 Andrea Tarlton,*

Mohammad Alimohammadi,† Marissa Penna-Martinez,‡ Gesine Meyer,‡

Anna L. Mitchell,x Earn H. Gan,x Eirik Bratland,{,‖ Sophie Bensing,†,#

Eystein S. Husebye,{,‖ Simon H. Pearce,x Klaus Badenhoop,‡ Olle Kampe,†,** and

Vincenzo Cerundolo*

The mechanisms behind destruction of the adrenal glands in autoimmune Addison’s disease remain unclear. Autoantibodies against

steroid 21-hydroxylase, an intracellular key enzyme of the adrenal cortex, are found in >90% of patients, but these autoantibodies

are not thought to mediate the disease. In this article, we demonstrate highly frequent 21-hydroxylase–specific T cells detectable in

20 patients with Addison’s disease. Using overlapping 18-aa peptides spanning the full length of 21-hydroxylase, we identified

immunodominant CD8+ and CD4+ T cell responses in a large proportion of Addison’s patients both ex vivo and after in vitro

culture of PBLs £20 y after diagnosis. In a large proportion of patients, CD8+ and CD4+ 21-hydroxylase–specific T cells were very

abundant and detectable in ex vivo assays. HLA class I tetramer–guided isolation of 21-hydroxylase–specific CD8+ T cells showed

their ability to lyse 21-hydroxylase–positive target cells, consistent with a potential mechanism for disease pathogenesis. These

data indicate that strong CTL responses to 21-hydroxylase often occur in vivo, and that reactive CTLs have substantial prolif-

erative and cytolytic potential. These results have implications for earlier diagnosis of adrenal failure and ultimately a potential

target for therapeutic intervention and induction of immunity against adrenal cortex cancer. The Journal of Immunology, 2014,

193: 2118–2126.

Autoimmune Addison’s disease is caused by the destructionof the adrenal cortex, and can occur either in isolation oras part of autoimmune polyendocrine syndromes type 1

or 2 (1–3). The target Ag for Addison’s disease was identifiedover 20 years ago by Winqvist et al. (4) as steroid 21-hydroxylase(21-OH), a key intracellular steroidogenic enzyme exclusivelyexpressed in the adrenal cortex. Rapidly, this seminal finding wastranslated into clinical practice; assay of 21-OH Abs is the mostimportant biomarker for autoimmune Addison’s disease, present in.90% of patients (5). 21-OH Abs are typically present years before

clinical disease is evident and may be found when testing individuals

who are at risk for developing Addison’s disease, that is, patients

who have another autoimmune disease or have a relative with

Addison’s disease. Preclinical adult patients with 21-OH Abs,

however, have a cumulative risk of only ∼20% of developing overt

Addison’s disease if adrenal function is normal at the start of the

observation (6). Thus, a significant proportion of individuals with 21-

OH Abs continue to remain disease free.Histological studies of adrenal glands from deceased Addison’s

disease patients show significant mononuclear cell infiltration into

the adrenal gland (7), and because 21-OH is an intracellular enzyme,

autoantibodies are unlikely to directly mediate destruction of the

adrenal gland. In addition, as demonstrated during pregnancy, 21-

OH Abs transferred from a mother with Addison’s disease did not

cause disease in the child (8). Instead, 21-OH Abs are more likely

to be an indication of T cell–mediated destruction of the adrenal

cortex, perhaps mediating or augmenting Ag presentation (9).It was first shown by Freeman et al. (10) that PBMCs from

Addison’s disease patients, but not controls, proliferated in re-

sponse to adrenal proteins, and this observation was followed by

a study demonstrating that PBMC proliferation and IFN-g secre-

tion occurred particularly in the presence of 21-OH (9). More

recently, Rottembourg et al. (11) established that a significant

proportion of HLA-B8+ patients have circulating T cells that are

specific for a dominant 21-OH peptide. However, a need remains

for comprehensive epitope mapping to show which epitopes on

21-OH are targeted by T cells in Addison’s disease patients with

different haplotypes and, importantly, whether these cells are

functionally capable of destroying the adrenal cortex.To address these questions, we used 18-mer overlapping syn-

thetic peptides spanning the entire 21-OH protein and demonstrated

that T cells from Addison’s disease patients, unlike those from

*Medical Research Council Human Immunology Unit, Medical Research CouncilWeatherall Institute of Molecular Medicine, Radcliffe Department of Medicine, Uni-versity of Oxford, Oxford OX3 9DU, United Kingdom; †Centre of Molecular Med-icine, Department of Medicine (Solna), Karolinska Institutet, 171 76 Stockholm,Sweden; ‡Division of Endocrinology, University Hospital Frankfurt, 60590 Frankfurtam Main, Germany; xInstitute of Genetic Medicine, Newcastle University, Newcastleupon Tyne NE1 3BZ, United Kingdom; {Department of Clinical Science, Universityof Bergen, N-5021 Bergen, Norway; ‖Department of Medicine, Haukeland UniversityHospital, 5021 Bergen, Norway; #Department of Molecular Medicine and Surgery,Karolinska Institutet, SE-171 77 Stockholm, Sweden; and **Science for Life Labo-ratory, Uppsala University 750 03, Uppsala, Sweden

