It is well established that TH17 cells differentiated in the presence of IL-6 and TGF-beta are not encephalitogenic. Instead, IL-23 treatment is necessary for the terminal differentiation of TH17 cells and acquisition of encephalitogenic potential. We observed that T-bet protein is not expressed in non-pathogenic TH17 cells, but its expression is restored in pathogenic TH17+IL-23 cells. Furthermore, Tbx21-/- TH17+IL-23 cells cannot induce EAE in WT recipients, which led us to hypothesize that T-bet induces expression of gene(s) which are necessary for triggering neuroinflammation by TH17+IL-23 cells. We have performed microarrays and T-bet specific ChIP-Seq analysis on IL-23 treated WT and T-bet deficient TH17 cells and obtained a list of candidate genes that are differentially regulated in the two groups. We are focusing specifically on a group of genes that were differentially expressed and were directly bound by T-bet in the promoter regions, or a group of genes that have been previously associated with a higher risk of developing MS in genome wide association studies. The experimental plan is to express the genes that are significantly downregulated in T-bet deficient TH17 cells by retroviral transduction, and test whether their expression can restore the pathogenic potential of Tbx21-/- TH17 cells in the adoptive transfer model of EAE. In addition to the gene complementation approach, we intend to silence the expression of a group of genes whose expression is higher in T-bet deficient TH17 cells than in WT controls. In this context, genes that have been associated with the immunosuppressive, regulatory or anergic phenotype are of a particular interest. For example, Ebi3 is a common subunit of two cytokines, interleukin (IL)-27 and IL-35. IL-27, which is produced by the cells of innate immune system, negatively regulates the differentiation of TH17 cells, and IL-35, a novel inhibitory cytokine, is specifically produced by regulatory T cells and is required for their maximal suppressive activity. We observed that Ebi3 transcripts are more abundant in T-bet deficient TH cells (TH0, TH1, TH17 and TH17 + IL-23) than in WT TH cells. Therefore, it is possible that CD4+ TH cells in T-bet knockout mice have more a regulatory than pathogenic phenotype, which could account for the resistance of Tbx21-/- mice to EAE. To directly address this question, we have crossed Tbx21-/- mice to Ebi3-/- mice, and we are functionally characterizing the CD4+ TH responses from WT, Tbx21-/-, Ebi3-/- and Tbx21-/-Ebi3-/- (double knockout) mice, and testing the susceptibility of the double knockout mice to EAE.
