Arthritogenic Th17 cells and impairment of Treg function in ZAC mice. (A and B) Frequency of splenic CD4⁺ and CD8⁺ T cells (A) and their cell numbers (B) in 8-wk-old BALB/c, ZAC, SKG, and W163A mice (top, n = 11 each). Frequency of Foxp3⁺ Treg among CD4⁺ T cells (bottom, A and B) or CD44^hi CD4⁺ Tconv cells are also shown (B). (C) Representative intracellular cytokine staining of splenic CD4⁺ T cells from ZAC and BALB/c mice (n = 5 each). (D) IL-4 mRNA expression levels of freshly isolated splenic CD4⁺ T cells or CXCR5^hiPD-1^hi Tfh cells from Peyer’s patches (PP) of ZAC and BALB/c mice. IL-4 expression relative to GAPDH as a reference by quantitative RT-PCR (n = 5 each). (E) PD-1 and CXCR5 staining of CD4⁺ T cells from PP of BALB/c and ZAC mice. (F) Arthritis scores (left) and frequencies of diarrheic mice (right) in ZAC and IL-17A^−/− ZAC mice (n = 23 each). (G) In vitro suppression assay with WT or ZAC Foxp3⁺ Treg cells with WT CD4⁺ Tconv at indicated ratios (n = 7 each). (H) Arthritis scores (left) and frequency of diarrheic mice (right) of ZAC mice intravenously injected with 0.4 or 1.2 × 10⁶ WT Foxp3⁺Treg cells at 6 wk of age (n = 7 each). (I) Ratios of splenic ZAC and WT Treg cells after 8 wk of WT Treg transfer as in H. WT Treg ratio in each chart. (J) Arthritis scores of RAG2^−/− recipients transferred with 1 × 10⁶ WT CD4⁺ T cells, 1 × 10⁶ ZAC CD4⁺ T cells, or 1 × 10⁶ ZAC CD4⁺ T and 2 × 10⁵ WT Foxp3⁺ Treg cells (n = 5 each). Mean ± SD in B, D, F–H, and J. Two-tailed unpaired Student’s t test in B, D, and G.