Introduction
Rheumatoid arthritis (RA) is associated with a twofold increase of non-Hodgkin's lymphoma [
1] and a threefold increase of Hodgkin's lymphoma [
2]. The effect of immunosuppressive drugs on the risk of lymphoma is debated. Most recent studies did not find an overall increased risk of non-Hodgkin's lymphoma in RA patients treated with methotrexate (MTX). Several reports, however, showed that MTX can rarely induce Epstein-Barr virus (EBV)-associated lymphoproliferation regressive after withdrawal of the drug [
3,
4].
Recent concerns about possible treatment effects and lymphoma have focused on anti-TNF drugs because of their profound immunoregulatory effect. A recent meta-analysis of randomized controlled trials of infliximab and adalimumab identified 10 cases of lymphoma (four cases in the randomized phase of the trials and six cases in the extension phase) in the treated groups (3,493 patient-years) and none in the placebo groups (1,512 patient-years) [
5]. Inflammatory activity of the underlying disease is the main risk factor of lymphoma in RA [
6], however, and anti-TNF therapy is used for patients with the most active disease. Results for three large cohorts of RA patients did not reveal any increased risk of lymphoma in RA patients receiving anti-TNF drugs versus RA patients receiving classical disease-modifying anti-rheumatic drugs (DMARDs). In most of these cohorts, however, increased risk of lymphoma persisted as compared with that in the general population [
7‐
9].
Cases of EBV-associated lymphoproliferation that regressed after withdrawal of MTX have been described [
3,
4]. Case reports of lymphoma associated or not with EBV, treated with anti-TNF drugs and regressing after withdrawal of therapy have also been reported [
10,
11]. These cases may mimic post-transplant lymphoproliferative disorder, a severe complication of EBV reactivation linked to impaired EBV control by CD8 T cells and arising in allograft recipients receiving immunosuppressive drugs [
12].
Taken together, such data provide reliable arguments to investigate a potential EBV reactivation during MTX and/or TNFα antagonist therapy as a possible first step of lymphoma induction. During primary EBV infection, specific cytotoxic CD8
+ T cells expand and recognize epitopes from lytic-cycle antigens and, to a lesser extent, from latent-cycle antigens. A small population of EBV-specific memory CD8
+ T cells further persists [
13] and plays a crucial role in the control of persistent EBV infection [
14]. An impaired EBV-specific T-cell response could constitute one of the first steps of lymphoma induction with immunosuppressive drug therapy.
The present study aimed to determine the EBV viral load and the specific effector CD8+ T-cell response against EBV antigens in patients with RA and spondylarthropathy (SpA) receiving MTX or anti-TNF drugs, to shed some light on a possible impaired EBV-specific T-cell response as the triggering mechanism of lymphomagenesis in this population.
Materials and methods
Study population
All studied subjects were seropositive for EBV. The present study consisted of two parts. In the cross-sectional first part of the study we investigated EBV-specific IFNγ-producing T cells at baseline (week 0) in 87 patients: 32 MTX naïve RA patients (mean age 60 ± 16 years, mean duration of disease 4.5 ± 6.6 years), 27 patients with RA receiving MTX who were not responders to the drug (mean age 53 ± 11 years, mean duration of disease 9.5 ± 10.5 years) and 28 patients with SpA (14 not receiving DMARDs and 14 receiving MTX; mean age 36 ± 11 years, mean duration of disease 9.6 ± 9.7 years). Patients with RA fulfilled the 1987 American College of Rheumatology criteria [
15] and those with SpA fulfilled the European Spondylarthropathy Study Group criteria [
16]. All RA patients were rheumatoid factor positive and/or anti-cyclic citrullinated peptide positive. The Disease Activity Score for 28 joints was 4.8 ± 1.2 in naïve RA patients and was 5.6 ± 1.2 in RA patients who were nonresponders to MTX. The Bath Ankylosing Spondylitis Disease Activity Index score [
17] was 55 ± 22 in SpA patients. The control group comprised 22 patients with mechanic radiculopathic conditions (mean age 47 ± 15 years).
From the 87 patients included in the cross-sectional part of the study, 62 underwent the second longitudinal part of the study for EBV-specific IFNγ-producing T cells after 3 months (week 12) of MTX or anti-TNF treatment. Forty patients (21 SpA and 19 RA) received anti-TNF drugs. All RA patients and 10/21 SpA patients had anti-TNF + MTX. Twenty-two MTX naive RA patients received MTX. EBV viral load data were also available for 67 patients and 15 control individuals at week 0, and for 52 patients at week 12.
