Download Increased Frequency of EBV-Specific Effector Memory CD8 T Cells

The Journal of Immunology
Increased Frequency of EBV-Specific Effector Memory CD8ⴙ
T Cells Correlates with Higher Viral Load in Rheumatoid
Arthritis1
Jan D. Lünemann,2* Oliver Frey,2† Thorsten Eidner,‡ Michael Baier,§ Susanne Roberts,*
Junji Sashihara,¶ Rudolf Volkmer,储 Jeffrey I. Cohen,¶ Gert Hein,‡ Thomas Kamradt,3†
and Christian Münz3,4*
EBV is a candidate trigger of rheumatoid arthritis (RA). We determined both EBV-specific T cell and B cell responses and
cell-associated EBV DNA copies in patients with RA and demographically matched healthy virus carriers. Patients with RA
showed increased and broadened IgG responses to lytic and latent EBV-encoded Ags and 7-fold higher levels of EBV copy
numbers in circulating blood cells. Additionally, patients with RA exhibited substantial expansions of CD8ⴙ T cells specific
for pooled EBV Ags expressed during both B cell transformation and productive viral replication and the frequency of CD8ⴙ
T cells specific for these Ags correlated with cellular EBV copy numbers. In contrast, CD4ⴙ T cell responses to EBV and T
cell responses to human CMV Ags were unchanged, altogether arguing against a defective control of latent EBV infection
in RA. Our data show that the regulation of EBV infection is perturbed in RA and suggest that increased EBV-specific
effector T cell and Ab responses are driven by an elevated EBV load in RA. The Journal of Immunology, 2008, 181:
991–1000.
R
heumatoid arthritis (RA)5 is a chronic inflammatory disease with an unknown etiology. Data from clinical trials
as well as from animal models demonstrate a role for
both T and B lymphocytes in RA pathogenesis (1, 2) and certain
HLA-DR class II alleles; for example, HLA-DRB1*01 and HLA-
*Laboratory of Viral Immunobiology, Christopher H. Browne Center for Immunology and Immune Diseases, Rockefeller University, New York, NY 10021; †Institut
für Immunologie, ‡Klinik für Innere Medizin III, and §Institut für Medizinische Mikrobiologie, Universitätsklinikum Jena, Jena, Germany; ¶Medical Virology Section,
Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and 储Institut für
Medizinische Immunologie, Charitè Universitätsmedizin Berlin, Berlin, Germany
Received for publication February 20, 2008. Accepted for publication May 13, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
J.D.L. is a recipient of the Dana Foundation and Irvington Institute’s Human Immunology Fellowship from the Cancer Research Institute and is supported by a Pilot
Grant from the National Multiple Sclerosis Society (PP1145) and an Institutional
Clinical and Translational Science Pilot and Collaborative Project Grant (to the Rockefeller University Hospital). C.M. is supported by the Dana Foundation’s Neuroimmunology Program, the Arnold and Mabel Beckman Foundation, the Alexandrine and
Alexander Sinsheimer Foundation, the Burroughs Wellcome Fund, the Starr Foundation, the National Cancer Institute (R01CA108609 and R01CA101741), the National Institute of Allergy and Infectious Diseases (RFP-NIH-NIAID-DAIDS-BAA06-19), the Foundation for the National Institutes of Health (Grand Challenges in
Global Health), and an Institutional Clinical and Translational Science Award (to the
Rockefeller University Hospital). T.K. is supported by the Deutsche Forschungsgemeinschaft (SFB 604 C5) and the Gemeinnützige Hertie-Stiftung (1.319.110/03/03).
J.S. and J.I.C. are supported by the intramural research program of the National
Institute of Allergy and Infectious Diseases. J.S. is partially supported by the Japan
Herpes Virus Infection Forum.
2
J.D.L. and O.F. contributed equally to this work and share first authorship.
3
T.K. and C.M. contributed equally to this work and share senior authorship.
4
Address correspondence and reprint requests to Dr. Christian Münz, Christopher H.
Browne Center for Immunology and Immune Diseases, Rockefeller University, Box
390, 1230 York Avenue, New York, NY 10021. E-mail address: munzc@
rockefeller.edu
5
Abbreviations used in this paper: RA, rheumatoid arthritis; EA, early Ags; EBNA,
EBV nuclear Ag; Flu-HA, influenza hemagglutinin peptide; HCMV, human CMV;
HD, healthy donors; SEB, Staphylococcus enterotoxin B; VCA, viral capsid Ag.
www.jimmunol.org
DRB1*0401 are the strongest risk-conferring genes (3). It is, however, unclear to date if pathogenic T cells in RA predominantly
recognize self or pathogen-derived Ags. The fact that concordance
rates for RA are only between 15 and 30% among monozygotic
twins strongly argues for environmental factors, such as pathogens
that trigger or perpetuate the disease (4).
EBV has been suspected for more than 25 years to be involved
in RA pathogenesis (5–9). EBV is a gammaherpesvirus that infects
⬎90% of the human adult population. After primary infection in
childhood or adolescence, EBV persists life-long in B lymphocytes. In latently infected B cells, only some viral proteins are
expressed and confer resistance to apoptosis, possibly preventing
activation-induced cell death of autoreactive B cells. The frequency of EBV-infected B cells in the blood is low (0.5–50 per
million) and stable over time. Periodically, activation of infected B
cells by Ag receptor triggering leads to reactivation of EBV into
lytic cycle for transmission of infectious virus in the saliva of
healthy virus carriers.
Strong T cell responses against lytic and latent EBV Ags can be
detected in healthy asymptomatic carriers and are critically important for the control of latent infection. CD4⫹ Th cell responses
against EBV nuclear Ag 1 (EBNA1) can be consistently detected
in healthy virus carriers. EBNA1-specific T cells produce IFN-␥,
and they can recognize and kill EBV-infected cells and thereby
prevent the outgrowth of EBV-transformed B cells or lymphoma
cells. EBV-specific CD8⫹ CTLs undergo massive clonal expansion during acute EBV infection when CD8⫹ T cells, recognizing
one particular EBV peptide, can comprise ⬎40% of all CD8⫹ T
cells in the blood (10).
Compared with healthy EBV carriers, patients with RA show
higher titers of IgG Abs specific for both latent and lytic EBVencoded Ags (11–13). Several groups reported that T cell responses to selected EBV Ags are altered in frequency (14 –16) and
are functionally impaired in RA patients (5, 6, 14). Higher frequencies of EBV-infected B cells (17) and up to 10-fold increased
992
EBV IN RA
Table I. Patients and healthy blood donors
Number
Number (female/male)
Age (years ⫾ SD)
Disease duration (years ⫾ SD)
Disease activity (RADAI ⫾ SD)
Disease activity (DAS28 ⫾ SD)a
RA
HD
25
19/6
58.75 ⫾ 9.138
12.9 ⫾ 7.9
3.7 ⫾ 1.42
4.0 ⫾ 0.97
20
8/12
58.16 ⫾ 6.35
n/a
n/a
n/a
a
The disease activity scores using 28 joint counts (DAS28) were obtained in 20
patients with RA.
viral copy number in circulating mononuclear cells (18) have also
been detected in RA patients. Herein, we investigated both EBVspecific T cell and B cell responses as well as EBV regulation in
25 patients with RA and 20 demographically matched healthy virus carriers. Our data show that EBV infection is perturbed in
patients with RA and support the concept that RA-associated immune dysfunctions drive enhanced EBV replication in B cells,
thereby stimulating increased EBV-specific effector T cell and Ab
responses.
