Figure 2. TGF-β3-induced T_H17 cells are highly pathogenic in inducing autoimmunity.

(a) Intracellular cytokine staining showing IL-17 expression of PMA+ionomycin stimulated naïve CD4⁺ T cells from C57BL/6 (wt) mice differentiated in vitro with TGF-β1-IL-6 or TGF-β3-IL-6 for four days. (b) ELISA showing IL-17 production from supernatants of naïve CD4⁺ T cells from C57BL/6 (wt) mice stimulated in vitro with TGF-β1, TGF-β3, TGF-β1-IL-6, TGF-β3-IL-6 for four days. (c,d) Quantitative RT-PCR of Rorc, IL-17a, IL-17f, IFNγ, Il23r and Il22 mRNA expression from wt naïve CD4⁺ T cells stimulated in vitro with TGF-β1-IL-6, TGF-β3-IL-6 or no cytokines for four days. (e) Flow cytometry analysis of IL-23R-GFP expression of naive CD4⁺ T cells from IL-23R-GFP knock-in mice differentiated in vitro with TGF-β1-IL-6, TGF-β3-IL-6 or no cytokines for four days (f) Mean clinical scores (disease incidence) in wild-type recipients 30 days after transfer of naïve CD4⁺ T cells (5×10⁶ cells) from 2D2 transgenic mice that were differentiated in vitro with TGF-β1-IL-6 or TGF-β3-IL-6. Statistical analysis by linear regression curve was performed and graphed including the 95% confidence band of the regression line (***p<0.001) (g) Quantification of CNS lesions in the meninges and parenchyma from recipient mice sacrificed on day 30 (**p<0.01). Data is pooled from three independent experiments (n=6). (h) TGF-β signaling PCR array analysis of Smad 1, 3, 5 from wt naïve CD4⁺ T cells differentiated with TGF-β1-IL-6 or TGF-β3-IL-6 for 24 hours. cDNA was prepared and processed according to manufacturer’s instruction (Sabioscience, CA). The graph illustrates the relative fold change in the Smads of TGF-β3-IL-6 condition compared to TGF-β1-IL-6. The dotted line represents the normalized expression of TGF-β1-IL-6 set at one for each of the Smads. The data is pooled from three independent replicates. (i) Flow cytometry analysis showing p-smad1&5 or p-smad3 expression from wt naïve CD4⁺ T cells stimulated with TGF-β1 or TGF-β3 for 30 minutes. Adoptive transfer data from (f) is a representative of four independent experiments. All data are a representative of more than three independent experiments, unless otherwise mentioned, with similar results. Statistical significance of *p<0.05, **p<0.01, or ***p<0.001 is indicated for the RT-PCR data. Error bars indicate mean ± s.d.