The present study was performed with approval of the local ethics committee (CPP Ile de France 7), and informed consent was obtained from all study participants.
Isolation of peripheral blood mononuclear cells
Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Ficoll-Hypaque 1.107 (Biochrom, Berlin, Germany). The PBMCs were then frozen in FCS containing 10% dimethyl sulfoxide (Sigma, Saint-Quentin Fallavier, France) and stored in liquid nitrogen until use.
Epstein-Barr virus peptides
A set of 39 9-mer latent-cycle peptides was used, corresponding to known human leukocyte antigen (HLA) class I-restricted cytotoxic T lymphocyte epitopes. Considering that the HLA status of our study patients and control individuals was unknown, these peptides were chosen as being recognized by a broad range of class I molecules [
18]. The latent-cycle peptides used were immunodominant sequences from EBNA1, EBNA3A, EBNA3B, EBNA3C and LMP2 already tested in four different laboratories [
18]. Lytic-cycle EBV antigens were represented by a BMLF 9-mer peptide and a collection of 47 overlapping 15-mer lytic-cycle peptides spanning the entire sequence of BZLF1 protein. The BMLF 9-mer is a peptide from the replicative phase of EBV previously reported to be an immunodominant HLA-A2-restricted epitope [
19,
20] (Table
1). Lyophilized peptides were dissolved in sterile water supplemented with 10% dimethyl sulfoxide at 40 μg/ml and were stored at -20°C. For peptide pulsing, target cells were incubated with peptides (final concentration 2 μg/ml). Individual responses to latent-cycle peptides and lytic-cycle peptides were summed and analyzed as a whole, and were also analyzed separately.
Table 1
Human leukocyte antigen class I-restricted cytotoxic T-lymphocyte Epstein-Barr virus epitopes
A2 | EBNA3A | 596 to 604 | SVRDRLARL |
A2.01 | EBNA3C | 284 to 293 | LLDFVRFMGV |
A2.01 | LMP2 | 329 to 337 | LLWTLVVLL |
A2.01 | LMP2 | 426 to 434 | CLGGLLTMV |
A2.01 | BMLF1 | 280 to 288 | GLCTLVAML |
A2.06 | LMP2 | 453 to 461 | LTAGFLIFL |
A3 | EBNA3A | 603 to 611 | RLRAEAQVK |
A11 | EBNA3B | 399 to 408 | AVFDRKSDAK |
A11 | EBNA3B | 416 to 424 | IVTDVSVIK |
A11 | LMP2 | 340 to 350 | SSCSSCPLSKI |
A23 | LMP2 | 131 to 139 | PYLFWLAAI |
A24 | EBNA3A | 246 to 253 | LYSIFFDY |
A24 | LMP2 | 419 to 427 | TYGPVFMCL |
A24.02 | EBNA3B | 217 to 225 | TYSAGIVKI |
A25 | LMP2 | 442 to 451 | VMSNTLLSAW |
A29 | EBNA3A | 491 to 499 | VFSDGRVAC |
A30.02 | EBNA3A | 176 to 184 | AYSSWMYSY |
B7 | EBNA3A | 502 to 510 | GPAPAGPIV |
B7 | EBNA3A | 379 to 387 | RPPIFIRRL |
B7 | EBNA3C | 881 to 889 | QPRAPIRPI |
B8 | EBNA3A | 158 to 166 | QAKWRLQTL |
B8 | EBNA3A | 325 to 333 | FLRGRAYGL |
B8 | BZLF1 | 190 to 197 | RAKFKQLL |
B27.02 | EBNA3B | 244 to 254 | RRARSLSAERY |
B27.02/.04/.05 | EBNA3C | 258 to 266 | RRIYDLIEL |
B27.04 | LMP2 | 236 to 244 | RRRWRRLTV |
B27.05 | EBNA3B | 149 to 157 | HRCQAIRKK |
B27.05 | EBNA3C | 249 to 258 | LRGKWQRRYR |
B27.05 | EBNA3C | 343 to 351 | FRKAQIQGL |
B35 | EBNA3A | 458 to 466 | YPLHEQHGM |
B35 | EBNA3B | 488 to 496 | AVLLHEESM |
B35 | BZLF1 | | EPLPQGQLTAY |
B35.01 | EBNA1 | 407 to 417 | HPVGEADYFEY |
B39 | EBNA3C | 271 to 278 | HHIWQNLL |
B44 | EBNA3B | 567 to 666 | VEITPYKPTW |
B44.02 | EBNA3C | 281 to 290 | EENLLDFVRF |
B44.02 | EBNA3C | 335 to 343 | KEHVIQNAF |
B44.03 | EBNA3C | 163 to 171 | EGGVGWRHW |
B60 | LMP2 | 200 to 208 | IEDPPFNSL |
B62 | EBNA3A | 406 to 414 | LEKARGSTY |
B62 | EBNA3B | 831 to 839 | GQGGSPTAM |
B62 | EBNA3C | 213 to 222 | QNGALAINTF |
ELISPOT assay
The ELISPOT-IFNγ assay was used to determine the frequency of T cells that produced IFNγ in response to a brief exposure to EBV antigens, as previously published [
21]. Briefly, nitrocellulose ELISPOT plates (Millipore, Guyancourt, France) were coated with anti-IFNγ antibody (1 μg/ml, 100 μl/well in PBS; 1-D1K; Mabtech, Sophia Antipolis, France). PBMCs were added in duplicate wells at 10
5 cells per well with 2 μg/ml peptide. The second biotinylated anti-IFNγ monoclonal antibody was then added (7-B6-biotin; Mabtech) and IFNγ secreting cells were revealed with an enzymatic reaction with streptavidin-conjugated alkaline phosphatase (Sigma-Aldrich, Saint-Quentin Fallavier, France).