Materials and Methods
Patients and healthy control individuals
Twenty-five patients with RA diagnosed according to the 1987 American
College of Rheumatology criteria (19) were recruited between March 2006
and April 2007 at the Department of Internal Medicine at the University of
Jena. At the time of blood collection, patients’ medical records were reviewed, and current medications, pertinent laboratory data, and RA disease
history were recorded by the treating physicians. Disease activity was assessed with a standardized patient questionnaire and a RA disease activity
index (in all 25 patients) (20) as well as the disease activity score using 28
joint counts (in 20 of 25 patients) were calculated. All patients had normal
white blood cell counts at the time of blood drawing. None of the patients
received more than 20 mg prednisolone. Ten of the patients were treated
with methotrexate, and six patients were under treatment with a TNF-␣blocking agent. One patient was treated with azathioprine and another with
mycophenolate mofetil. In a subgroup analysis, we compared patients receiving immunosuppressive therapy (methotrexate, anti-TNF, azathioprine, mycophenolate mofetil) (n ⫽ 14/25) with those not receiving immunosuppressive or immunomodulatory therapy (n ⫽ 11/25) compared
with healthy donors and noted no significant differences in any parameter
tested. Age- and sex-matched healthy donors were recruited from a family
physician’s private practice. We excluded patients with autoimmune or
other chronic inflammatory disease, metabolic disorders, and a recent history of infection. Not all patients and controls were included in every
analysis; rather, subsets of both groups were chosen as indicated in the
results section and figure legends. Demographic and clinical characteristics
are given in Table I. The study was approved by the local Institutional
Review Board, and all subjects provided informed consent.
ELISA and Western blot
The detection of EBV and human CMV (HCMV)-specific IgG Abs was
performed by ELISA following the manufacturer’s instructions (Dade Behring/Siemens). The ELISA plate for detection of EBV-specifc IgG was
coated with a defined mixture of relevant virus Ags, which included
epitopes derived from early Ags (EA), viral capsid Ag (VCA), and
EBNA1. The Western blot for the distinct detection of recombinant EBVencoded Ags (EAp54, EAp138, VCAp23, VCAp18, EBNA1) was performed according the manufacturer’s instructions (Mikrogen). The ELISA
plate for detection of HCMV-specifc IgG was coated with cell lysates from
HCMV-infected cells. To determine anti-EBNA1 IgG isotype titers (21),
the C-terminal domain of EBNA1 (aa 458 – 641) was recombinantly expressed with the expression vector pET15b (Novagen and a gift of Drs.
Dan Zhang and Michael O’Donnell, New York, NY) in Escherichia coli
BL21 (DE3) pLysS cells. Production was induced with 1 mM isopropyl
␤-D-thiogalactoside (Invitrogen). The protein was purified and the identity
determined by Western blot analysis with EBNA1-specific Ab (MAB8173;
Chemicon International). Ninety-six-well polystyrene plates (Nalgene
Nunc International) were coated with 1 ␮g/well of rEBNA1458 – 641 protein
in PBS or PBS alone overnight at 4°C. Plates were blocked with 200
␮l/well 5% nonfat milk powder for 30 min, followed by 30 min in PBS
containing 5% BSA. Test plasma samples, diluted 1/10, 1/100, 1/200,
1/500, 1/1000, and 1/2000 in 3% BSA, were added for 30 min at room
temperature. Plates were washed three times with TBST (10 mM Tris, 140
mM NaCl, 0.05% Tween 20). Biotin mouse anti-human IgG1, IgG2, and
IgG3 Abs (BD Pharmingen) were added at 1/1000 and the anti-human
IgG4 Ab was added at 1/5000 in TBST for 30 min at room temperature.
After plates were washed three times in TBST, avidin-bound HRP was
added for 20 min at room temperature, followed by tetramethylbenzidine
substrate (R&D Systems) to develop the reaction for 10 min at room temperature and 1 M H2SO4 to stop the reaction. Plates were read in a microplate reader (Dynex Technologies). Samples were processed blinded to the
clinical diagnosis. Titers were defined by the 10% effect concentration
(EC10) value of individual titration curves.
EBNA1 membranes
Cellulose-bound peptides were prepared according to the standard SPOT
synthesis protocol (22) by a MultiPep SPOT-robot (Intavis Bioanalytical
Instruments) on a ␤-alanine-modified cellulose membrane (23). Altogether,
211 12-mer peptides, overlapping by 9 aa, were covalently linked to the
membrane via two ␤-alanine residues. The membrane was activated with
96% ethanol for 10 min, washed three times with TBS for 10 min, and
blocked with blocking buffer (Sigma-Aldrich) for 3 h at 4°C. After an
additional washing step, the membrane was incubated with serum samples
diluted 1/1000. Bound Abs were detected using a rabbit anti-human IgGalkaline phosphatase Ab (DakoCytomation) in a 1/1000 dilution. As a color
substrate, 5-bromo-4-chloro-3-indolyl phosphate/NBT (Sigma-Aldrich)
was used. The intensity of the spots was semiquantitatively evaluated on a
0 –3 point scale by an investigator blinded to the origin of the sample.
Peptide preparation
Peptides were synthesized by the Proteomics Resource Center, Rockefeller
University. All peptides were created using a Protein Technologies Symphony multiple-peptide synthesizer (Rainin Instrument) on Wang resin
( p-Alkoxy-benzyl alcohol resin; Bachem and Midwest Bio-Tech) using
N-Fmoc (9-fluorenylmethyloxycarbonyl) nitrogen terminal-protected
amino acids (AnaSpec). Couplings were conducted using HBTU (2-(1Hbenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) and
HOBT (1-hydroxybenzotriazole) in NMP (N-methylpyrrolidinone) as the
primary solvent. Simultaneous resin cleavage and side-chain deprotection
were achieved by treatment with concentrated, sequencing-grade trifluoroacetic acid with triisopropylsilane, water, and also ethanedithiol (if indicated by Cys or Met in sequence) added as ion scavengers in a ratio of
95:2:2:1 (all chemical reagents were purchased from Fisher Scientific,
Fluka, and AnaSpec). Peptides were then released in 8 M acetic acid,
filtered from resin, rotary evaporated, and redissolved in HPLC-grade water
for lyophilization. All crude lyophilized products were subsequently analyzed
by reverse-phase HPLC (Waters Chromatography) using a Merck Chromolith
Performance C18 column. Individual peptide integrity was determined and
verified by MALDI mass spectrometry using a PerkinElmer/Applied Biosystems Voyager spectrometer system. Overlapping peptides of 12- to 22-aa
length (average of 15-aa length) with 11-aa overlap were designed for the
EBNA1400 – 641 sequence of the B95-8 EBV strain using the peptide-generator
tool of the HIV sequence database at the Los Alamos National Laboratory
(PeptGen: HIV Molecular Immunology Database; http://www.hiv.
lanl.gov/content/sequence/PEPTGEN/peptgen.html (accessed October 29,
2003)). Ten peptides (11 for pool no. 5) were combined in each of five
different pools (see Table III). For CD8⫹ T cell epitopes from EBV and
HCMV, we synthesized nonamer peptides that are part of the CEF
control peptide pool of the National Institutes of Health AIDS Research
and Reference Reagent Program (24) (Table II). Where indicated, influenza HA306 –318 (PKYVKQNTLKLAT) served as a control peptide.
