Fig. 2. NR4A1 is required for T cell tolerance formation.

a, Experimental strategy for OT-II peptide-induced CD4⁺ T cell tolerance in vivo. CFA, complete Freund’s adjuvant; i.p., intraperitoneally; i.v., intravenously. b, Flow cytometry measurement of NR4A1 expression in sorted donor-derived T cells after restimulation with OT-II peptide-loaded APCs for 3 h. c, Quantification of NR4A1 expression (mean fluorescence intensity; MFI) in naive T cells and donor-derived T cells from both activated and tolerant groups, after restimulation with OT-II peptide-loaded APCs. d, ELISA measurement of IL-2 from Nr4a1^+/− and Nr4a1^−/− CD4⁺ T cells activated either by anti-CD3 alone or by anti-CD3 and anti-CD28 in a dosage-dependent manner. e, Flow cytometry analysis of IFNγ, IL-2 and FOXP3 expression in wild-type and Nr4a1^−/− OT-II T cells in spleens in the context of peptide-induced tolerance. f , Quantification of donor-derived wild-type and Nr4a1^{−/ −} OT-II T cells. g, Body weight measurement (shown as ratio compared with original body weight) of Rag1^−/− mice after receiving wild-type or Nr4a1/^−/− naive CD4⁺ CD45RB^hi T cells. h, Flow cytometry analysis of donor-derived T cells in lamina propria (LP) four weeks after T cell transfer. KO, knockout. Eight-week-old mice; n = 3 (c, d); n = 4 (f–h). *P < 0.05, **P < 0.01 (two-sided unpaired Student’s t-test). Data are representative of at least two individual experiments and are presented as mean ± s.d.