1A.D. and J.-L.C. made equal first author contributions; D.S. and F.D. made equalsecond author contributions.

Received for publication January 9, 2014. Accepted for publication June 10, 2014.

This work was supported by Euradrenal (Grant 201167), by the Wellcome Trust (Grant084923 to V.C.), by Cancer Research UK (Program Grant C399/A2291 to V.C.), by theMedical Research Council (to V.C.), by the Swedish Research Council, by the Torstenand Ragnar Soderberg Foundations, by the NovoNordisk Foundation (to O.K.), and bythe European Commission (EU-FP7 Grant 241447 NAIMIT, to K.B.).

Address correspondence and reprint requests to Dr. Vincenzo Cerundolo, WeatherallInstitute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX39DS, U.K. E-mail address: vincenzo.cerundolo@imm.ox.ac.uk

The online version of this article contains supplemental material.

Abbreviations used in this article: ICS, intracellular staining; 21-OH, 21-hydroxylase.

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

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400056

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Table

I.Patientcharacteristics,21-O

HAbtiters,andHLA

haplotypes

Patient

Gender

Ageat

Diagnosis

Disease

Durationat

Sam

pling(y)

21-O

HAb

Titer

HLA-A

HLA-B

HLA-C

HLA-D

RB1

HLA-D

QB1

Patient1

F59

0.5

++

A*02

B*07,B*15

C*03,C*07

DRB1*04,DRB1*09

DQB1*03

Patient2

F40

2++

A*01,A*03

B*08,B*18

C*05,C*07

DRB1*03

DQB1*02

Patient3

F18

2++

A*01,A*32

B*07,B*08

C*07

DRB1*03,DRB1*15

DQB1*02,DQB1*06

Patient4

F35

1++

A*01

B*08

C*07

DRB1*03

DQB1*02

Patient5

F52

1++

A*01,A*02

B*04,B*08

C*03,C*07

DRB1*03,DRB1*04

DQB1*02

Patient6

F23

1++

A*01,A*03

B*04,B*08

C*03,C*07

DRB1*03,DRB1*04

DQB1*02,DQB1*03

Patient7

F17

1++

A*02,A*68

B*08,B*44

C*05,C*07

DRB1*03,DRB1*15

DQB1*02,DQB1*06

Patient8

F18

2ND

A*01

B*08,B*15

C*07

DRB1*03,DRB1*11

DQB1*02,DQB1*03

Patient9

M22

2++

A*01,A*03

B*07,B*08

C*07

DRB1*03,DRB1*15

DQB1*02,DQB1*06

Patient10

F54

4++

A*01,A*24

B*08,B*35

C*04,C*07

DRB1*03,DRB1*07

DQB1*02,DQB1*03

Patient11

F36

1++

A*02,A*26

B*07,B*15

C*03,C*07

DRB1*03,DRB1*12

DQB1*03

Patient12

F56

1++

A*02

B*08,B*15

C*03,C*07

DRB1*04

DQB1*03

Patient13

M25

3++

A*02,A*24

B*27,B*44

C*02,C*05

DRB1*04

DQB1*04,DQB1*05

Patient14

M17

2+++

A*01,A*03

B*08,B*15

C*03,C*07

DRB1*08,DRB1*14

DQB1*02,DQB1*04

Patient15

M18

2+

A*01,A*02

B*27,B*44

C*02,C*05

DRB1*03,DRB1*08

DQB1*03,DQB1*06

Patient16

F45

4.5

+A*32,A*66

B*07,B*35

C*04,C*07

DRB1*04,DRB1*13

DQB1*03,DQB1*05

Patient17

F24

3.5

+++

A*02

B*35,B*37

C*02

DRB1*04,DRB1*16

DQB1*03,DQB1*05

Patient18

F18

2++

A*01,A*02

B*08,B*40

C*07

DRB1*04,DRB1*15

DQB1*02,DQB1*03

Patient19

F39

17

++

A*01,A*02

B*08,B*40

C*03,C*07

DRB1*03,DRB1*12

DQB1*02,DQB1*06

Patient20

F33

19

++

A*31

B*40,B*51

C*03,C*15

DRB1*03,DRB1*15

DQB1*02,DQB1*03

Control1

__

N/A

N/A

A*02,A*03

B*07,B*44

C*05,C*07

DRB1*04,DRB1*15

DQB1*03,DQB1*06

Control2

__

N/A

N/A

A*02,A*29

B*44

C*05,C*16

DRB1*07,DRB1*12

DQB1*02,DQB1*03

Control3

__

N/A

N/A

A*02,A*11

