**Methrotexate Treatment Inmunomodulates Abnormal Cytokine Expression by T CD4 Lymphocytes Present in DMARD-Naïve Rheumatoid Arthritis Patients**

**Jorge Monserrat Sanz 1,2,\*, Cristina Bohórquez 3, Ana Maria Gómez 1, Atusa Movasat 3, Ana Pérez 2,3, Lucía Ruíz 3, David Diaz 1,2, Ana Isabel Sánchez 3, Fernando Albarrán 3, Ignacio Sanz <sup>4</sup> and Melchor Álvarez-Mon 1,2,3,\***


Received: 27 July 2020; Accepted: 14 September 2020; Published: 18 September 2020

**Abstract:** CD4+T-lymphocytes are relevant in the pathogenesis of rheumatoid arthritis (RA), however, their potential involvement in early RA remains elusive. Methotrexate (MTX) is a commonly used disease-modifying antirheumatic drug (DMARD), but its mechanism has not been fully established. In 47 new-onset DMARD-naïve RA patients, we investigated the pattern of IFNγ, IL-4 and IL-17A expression by naïve (TN), central (TCM), effector memory (TEM) and effector (TE) CD4<sup>+</sup> subsets; their STAT-1, STAT-6 and STAT-3 transcription factors phosphorylation, and the circulating levels of IFNγ, IL-4 and IL-17. We also studied the RA patients after 3 and 6 months of MTX treatment and according their clinical response. CD4+T-lymphocyte subsets and cytokine expression were measured using flow cytometry. New-onset DMARD-naïve RA patients showed a significant expansion of IL-17A+, IFNγ<sup>+</sup> and IL-17A+IFNγ<sup>+</sup> CD4+T-lymphocyte subsets and increased intracellular STAT-1 and STAT-3 phosphorylation. Under basal conditions, nonresponder patients showed increased numbers of circulating IL-17A producing TN and TMC CD4+T-lymphocytes and IFNγ producing TN, TCM, TEM CD4+T-lymphocytes with respect to responders. After 6 months, the numbers of CD4+IL-17A<sup>+</sup>TN remained significantly increased in nonresponders. In conclusion, CD4+T-lymphocytes in new-onset DMARD-naïve RA patients show IL-17A and IFNγ abnormalities in TN, indicating their relevant role in early disease pathogenesis. Different patterns of CD4<sup>+</sup> modulation are identified in MTX responders and nonresponders.

**Keywords:** naïve rheumatoid arthritis; CD4+ T-lymphocytes; methotrexate response; IFNγ, IL-17A; STAT expression
