ZAC Tconv and Treg cells exhibit highly activated and inflammatory phenotypes with impaired Treg function. (A) Cell number of total splenocytes (left) and splenic Foxp3⁺CD4⁺ T cells (right) in 8-wk-old BALB/c, SKG, ZAC, and W163A mice (n = 11 each). (B and C) Representative FACS staining of indicated molecules expressed by splenic Foxp3⁻CD4⁺ T cells (B) and Foxp3⁺CD4⁺ Treg cells (C) from ZAC and BALB/c mice (n = 5). (D) Representative intracellular cytokine staining of splenic CD4⁺ T cells from BALB/c, ZAC, and IL-17A KO ZAC mice after PMA/ionomycin stimulation (n = 3). (E) In vitro suppression assay with WT or W163A Foxp3⁺ Treg cells with indicated WT CD4⁺ Tconv:Treg ratios (n = 7 each). (F) Representative plots of splenic ZAC and WT thy1.1⁺ Treg cells after 8 wk of WT Treg transfer as in Fig. 3 H. See also Fig. 3 I. (G and H) RAG2^−/− mice were transferred with WT CD4⁺ T cells, ZAC CD4⁺ T cells, or co-transferred with ZAC CD4⁺ T cells and WT Foxp3⁺CD4⁺ Treg cells from FIG BALB/c mice as in Fig. 3 I. Spleen (right) and spleen weight (left) after 10 wk of transfer (G), and frequency of diarrheic mice (H; n = 5 each). Scale bar: 3 mm in G. Two-tailed unpaired Student’s t test and mean ± SD in A, E, and G.