**1. Introduction**

The immune system plays a relevant role in the pathogenesis of rheumatoid arthritis (RA) [1,2]. However, the involvement of CD4+T-lymphocytes is only partially understood. CD4+T-lymphocytes form a regulatory and functionally diverse population of the immune system. This cell heterogeneity

includes different patterns of cytokine secretion and stages of differentiation/activation [3–6]. CD4+T-lymphocytes subsets are characterized by their ability to produce cytokines such as IFNγ, IL-4 or IL-17A, and they are named Th1, Th2 and Th17, respectively [7,8]. Different signals are involved in promoting development, but the signal transducer and activator of transcription (STAT) family of proteins appears to be critical for the activation of the subset-characteristic transcription factors [9]. The binding of these STATs can activate lineage-specific enhancers associated with alternative cell fates [10]. STAT-1, STAT-6 and STAT-3 are recognized as essential for T-bet, Gata3 and RORc activation, which promote Th1, Th2 and Th17 cell differentiation, respectively.

Based on their distinctive functional and phenotype patterns, CD4+T-lymphocytes are divided into CD4<sup>+</sup> naïve (TN), central-memory (TCM), effector-memory (TEM) and effector (TE) T subsets [11]. CD4<sup>+</sup>TN exhibits noneffector functions, while CD4<sup>+</sup>TCM can rapidly proliferate and express multiple different effector molecules such as cytokines after being stimulated by antigens, and exhibit diminished activation requirements [11–13]. CD4<sup>+</sup>TEM produce effector cytokines but have limited proliferative capacity, and CD4<sup>+</sup>TE are at a final differentiation stage and share high levels of cytokine production [14]. The requirements for the activation, proliferation and survival of these subsets are different, as well as their capacity to enter lymphoid and inflamed nonlymphoid tissues [15].

RA patients show several abnormalities in circulating CD4+T-lymphocytes including an imbalance between circulating Th1, Th2 and Th17 subsets and abnormal serum levels of their hallmark IFNγ, IL-4 and IL-17A cytokines [16,17]. However, contradictory results have been published on the percentages of these T cell subsets and of the cytokine concentration in RA patients [18–23]. Several factors might be involved in this variability, including the clinical stage of the disease and the previous and active treatments. There is also evidence of an abnormal regulation of the expression and phosphorylation of the Th1, Th2 and Th17 inducers STAT-1, STAT-6 and STAT-3, respectively [24–26]. The treatment of RA has dramatically improved in recent decades by the introduction and use of methotrexate (MTX) [27]. MTX has become the most commonly used disease-modifying antirheumatic-drug (DMARD) in RA, but its mechanism of action remains elusive. Additionally, controversial effects have described the concerning results of MTX in the CD4+T-lymphocytes distribution and activity of the Th1, Th2 and Th17 subsets [16,19,22,23]. Thus, the analysis of Th1, Th2 and Th17 subsets in new-onset DMARD-naïve RA patients may clarify the role of these cells in the pathogenesis of the disease and the study of the immunomodulatory and clinical effects of MTX treatment may favor the understanding of the heterogeneity in the response to this DMARD.

In this work, in a homogenous population of new-onset DMARD-naïve RA patients, we have investigated the pattern of IFNγ, IL-4 and IL-17A expression by TN and TCM, TEM and TE CD4+T-lymphocytes. We have also studied the expression and phosphorylation of the Th1, Th2 and Th17 transcriptional factors, STAT-1, STAT-6 and STAT-3, as well as the circulating levels of IFNγ, IL-4 and IL-17A. Furthermore, we have followed the patients during the first six month of MTX treatment and stratified them according to the clinical response attained.

### **2. Results**

#### *2.1. Patient Demographic Characteristics*

Table 1 shows the baseline characteristics of the 47 new-onset DMARD-naïve RA patients who eventually became responders (*n* = 31) or nonresponders (*n* = 16) after six months of MTX treatment. No significant differences were observed in terms of age, sex and clinical variables examined between both groups of patients. We analyzed the evolution of C-reactive protein (CRP), disease activity score of 28 (DAS28) and the Health Assessment Questionnaire (HAQ) in both groups of patients at a 6-month follow-up. After six months of MTX treatment, the responders, however, showed a significant reduction in CRP from 16.12 ± 6.39 to 4.90 ± 2.31 mg/dl, in DAS28 from 3.62 ± 0.49 to 2.23 ± 0.41, and in HAQ from 0.76 ± 0.56 to 0.47 ± 0.26. The nonresponders also showed a significant reduction in CRP,

from 16.57 ± 5.33 to 9.09 ± 4.18 mg/dL. The reductions in DAS28 from 3.69 ± 0.46 to 3.61 ± 0.25 and in HAQ from 0.78 ± 0.79 to 0.72 ± 0.65 were not statistically significant.


**Table 1.** Patient demographics and clinical and biological characteristics at baseline.

CRP, C-reactive protein; anti-CCP, anticyclic citrullinated peptide antibody; DAS28, Disease Activity Score 28; HAQ, Health Assessment Questionnaire.
