Introduction
The ideal approach for predicting and monitoring the outcomes of treatment in rheumatoid arthritis (RA) remains elusive. The advent of intensive, goal-directed treatment strategies, employing combinations of synthetic as well as biologic disease-modifying antirheumatic drugs (DMARDs), has substantially improved the prognosis of this disease [
1]. However, many patients still fail to achieve low disease activity or remission [
2‐
4]. Failure to completely abrogate inflammation puts patients at risk for disease progression, including joint destruction, disability, impaired quality of life, cardiovascular disease, and premature death [
5,
6].
The complexity of this issue emerges with the realization that, at times, discriminating clinical signs and symptoms of active joint inflammation from non-inflammatory joint disease or chronic pain syndromes is challenging [
7‐
9]. With many synthetic and biologic DMARDs now available, our limited ability to predict and to efficiently judge the likely outcomes of DMARD therapy represents a critical barrier to the development of more effective treatment strategies using these agents.
Recently, we have established an approach of immune-response profiling for the discovery of complex biomarkers predictive of treatment response [
10]. Although the levels of serum cytokines have proven to be disappointing for use as disease biomarkers [
10‐
14], our work has suggested that immune response signatures may provide greater biologically relevant information. The results of our published studies demonstrate proof of principle that our approach, based on multiplexed analysis of
ex vivo cytokine production in response to broad stimulation, can identify profiles of immune function associated with radiographic joint damage as well as myocardial disease in patients with RA [
10,
15]. Further, the discovery of a correlation between a profile of immune response to Epstein-Barr virus (EBV) and human cytomegalovirus (CMV) and the severity of radiographic joint destruction in RA demonstrates the relevance of the data generated to the investigation of pathogenesis [
16]. The purpose of this study was to identify immune response signatures that are associated with treatment outcomes in patients with early RA.
Methods
Study design and participants
A 24-week prospective observational cohort study of patients with newly diagnosed RA was performed at our institution. Consecutive patients with a new diagnosis of inflammatory arthritis during the enrollment period of July 2008 to December 2010 were referred for screening by rheumatologists in our division. At conception of this study, the available classification criteria for RA were the 1987 criteria, which were too insensitive in early disease for use in this study (which took place before the 2010 revised classification criteria were published) [
17]. Therefore, the Leiden early RA prediction rule was used [
18,
19]. Eligible patients were required to have a score ≥8 on the early RA prediction rule and to be starting their initial treatment with conventional DMARDs within 3 weeks of diagnosis. Patients who were prescribed biologic agents were ineligible. All patients (n = 71) participated in research study visits at baseline and after 21 to 24 weeks of follow up. Patients were required to provide written informed consent prior to study participation. The institutional review board of the Mayo Foundation approved this study.
Data collection
During each research study visit, one consultant rheumatologist (JMD) performed a standardized clinical evaluation of the patient, consisting of the 68-tender joint count, the 66-swollen joint count and the physician global assessment (0 to 100 mm). All patients completed the study questionnaire, which included visual analog scales (0 to 100 mm) for the levels of pain, fatigue and the patient global assessment of disease activity, and the Health Assessment Questionnaire (HAQ) disability index [
20,
21]. The dates of initiation/change, dosages, and route (that is, oral or intra-articular) of DMARDs or corticosteroids were obtained from the patients and confirmed in the most recent outpatient medication list. Medical records were reviewed to collect relevant demographic information, symptom duration at baseline, body mass index (kg/m
2), smoking status (current, former, or never), and test results for rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPA). C-reactive protein (CRP) was measured by turbidometric assay (Roche Diagnostics, Indianapolis, Indiana, USA). Enzyme-linked immunosorbent assays were done to assess past CMV infection or exposure using the VIDAS® CMV IgG (bioMerieux, Inc., Marcy l'Etoile, France), and multiplexed immunoassays were done to assess past EBV infection using the BioPlex™ 2200 System assessing EBV IgG and IgM (Bio-Rad Laboratories, Hercules, California, USA).
Definition of outcome measures
Clinical disease activity was defined by the Disease Activity Score in 28 joints (DAS28), based on the four-variable version using CRP [
22,
23]. Physical disability was defined by the HAQ disability index. Treatment response was defined as the difference (Δ) between the baseline and 21- to 24-week values for the DAS28 and HAQ disability index. A clinically meaningful improvement in the DAS28 was defined as a decrease ≥1.2.