Intracellular cytokine staining assay
Heparinized whole blood (0.5 ml) was stimulated with peptide mixtures for
6 h in the presence of 1 ␮g/ml of costimulatory mAbs to CD28 (L293) and
CD49d (L25) (BD Biosciences) and brefeldin A at a concentration of 10
␮g/ml (Sigma-Aldrich). EBNA1 peptide pools were added at a final concentration of 3.5 ␮M per peptide per reaction. Immunodominant T cell
epitopes from EBV and HCMV proteins were added at a final concentration of 1 ␮M per peptide. Negative controls included costimulatory Abs
and brefeldin A at the same concentration without peptides. Staphylococcus enterotoxin B (SEB; 1.5 ␮g/ml) stimulation served as a positive control. After 6 h of incubation at 37°C in 5% CO2, each sample received 4 ␮l
of 0.5 M EDTA and 4.5 ml FACS lysing buffer (BD Immunocytometry
Systems) before storage at ⫺80°C overnight. After thawing, cells were
resuspended in 0.5 ml of permeabilization solution (0.25% BSA, 0.02%
The Journal of Immunology
993
Table II. HLA class I-restricted EBV- and HCMV-encoded Ags
Ag
Sequence
Restriction
Virus
EBNA3A (158 –166)
EBNA3A (325–333)
EBNA3A (379 –387)
EBNA3A (458 – 466)
EBNA3A (603– 611)
EBNA3B (416 – 424)
EBNA3C (258 –266)
EBNA3C (281–290)
BZLF1 (190 –197)
BRLF1 (28 –37)
BRLF1 (134 –143)
BRLF1 (148 –156)
BMLF1 (259 –267)
HCMV pp65 (417– 426)
HCMV pp65 (495–503)
HCMV pp65 (512–521)
QAKWRLQTL
FLRGRAYGL
RPPIFIRRL
YPLHEQHGM
RLRAEAQVK
IVTDFSVIK
RRIYDLIEL
EENLLDFVRF
RAKFKQLL
DYCNVLNKEF
ATIGTAMYK
RVRAYTYSK
GLCTLVAML
TPRVTGGGAM
NLVPMVATV
EFFWDANDIY
HLA-B8
HLA-B8
HLA-B7
HLA-B35
HLA-A3
HLA-A11
HLA-B27
HLA-B44
HLA-B8
HLA-A24
HLA-A11
HLA-A3
HLA-A2
HLA-B7
HLA-A2
HLA-B44
EBV
EBV
EBV
EBV
EBV
EBV
EBV
EBV
EBV
EBV
EBV
EBV
EBV
HCMV
HCMV
HCMV
Proliferation assay by CFSE dilution
PBMCs were isolated from blood samples via density centrifugation.
PBMCs were washed in PBS and incubated at 37°C in 1.25 ␮M CFSE
(Molecular Probes) in PBS at a concentration of 107 cells per ml for 10
min. Cells were washed in PBS and resuspended in RPMI 1640 with 5%
PHS at a concentration of 1 ⫻ 106 cells per ml. PBMCs were distributed
in 1 ml at 1 ⫻ 106 cells per well into 48-well plates. Cells were stimulated
with the respective pools of peptides with a final concentration of 3.5 ␮mol
per EBNA1 peptide and 1 ␮mol of the other EBV-encoded and HCMV
pp65-derived Ags. SEB was used as a positive control at a final concentration of 1.5 ␮g/ml. At the conclusion of a 6-day incubation at 37°C and
5% CO2, cells were harvested and washed once in PBS and stained with the
indicated combinations of directly fluorochrome-labeled Abs against CD3,
CD8, CD62L (SK11), CD27 (CLB-27/1), CD28, and CD45RO (all from
Invitrogen or BD Pharmingen) for 30 min at 4°C. The cells were washed
once with PBS and resuspended in 200 ␮l FACS buffer before FACS
analysis. The samples were measured on a BD LSR II flow cytometer.
Gating and calculations for precursor frequencies were performed with
FlowJo software. The frequencies of proliferating Ag-specific T cells were
determined by subtracting the background frequency from the frequency of
Ag-stimulated positive samples.
Luminex assay
sodium azide, 0.5% saponin in PBS) and left at room temperature for 10
min. After an additional centrifugation, the permeabilization solution was
decanted and cells were stained with directly fluorochrome-labeled Abs
against CD3 (S4.1; Invitrogen), CD4 (S3.5; Invitrogen), CD8 (RPA-T8),
IFN-␥ (25723.11), and CD45RO (UCHL-1; all from BD Pharmingen) for
15 min at room temperature. After two washes, cells were resuspended in
200 ␮l of FACS buffer solution (0.25% BSA and 0.02% sodium azide in
PBS). At least 50,000 events were collected on a BD LSR II flow cytometer (BD Biosciences) by gating on CD3⫹ lymphocytes. Frequencies of
CD3⫹-gated CD4⫹ and CD8⫹ Ag-specific IFN-␥ producing T cells were
calculated using FlowJo software (Tree Star). According to criteria previously used in ELISPOT analyses (25), a positive response required a frequency at least 2-fold above background (no Ag) and at least 10 IFN-␥⫹
events. The frequencies of Ag-specific T cells were determined by subtracting the background frequency from the frequency of Ag-stimulated
positive samples.
A
Cell supernatants from growing T cell cultures were analyzed at day 6 for
cytokines and using the Protein Multiplex Immunoassay kit (Biosource
International) as per the manufacturer’s protocol. In brief, Multiplex beads
were vortexed and sonicated for 30 s, and 25 ␮l was added to each well and
washed twice with wash buffer. The samples were diluted 1/2 with assay
diluent and loaded onto a Multiscreen BV 96-well filter plate (Millipore)
with 50 ␮l of incubation buffer already added to each well. Serial dilutions
of cytokine standards were prepared in parallel and added to the plate.
Samples were then incubated on a plate shaker at 600 rpm in the dark at
room temperature for 2 h. The plate was applied to a MultiScreen Vacuum
Manifold (Millipore) and washed twice with 200 ␮l of wash buffer. Biotinylated anti-human MultiCytokine Reporter (100 ␮l; Biosource International) was added to each well. The plate was incubated on a plate shaker
at 600 rpm in the dark at room temperature for 1 h. The plate was applied
to a MultiScreen Vacuum Manifold and washed twice with 200 ␮l of wash
buffer. Streptavidin-PE was diluted 1/10 in wash buffer, and 100 ␮l was
EBV
IgG Titer
1600
CMV
40000
1200
30000
800
20000
400
10000
0
0
HD
B
HD
RA
RA
EBNA1
VCAp18
VCAp23
EAp138
EAp54
RA, seropositive (%)
84
100
100
24
32
HD, seropositive (%)
82
100
100
0
0
p-value
n.s.
n.s.
n.s.