The number of specific T-cell responders per 106 PBMCs was calculated after subtraction of the background, which corresponded to the mean value of IFNγ spots associated with nonstimulated PBMCs (PBMCs in the presence of medium alone). Results were expressed as spot-forming cells (SFCs) per 106 PBMCs and were calculated for each pool of peptides as follows:
SFCs/106 PBMCs = 10 x (mean SFCs/105 cells from two antigen-stimulated wells - mean SFC/105 cells from four unstimulated wells).
Results were presented as the individual response to the set of latent-cycle peptides (9-mer peptides), to the set of lytic-cycle peptides (BMLF 9-mer peptide added with the 15-mer lytic-cycle peptides) or to both sets.
Wells were counted as positive if they contained at least 50 SFCs/106 PBMCs and exhibited at least twofold the mean value of the background (per million PBMC). The median number of IFNγ-producing PBMCs in the presence of medium alone (background) was zero spots/well (range 0 to 4).
Epstein-Barr virus load in peripheral blood mononuclear cells
The level of EBV DNA copies in PBMCs was measured by Taqman real-time quantitative PCR as previously described [
22]. For each quantification, 500,000 to 10
6 PBMCs were thawed and DNA extractions were further performed. The PCR primers were selected to amplify a 121 bp product in the thymidine kinase gene. A pcDNA 3.1 vector (Invitrogen, Groningen, the Netherlands) containing one copy of the EBV target region was used as standard for EBV quantification. The level of albumin DNA copies in PBMC samples estimated by real-time PCR was used as the endogenous reference to normalize the variations in PBMC number or DNA extraction. All standard dilutions, control samples and PBMC samples were run in parallel and in duplicate for EBV and albumin DNA quantifications. The normalized value of the cell-associated EBV DNA load corresponding to the ratio EBV average copy number/albumin average copy number × 2.10
6 was finally expressed as the number of EBV DNA copies per 10
6 PBMC.
Statistical analysis
Results are given as the percentage of patients with positive EBV T-cell response, as well as the mean response ± standard deviation. Statistical analyses involved use of StatView 5.0 (Abacus Concepts, Berkeley, CA, USA). Nonparametric tests were used. Comparisons between groups involved the Kruskal-Wallis test. Cross-sectional comparison of EBV T spots or the EBV copy number distribution involved the Mann-Whitney rank-sum test. Longitudinal comparison of EBV T spots or the EBV copy number between week 0 and week 12 involved the Wilcoxon test. Correlation studies involved Spearman's correlation. P < 0.05 was considered statistically significant.
Discussion
The present large cross-sectional and longitudinal study showed no abnormality in EBV viral load or EBV-specific T-cell response in patients with RA or SpA at baseline or after treatment with MTX or anti-TNF drugs.
In contrast to other studies [
23,
24], we did not find increased EBV viral load in PBMCs of patients with RA or SpA. A casual high EBV DNA prevalence in our control group (80%) could account for the differing results, and/or the highly sensitive PCR used in our study might explain such differences. Our cross-sectional study revealed no significant differences between patients and control individuals in the proportion of subjects with positive EBV-specific IFNγ-producing T cells in PBMCs, the mean number of SFCs or the SFC distribution. Two studies have assessed the EBV-specific T-cell response in PBMCs of RA patients [
25,
26]. The first study found no difference between 49 RA patients and 26 control individuals in the frequency of T cells directed against two immunodominant EBV peptides, but did observe a reduced ability to produce INFγ in RA patients; the effect of immunosuppressive treatment was not assessed [
25]. In the second study, EBV-specific effector CD8 T cells were higher in number in RA patients (n = 25) than in control individuals (n = 20), but this study concerned a low number of patients and was only cross-sectional [
26]. Actually, this increase in IFNγ secreted T cells was related to increased viral load, which we did not observe in our study.