B*44

C*04,C*05

DRB1*04,DRB1*13

DQB1*03,DQB1*06

Control4

__

N/A

N/A

A*02,A*03

B*07,B*57

C*06,C*07

DRB1*07,DRB1*15

DQB1*06

Control5

__

N/A

N/A

A*02,A*32

B*08,B*14

C*07,C*08

DRB1*03,DRB1*07

DQB1*02

Control6

__

N/A

N/A

A*23,A*25

B*13,B*44

C*05,C*06

DRB1*04,DRB1*07

DQB1*02,DQB1*03

Control7

__

N/A

N/A

A*24

B*07,B*35

C*04,C*07

DRB1*03,DRB1*13

DQB1*02,DQB1*06(1)

21-O

HAbtiters

wereclassified

as+(11–100),++(101–1000),and+++(1001–10,000).

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healthy controls, responded to the pool of 21-OH peptides. Suchresponses were mainly dominated by MHC class I–restrictedCD8+ T cells and focused on immunodominant regions on 21-OH.We extended these findings by demonstrating that HLA-A2–restricted 21-OH–specific CD8+ T cell clones generated from anAddison’s patient are capable of lysing HLA-A2 target cellstransduced with lentiviral vectors encoding the full-length 21-OHprotein and HLA-A2+ 21-OH+ tumor cells, thus providing im-portant insights into the pathogenesis and progression of Addi-son’s disease.

Materials and MethodsPatients and controls

Blood samples were taken from 21-OH Ab–positive Addison’s diseasepatients attending hospital clinics in Sweden, Norway, Germany, and theU.K., and their characteristics are shown in Table I. Eight patients wereanalyzed from Bergen, Norway (E.H.); seven patients from Newcastle,U.K. (S.H.P.); four from Frankfurt am Main, Germany (K.B.); and onefrom Stockholm, Sweden (S.B.). Local ethical approval was verified andapproved by the ethical committee for the EURADRENAL studies, andinformed consent was obtained. Inclusion criteria for patients werea clinical diagnosis of primary adrenal insufficiency and the presence of21-OH Ab at the onset. For comparison, blood samples from adult healthyvolunteers from the U.K. and Norway were used, and all the volunteerswere anonymized.

Patient HLA haplotype

DNA was extracted from patients’ PBMCs for haplotyping, using theQIAGEN DNeasy Kit, and haplotyping was conducted by the sequencingfacility at the Weatherall Institute for Molecular Medicine, Oxford, U.K.

21-OH peptides

Overlapping 18-aa peptides that span the whole 21-OH sequence weresynthesized by facilities at the Weatherall Institute of Molecular Medicineand are listed below in Supplemental Table I. Peptides were dissolved inDMSO at a concentration of 40 mg/ml and were used either individually oras a combined pool.

T cell culture assay

Patient cells were stimulated at 6 3 106 per well with 1 mg/ml 21-OHpeptide pool in RH-10 (RPMI 1640, pH 7.4, supplemented with 10%human serum, 1% penicillin/streptomycin, 1% L-glutamine, 1% nones-sential amino acids, 1% sodium pyruvate, 1% HEPES, and 0.1% 2-ME).Cells were initially pulsed in 200 ml for 1 h before the volume was in-creased to 2 ml and IL-7 was added at 25 ng/ml. On day 3, cells were fedwith RH-10 containing 1000 IU/ml IL-2 (Pharmacia) and continued to befed and/or split as necessary with IL-2–containing medium until day 13,when they were washed to remove IL-2 and rested overnight in RH-10, inpreparation for the intracellular staining (ICS) assay.