0.03
0.006
FIGURE 1. Increased EBV-specific IgG titer and frequent IgG recognition of EBV-encoded early Ags in RA. A, RA patients show increased IgG
responses to EBV- (Mann-Whitney U test: p ⫽ 0.03) but not to HCMV-encoded Ags. Displayed are titers of Abs specific for EBV-encoded EA, VCA,
and EBNA1 and specific for HCMV-infected cell lysates. B, EBV IgG-containing sera were subsequently tested for their Ag specificity by immunoblotting.
While VCA- and EBNA1-specific Abs were present in the majority of both RA patients and healthy virus carriers, EBV-EA targeting IgG were exclusively
detectable in RA sera (Fisher’s exact test: p ⫽ 0.03 for EAp138; p ⫽ 0.006 for EAp54).
994
added directly to each well. The plate was incubated on a plate shaker at
600 rpm in the dark at room temperature for 30 min. The plate was then
applied to the vacuum manifold, washed twice, and each well was resuspended in 100 ␮l wash buffer and shaken for 1 min. The assay plate was
then transferred to the Bio-Plex Luminex 100 XYP instrument (Millipore)
for analysis. Cytokine concentrations were calculated using Bio-Plex Manager 3.0 software with a five-parameter curve-fitting algorithm applied for
standard curve calculations.
EBV IN RA
Table III. EBNA1 peptides
Peptide
Pool No.
1
Viral loads
EBV DNA was quantified from PBMCs by quantitative real-time PCR
using a TaqMan PCR kit and an Applied Biosystems model 7500 sequence
detector (26). DNA was extracted from PBMCs using the QIAamp DNA
blood mini kit (Qiagen) following the manufacturer’s protocol. A region
from the BamHI W fragment of EBV was amplified using primers 5⬘GGACCACTGCCCCTGGTAAA-3⬘ and 5⬘-TTTGTGTGGACTCCTG
GGG-3⬘ and detected with fluorogenic probe 5⬘-FAM-TCCTGCAGCTA
TTTCTGGTCGCATCA-TAMRA-3⬘. The human bcl-2 gene was
amplified using primers 5⬘-CCTGCCCTCCTTCCGC-3⬘ and 5⬘-TGCAT
TTCAGGAAGACCCTGA-3⬘ and detected with fluorogenic probe 5⬘-FAMCTTTCTCATGGCTGTCC-TAMRA-3⬘. The EBV BamHI W fragment
copy number per cell was calculated using the formula n ⫽ 2 ⫻ W/B,
where n is the EBV BamHI W copy number/cell, W is the EBV BamHI W
copy number, and B is the bcl-2 copy number. All samples were tested in
at least duplicate, and the mean results were determined.
2
3
Statistical analysis
Statistics were performed using commercial software (Prism 4, GraphPad
Software). Comparisons between RA patients and healthy donors were
based on the nonparametric Mann-Whitney U test. Categorical differences
between the two cohorts were analyzed by Fisher’s exact test. For correlation analyses between clinical disease parameters and T cell responses,
we used the nonparametric Spearman correlation.
Results
4
Increased EBV-specific IgG titer and frequent IgG recognition
of EBV-encoded early Ags in RA
First, we evaluated IgG Ab responses to EBV- and HCMV-encoded Ags in 25 patients with RA compared with 20 demographically matched healthy donors (HD). The ELISA plate for detection of EBV-specific IgG was coated with a mixture of epitopes
derived from EA, VCA, and EBNA1. The ELISA plates for the
detection of HCMV-specific IgG were coated with lysates from
HCMV-infected cells and noninfected control lysates. Differences
in Ab reactivities against infected compared with noninfected cell
lysates are reported. As shown in Fig. 1, both patients with RA and
HD were universally infected with EBV. HCMV responses could
be detected in 73% of HD and 69% of RA patients, respectively.
EBV-specific, but not HCMV-specific, IgG titers were significantly higher in the RA cohort ( p ⫽ 0.03) (Fig. 1A). Next, we
tested the Ag specificity of EBV targeting IgG by immunoblotting.
Healthy donors as well as RA patients were both universally seropositive for EBNA1 and viral capsid-specific IgG. In contrast,
IgG responses to EBV-EA were only observed in RA patients but
not in healthy donors (Fig. 1B). These data show that patients with
RA exhibit elevated IgG responses toward EBV and a broader
recognition of EBV-encoded Ags compared with healthy EBV carriers, whereas immune responses against HCMV were similar in
both groups.
Expansion of EBV-specific CD8⫹ immediate effector T cells in
RA
Employing an ex vivo flow cytometry-based intracellular IFN-␥
staining assay, we determined EBV- and HCMV-specific CD4⫹
and CD8⫹ T cell responses in 25 patients and 20 healthy virus
carriers. For restimulation of PBMCs, either a mixture of MHC
class I-restricted T cell epitopes derived from three latent
(EBNA3A, EBNA3B, EBNA3C) and three lytic (BZLF1, BRLF1,
and BMLF1) Ags (Table II) or a peptide library of the C-terminal
5
Description
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
EBNA1
(400 – 414)
(405– 418)
(409 – 422)
(413– 429)
(420 – 434)
(425– 439)
(430 – 451)
(435– 451)
(442– 458)
(449 – 461)
(452– 465)
(456 – 469)
(460 – 474)
(465– 478)
(469 – 482)
(473– 487)
(478 – 491)
(482– 496)
(487–503)
(494 –508)
(499 –510)
(501–514)
(505–518)
(509 –522)
(513–527)
(519 –532)
(523–536)
(527–541)
(532–544)
(535–548)
(539 –554)
(545–559)
(549 –563)
(554 –566)
(557–571)
(562–576)
(566 –580)
(571–584)
(575–588)
(579 –593)
(584 –597)
(588 – 600)
(591– 604)
(595– 609)
(600 – 614)
(605– 619)
(610 – 625)
(616 – 629)
(620 – 634)
(625– 639)
(630 – 641)
Sequence
PGRRPFFHPVGEADY
FFHPVGEADYFEYH
VGEADYFEYHQEGG
DYFEYHQEGGPDGEPDV
EGGPDGEPDVPPGAI
GEPDVPPGAIEQGPA
PPGAIEQGPADDPGEGPSTGPR
EQGPADDPGEGPSTGPR
PGEGPSTGPRGQGDGGR
GPRGQGDGGRRKK
GQGDGGRRKKGGWF
GGRRKKGGWFGKHR
KKGGWFGKHRGQGGS
FGKHRGQGGSNPKF
RGQGGSNPKFENIA
GSNPKFENIAEGLRA
FENIAEGLRALLAR
AEGLRALLARSHVER
ALLARSHVERTTDEGTW
VERTTDEGTWVAGVF
DEGTWVAGVFVY
GTWVAGVFVYGGSK
AGVFVYGGSKTSLY
VYGGSKTSLYNLRR
SKTSLYNLRRGTALA
NLRRGTALAIPQCR
GTALAIPQCRLTPL
AIPQCRLTPLSRLPF
RLTPLSRLPFGMA
PLSRLPFGMAPGPG
LPFGMAPGPGPQPGPL
PGPGPQPGPLRESIV
PQPGPLRESIVCYFM
LRESIVCYFMVFL
SIVCYFMVFLQTHIF
FMVFLQTHIFAEVLK
LQTHIFAEVLKDAIK
FAEVLKDAIKDLVM
LKDAIKDLVMTKPA
IKDLVMTKPAPTCNI
MTKPAPTCNIRVTV
APTCNIRVTVCSF
CNIRVTVCSFDDGV
VTVCSFDDGVDLPPW
FDDGVDLPPWFPPMV
DLPPWFPPMVEGAAA
FPPMVVEGAAAEGDDG
EGAAAEGDDGDDGDE
EGDDGDDGDEGGDGD
DDGDEGGDGDEGEEG
GGDGDEGEEGQE
domain of EBNA1 (aa 400 – 641) was chosen. The MHC class
I-restricted T cell epitopes were selected based on their previous
identification as immunodominant CD8⫹ T cell epitopes (27),
whereas EBNA1 was chosen as a dominant EBV-encoded CD4⫹
T cell Ag in healthy virus carriers (Table III) (27–29). MHC class
I-restricted HCMV pp65-derived peptides were used as control
Ags (Table II) (24, 26, 30). The mixture of MHC class I-restricted
T cell epitopes constitutes the EBV- and HCMV-derived portion
of the control peptide pool, which is used as positive controls in
immunomonitoring studies of patients infected with HIV and contains CD8⫹ T cell epitopes recognized by most individuals (24).
