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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Immunol. Author manuscript; available in PMC Jul 15, 2009.
Published in final edited form as:
PMCID: PMC2570434
NIHMSID: NIHMS68638

Increased Frequency of EBV-Specific Effector Memory CD8+ T Cells Correlates with Higher Viral Load in Rheumatoid Arthritis1

Abstract

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 CD8+ T cells specific for pooled EBV Ags expressed during both B cell transformation and productive viral replication and the frequency of CD8+ T cells specific for these Ags correlated with cellular EBV copy numbers. In contrast, CD4+ 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.

Rheumatoid 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-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 (59). 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-γ, 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 EBV-encoded Ags (1113). Several groups reported that T cell responses to selected EBV Ags are altered in frequency (1416) and are functionally impaired in RA patients (5, 6, 14). Higher frequencies of EBV-infected B cells (17) and up to 10-fold increased viral copy number in circulating mononuclear cells (18) have also been detected in RA patients. Herein, we investigated both EBV-specific 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-α-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.

Table I
Patients and healthy blood donors

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 EBV-encoded 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 β-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 μg/well of rEBNA1458–641 protein in PBS or PBS alone overnight at 4°C. Plates were blocked with 200 μl/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 β-alanine-modified cellulose membrane (23). Altogether, 211 12-mer peptides, overlapping by 9 aa, were covalently linked to the membrane via two β-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 IgG-alkaline 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-(1H-benzotriazole-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.

Table II
HLA class I-restricted EBV- and HCMV-encoded Ags
Table III
EBNA1 peptides

Intracellular cytokine staining assay

Heparinized whole blood (0.5 ml) was stimulated with peptide mixtures for 6 h in the presence of 1 μg/ml of costimulatory mAbs to CD28 (L293) and CD49d (L25) (BD Biosciences) and brefeldin A at a concentration of 10 μg/ml (Sigma-Aldrich). EBNA1 peptide pools were added at a final concentration of 3.5 μM per peptide per reaction. Immunodominant T cell epitopes from EBV and HCMV proteins were added at a final concentration of 1 μM per peptide. Negative controls included costimulatory Abs and brefeldin A at the same concentration without peptides. Staphylococcus enterotoxin B (SEB; 1.5 μg/ml) stimulation served as a positive control. After 6 h of incubation at 37°C in 5% CO2, each sample received 4 μl 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% 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-γ (25723.11), and CD45RO (UCHL-1; all from BD Pharmingen) for 15 min at room temperature. After two washes, cells were resuspended in 200 μl 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-γ 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-γ+ events. The frequencies of Ag-specific T cells were determined by subtracting the background frequency from the frequency of Ag-stimulated positive samples.

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 μM 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 μmol per EBNA1 peptide and 1 μmol of the other EBV-encoded and HCMV pp65-derived Ags. SEB was used as a positive control at a final concentration of 1.5 μg/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 μl 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

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 μl 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 μl 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 μl of wash buffer. Biotinylated anti-human MultiCytokine Reporter (100 μl; 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 μl of wash buffer. Streptavidin-PE was diluted 1/10 in wash buffer, and 100 μl was 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 μl 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.

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′-TTTGTGTGGACTCCTGGGG-3′ and detected with fluorogenic probe 5′-FAM-TCCTGCAGCTATTTCTGGTCGCATCA-TAMRA-3′. The human bcl-2 gene was amplified using primers 5′-CCTGCCCTCCTTCCGC-3′ and 5′-TGCATTTCAGGAAGACCCTGA-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.

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

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.

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 ...

Expansion of EBV-specific CD8+ immediate effector T cells in RA

Employing an ex vivo flow cytometry-based intracellular IFN-γ 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 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) (2729). 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 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-γ-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, 3033), 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-γ-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%).

FIGURE 2
Increased Frequency of EBV-specific CD8+ immediate effector T cells in RA. A, Ex vivo flow cytometry-based intracellular IFN-γ staining assay to determine the frequency of EBV- and HCMV-specific T cells in RA and HD. Depicted is the analysis of ...

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 multiparameter flow cytometry-based CFSE dilution assay. In line with our finding on ex vivo IFN-γ 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 patients and three healthy virus carriers, thus not reaching statistical significance.

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 EBV-encoded immunodominant ...

We further characterized the phenotype of CFSE-diluted EBV-specific 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, 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.

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 ...

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 requirements (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 EBV-specific 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-γ 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 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-γ), Th2 (IL-13), and Th17 (IL-17) polarization as well as for IL-2 and IL-10 production. Of these, only IFN-γ 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-γ: 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-γ was moderately increased in RA-derived positive cultures stimulated with immunodominant CD8+ T cell epitopes (p = 0.03). These data are consistent with the observation that Th1 polarization and IFN-γ 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-γ 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/alanine-rich 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 located 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 EBV-seronegative 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.

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 ...

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-γ-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.

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 ...

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-γ-producing CD8+ 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 (4346). 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-mediated 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 EBV-specific 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.

Footnotes

1J.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-BAA-06-19), 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 Forschungsge-meinschaft (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.

5Abbreviations 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.

Disclosures

The authors have no financial conflicts of interest.

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