Figure 5: T-bet-deficient T cells are hyper-responsive to IL-23.

C57BL/6 Rag1^−/− mice were injected i.p. with either WT or Tbx21^−/− CD4⁺CD25^-CD45RB^hi T cells or a 1:1 mixtures of WT (CD45.1⁺) and Tbx21^−/−(CD45.2⁺) CD4⁺CD25⁻CD45RB^hi T cells. Mice were killed when recipients of Tbx21^−/− T cells developed clinical signs of disease (∼5 weeks) and assessed for intestinal inflammation. (a) Colitis scores. (b) Frequencies of IL-17A⁺ and/or IFN-γ⁺ cells in the colon of mice receiving mix of WT and Tbx21^−/− CD4⁺ T cells. (c) CD4⁺ T cells were purified by flow cytometry from the inflamed colon of mice receiving a mix of WT and Tbx21^−/− CD4⁺ T cells and mRNA levels of indicated genes were analysed by quantitative reverse transcription PCR (qRT-PCR). (d) ChIP-qRT-PCR for the occupancy of H3K4me3 or RNA Pol II around the TSS of indicated genes in CD4⁺ T cells purified as described in c. (e) Immunoblot for pY705-STAT3, total STAT3, EOMES, Runx3 and Actin on whole cells lysates of CD4⁺ T cells purified as in c, left unstimulated or stimulated with IL-23 for 12 h. (f) Concentrations of indicated cytokines secreted into culture medium by CD4⁺ T cells purified and stimulated as described in e. Data in a–c and f represent results from one of two independent experiments (n=5 for WT, n=6 for Tbx21^−/−, n=6 for WT+Tbx21^−/−). Bars are the mean and each symbol represents an individual mouse. Bars are the mean and error bars represent s.e.m. Data in d are pooled results from two independent experiments, bars are the mean and error bars represent s.d. *P<0.05, **P<0.01, ***P<0.001 as calculated by Mann–Whitney U test.