PMBCs were analyzed for intracellular cytokine production after
6 h of stimulation with peptide Ags or SEB as positive control. As
shown in Fig. 2, RA patients demonstrated substantial expansions
of CD8⫹ T cells specific for both EBV-encoded immunodominant
The Journal of Immunology
995
A
w/o
105
0.19
SEB
105
0.088
HCMVpp65
105
1.34
0.23
EBV
105
0.2
EBNA1pool1-5
0.091
105
0.23
104
104
104
104
104
103
103
103
103
103
102
102
102
102
0
0
0 102
103
104
105
105
0.18
0
0 102
103
104
105
105
0.36
0.21
104
105
0.18
105
0.3
103
104
105
0 102
0.19
105
0.32
104
104
104
104
103
103
103
103
103
102
102
102
102
0
0 102
103
104
105
0
0 102
103
104
105
103
104
105
104
105
0.23
0.25
102
0
0
0 102
103
EBNA1pool5
104
0
0.27
0
0 102
EBNA1pool4
EBNA1pool3
105
0.21
103
0.15
102
0
0 102
EBNA1pool2
EBNA1pool1
IFN-γ
1.3
0 102
103
104
105
0 102
103
104
105
CD8
B
EBNA1
EBV
CD4+ T cells
IFN-γ producing T cells
CD8+ T cells
*
0.6
0.2
FLU-HA
HCMVpp65
CD8+ T cells
*
0.8
CD8+ T cells
3
SEB
0.2
CD4+ T cells
CD8+ T cells
CD4+ T cells
8
25
0.5
20
0.6
0.4
0.1
0.3
6
2
0.1
15
0.4
4
1
10
0.2
0.2
0.0
0.0
0.1
2
5
0
0.0
0.0
HD
RA
HD
RA
0
0
HD
RA
HD
RA
HD
RA
HD
RA
HD
RA
FIGURE 2. Increased Frequency of EBV-specific CD8⫹ immediate effector T cells in RA. A, Ex vivo flow cytometry-based intracellular IFN-␥ staining
assay to determine the frequency of EBV- and HCMV-specific T cells in RA and HD. Depicted is the analysis of CD3⫹ T cells from a representative RA
patient with a strong CD8⫹ T cell response to EBV-encoded Ags. MHC class I-restricted HCMV pp65-derived peptides were used as control Ags (Table
II). MHC class I-restricted T cell epitopes derived from three latent (EBNA3A, EBNA3B, EBNA3C) and three lytic (BZLF1, BRLF1, BMLF1) Ags
(designated as EBV) were selected based on their previous identification as immunodominant CD8⫹ T cell epitopes (Table II). The C-terminal domain of
EBNA1 (aa 400 – 641), toward which most healthy virus carriers develop a CD4⫹ T cell response, was chosen as a dominant EBV-encoded CD4⫹ T cell
Ag, and a total of 51 overlapping peptides were distributed into five different pools (Table III). PMBCs were analyzed for intracellular cytokine production
after 6 h of stimulation with peptide Ags or SEB as positive control. B, Patients with RA showed substantial expansions of CD8⫹ T cells specific for both
EBV-encoded immunodominant MHC class I-restricted Ags (mean frequency in HD vs RA: 0.11% vs 0.28% of all circulating CD8⫹ T cells; p ⫽ 0.0029)
and EBNA1 (mean frequency in HD vs RA: 0.03% vs 0.23% of all circulating CD8⫹ T cells; p ⫽ 0.03), indicating that IFN-␥-producing, EBV Ag-specific,
effector CD8⫹ T cells are expanded in RA. The frequency of EBNA1-specific CD4⫹ T cells among all CD4⫹ lymphocytes (mean frequency in HD vs RA:
0.03% vs 0.05%) and HCMV pp65-specific CD8⫹ T cells among all CD8⫹ lymphocytes (mean frequency in HD vs RA: 0.72% vs 0.54%) did not
significantly differ between patients and controls. Additionally, frequencies of CD4⫹ T cells specific for Flu-HA, chosen for its reported promiscuous MHC
class II binding and its immunodominance in humans, did not differ between 14 healthy donors and 15 RA patients who were tested for Flu-HA recognition
(mean frequency in HD vs RA: 0.04% vs 0.06%). Following SEB stimulation, patients and controls showed similar frequencies of IFN-␥-producing CD8⫹
T cells (mean frequency in HD vs RA: 7.1% vs 5.2%) and CD4⫹ T cells (mean frequency in HD vs RA: 1.9% vs 2.7%).
MHC class I-restricted Ags (mean frequency in HD vs RA: 0.11%
vs 0.28% of all circulating CD8⫹ T cells; p ⫽ 0.0029) and EBNA1
(mean frequency in HD vs RA: 0.03% vs 0.23% of all circulating
CD8⫹ T cells; p ⫽ 0.03), indicating that IFN-␥-producing, EBV
Ag-specific, effector CD8⫹ T cells are expanded in RA. Notably,
the frequency of EBNA1-specific CD8⫹ T cells was at the detection limit of the ex vivo cytokine staining assay in healthy virus
carriers (26, 30 –33), whereas these cells were clearly detectable in
patients with RA. The frequency of EBNA1-specific CD4⫹ T cells
among all CD4⫹ lymphocytes (mean frequency in HD vs RA:
0.03% vs 0.05%) and HCMV pp65-specific CD8⫹ T cells among
all CD8⫹ lymphocytes (mean frequency in HD vs RA: 0.72% vs
0.54%) did not significantly differ between patients and controls.
