Recombinant human tgfβ

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).

Toxoplasma gondii tachyzoites were maintained in HEp-2 cells in MEM. Tachyzoites for the experiments were prepared by centrifugation of the supernatant of cell culture and resuspension of the parasites in phosphate-buffered solution (PBS). Pregnant mice were inoculated via I.P. injection with 400 tachyzoites in 200 μl of aseptic PBS on gd 8. The normal control group was inoculated with 200 μl of aseptic PBS at the same time. The infected mice received recombinant human TGF-β (0.75 μg in 200 μl of PBS; clone: 2Ar2; Abcam, United Kingdom) or TGF-β-neutralizing antibody (5 μg in 200 μl of PBS; clone: ab64715; Abcam, United Kingdom), all without LPS or any carrier, or the same volume of sterile PBS via tail vein injection on gd 7 and gd 9. The volume of TGF-β or TGF-β-neutralizing antibody injected in the mice was determined with reference to a previous study (Zhang et al., 2010 ), as well as our preparatory experiments.