*2.2. New-Onset DMARD-Naïve RA Patients Show An Expansion of CD4*+*IL-17A*<sup>+</sup> *and CD4*+*IFN*γ<sup>+</sup> *T-Lymphocytes*

We investigated IL-17A, IFNγ and IL-4 expression by circulating CD4+T-lymphocyte subsets from 47 new-onset DMARD-naïve RA patients and 29 HCs (healthy controls) before starting MTX treatment and during the initial 6 months of treatment (Figure 1). There were no significant differences either in the number or in the percentage of circulating T-lymphocytes or CD4+T-lymphocytes between RA patients and HCs at baseline (T-lymphocytes: 2801.99 ± 380.15 vs. 2002.15 ± 347.12 cells/μL and 49.47 <sup>±</sup> 2.52 vs. 52.88 <sup>±</sup> 5.95%; CD4<sup>+</sup> T-lymphocytes: 1131.60 <sup>±</sup> 174.23 vs. 825.20 <sup>±</sup> 79.84 cells/μL and 37.13 ± 2.17 vs. 40.29 ± 4.69%, respectively).

Next, we investigated the IL-17A, IFNγ and IL-4 expression by PMA (phorbol-12-myristate-13 acetate)-activated CD4+T-lymphocytes from each individual. RA patients had significantly increased CD4+IL-17A+T-lymphocyte numbers with respect to HCs (Figure 1a, panel B). The percentage of CD4+IL-17A<sup>+</sup> cells in the CD4+T-lymphocyte population was also increased in patients (Figure 1a, panel E). This CD4+IL-17A<sup>+</sup> lymphocyte expansion could mainly be explained by a significant broadening of the CD4+IL-17A<sup>+</sup>TN and CD4+IL-17A<sup>+</sup>TCM lymphocytes (Figure 1a, panel E).

RA patients also showed a significant increment in the numbers of CD4+IFNγ<sup>+</sup> cells with an expansion of the CD4+IFNγ<sup>+</sup> TEM and CD4+IFNγ<sup>+</sup> TE lymphocytes (Figure 1a, panel A). However, there were no significant differences in the percentages of IFNγ+-producing cells in the different CD4+T-lymphocyte subsets between patients and HCs (Figure 1a, panel D). The numbers or percentages of CD4+IL-4+T-lymphocytes were similar in both groups of subjects (Figure 1a, panel C and F). A representative dot plot of IL-17A, IFNγ and IL-4 expression by CD4<sup>+</sup> T-lymphocytes is shown in Figure 1b.

**Figure 1.** Intracellular IFNγ, IL-17A, IL-4 expression by the different activation/differentiation stages of T CD4<sup>+</sup> lymphocytes from rheumatoid arthritis (RA) patients. Note: (**a**) Data represent numbers (nº/γl) (A, B and C panels) and percentages (%) (D, E and F panels) of total CD3+CD4<sup>+</sup> (Th), and the TNaïve, TCM, TEM and TE CD4<sup>+</sup> lymphocyte subsets that express intracellular IL-17A, IFNγ and IL-4 after in vitro phorbol-12-myristate-13-acetate (PMA) stimulation in disease-modifying antirheumatic drug (DMARD)-naïve RA patients ( ) and healthy controls ( ). % (percentages) refers to total population of the indicated lymphocytes. All values are expressed as the mean cell numbers ± S.E.M. \*, *p* < 0.05 for RA patients vs. healthy controls. (**b**) Panel 1. The first dot plots represent the selected gates and percentages of TN, TCM, TEM and TE CD4<sup>+</sup> T-lymphocytes in two representative situations: a healthy control and RA patient at baseline before Methotrexate (MTX) treatment. Panel 2, 3 and 4. Dot plots represent the percentages of IL-17A, IFNγ and IL-4-producing CD4<sup>+</sup> T cells in the presence and absence of PMA stimulation in the two representative cases described in panel 1. R1 and R2 represents the regions that include the TNaïve and TEM CD4<sup>+</sup> lymphocyte subsets, respectively.

Interestingly, the numbers of TN and TMC CD4+T-lymphocytes expressing both IL-17A and IFNγ were significantly increased in patients compared with HCs (Figure 2a).

**Figure 2.** Intracellular IFNγ+IL-17A<sup>+</sup> double-positive expression by CD3<sup>+</sup>, TN and TMC CD4<sup>+</sup> lymphocytes in RA patients. Note: (**a**) Data represent numbers (nº/μL) of CD4<sup>+</sup>, TNaïve and TCM CD4<sup>+</sup> lymphocytes in ( ) DMARD-naïve RA patients and ( ) healthy controls. All values are expressed as the mean cell numbers ± S.E.M. \*, *p* < 0.05 for RA patients vs. healthy controls. (**b**) Dot plots represent the percentages of IFNγ+IL-17A<sup>+</sup> double-positive expression by CD4<sup>+</sup> T-lymphocytes after in vitro PMA stimulation in two representative situations: a healthy control and a RA patient at baseline before MTX treatment.

Next, we investigated the expression and phosphorylation of STAT-1, STAT-3 and STAT-6 transcription factors in CD4<sup>+</sup> T-lymphocytes from patients and HCs (Figure 3). Patients showed an increased percentage of phosphorylated STAT-1 and STAT-3 protein in the four different CD4+T-lymphocyte subsets analyzed with respect to HCs. Simultaneously, each CD4+T-lymphocyte subset from patients showed normal STAT-6 phosphorylation. Total STAT-3 protein was significantly increased in the four CD4+T-lymphocyte subsets from patients with respect to the HCs, while total STAT-6 protein was significantly increased in the CD4<sup>+</sup> and TN and TCM subsets and total STAT-1 protein was normal.

Finally, we investigated the IL-17A, IFNγ and IL-4 serum levels from patients and HCs (Figure 4). We found that patients had significantly increased levels of IL-17A and IFNγ, but normal IL-4 concentrations.

**Figure 3.** STAT-1, STAT-3 and STAT-6 phosphorylation and total protein expression by T CD4<sup>+</sup> lymphocyte subsets from new-onset DMARD-naïve RA patients. Note: (**a**) Data represent the mean florescence intensity (MFI) and percentage (%) of the total and phosphorylated proteins, respectively,

in CD3+CD4<sup>+</sup>, TNaïve, TEM, TE and TCM CD4+T-lymphocytes of ( ) DMARD-naïve RA patients and ( ) healthy controls. All values are expressed as the mean MFI or percentage ± S.E.M. \*, *p* < 0.05 for RA patients vs. healthy controls. (**b**) Histograms represent the mean fluorescence intensity (MFI) of STAT-1, STAT-3 and STAT-6 total protein stimulated with IFNγ, IL-6 and IL-4, respectively, and their negative controls, in two representative cases: a healthy control and a RA patient at baseline before MTX treatment. (**c**) Histograms represent the percentages of STAT-1, STAT-3 and STAT-6 phosphorylation stimulated with IFNγ, IL-6 and IL-4, respectively, and their negative controls in two representative cases: a healthy control and an RA patient at baseline before MTX treatment.

**Figure 4.** IL-17A, IFNγ and IL-4 serum levels in new-onset DMARD-naïve RA patients. Data represent the mean value serum levels of IL-17A, IFNγ and IL-4 in ( ) RA patients and ( ) as healthy controls (panels **A**, **B** and **C**). All values are expressed as the mean serum levels ± S.E.M. \*, *p* < 0.05 for RA patients vs. healthy controls.