Additionally, frequencies of CD4⫹ T cells specific for an influenza
hemagglutinin peptide (Flu-HA, aa 306 –318), chosen for its reported promiscuous MHC class II binding and its immunodominance in humans (34), did not differ between 14 healthy donors
and 15 RA patients, who were tested for Flu-HA recognition
(mean frequency in HD vs RA: 0.04% vs 0.06%). Following SEB
stimulation, patients and controls showed similar frequencies of
IFN-␥-producing CD8⫹ T cells (mean frequency in HD vs RA:
7.1% vs 5.2%) and CD4⫹ T cells (mean frequency in HD vs RA:
1.9% vs 2.7%).
These findings appeared to be independent of the patients’ treatment status, since both untreated (n ⫽ 11/25) and treated (n ⫽ 14/25)
patients as well as patients receiving anti-TNF therapy (n ⫽ 6)
showed significantly higher frequencies of EBV Ag-specific CD8⫹ T
cells compared with healthy controls. In contrast, frequencies of
CD4⫹ T cells specific for EBNA1, CD8⫹ T cell responses to HCMV
pp65, and CD4⫹ as well as CD8⫹ T cell responses to SEB were
similar between untreated and treated patients vs controls (data not
shown). Collectively, these data indicate that the frequency of circulating CD8⫹ T cells, targeting both lytic and latent EBV-encoded
Ags, is specifically increased in patients with RA.
Effector and central memory compartmentalization of
EBV-specific CD8⫹ T cells in RA
To identify which CD8⫹ T cell subsets contribute to the increased
response in RA, we determined proliferative responses to the respective viral Ags in 25 RA patients and 20 healthy controls using a
996
EBV IN RA
FIGURE 3. Increased proliferative capacity of
lytic and latent EBV Ag-specific CD8⫹ T cells in
RA. Employing a flow cytometry-based CFSE dilution assay, we detected that patients with RA showed
substantially higher proliferative responses to EBVencoded immunodominant CD8⫹ T cell epitopes, but
not to HCMV pp65 (p ⫽ 0.004). EBNA1-specifc
CD8⫹ T cell proliferation tended also to be higher in
RA, but it was only detectable in six patients and
three healthy virus carriers and did not reach statistical significance. EBNA1-specific CD4⫹ T cell proliferation was similar in RA patients and in healthy
virus carriers.
multiparameter flow cytometry-based CFSE dilution assay. In line
with our finding on ex vivo IFN-␥ production, patients with RA
showed substantially higher proliferative responses to EBV-encoded
immunodominant CD8⫹ T cell epitopes ( p ⫽ 0.004), but not to
HCMV pp65 (Fig. 3). EBNA1-specific CD8⫹ T cell proliferation
tended also to be higher in RA, but it was only detectable in six
A
SEB
CD8
No Antigen
patients and three healthy virus carriers, thus not reaching statistical
significance.
We further characterized the phenotype of CFSE-diluted EBVspecific CD8⫹ T cells using markers indicative for central and
effector memory compartments (CD45RO, CD62L) as well as for
the costimulatory molecules CD28 and CD27 (30). As expected,
HCMVpp65
EBV
EBNA1
0.91
17.7
0.71
2.49
0.65
0.55
21.6
0.36
1.58
0.74
CFSE
11.1
8.05
2.4
CD28
0.086
CD27
11.3
CD62L
0.84
CD45RO
CD8+ T cells:
6.94
3.94
CFSE
B
SEB
CD8
No Antigen
HCMVpp65
EBV
EBNA1
0.75
32.6
1.25
0.87
1.67
0.6
14.2
0.61
0.33
0.7
CFSE
3.66
3.48
0.95
3.98
CD28
1.85
CD27
1
2.89
CD62L
CD45RO
CD8+ T cells:
0.41
CFSE
⫹
FIGURE 4. Expanded EBV-specific CD8 T cells in RA predominantly originate from the CD45RO⫹CD62L⫺ effector memory T cell pool. The
phenotype of CFSElow EBV-specific CD8⫹ T cells was determined using markers indicative for central and effector memory compartments (CD45RO,
CD62L) as well as for differentiation and costimulation (CD27, CD28). A, Nearly all EBV-specific T cells originated from the memory pool (CD45RO⫹).
Most expanded CD8⫹ T cells specific for immunodominant MHC class I-restricted epitopes lacked CD62L expression identifying them as effector memory
T cells. B, Proliferating CD8⫹ T cells specific for EBNA1 consisted of both CD62L⫹ and CD62L⫺ memory T cells. As expected, most CD8⫹ T cells
specific for EBV Ags were CD27⫹CD28⫹, and we did not detect any statistically significant difference in the frequency of CD27 or CD28 expression by
EBV-specific CD4⫹ and CD8⫹ memory T cells between patients and healthy volunteers.
The Journal of Immunology
aa
1
61
121
181
241
301
361
421
481
541
601
B
3.0
Intensity of spots (AU)
aa
1
61
121
181
241
301
361
421
481
541
601
RA
2.5
2.0
1.5
1.0
0.5
0.0
0
0.0
HD
Intensity of spots (AU)
A
997
50
100
150
200
0.5
1.0
1.5
2.0
2.5
3.0
C
msdegpgtgpgnglgekgdtsgpegsggsgpqrrggdnhgrgrgrgrgrgggrpgapggs
gsgprhrdgvrrpqkrpscigckgthggtgagagaggagaggagagggagagggaggagg
aggagagggagagggaggaggagagggagagggaggagagggaggaggagagggagaggg
aggagagggaggaggagagggagaggaggaggagaggagagggaggaggagaggagagga
gaggagaggaggagaggaggagaggaggagagggaggagaggaggagaggaggagagga
ggagaggaggagagggagaggagaggggrgrggsggrgrggsggrgrggsggrrgrgrer
arggsrerargrgrgrgekrprspssqssssgspprrpppgrrpffhpvgeadyfeyhqe
ggpdgepdvppgaieqgpaddpgegpstgprgqgdggrrkkggwfgkhrgqggsnpkfen
iaeglrallarshverttdegtwvagvfvyggsktslynlrrgtalaipqcrltplsrlp
fgmapgpgpqpgplresivcyfmvflqthifaevlkdaikdlvmtkpaptcnirvtvcsf
ddgvdlppwfppmvegaaaegddgddgdeggdgdegeegqe
N-terminal
GA-repeat
C-terminal
D
HD
RA
EBNA1-IgG1
1500
*
1000
500
0
HD
RA
EBNA1-IgG2
EBNA1-IgG4
1500
1500
1000
1000
500
500
0
HD
RA
0
HD
RA
FIGURE 5. Broadened specificity and IgG1 polarization of EBNA1-specific IgG responses in RA. A, Epitopes recognized by EBNA1-specific IgG were
identified using a membrane carrying spots of 211 covalently linked overlapping dodecamer peptides that cover the entire sequence of EBNA1 (aa 1– 641).