FACS staining

Cells were stained for intracellular cytokine secretion after 14 d in culture. Atotal of 200,000 cells were pulsed with individual peptides or the pool ofpeptides at 10 mg/ml for 5 h, with 10 mg/ml brefeldin A (eBioscience)added after the first hour to prevent cytokine secretion into the supernatant.Cells were washed and stained for the surface markers CD8 APC-H7, CD4PerCP, and CD3 V500 (BD) together with LIVE/DEAD viability marker(Invitrogen) diluted in PBS for 30 min on ice, then washed and fixed withfixation solution (eBioscience) for a further 30 min on ice or left overnight

FIGURE 1. 21-OH–specific T cells are detectable in Addison’s disease

patients. The production of IFN-g from CD8+ (A) and CD4+ (B) T cells in

response to 21-OH was assessed by culturing PBMCs from healthy con-

trols or Addison’s patients for 14 d in the presence of a pool of peptides

spanning the full-length 21-OH protein, followed by stimulation for 5 h

with the 21-OH peptide pool (peptide pool) or with DMSO. T cells were

surface stained for CD4 and CD8 expression, then stained intracellularly

for the production of IFN-g. Because the two populations compared in (A)

and (B) (patients versus controls) have unequal variances, p values were

determined using an unpaired t test with Welch’s correction: *p = 0.0294,

***p = 0.0008. Each dot corresponds to a different patient. Different

symbols were used for controls (:) and patients (d).

FIGURE 2. 21-OH–specific T cells can be identified for many years

after diagnosis in the peripheral blood of Addison’s disease patients. 21-

OH–specific T cell responses, as defined by the frequency of IFN-g–pos-

itive T cells upon stimulation with the 21-OH peptide pool, were analyzed

at different time points after diagnosis in Addison’s disease patients. (A)

indicates the frequency of 21-OH–specific CD8+ T cells, whereas (B)

indicates 21-OH–specific CD4+ T cells. Each symbol represents a different

patient, corresponding to the order shown in Table I.

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at 4˚C. Cells were permeabilized by washing with permeabilization buffer(eBioscience) and stained with the intracellular Ab IFN-g FITC (eBio-science) diluted in the permeabilization buffer for 30 min on ice. Cellswere washed with PBS and resuspended in FACS buffer for acquisition byflow cytometry. The presence of CD107 on the T cell surface was assessedby the addition of PE-conjugated CD107 (BD) Ab at the same time asbrefeldin A. CD4+ and CD8+ T cell responses to the peptide diluent DMSOwere negligible and ,0.001%.

Flow cytometry and gating strategy

Cells were acquired by flow cytometry on FACSCanto II and samples wereanalyzed using FlowJo (TreeStar). Cells were gated to remove doublets,then separated into Live CD3+ CD4+ or CD8+ cells and analyzed separatelyfor their cytokine positivity.

Ex vivo ELISPOT assay

Plates were coated overnight at 4˚C with 4 mg/ml capture Ab (MabTech) incoating buffer (Sigma-Aldrich). Plates were washed twice with RPMI1640, then incubated for 1 h at 37˚C with 200 ml blocking buffer (RPMI1640 + 10% human serum). After washing plates three times, cells wereadded in triplicate at 500,000 per well with 10 mg/ml peptide in X-Vivo 15serum-free medium (Lonza) and incubated for 18 h at 37˚C. Plates werewashed with wash buffer (distilled water + 0.05% Tween-20) followed bydistilled water before adding 0.2 mg/ml biotin detection Ab (MabTech)diluted in PBS, then incubated for 2 h at 37˚C. Plates were washed again,and 1 mg/ml Streptavidin-ALP (MabTech) diluted in PBS was added, andplates were then incubated for a further hour at room temperature. Finally,plates were washed once more before developing by adding 100 ml sub-strate solution and incubated for 10 min in the dark at room temperature.

Development was stopped by rinsing plates thoroughly with cold waterand the plates were left to dry completely before counting spots using anAID EliSpot Reader.

HLA-class I tetramers

Monomers were made by refolding the HLA-A2 or HLA B8 H chain and b2-microglobulin proteins with the 21-OH342–350 peptide (LLNATIAEV) or the21-OH428–435 peptide (EPLARLEL) together at 4˚C for 40 h (12). The refoldmixture was then concentrated to ∼8 ml using a nitrogen gas–pressured stircell with a presoaked ultrafiltration 150-mm membrane (Millipore) beforebeing biotinylated with a BirA enzyme overnight, followed by fast proteinliquid chromatography separation to remove aggregates. The concentrationof the eluted monomer was measured by BCA assay, and aliquots werestored at 280˚C. Monomers were tetramerized using Streptavidin-APC(eBioscience) before being used to stain T cells.

Granzyme B ELISA

Granzyme B secretion was measured by ELISA in the supernatant afterovernight coculture of 20,000 CTLs with 40,000 target cells. ELISA plateswere precoated overnight with anti-Granzyme B Ab before being washedand loaded with supernatants overnight at 4˚C. Plates were washed and in-cubated with the biotin Ab followed by ExtrAvidin Peroxidase and developedusing TMB (3,39,5,59-tetramethylbenzidine) solution (Sigma-Aldrich).

