Dox dose-dependent expression of ZAP-70 and elicitation of autoimmune diseases in Dox-low Tet-on ZAP mice. (A) Scheme of Tet-inducible human WT ZAP-70 double transgenic mice. rtTA, reverse tetracycline activator; TRE, Tet-responsive element; PminCMV, minimum CMV promoter. (B) Correlation between percent GFP⁺ cells among CD3⁺ splenocytes of rtTA⁺ hZAP-70⁺ mice and Dox concentration in the culture. Representative result of three independent experiments. (C) Splenocytes of rtTA⁺ hZAP-70⁺ BALB/c mice with intact endogenous ZAP-70 were cultured with Con A (2 μg/ml) for 3 d in the presence of indicated concentrations of Dox. The percentages of GFP⁺ cells and their MFIs (in parentheses) for CD3⁺ T cells were shown. Representative result of three independent experiments. (D) Correlation between GFP expression and the amount of ZAP-70 assessed by intracellular staining of both human and mouse ZAP-70 in CD4⁺ T cells from rtTA⁺ hZAP-70⁺ BALB/c mice by specific mAb. Numbers indicate MFIs. (E) Representative photos of the ear and the tail of Tet-on ZAP or BALB/c mice treated with low-dose Dox for 5 wk. Note severe dermatitis in the ear and the tail, and systemic debilitation of the mice. Scale bar: 10 mm. (F) FACS staining of indicated molecules for splenic Foxp3⁺ CD4⁺ Treg cells in Tet-on ZAP mice Dox-treated for 10 wk, as in Fig. 8 I. Percentages of positively stained cells and their MFIs (in parentheses) are also shown. (G) TCRβ, Foxp3, and GFP expression in freshly isolated thymocytes from BALB/c and Tet-on ZAP mice Dox-treated for 9 wk. Shaded area indicates isotype control. Representative data of more than five experiments. (H) GFP expression in Dox-treated Tet-on ZAP splenocytes in the mice shown in G. Representative result of three independent experiments. (I) Expression of ZAP-70 in thymocytes, splenic CD4⁺, and splenic CD8⁺ T cells, detected by intracellular staining with anti-ZAP-70, in Dox-treated Tet-on ZAP mice (n = 3 each, mean ± SD). (J) Surface expression of TCRβ, CD5, CD69, and HSA (CD24) on DP and CD4SP thymocytes in Tet-on ZAP mice treated with high or low dose Dox as in G. Percentages of positive cells are shown. Representative result of two independent experiments. (K) Relative protein expression levels of ZAP-70 in T cells compared to WT BALB/c. R.U., relative unit.