Each spot contains a linear synthetic peptide of 12-aa length bound to a continuous cellulose membrane. Representative examples from one RA patient and
one healthy donor (HD) are shown in A. The relative spot intensities correlate with the amount of bound serum Abs. B, Mean relative spot intensities for
12 HD (lower panel) and 12 RA (upper panel) patients. IgG Abs from healthy donors recognized predominantely the glycine/alanine-rich repeat (GA)
domain of EBNA1 (aa 88 –323). In contrast, IgG Abs form RA patients recognized a much broader array of epitopes, including epitopes in the arginineenriched flanking regions of the GA-domain and in the C-terminal domain of EBNA1. The differentially recognized spots are outlined in black in B, and
the corresponding primary protein sequence of EBNA1 is underlined in C. D, RA patients showed selectively increased titers of EBNA1 (aa 458 – 641)specific IgG1 (p ⫽ 0.02) but not IgG2 or IgG4. EBNA1-specific IgG3 Abs could not be detected in HD or RA patients (data not shown).
nearly all EBV-specific CD8⫹ T cells originated from the
CD45RO⫹ memory compartment.
The CD8⫹ T cells specific for EBNA1 consisted of CD62L⫹
and CD62L⫺ memory T cells (Fig. 4B) and thus did not differ in
this respect from EBNA1-specific CD4⫹ T cells, in which both
CD62L⫹ central memory and CD62L⫺ effector memory T cell
subsets contributed approximately equally to EBNA1-specific
CD4⫹ T cell proliferation in patients (data not shown) and healthy
controls (30). The phenotypes described above were consistently
found in all donors showing CD4⫹ and/or CD8⫹ T cell proliferative
responses to EBV-encoded Ags, with no statistically significant difference found between RA patients and healthy virus carriers.
Latent EBV Ag-specific CD4⫹ and CD8⫹ memory T cells accumulate within a CD27⫹CD28⫹ differentiation compartment during primary infection and remain enriched within this compartment throughout the persistent phase of infection (35, 36). CD8⫹
T cells specific for lytic cycle Ags accumulate within both
CD27⫹CD28⫹ and CD27⫹CD28⫺ compartments, indicating a
difference in differentiation states and/or costimulatory require-
ments (37). Stimulation with immunodominant CD8⫹ T cell
epitopes resulted in expansion of a minor CD27⫹CD28⫺ population, which also lacked CD62L expression, indicating further differentiated effector memory T cells (38) (Fig. 4A). Most proliferating EBNA1-specific CD8⫹ T cells in our cohort of RA patients
and healthy donors originated from the CD27⫹CD28⫹ compartment (Fig. 4B). We did not detect any statistically significant difference in the frequency of CD27 or CD28 expression by EBVspecific CD4⫹ and CD8⫹ T cells between patients and healthy
volunteers (data not shown). These data do not support the concept
of insufficient T cell responses to EBV in RA. The preferential
expansion and increased frequency of effector memory and IFN-␥
producing CD8⫹ T cells specific for EBV proteins expressed during viral replication, however, suggests an increased availability of
these Ags due to higher viral replication in patients with RA.
Cytokine profiling of EBV-specific T cell immunity in RA
To determine whether patients with RA show qualitatively altered
EBV-specific T cell cytokine profiles compared with healthy virus
998
1.0
Frequency of CD8+ T cells specific for
pooled viral antigens
Viral load
15000
(EBV copy number per 106 PBMC)
FIGURE 6. Increased EBV loads
correlate with higher frequencies of
EBV-specific CD8⫹ T cells in RA. A,
EBV viral loads are 7.3-fold higher in
RA patients compared with healthy virus carriers (p ⫽ 0.02; unpaired t test
with Welch’s correction). EBV DNA
was quantified from PBMC by quantitative real-time PCR in relation to
bcl-2 expression. All samples were
tested at least in duplicate, and the
mean values are shown. Bars and error
bars represent means and SEM, respectively. B, RA patients with high EBV
copy numbers show higher frequencies
of IFN-␥-producing EBV-specific
CD8⫹ T cells specific for MHC class
I-restricted, pooled lytic, and latent
EBV Ags (Spearman r ⫽ 0.53; p ⫽
0.02).
EBV IN RA
10000
5000
0
HD
carriers, we analyzed supernatants of EBV-specific T cell cultures
obtained at day 6 after primary proliferation for the composition of
cytokines indicative of Th1 (IFN-␥), Th2 (IL-13), and Th17 (IL17) polarization as well as for IL-2 and IL-10 production. Of these,
only IFN-␥ and IL-2 were detectable at low levels in both RA- and
HD-derived cell cultures stimulated with MHC class I-restricted
EBV Ags (mean concentrations for IFN-␥: 41 pg/ml (HD) vs 323
pg/ml (RA); mean concentrations for IL-2: 58 pg/ml (HD) vs 86
pg/ml (RA) as measured in 14 representative positive T cell cultures), and production of IFN-␥ was moderately increased in RAderived positive cultures stimulated with immunodominant CD8⫹
T cell epitopes ( p ⫽ 0.03). These data are consistent with the
observation that Th1 polarization and IFN-␥ production predominate in EBV-specific T cell immunity and do not argue for a
qualitatively altered T cell response to EBV in patients with RA.
Broadened specificity and IgG1-polarization of EBNA1-specific
IgG responses in RA
To further assess the relative activity of EBV-specifc Th1 cells in
vivo, we recombinantly expressed the immunogenic C terminus of
EBNA1 (aa 458 – 641) in an eukaryotic expression system and
analyzed EBNA1-targeting IgG isotype-specific responses in 25
patients and 20 healthy EBV carriers (21). IFN-␥ can skew human
Ab responses toward the IgG1 opsonizing and complement-fixing
Ig subclass and IL-4 toward the allergy-related IgG4 and IgE subclasses (21). As depicted in Fig. 5D, patients with RA showed
significantly increased titers of EBNA1-specific IgG1 ( p ⫽ 0.02),
but not IgG2 or IgG4. EBNA1-specific IgG3 Abs could not be
detected in HD or RA patients (data not shown). IgG1 was the
most frequently detected isotype in all individuals tested, possibly
reflecting the Th1 polarization of EBNA1-specific T cell immunity. IgG4 responses to EBNA1 were detected in a minor subgroup
of patients and controls, with no statistically significant differences
found between both cohorts. We next determined target epitopes of
EBNA1-specific IgG in 12 patients and 12 controls by using membranes, on which 211 covalently linked and overlapping dodecamer peptides, covering the entire sequence of EBNA1 (aa 1– 641),
had been spotted (22, 23). As shown in Fig. 5, the glycine/alaninerich repeat (GA) domain of EBNA1 (aa 88 –323) was predominantly recognized by IgG Abs from healthy virus carriers. In contrast, IgG Abs from RA patients recognized a much broader array
of epitopes. Differentially recognized epitopes were primarily lo-
RA
Spearmen r = 0.53
0.8
p = 0.02
0.6
0.4
0.2
0.0
101
102
103
104
105
Viral Load
cated in the arginine-enriched flanking regions of the GA domain
(EBNA1 aa 33– 89 and EBNA1 aa 324 – 402) and in the C-terminal
domain of EBNA1 (EBNA1 aa 421–527) (Fig. 5B). None of these
epitopes was exclusively recognized by RA patients or by healthy
virus carriers. Membranes incubated with control sera from EBVseronegative donors did not show any positive spots (data not
shown). These data support the concept that patients with RA show
increased as well as broadened EBV-specific immune responses.