Our longitudinal results did not reveal any influence of immunosuppressive treatment on the EBV viral load. These results are in accordance with several studies on Crohn disease or RA patients assessing the evolution of EBV viral load under immunosuppressive treatment [
27‐
29]. To the best of our knowledge, no published study has specifically evaluated the longitudinal effect of MTX and anti-TNF drug on EBV-specific T-cell effector functions in patients with RA or SpA. At 3-month follow-up, neither MTX treatment in RA patients nor anti-TNF therapy in RA and/or SpA patients modified these effector functions, regardless of the EBV peptide used for pulsing – latent-cycle peptides or lytic-cycle peptides or the full set of peptides. The lack of increase in the EBV viral load during the same period in all groups of patients agrees with the preserved specific T-cell effector function, which was confirmed by a global correlation between EBV viral load and EBV-specific T-cell response.
Interestingly, in five patients treated with anti-TNF, an inadequate
in vitro EBV-specific IFNγ production was observed after specific pulse with EBV peptides despite an
in vivo high viral load. Among those patients, two different profiles were observed. The first profile corresponded to patients without any IFNγ production in spite of high EBV viral loads (>1,000/10
6 PBMCs), both at week 0 and week 12. In such cases, the lack of adequate HLA for presenting one of the EBV peptides probably accounted for the absence of IFNγ production after specific pulse. The second profile corresponded to two patients having EBV-specific T-cell IFNγ production at baseline but a discordant evolution between an IFNγ secreted T-cell decrease and an EBV viral load increase after 12 weeks of treatment. In these two patients, immunosuppressive therapy might have impaired EBV-specific T-cell effector functions leading to the lack of control of the EBV viral load. These two patients having been treated with the association of anti-TNF antibody and MTX makes it impossible to differentiate a possible effect of one drug individually. Nevertheless, we never saw profound discrepancies, such as those observed in a pediatric sample in whom post-transplant lymphoproliferative disorder developed after liver transplantation [
30]. Since we analyzed data only 12 weeks after the introduction of immunosuppressive treatment, however, we cannot exclude that MTX or anti-TNF therapy could induce impaired EBV control after longer-term treatment. This relative short time duration of immunosuppressive treatment exposure might be considered as a limitation of our study. Nevertheless, post-transplant lymphoproliferative diseases occurring in children, for example, have been reported to occur after a short-term exposure to immunosuppressive treatments (median delay of 12 weeks, range 6 to 56 weeks) [
30]. Moreover, with the same methodology (ELISPOT assay), we detected a significant decrease of the specific anti-tuberculosis T-cell response in patients after 12 weeks of anti-TNF therapy [
31]. Lastly, in the present study, patients treated with MTX were treated for several years on average, and their results were no different from the MTX naïve patients at baseline.
Conclusions
In patients with RA or SpA, short-term (3-month) exposure to MTX or anti-TNF therapy does not alter the EBV viral load or the EBV-specific T-cell response. These findings are rather reassuring in light of a suggested increased risk of EBV-associated lymphoma in patients receiving immunosuppressive therapy. Long-term follow-up of the EBV-specific T-cell response, however, is necessary. Moreover, control of EBV is only one mechanism of control of lymphomagenesis and the different epidemiologic studies currently available do not eliminate the possibility of increased risk of non-EBV-associated lymphoma in patients receiving immunosuppressive therapy.
Acknowledgements
The set of EBV peptides was kindly provided by Prof. D Olive (INSERM Action-Thématique-Concertée). The authors thank Dr Martine Sinet and Prof. Martine Raphael for helpful discussions. The present work was supported by Assistance Publique Hôpitaux de Paris, Département de la Recherche Clinique, Paris, France (Contrat d'Initiation à la Recherche Clinique) and by Société Française de Rhumatologie.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
XM was responsible for the study design, manuscript preparation and interpretation of the data. CM-R was responsible for sample blood collection, manuscript preparation, interpretation of data and statistical analyses. NG performed the ELISPOT assays and statistical analyses. CA performed the EBV quantitative PCR. JS, MI and SP contributed to the blood sample collection. AU and IG contributed to the ELISPOT assay analyses. GC and AV contributed to interpretation of the data.