21-OH lentivirus constructs

RNA was extracted from 21-OH–expressing NCI H295 cells using theQIAGEN RNeasy Kit and cDNA synthesized using the RETROSCRIPTkit. The 21-OH fragment was amplified from prepared cDNA using theforward primer 59-CGCGGATCCACCATGCTGCTCCTGGGCCTGCTG-

FIGURE 3. Immunodominance and epitope map-

ping of 21-OH–specific T cell responses. Percentage of

21-OH–specific CD8+/CD3+ (A) and CD4+/CD3+ (B)

T cells, as defined by IFN-g ICS. T cell responses were

measured after in vitro T cell expansion with 21-OH

overlapping peptides spanning the full-length 21-OH

protein, followed by stimulation with either individual

peptides (amino acid sequence shown in the figure) or

pool of all peptides (peptide pool). Negative control

samples in DMSO (no peptide) are shown in each

panel. Each symbol represents a different patient,

corresponding to the order shown in Table I. The hor-

izontal straight line indicates the cutoff value for pos-

itive responses, as defined by percentage values above

the mean value of the percentage of IFN-g secretion

plus three times the SD. Such values were 0.061% and

0.066% for CD8+ and CD4+ T cell responses, respec-

tively.

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39 and the reverse primer 59-CCGCTCGAGCTGGCTCTGGCCCGGGC-TGTG-39, containing the BamHI and XhoI restriction sites. The insert wasdigested using BamHI and Xho1 and ligated into a GFP lentiviral vector,which was then used to transform competent DH5alpha bacteria.

Lentiviral particles were made by combining the GFP–21-OH lentivector,pMDG, and the Gag-pol expressor with Fugene 6 transfection reagent(Promega), then adding dropwise to 293T cells, which had been grown to90% confluency. Supernatants containing viral particles were collected after2 d and used to transduce B cells. Cells were sorted for GFP positivity andcultured for a further 2 wk before being used in functional assays.

VITAL assay

The VITAL assay was performed as previously described (13). Briefly,150,000 21-OH–GFP transduced cells were placed together with 150,000untransduced B cells that had been labeled with CellTracker Orange CMTMR(5-(and -6)-(((4-chloro-methyl)benzoyl)amino)tetramethylrhodamine; Invi-trogen) in flat-bottomed 96-well plates and cocultured with 21-OH–specific

T cell clones overnight at different effector:target ratios. Cells were thenwashed and stained with Invitrogen LIVE/DEAD dye and acquired by flowcytometry to determine killing of target cells. Equal numbers of CMTMR-labeled untransduced B cells were placed in a well containing the 21-OH–GFP B cells and cocultured with 21-OH342–350 T cell clone at different ef-fector:target ratios. The target cell survival was calculated as the percentageof GFP-positive cells compared with the CMTMR-positive cells, and thetarget cell lysis was calculated as 100 minus the cell survival percentage.

ResultsHigh frequency of 21-OH–specific CD8+ and CD4+ T cells inPBMCs from Addison’s disease patients

To assess the overall T cell responses to 21-OH in Addison’sdisease patients, PBMCs were obtained from Addison’s diseasepatients with a high titer of 21-OH–Ab. The HLA haplotype

FIGURE 4. Ex vivo 21-OH–specific T cell responses can be identified for many years after diagnosis in the peripheral blood of Addison’s disease

patients. (A) 21-OH–specific T cell responses were analyzed from PBMCs in ex vivo ELISPOT assays against the pool of the 21-OH peptides at different

time points after diagnosis in Addison’s disease patients. Each symbol represents a different patient, corresponding to the order shown in Table I. (B)

Cumulative ex vivo ELISPOT data from PBMCs from Addison’s disease patients and healthy controls. 21-OH–specific T cell responses were analyzed

from PBMCs in ex vivo ELISPOT assays from Addison’s disease patients and healthy controls tested against the pool of the 21-OH peptides. The mean

number of spots6 SD is indicated. (C) Ex vivo and cultured ELISPOT from CD4+ and CD42 T cells. 21-OH–specific T cell responses were analyzed from

sorted CD4+ and CD42 T cells purified from PBMCs of patient 5 and patient 15 and tested either in ex vivo assays (ex vivo) or after culture for 2 wk with

the pool of the 21-OH peptides (cultured). ELISPOT assays were performed against the pool of the 21-OH peptides. Background responses in the absence

of peptides are shown (cells only).