EBV copy numbers in circulating blood cells are 7-fold
increased in RA
We next quantified levels of cell-bound viral genomes in circulating blood cells in patients and controls and compared frequencies
of EBV-specific T cells with viral loads. To this end, we determined EBV DNA copy numbers in PBMCs by real-time PCR
using bcl-2 DNA controls to determine copy numbers per genome
in 19 patients and 13 healthy EBV carriers (26). EBV DNA was
detectable in 13 of 19 (68%) RA patients and in 6 of 13 (46%)
healthy donors from whom PBMCs were accessible for viral load
quantification. RA patients showed 7.3-fold increased levels of
cell-associated EBV DNA copies (mean 1,397 for HD vs 10,146
for RA per 106 PBMC, respectively) (Fig. 6). We found that RA
patients with high EBV copy numbers showed higher frequencies
of IFN-␥-producing EBV-specific CD8⫹ T cells specific for MHC
class I-restricted, pooled lytic, and latent EBV Ags (Spearman r ⫽
0.53; p ⫽ 0.02) (Fig. 6, right panel). Comparisons of viral loads
and CD4⫹ T cell frequencies did not show any statistically significant correlations. There was no significant correlation between
clinical disease activity as assessed by the disease activity score
using 28 joint counts (DAS28) and EBV copy numbers. Furthermore, proliferating T cell frequencies to EBV-encoded Ags did not
correlate with EBV copy numbers in RA patients and healthy virus
carriers.
Discussion
Our study demonstrates that RA patients have elevated CD8⫹ T
cell and B cell responses to pooled lytic and latent EBV Ags that
are involved in both B cell transformation and productive viral
replication. We did not find evidence for a defective T cell control
of EBV infection in RA. In contrast, higher levels of cell-associated viral genomes in circulating blood cells correlated with increased frequencies of EBV-specific and IFN-␥-producing CD8⫹
The Journal of Immunology
T cells, suggesting that increased viral replication drives enhanced
EBV-specific immune responses in RA.
EBV manipulates the human B cell compartment to achieve
persistence in memory B cells and is strongly regulated by and
responsive to the biology of its main host cell. It is thought that
EBV initially infects extrafollicular naive B cells in tonsils after
transmission by saliva exchange (39). By driving B cells into
activation and proliferation, upon which they home to germinal
centers and then differentiate with the help of EBV Ags into memory B cells, the virus gains access to a long-lived host cell reservoir
and persists in the absence of EBV protein expression with the
exception of EBNA1 during homeostatic B cell division (40). Ag
stimulation and/or receipt of a plasma cell differentiation signal
drive occasional reactivations into the viral lytic cycle (41). It has
previously been proposed that B cell dysfunction in autoimmune
diseases associated with prominent autoantibody production alters
regulatory mechanisms of EBV persistence, since patients with
systemic lupus erythematosus show aberrant expression of viral
latent and lytic genes in the blood, as well as abnormally high
frequencies of circulating EBV-infected cells associated with disease flares (42). Autoantigen-specific B cells are normally neutralized or controlled by several tolerance checkpoints during B cell
development (43– 46). Patients with RA exhibit defects in central
B cell tolerance mechanisms, resulting in higher frequencies of
circulating autoreactive B cells (47) and rheumatoid factor, which
consists of autoantibodies against the constant region of IgG Abs
and can be detected in ⬃80% of RA patients (48). Rheumatoid
factor has shown to induce EBV replication in B cells via BCR
stimulation in vitro (49), and it is therefore conceivable that higher
viral loads in blood cells result from frequent B cell autoantigen
recognition in RA instead of being a consequence of impaired T
cell-mediated immune responses to EBV.
The hypothesis that autoreactivity partly contributes to increased levels of EBV replication by triggering EBV release from
autoreactive EBV-infected B cells fits well with the finding that
lytic EBV Ag-specific T cell responses are increased in RA. CD4⫹
and CD8⫹ T cells against lytic EBV Ags have previously been
isolated from inflamed joints of RA patients (50, 51), indicating
that EBV-specific T cells are not only elevated in peripheral blood,
but that they also infiltrate inflamed joints in RA. In agreement
with these previous studies, we demonstrate that most EBV-specific CD8⫹ T cells belong to the effector memory compartment
and lack homing markers to secondary lymphoid tissues like
CD62L (38), and they therefore might preferentially home to sites
of autoimmune inflammation.
Organized lymphoid structures that resemble secondary lymphoid organs are frequently found in synovial tissues from RA
patients (52). Analyses of the rearranged Ig V genes of B cells that
were isolated from biopsies of these tertiary lymphoid structures in
patients with RA have shown that B cells undergo Ag-driven
clonal expansion and somatic hypermutation at these ectopic follicles (53, 54). Similar structures are found in the CNS of patients
with multiple sclerosis (55), another autoimmune disease associated with altered immune responses to EBV (26), and it has recently been reported that ⬎40% of brain-infiltrating B cells and
plasma cells are infected with EBV and show expression of primarily latent viral Ags spatially associated with expansions of infiltrating CD8⫹ T cells (56). Instead of latent Ags, expression of
lytic EBV Ags has been described in the synovial tissue of RA
patients (57) and could enhance inflammation via local restimulation of T cells directed against these Ags. Therefore, even though
elevated EBV-specific immune cell responses might represent an
epiphenomenon in response to increased Ag load in circulating
blood cells in RA, they could nevertheless augment T cell-medi-
999
ated tissue damage and contribute to a local proinflammatory environment by recognizing EBV gene products in inflamed joints.
An implication of this line of thought is that deregulated EBVspecific immune responses and increased viral titers in RA can be
normalized by clinically effective therapeutic B cell depletion (58).
It will therefore be of interest to longitudinally study EBV-specific
T and B cell responses in RA patients during B cell-depleting
rituximab treatment and to evaluate the validity of EBV-associated
immune parameters as potential biomarkers for treatment responsiveness in RA (59).
In conclusion, we have demonstrated perturbations of EBV infection in patients with RA. The altered immune recognition of
EBV in RA is not restricted to one specific Ag but involves a
mixture of both lytic and latent viral gene products expressed during viral replication. Expanded CD8⫹ T cell responses to these
Ags positively correlate with increased viral loads in circulating
blood cells. This suggests that RA-associated immune dysfunctions drive enhanced EBV replication in B cells and thereby stimulate EBV-specific T cell responses. Future studies will need to
address whether specific B cell abnormalities in RA correlate with
changes in the regulation of EBV infection observed in these patients. Our data indicate that investigating the biology of EBV
infection in the context of autoimmunity has the potential to provide new insights into mechanisms of EBV regulation and the
pathogenesis of autoimmune diseases.
Acknowledgments
We gratefully acknowledge Drs. J. Frey and H. Jödicke for their help with
the recruitment of the healthy donors. We thank all of our patients for their
continual cooperation.
Disclosures
The authors have no financial conflicts of interest.
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