Reduced ZAP-70 expression in the thymus and functional Treg impairment in the periphery are essential for inducing autoimmunity. (A and B) Donor CD4SP TCRβ^hi thymocytes (3 × 10⁵) from Dox 2.0– or 0.5–treated Tet-on ZAP mice for 4 wk were transferred to RAG2^−/− mice, then treated with Dox 0.5 food for 12 wk and assessed for indicated diseases. Correlation between GFP MFIs of donor thymocytes and arthritis severity (A), or the occurrences of indicated diseases in recipient RAG2^−/− mice (B). (C–E) Adoptive transfer of CD4⁺ or CD4⁺CD25⁻ splenic T cells (3 × 10⁵) from Dox-treated Tet-on ZAP mice into RAG2^−/− mice. Percent survival (C), incidence of histologically evident autoimmune diseases (D), and histological scores of autoimmune/inflammatory diseases (E) in RAG2^−/− recipient mice 12 wk after cell transfer. (F) CD4⁺ or CD4⁺CD25⁻ splenic T cells (3 × 10⁵) from individual Tet-on ZAP mice treated with either Dox 0.5 (left) or 2.0 (right) food for 5 wk or whole splenocytes from untreated Dox (−) Tet-on ZAP mice were transferred to RAG2^−/− mice, then treated with either Dox 0.5 or 2.0 food for 11–12 wk and monitored for arthritis severity (n = 3). (G) CD25^hi CD4⁺ T cells (3 × 10⁵) from Dox 0.5 Tet-on ZAP or BALB/c mice were transferred to 4-wk-old Tet-on ZAP recipient mice 3 d after starting Dox 0.5 food treatment and monitored arthritis severity. Logrank test in C, Fisher’s exact test in D, Mann-Whitney U test in E, two-tailed unpaired Student’s t test in G. Mean ± SD in E–G.