Tofacitinib

T cells from the lymph nodes of wild-type and Ptpn22^−/− mice were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM, Invitrogen) supplemented with 10% FCS, L-glutamine, antibiotics and 50 μM 2-mercaptoethanol. The agonist peptide SIINFEKL (N4) and partial agonist SIITFEKL (T4) (Peptide Synthetics) were added to culture medium at concentrations stated in figure legends. Recombinant human TGFβ (R&D) was added to cultures at 5 ng/ml unless otherwise stated and, where indicated, a TGFβ inhibitor (SB431542, 2.5 μM, Abcam), Jak3 inhibitor (tofacitinib, 200 nM, Tocris Bioscience), or recombinant human IL-2 (Peprotech) were also added. Transwell experiments were performed using HTS Transwell 96 system 0.4 μm pore polycarbonate membrane (Corning). For measurement of IL-2, cells were stimulated with peptide ± TGFβ in the presence of blocking CD25 mAb (Biolegend) in order to prevent IL-2 consumption. IL-2 in culture supernatants was measured using the Mouse IL-2 ELISA Ready-SET-Go!^® kit (eBioscience).

Cl-amidine (Calbiochem, Darmstadt, Germany), the TLR4 agonist lipopolysaccharide (ultrapure LPS; Invivogen, San Diego, CA, USA), SB203580, U0126, SP600125, BAY 11-7085, PI-103 (Adooq, Irvine, CA, USA), Tofacitinib, niclosamide (Tocris, Bristol, UK), and T-5224 (APExBIO, Houston, TX, USA) were purchased commercially. Unless otherwise stated, all chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ten or twenty millimoles stock solution of Cl-amidine was dissolved in phosphate buffer solution (PBS), and other inhibitors were dissolved in DMSO. PBS or DMSO were used as vehicle controls in each experiment.