Mouse work was performed at Harvard Medical School under specific-pathogen-free conditions using littermate controls. The strains used were C57BL/6J, B6.Aire−/−, B6.Foxn1-cre, B6.Ctla4-flox, and B6.Rag1−/−. Additional details are available in SI Appendix, Materials and Methods .
Isolation of mTECs. mTECs were isolated according to published protocols (4 (link)). Briefly, thymi were dissected, chopped, and incubated for 15 min in Dulbecco’s Modified Eagle Medium ( Gibco) plus 2% fetal calf serum ( Gibco), 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( Lonza), 0.5 mg/mL collagenase (Sigma), and 0.1 mg/mL DNase (Sigma), then for 15 min in the same buffer plus 0.5 mg/mL collagenase/dispase (Roche) and 0.1 mg/mL DNase, and then briefly with 10 mM ethylenediaminetetraacetic acid. Cells were stained with primary antibodies against CD45, Ly51, MHC-II molecules, and/or CD80 (all BioLegend). In some cases, cells were fixed using fixation/permeabilization buffer (eBioscience) and stained with anti-CTLA-4 antibody (BioLegend) in permeabilization buffer (eBioscience).
Isolation of mTECs. mTECs were isolated according to published protocols (4 (link)). Briefly, thymi were dissected, chopped, and incubated for 15 min in Dulbecco’s Modified Eagle Medium ( Gibco) plus 2% fetal calf serum ( Gibco), 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( Lonza), 0.5 mg/mL collagenase (Sigma), and 0.1 mg/mL DNase (Sigma), then for 15 min in the same buffer plus 0.5 mg/mL collagenase/dispase (Roche) and 0.1 mg/mL DNase, and then briefly with 10 mM ethylenediaminetetraacetic acid. Cells were stained with primary antibodies against CD45, Ly51, MHC-II molecules, and/or CD80 (all BioLegend). In some cases, cells were fixed using fixation/permeabilization buffer (eBioscience) and stained with anti-CTLA-4 antibody (BioLegend) in permeabilization buffer (eBioscience).