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revealed a high proportion of patients expressing HLA-A2, HLA-A1, and HLA-B8 (Table I). The frequency of 21-OH–specificCD4+ and CD8+ T cells was then measured by ICS for IFN-g inresponse to restimulation with the individual peptides or with thepool of peptides for 13 d.We found that most of the 20 Addison’s disease patients ana-

lyzed in this study had high numbers of 21-OH–specific CD8+ andCD4+ T cells, compared with the baseline values found in healthycontrols (Fig. 1). Of note, although the magnitude of 21-OH–specific CD8+ T cell responses appeared greater than the magni-

tude of 21-OH–specific CD4+ T cell responses (Fig. 1) becausethese experiments were performed after stimulating PBMCs oncein vitro with a pool of overlapping 18-mer peptides, the relativefrequency of 21-OH–specific CD8+ and CD4+ T cell responsescannot be compared.To assess the longevity of 21-OH–specific T cell responses, we

compared the frequency of 21-OH–specific T cell responses inpatients who had been diagnosed with Addison’s disease for up to20 y (Fig. 2). We observed that although the magnitude of the 21-OH–specific T cell response decreased over time, we were still

FIGURE 5. Addison’s disease patients

show strong ex vivo T cell responses to 21-

OH. Ex vivo ELISPOT assay with PBMCs

from Addison’s disease patients (top panel)

or healthy controls (lower panel) upon

stimulation with pool of overlapping pep-

tides spanning the full-length 21-OH protein

(peptide pool) or indicated peptides. Num-

bers of spots per million cells are shown on

the y-axis. Results from three representative

patients are shown. Bars represent SEM.

FIGURE 6. Identification of the immu-

nodominant optimal-length 21-OH337–354

peptide. PBMCs from patient 7 were rested

overnight and stimulated with the indicated

peptides in duplicate wells in an ELISPOT

assay, and spots per million cells were

counted.

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able to detect responses in the majority of our samples from 1 to2 y since diagnosis.

T cell responses are targeted to immunodominant 21-OHregions

Having established the presence of high frequency CD8+ and CD4+

T cell responses to 21-OH in patients with Addison’s disease, wenext mapped the peptide epitopes recognized by the 21-OH–specificT cells. PBMCs that had been expanded with the pool of 21-OH

peptides were tested against each individual overlapping peptidebefore measuring the IFN-g secretion by ICS and flow cytometry.The majority of patients revealed CD8+ T cells capable of recog-nizing 21-OH337–354 and/or 21-OH428–445 (Fig. 3A, SupplementalFig. 1A). In contrast, a number of CD4+ T cells recognized thepeptide 21-OH 207–224 (Fig. 3B, Supplemental Fig. 1B).

T cell responses to dominant 21-OH peptides are detectable exvivo

Similar results were observed by ex vivo ELISPOT assays usingpatients’ PBMCs, including both 21-OH–specific CD4+ and CD8+

T cell responses (Figs. 4, 5). Although the recall assay response isindicative of a memory T cell response to 21-OH in Addison’spatients, this assay remains semiquantitative and does not representthe actual frequency of 21-OH–specific T cells in Addison’s diseasepatients. To gain a better understanding of the magnitude of 21-OH–specific T cells, we performed ex vivo ELISPOT assays induplicate with the immunodominant peptide identified duringthe recall assay (Figs. 4, 5). We found that the patients showedresponses in the ex vivo assay that were specific to the same pep-tides recognized in the recall assay (Fig. 5), demonstrating that theT cell responses were not being biased to particular peptides duringthe bulk culturing with the pool of peptides. The ex vivo frequencyof 21-OH–specific T cell responses (ranging from ∼0.001–0.01%)was similar to melanoma-specific CD8+ T cell responses observedin cancer patients (12). As shown above, we confirmed the lon-gevity of 21-OH–specific responses, as ex vivo ELISPOT assaysdemonstrated the presence of 21-OH–specific responses many yearsafter diagnosis (Fig. 4A). Cumulative ex vivo ELISPOT data areshown in Fig. 4B. To further characterize the relative frequency ofCD8+ and CD4+ 21-OH–specific T cell responses in ex vivo assays,CD4+ and CD4- T cells were sorted from PBMCs from patient 5and patient 15 and tested in ex vivo ELISPOTassays for their abilityto recognize the pool of 21-OH peptides (Fig. 4C). The results inFig. 4A showed that PBMCs from patient 15 have a detectable exvivo ELISPOT response against the pool of 21-OH peptides,whereas PBMCs from patient 5 have a much lower frequency of 21-OH–specific T cells. Separation of CD4+ and CD42 T cells showedthat the ex vivo 21-OH–specific T cell response seen in patient 15was mainly dominated by CD42 21-OH–specific T cells, as 21-OH–specific CD4+ T cell responses could be detected only afterenriching T cell cultures for CD4+ T cells (Fig. 4C).