Immune-response profiling
Immune-response profiling was performed on samples obtained from all 71 patients at the baseline visit and from a subset of 43 patients at the 21- to 24-week visit. Detailed methods of our approach to profiling the systemic immune response were previously described [
10]. Briefly, peripheral blood mononuclear cells (PBMC) from patients were cultured in the presence of a panel of six stimuli, or in media alone, for 48 hours. Stimuli used were immobilized anti-CD3/anti-CD28 monoclonal antibodies (anti-CD3/anti-CD28), combined lysates of purified CMV and EBV, containing both viral peptides and DNA, phytohemagglutinin (PHA), phorbol myristate acetate with ionomycin (PMA/ionomycin), a mixture of staphylococcal enterotoxins A and B (SEA/SEB), and CpG oligonucleotides. At 48 hours of culture, cell-free supernatants were removed and frozen for subsequent cytokine analysis.
The production of cytokines in the supernatants was analyzed using the MSD® 96-well MULTI-SPOT® Human Cytokine Assays tissue culture kit (Meso Scale Discovery (MSD), Rockville, Maryland, USA). The cytokine panel included IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-10, IL-12, IL-13, IL-17A, IFN-γ, TNF-α, monocyte chemoattractant protein (MCP)-1 (CCL2), monocyte inflammatory protein (MIP)-1β (CCL4), granulocyte colony stimulating factor (G-CSF), and granulocyte monocyte colony stimulating factor (GM-CSF).
Statistical analysis
The baseline characteristics were analyzed using descriptive statistics, including mean (SD) or median (range) as appropriate. All statistical tests were two-sided; the significance level was set at 0.05 for all analyses. Paired
t-tests were used to assess changes in characteristics between baseline and follow-up visits. Mixed models were used to normalize the cytokine data and adjust for age and sex as previously described [
10]. Principal components analysis (PCA) was used to derive immune-response profiles based on the first and second principal components of
ex vivo cytokine production for each stimulus [
16]. This analytic technique provided a relative weighting of cytokine importance in each profile and quantitatively summarized the information in immune-response profiles as PCA scores (rescaled 0 to 100 for interpretation) for subsequent screening.
Spearman methods and Wilcoxon rank sum tests were used to test for associations of both the baseline immune response PCA scores and the changes in these scores from baseline to 21 to 24 weeks with the changes in the clinical disease activity measures from baseline to 21 to 24 weeks. For immune profiles significantly associated with treatment response, partial Spearman correlation was used to adjust for potential confounding factors, including age, sex, body mass index, smoking status, RF status, ACPA status, CMV immunoglobulin (Ig)G, EBV IgG, methotrexate use, and prednisone use.
Discussion
We report the discovery of a T-cell immune-response signature associated with CMV immunity that is predictive of inadequate response to initial DMARD therapy among patients with early RA. Specifically, higher production of both type 1 (for example, IFN-γ) and type 2 (for example, IL-4) T-cell cytokines by PBMC, in response to stimulation with combined CMV/EBV lysate ex vivo, is predictive of inadequate DAS28 response to initial DMARD therapy over 21 to 24 weeks. Because the CMV/EBV immune-response signature was found to correlate to CMV IgG serology, the observed association with treatment response is likely mediated by CMV rather than EBV immunity. Further, we show that DMARD-induced amelioration of clinical disease activity correlates with augmentation of the CMV-related immune-response signature.
With further development, this signature could potentially be useful as a predictive biomarker for individualizing the management of early RA. Because the patients with high baseline CMV responsiveness experienced inferior outcomes of initial DMARD therapy, future studies should investigate whether a more aggressive treatment strategy in these patients could lead to more favorable outcomes. For example, the hypothesis could be tested that patients with the high CMV-specific T-cell immune-response signature would have better clinical response to therapy combining methotrexate and a TNF antagonist. The significance of this scenario is highlighted by the knowledge that 50 to 70% of patients fail to respond to initial methotrexate monotherapy [
2,
24], yet currently there are no biomarkers or prediction models that reliably and accurately identify these individuals [
24‐
27]. The advent of new techniques for individualizing initial therapy could improve outcomes for patients with RA, by inducing clinical remission earlier and/or by sparing patients trials of costly and risky medications to which they are pre-destined to respond unfavorably [
28,
29].
The reported immune-response signature clearly requires further refinement and validation. First, we must verify that CMV is the target of the immune response in our signature that is predictive of treatment outcome, by evaluating profiles of cytokine response to CMV and EBV separately. Removing EBV from the CMV immune response assay could conceivably reduce both technical and biological variability in the response profile, resulting in a more robust and informative assay. Second, we must determine if this signature is associated with the clinical response to specific DMARDs or whether it is predictive of response to disease-modifying therapy in general. In this regard, a limitation of this study is the heterogeneity among patients in the selection of DMARD therapy. Therefore, it will be of interest to determine if the CMV-related immune-response signature is predictive of successful response to treatment with a TNF antagonist or other biologic agent using a defined protocol. Third, we recognize that the CMV-related immune-response signature as undertaken in this study would be challenging to translate to clinical settings, so further work is necessary to develop a practical, reliable, and scalable assay based on our approach. These crucial studies are currently underway at our institution.
