*4.2. Detection of Th17 and Treg Cells Using Flow Cytometry.*

Using Ficoll-Paque (GE Healthcare Bio-Sciences, Uppsala, Sweden) and performing density gradient centrifugation, we were able to isolate PBMC. In the next step, cells were cultured in RPMI 1640 (Invitrogen, Paisley, UK), 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Thermofisher, USA), 100 U/mL penicillin, and 100 ug/mL streptomycin (Sigma-Aldrich, Saint Louis, MO, USA) for 12 h. After that period, cells were harvested and stained for particular membrane antigens using anti-CD4 APC-Cy7, anti-CD25 PE, anti-CD127 FITC, anti-CCR6 APC, and anti-CXCr3 PE-Cy7 murine Abs. Cells were washed, then they were acquired, analyzed, and sorted using a FACSAria cell sorter/cytometer and Diva software. By using 7AAD staining, we were able to eliminate dead cells from analysis (Figure S3). All reagents used in the flow cytometry were purchased from Becton Dickinson (San Jose, CA, USA).

### *4.3. Isolation of Total RNA from Th17 and Treg Cells*

Total RNA was isolated from earlier sorted Th17 and Treg cells using the miRNeasy Micro Kit (Qiagen, Germantown, MD, USA). The quantity and quality of isolated RNA were evaluated by the Quawell Q5000 spectrophotometer. Total RNA isolated during this procedure was used to perform the reverse transcription reaction using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA). We stored synthesized cDNA at −20 ◦C for the next step. Preamplification of cDNA from this reverse transcription was performed using TaqMan® PreAmp Master Mix Kit (Applied Biosystem, Carlsbad, CA, USA) according to the manufacturer instructions. The rest of the isolated total RNA from Th17 and Treg cells were used to perform reverse transcription reaction using the miRCURY LNA RT kit (Qiagen) according to the manufacturer instructions. Both reverse transcriptions were conducted in a thermocycler (SensoQuest Labcycler 48 s, Göttingen, Germany).

#### *4.4. Quantitative Real-Time PCR*

We used following TaqMan primer and probes (Applied Biosystems, Foster City, CA, USA) SMAD3 (Hs00969210\_m1), STAT3 (Hs00374280\_m1), STAT5a (Hs00559647\_m1), SOCS1 (Hs00705164\_m1), GAPDH (Hs02786624\_g1), and RPLO (Hs99999902\_m1). The following assays were used in microRNA experiment: hsa-miR-24-3p (QG-339306\_YP00204260), hsa-miR-26a-5p (QG-339306\_YP00206023), hsa-miR-31-5p (QG-339306\_YP00204236), hsa-miR-100-5p (QG-339306\_YP00205689), hsa-miR-126-3p (QG-339306\_YP00204227), hsa-miR-146a-5p (QG-339306\_YP00204688), hsa-miR-155-5p (QG-339306\_YP00204308), hsa-miR326 (QG-339306\_YP00204512), SNORD48 (hsa) (QG-339306\_YP00203903), and U6 snRNA (has, mmu) (QG-33906\_YP00203907). To prepare quantitative Real-Time PCR, we used TaqMan Gene Expression Master Mix (Applied Biosystems); and for quantitative with microRNA, we used miRCURY SYBR Green PCR Kit (Qiagen). Quantitative Real-Time PCR was performed on real-time cycler (Quant Studio 5, Applied Biosystem, Foster City, CA, USA). Each sample was

analyzed in duplicates. From that, we took the mean Ct value, and we used it in the next steps of the analysis. Ct values higher than 35 were taken out of analysis and considered below quantification. The housekeeping gene has been selected and relative expression was calculated by ΔΔCt method or ΔCt method normalized to RPLO; in the case of microRNA, SNORD48 was used as a reference.