Functional evidence that 21-OH–specific CD8+ T cells are Ag-specific cytotoxic lymphocytes

Specificity and HLA restriction of 21-OH–specific T cell responseswere confirmed by staining 21-OH–specific T cell lines and cloneswith HLA A2 and HLA B8 tetramers loaded with the peptides 21-OH342–350 and 21-OH428–435, respectively (Supplemental Fig. 2).HLA-A2 tetramer sorted 21-OH342–350–specific CD8+ T cellswere assayed for their ability to recognize 21-OH–expressingcells. First, we used overlapping 9 mers spanning the immuno-dominant peptide 21-OH337–354 to identify the epitope. The pep-tide 21-OH342–350 (LLNATIAEV) elicited the strongest response(Fig. 6). Then, using HLA-A2 tetramers loaded with the peptide21-OH342–350, we were able to sort a panel of CD8+ T cell linesand clones (Supplemental Fig. 2) and tested their specificity andfunctional activity (Figs. 7, 8, and Supplemental Fig. 3).HLA-A2/21-OH342–350 tetramer+ CD3+CD8+ T cells recog-

nized targets pulsed with nanomolar concentrations of the 21-OH342–350 peptide, but not with irrelevant peptides (SupplementalFig. 3 and data not shown). These data provide direct evidencethat HLA-A2/21-OH342–350 peptide tetramer+ CD3+CD8+ T cellpopulations derived from PBMCs of Addison’s disease patients

FIGURE 7. Ability of 21-OH–specific T cells to lyse target cells

expressing full-length 21-OH protein. HLA-A2–restricted 21-OH342–350–

specific T cell clone was coincubated overnight at indicated E:T ratios with

B cells either transduced with lentiviral vector encoding the full-length 21-

OH protein and GFP or with CMTMR-labeled untransduced B cells. (A)

shows FACS staining. Top two panels, Target cells in the absence of the

21-OH342–350–specific T cell clone. Bottom four panels, Cocultures be-

tween T cell clone and target cells at the indicated E:T ratios. Increasing

numbers of T cells are shown in the bottom left corner of each dot plot.

The percentage of GFP+ and CMTMR+ labeled cells is shown in each dot

plot. (B) shows percentage of specific lysis of 21-OH–positive target cells.

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contain HLA-A2–restricted CD8+ T cells recognizing the peptide21-OH342–350. In addition, T cells were able to lyse HLA A2+

target cells transduced with a lentiviral vector encoding the full-length 21-OH, thus confirming their cytotoxic capacity (Fig. 7).To further confirm the functional capability of 21-OH342–350–

specific T cells, we cocultured the 21-OH342–350–specific HLA-A2–restricted CD8+ T cell clone with the HLA-A2–expressingtumor line NCI H295 (14) and found an increase in CD107 ex-pression and granzyme B secretion in the supernatant, asmeasured by ELISA. NCI H295 recognition was reduced in the pre-sence of the HLA-A2–blocking Ab BB7.2 (Fig. 8).

DiscussionIn this study we show strong and long-lasting T cell responses againsta few specific peptides derived from 21-OH, the major adrenal cortexcell autoantigen. We have measured the frequency, phenotype, andfunctional activity of 21-OH–specific CD8+ and CD4+ T cells in thePBMCs of Addison’s patients directly ex vivo and after in vitro cul-ture. Our results show that 21-OH–specific CD8+ and CD4+ T cellsare often present in high numbers in PBMCs, are Ag experienced, andare capable of massive expansion when exposed to the appropriatecytokines, generating highly cytolytic T cell populations. The abilityof 21-OH–specific CD8+ T cell clones to recognize endogenous 21-OH protein may therefore be directly linked to the progression ofAddison’s disease.It has previously been established that T cells target autoantigens

and cause destruction in other organ-specific autoimmune diseases,

such as type 1 diabetes, myasthenia gravis, and multiple sclerosis(15, 16). However, it has often been difficult to characterize suchautoreactive T cells, as they are present at a very low frequency inperipheral blood. Addison’s disease offers an opportunity to betterunderstand why the immune system in organ-specific autoimmu-nity often targets intracellular proteins with a tissue-specific ex-pression and often key enzymatic functions in the tissues involved—for example, thyreoperoxidase in autoimmune thyroiditis, glutamicacid decarboxylase in type 1 diabetes, or steroid side-chain cleavageenzyme in autoimmune oophoritis (17).The high frequency of 21-OH–specific T cells, comparable to the