The findings of this study imply that CMV-related T-cell immunity may interact with the pathophysiology of RA. A potential unifying hypothesis for our findings must take into account not only that higher baseline CMV immune responsiveness is predictive of inadequate DMARD treatment response, but also that increasing CMV immune responsiveness from baseline to follow up is associated with good clinical response to DMARD therapy. Previous studies have demonstrated that patients with RA often have impairments of systemic T-cell function. For example, production of IFN-γ or IL-2 in response to mitogens has generally been found to be significantly lower in patients with active RA compared to inactive RA or healthy control subjects [
30‐
32]. In contrast, Pierer
et al. have reported a significantly higher magnitude of CD4+ IFN-γ-secreting T cells in response to CMV pp65 or CMV lysate in patients with RA compared to controls [
33]. Effective therapy for RA has been found to ameliorate the impaired T-cell responsiveness of IFN-γ production seen in RA patients [
30,
31,
34]. However, our data suggest that the association between T-cell responsiveness and treatment outcome was specific for the CMV stimulus and not to other T-cell stimuli, including CD3/CD28, PHA, CPG, and PMA/ionomycin. Previous studies have observed the general phenomenon of RA T-cell hypo-responsiveness using these non-specific stimuli [
30,
35,
36].
Rather, the findings of this study point to an interaction of rheumatoid disease specifically with CMV immunity. The significant correlation with CMV IgG suggests that the CMV-induced T-cell immune response signature is mediated by memory T cells. The nature of the CMV stimulus and pattern of T-cell cytokines suggest that this signature is mediated by CD4+ cells. Previous studies have shown that a subset of patients with RA has expanded pools of CMV-specific CD4+ IFN-γ-producing T cells in the peripheral blood [
33,
37,
38]. The distribution of CD4+ memory T cells against CMV is skewed to higher frequencies of cells in the peripheral blood than synovial fluid [
39]. These cells generally are thought to have a highly differentiated phenotype, lacking expression of the co-stimulatory molecule CD28, which has been associated with RA extra-articular manifestations and cardiovascular disease [
40,
41]. Positive CMV IgG and increased CMV-specific CD4+ CD28
null T cells have been reported to correlate with structural joint damage [
33]. With respect to the relationship between improvement in clinical disease activity and the augmentation of the CMV response, our data suggest that attenuation of inflammation affects the systemic CMV-specific memory T-cell pool. Speculatively, this could arise due to changes in the number, function, or phenotype of CMV-specific memory T cells circulating in the blood [
42,
43]. Due to the limitations of this discovery-oriented study, future research is needed to clarify the underlying immune mechanisms and implications of our findings.
We report that among patients who were treated with methotrexate during follow up and who attained clinically meaningful DAS28 improvement, the CMV-related T-cell immune response signature was observed to increase significantly from baseline to follow up. In contrast, patients who were treated with non-methotrexate DMARDs or who did not achieve a meaningful DAS28 response generally had a decline in their CMV-associated T-cell response. The mechanism of this finding is unclear, and the observational design of this study precludes causal assessment of treatment effects. However, a possible implication is that various DMARDs may have different effects on CMV T-cell responses. Such heterogeneity of treatment effects on CMV immunity is potentially significant in view of the aforementioned association between CMV IgG and disease progression, suggesting some DMARDs may not sufficiently antagonize the contribution of CMV immunity to disease, leading to inadequate treatment response and disease progression.
Competing interests
Mayo Foundation has submitted a patent application for the technique of multi-cytokine response profiling described in this article for assessing RA outcomes. JMD, KLK, and SEG are listed as inventors of technology in that patent application.
Authors’ contributions
JMD obtained research funding, designed the study and analysis plan, recruited all patients, directed data interpretation, and primarily drafted and revised the manuscript. KLK conceived of the immune response profiling methodology, contributed to study design, and participated in data interpretation and manuscript preparation. MAS performed all immune-response profiling assays. ABG, CSC, and TMT performed the statistical analysis. ELM and SEG contributed to the study design, data analysis and interpretation, and manuscript revision. All authors have critically analyzed and approved the final submitted version of the manuscript.