Il 21

Fluoxetine hydrochloride (Tocris Bioscience, USA) was reconstituted in phosphate buffered saline (PBS). Digoxin (Sigma, USA) was dissolved in dimethyl sulfoxide and further diluted appropriately in PBS to get the desired concentration at the time of injection. Peptides viz. MOG(35-55), PLP(131-151) acquired from Anaspec, USA used for disease induction were dissolved in PBS and emulsified in Freund's complete adjuvant (Sigma, USA). Antibodies anti-CD3, anti-CD4, PE-anti-TCRβ, APC-anti-IFNγ, FITC-anti-IL-17A, anti-IL-4, anti-IL-23, Alexa Fluor 488-anti-rat, Alexa Fluor 555-anti-rabbit, cytokines, IL-12, IL-17A, IL-21, IL-23, TGFβ, IL-6 were purchased from BD Pharmingen, Abcam, Cell Signaling Technology, and RnD Technologies. Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (certified) were procured from Gibco, USA.

The procedure of cell staining and flow cytometry was previously described (5 (link)). For intracellular staining of IL-21, gastric CD4⁺T cells were restimulated with 1 μg/ml anti-CD3 Ab (Clone UCHT1, BD), 1 μg/ml anti-CD28 Ab (Clone CD28.2, BD), and 3 μg/ml brefeldinA (eBioscience, CA, USA) for 12 h. The intracellular fixation/permeabilization buffer set (eBioscience, CA, USA) was used in IL-21 staining. Flow cytometric detection was performed with FACS Canto II (BD, NJ, USA). Data was analyzed by the FlowJo software (Tree Star). The anti-human antibodies used in this study were purchased from Biolegend, BD Biosciences, or Miltenyi Biotec, namely: CD3 (Clone UCHT1, BioLegend), CD4 (Clone OKT4, BioLegend), GITR (Clone 108-17, BioLegend), GITRL (Clone REA841, Miltenyi), IL-21 (Clone 3A3-N2.1, BD), BCL6 (Clone 7D1, BioLegend), STAT3 Phospho (Ser727) (Clone A16089B, BioLegend), BTLA (Clone MIH26, BioLegend), CXCR5 (Clone J252D4, BioLegend), and PD-1 (Clone EH12.2H7, BioLegend).

Single-cell suspensions were stained with antibodies against CD45, CD3, CD4, PD-1, CD25, interferon (IFN)-γ, IL-17, IL-9, IL-4, FOXP3 (eBioscience), and IL-21 (BD Biosciences) according to the manufacturers’ instructions. The dose of each of the above antibodies was 5 µl/test. To detect the cytokine profile, cells were stimulated with Leukocyte Activation Cocktail (2 µl/ml, BD Biosciences) for 5 h before flow cytometric analysis. For intracellular staining, cells were stained with the surface markers listed above and fixed in Fix/Perm Buffer (eBioscience), followed by permeabilization in the presence of antibodies specific for intracellular markers. All the reagents were purchased from BioLegend unless otherwise indicated. The stained cells were acquired on a Cytoflex flow cytometer (Beckman Coulter), and the analysis was performed using FlowJo software (TreeStar).