frequency of tumor-specific T cells observed in melanoma patients(12), as well as their long-term persistence after diagnosis is there-fore unexpected and consistent with the possibility that 21-OH–specific T cells are continuously stimulated in vivo by mechanismsthat need to be further investigated. It is tempting to speculate thatthis may be caused by nonadrenal expression of 21-OH (18) or thatsome remnants of the adrenal cortex persist within Addison’s dis-ease patients, perhaps owing to continued proliferation from residualadrenal progenitor or stem cells, either of which could continue tostimulate the 21-OH–specific T cells (19, 20). Alternatively, thelong-term persistence of 21-OH–specific CD8+ and CD4+ T cellsmay be due to cross-reactive viral or bacterial epitopes, as suggestedfor other autoimmune disorders (21). It is not possible to draw firmconclusions about the relative difference in frequency of 21-OH–specific CD8+ and CD4+ T cells, as the majority of the experimentsto compare the frequency of 21-OH–specific T cells were performed

FIGURE 8. Functional activity of 21-OH–specific CD8+ T cells. (A) ELISA with granzyme B–specific Ab from supernatant of the HLA-A2–restricted

21-OH342–350–specific T cell clone cocultured with the HLA-A2+ 21-OH–expressing tumor cell line NCI H295 in the presence or absence of the HLA-A2–

blocking Ab BB7.2. Positive control with NCI H295 cells pulsed with peptide 21-OH342–350 is shown. (B) Increased expression of CD107 on the surface of

the HLA-A2–restricted 21-OH342–350–specific T cell clone cocultured with the HLA-A2+ 21-OH–expressing tumor cell line NCI H295 in the presence or

absence of the HLA-A2–blocking Ab BB7.2. Positive control with NCI H295 cells pulsed with peptide 21-OH342–350 is shown. The results are repre-

sentative of two experiments.

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after stimulating PBMCs in vitro with a pool of overlapping 18-merpeptides spanning the full-length 21-OH protein. The apparenthigher frequency of CD8+ 21-OH–specific T cells is consistent withprevious observations demonstrating higher frequency of Ag-specific CD8+ T cells specific for self (22), virus (23), and cancerepitopes (24). However, a higher frequency of virus-specific CD4+

T cells has also been documented (25).Of interest, we found that T cell responses in Addison’s patients

are clustered to just a few immunodominant 21-OH epitopes. Aprevious report had identified the HLA-B8–restricted epitope 21-OH431–438 (11). Our results have confirmed and extended thesefindings by demonstrating that the response to peptide 21-OH431–

438 is immunodominant, as it was detected in a large proportion ofHLA B8+ patients. In addition, we identified a further HLA-A2–restricted dominant epitope at position 21-OH342–350.Previous studies have indicated an association between Addi-

son’s disease and the HLA DRB1*04- DQA1* 0301- DQB1*0302 haplotype (as reviewed in Ref. 26) and the HLA DRB1*0404–restricted recognition of a peptide spanning the region 21-OH342–361

(9). The results of our study, although confirming the presence of21-OH–specific CD4+ T cell responses, highlighted a significantlylarger frequency of HLA-class I–restricted CD8+ T cell response,with a strong bias toward T cell responses restricted by HLA-A2,HLA-A1, and HLA-B8.Because only a minority of individuals with 21-OH Ab develop

disease within an observation period of #5 y (6), future inves-tigations of T cell responses in these individuals and the ability topredict disease progression are of major importance, as diagnosticand therapeutic opportunities could result. A better understandingof the initiation of T cell responses at the molecular level inAddison’s disease may also open up a way to induce responsesagainst adrenal cortex cancer, a 21-OH–positive tumor with verypoor prognosis often afflicting younger people.In conclusion, we have identified immunodominant regions of

21-hydroxylase and have shown that 21-OH–specific T cells rec-ognize endogenous 21-OH protein and may therefore be directlylinked to the progression of Addison’s disease. Unexpectedly, thepersistence for many years of 21-OH T cell responses in the faceof destroyed adrenal glands without remaining endogenous steroidproduction indicates the presence of remaining Ag stimulationperhaps by ectopic expression of 21-OH in other tissues or bypathogens expressing a cross-reactive epitope continuously stim-ulating the 21-OH–specific T cells.

AcknowledgmentsWe thank Tim Rostron for haplotyping of patient samples, Zhanru Yu for

synthesizing all the peptides used in this article, and Hemza Ghadbane, Uzi

Gileadi, and Yanchun Peng for technical support and reagents.

DisclosuresThe authors have no financial conflicts of interest.

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